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Drug Research

ACTA POLONIAEPHARMACEUTICAVOL. �7�4� � No. �5 September/October 201�7 � � � � � � ISSN 2353-5288

EDITOR-in-CHIEFAleksander P. Mazurek

National Medicines Institute, The Medical University of Warsaw

ASSOCIATE EDITORS

Jacek BojarskiMedical College, Jagiellonian University, KrakÛw

Micha≥ TomczykMedical University, Bia≥ystok

MANAGING EDITOR

Hanna PlataEditorial office, Polish Pharmaceutical Society, Warsaw

EXECUTIVE EDITORIAL BOARD

The Medical University of Warsaw

The Medical University of GdaÒsk

The Medical University of Warsaw

K. Marcinkowski University of Medical Sciences, PoznaÒ

The Medical University of Wroc≥aw

Polish Pharmaceutical Society, Warsaw

Czech Pharmaceutical Society

Charles Sturt University, Sydney

Pharmazeutisches Institut der Universit‰t, Bonn

DOV Pharmaceutical, Inc.

Semmelweis University of Medicine, Budapest

Boøenna GutkowskaRoman KaliszanJan PacheckaJan PawlaczykJanusz PlutaWitold WieniawskiPavel KomarekHenry Ostrowski-MeissnerErhard RˆderPhil SkolnickZolt·n Vincze

This Journal is published bimonthly by the Polish Pharmaceutical Society (Issued since 1937)

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Impact factor IF (2015): 0.877 (5-Year IF = 1.121)MNiSW score (2016): 15.00Index Copernicus ICV (2015): 127.01

Acta Poloniae Pharmaceutica ñ Drug Research

Volume 74, Number 5 September/October 2017

CONTENTS

REVIEW

1321. Iqbql Azhar, Ejaz Basheer, Zeeshan Javaid, Kashif Sohai, A review of emerging analytical techniques for standardization Mahmood Sheikh of herbal medicinal products.

1327. Nighat Fatima, Syed Aun Muhammad, Muneer Fungal metabolites and antimalarial drug discovery; a review.Ahmed Qazi, Ziaulallah Shah, Abida K. Khan, Aneela Maalik, Hummera Rafique, Abdul Mannan, Muhammad Dawood, Amara Mumtaz

ANALYSIS

1343. Aleksandra Groman, Eløbieta Stolarczyk, Marta Jatczak, Development and validation of gas chromatography methods Eløbieta Lipiec-Abramska for the control of volatile impurities in the pharmaceutical

substance dutasteride.

1353. Kanakapura Basavaiah, Nagaraju Swamy, Simple and sensitive spectrophotometric assay of isoniazid Umakanthappa Chandrashekar in pharmaceuticals using permanganate, methyl orange and

indigo carmine as reagent.

1365. Krzesimir Ciura, Joanna Nowakowska, Piotr Kawczak, Quantitative structure-retention relationships and Katarzyna Ewa Greber, Micha≥ Jan Markuszewski quantitative structure-activity relationships study of macrolide

antibiotics on micellar thin layer chromatography.

1373. Marek SzlÛsarczyk, Iwona Nowakowska, Robert Piech, Voltammetric determination of trace elements W≥odzimierz Opoka, Jan Krzek (Cu, Pb, Zn) in peloid-based pharmaceuticals.

1379. Jerzy Falandysz, Maria ChudziÒska, Danuta Bara≥kiewicz, Occurrence, variability and associations of trace metallic Martyna Saba, Yuan-Zhong Wang, Ji Zhang elements and arsenic in sclerotia of medicinal Wolfiporia

extensa from polymetallic soils in Yunnan, China.

DRUG BIOCHEMISTRY

1389. Nusrat Shaheen, Muhammad Uzair, Bashir Ahmad Ch, In vitro cytotoxicity of Sanchezia speciosa extracts on humanAlamgeer epithelial cervical cancer (HeLa) cell line.

1395. Seyyed Abbas Zojaji, Ahmad Ghorbani, Faezeh Ghasemi, Exposure to forskolin improves trans-differentiation of boneReza Shafiee-Nick marrow stromal cells into insulin producing cells; generating cells

with better responses to physiological and pharmacological agents

DRUG SYNTHESIS

1405. Aliya Ibrar, Shamyla Nawazish, Imtiaz Khan, Tayyaba Triazolothiadiazines as a new class of efficient DNA intercalatingNoor, Bushra Uzair, Syed Majid Bukhari, agents.Farhan A. Khan, Asma Zaidi

1413. Ritchu Sethi, Sandeep Arora, Deepika Saini, Design and synthesis of N-(benzimidazol-1-yl methyl)-benzamide Thakur Gurjeet Singh, Sandeep Jain derivatives as anti-inflammatory and analgesic agents.

1427. Usama Fathy, Rasha S. Gouhar, Hanem M. Awad Design, synthesis and anticancer activity of some novel 5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one derivatives.

1437. Marwa F. Ahmed, Dareen Jaiash, Amany Belal Synthesis and design of new bromoquinazoline derivatives with anti-breast cancer activity.

1447. Justyna Øwawiak, Jacek Kujawski, Lucjusz Zaprutko Structure - tuberculostatic activity evaluation among isomeric bicyclic nitroimidazoles.

1453. Ebtehal S. Al-Abdullah, Nadia G. Haress, Mogedda E. Synthesis and antimicrobial evaluation of some new quinaldine Haiba, Neama A. Mohamed, Amany Z. Mahmoud derivatives.

NATURAL DRUGS

1465. Masood-Ur-Rehman, Naveed Akhtar Formulation of a cream containing extract of Bombax ceiba and its ex vivo characterization under various storage conditions.

1471. Tayyeba Rehman, Saeed Ahmad, Waheed Mumtaz Evaluation of antibacterial and carbonic anhydrase inhibitory Abbasi, Ashfaq Ahmad, Muhammad Bilal, Muhammad potential of methanolic extract of Nardostachys jatamansiMohsin Zaman, Aymen Owais Ghauri, Adeel Arshad, (D. Don) DC rhizomes.Khalid Akhtar

1477. Alamgeer, Khadija, Zain Nasir, Muhammad Naeem Evaluation of hepatoprotective potential of Euphorbia prostrataQaisar, Ambreen Malik Uttra, Kifayat Ullah Khan, ait extract against chemically induced hepatotoxicity in albino Ikram Ullah Khan, Muhamamd Saleem, Haseeb Ahsan, mice.Hira Asif, Amber Sharif, Waqas Younis, Huma Naz

1485. Sadia Javed, Amair Raza, Saqib Mahmood, Munazzah Strain improvement of Aspergillus terreus for hyper-productionMeraj, Farah Naz, Sameera Hassan of lovastatin and immobilization of mutant atu-06 strain for

repeated batch culture process.

1493. Leila Smail, Sihem Berdja, Boualem Saka, Samia Therapeutic effect of Brassica rapa var rapifera in type 2 diabetes Neggazi, Saliha Boumaza, Yasmina Benazzoug, induced by a high fat-sucrose diet of Psammomys obesus.Ghouti Kacimi, Lynda Boudarene, Souhila Aouichat Bouguerra

PHARMACEUTICAL TECHNOLOGY

1501. Grzegorz Do≥Íga, Zbigniew MarczyÒski, Mechanistic models in the assessment of drotaverine Marian M. Zgoda hydrochloride solubility after tablet disintegration in the presence

of selected excipients in pharmacopoeial acceptor fluid.

1513. Aqeel Aslam, Sajid Bashir, Babar Murtaza Formulation, development and in vitro evaluation of a novel sustained release hydrodynamically balanced gastroretentive dosage form for venlafaxine HCl.

1527. Tadeusz W≥adys≥aw Hermann Application of Laplace transforms to a pharmacokinetic opentwo-compartment body model.

1533. Aleksandra Amelian, Emilia SzymaÒska, Formulation and characterization of loratadine containing Katarzyna Winnicka orodispersible lyophilizates and films.

1543. Gurjeet Singh, Ghanshyam Das Gupta, Shailesh Sharma Effect of polymer and method on particle size and crystallinity of olmesartan medoxomil during the formulation of solid dispersions.

1563. Nayab Tahir, Asadullah Madni, Prince Muhammad Formulation and compatibility assessment of PLGA/lecithin basedKashif, Mubashar Rehman, Ahmed Raza, Muhammad lipid-polymer hybrid nanoparticles containing doxorubicin. Imran Khan, Muhammad Abdur Rahim, Abdul Jabar

PHARMACOLOGY

1573. Bushra Nasir, Nazar Muhammad Ranjha, Muhammad Pharmacokinetic studies of 5-fluorouracil in rabbit plasma.Hanif, Saira Nasir, Ghulam Abbas, Amina Afzal, Sheikh Abdur Rashid, Furqan Iqbal

1579. £ukasz Dobrek, Beata Skowron, Agnieszka Baranowska, Nephrotoxicity of a single dose of cyclophosphamideKatarzyna Ciesielczyk, Marta KopaÒska, Piotr Thor and ifosfamide in rats.

1591. Asta Kubiliene, Audrius Sveikata, Andrejus Zevzikovas, Investigation into pharmacokinetic properties of active alkaloidIlona Sadauskiene, Leonid Ivanov ibogaine and its metabolite noribogaine.

GENERAL

1599. Katarzyna Anna Grucha≥a and Agnieszka Zimmermann Law and economics of branded-to-generic, generic-to-generic and generic-to-branded substitution process in Poland.

1607. Orsolya Somogyi, Rom·na ZelkÛ The efficacy of written information about the application rules of transdermal patches - reading comprehension questionnaire survey in hungarian community pharmacies.

1613. Magdalena Waszyk - Nowaczyk, Anna Luczak, Evaluation of opinions on community pharmacy-based healthDawid Michalak, Joanna Kaczmarczyk, Halina Myrda, screenings for common chronic diseases.Micha≥ Michalak, Anna Ratka

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 5 pp. 1321ñ1326, 2017 ISSN 0001-6837Polish Pharmaceutical Society

Herbal medicines are widely accepted way oftreatment in both developed and developing coun-tries, WHO revealed that 80% of world populationstill uses herbs and other traditional medicine fortheir primary ailments (1). A 2012 survey revealedthat 32.2% of adult population consumes HerbalMedicine Products (HMP) in the United States ofAmerica (2). Moreover, this trend is not confined toNorth America, even in Scandinavian countries, forinstance, in Norway 39.7% of 600 surveyed preg-nant women depend upon herbal products, mostcommonly ginger, iron-rich herbs, Echinacea andcranberry (3). Although a very diverse range ofherbal medicines are in use across the continents,but unfortunately the traditional herbal productshave not been officially recognized. This indiffer-ence towards herbal products might be due to pro-crastination in this field or because of insufficientresearch. The quality, safety and efficacy data tolegitimize the herbal drug according to western

medicine regulatory authorities is inadequate andnot up to mark in comparison with prevailing allo-pathic medicinal system. The main enigma of herbalmedicine is their assorted chemical composition,which is due to multiple factors, these factor variesfrom botanical species, chemo types, parts in use(root, rhizomes, twigs, seeds leaf etc.). In addition,process of collection, drying, curing, atmosphericmoisture, day latitude, altitude, variability in har-vesting and geographical conditions are also respon-sible for variation in contents of herbal drugs (4). Ina nut shell a herbal medicine is diverse in its chemi-cal composition from batch to batch, which eventu-ally leads to significant pharmacological activityvariation, this activity divergence may lead to phar-macodynamics variability or pharmacokinetics dif-ference within the same batch or in different batch-es of same finished herbal product (5).

In order to overcome these possible pitfalls insafety, efficacy, authentication and for establish-

REVIEW

A REVIEW OF EMERGING ANALYTICAL TECHNIQUES FOR STANDARDIZATION OF HERBAL MEDICINAL PRODUCTS

IQBQL AZHAR1, EJAZ BASHEER2*, ZEESHAN JAVAID2, KASHIF SOHAIL2

and MAHMOOD SHEIKH1

1Department of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Pakistan2Akson College of Pharmacy, Mirpur University of Science & Technology, Mirpur AJ&K, Pakistan

Abstract: Herbal Medicine Products (HMP) are now in limelight across the globe by virtue of their particulartheory and long history of practice. But inadequate and uncontrolled quality control procedures are major hur-dles in their official recognition and prevalence as an established medical system. During hey days of herbaldrugs, herbal practitioners carefully formulate and dispense the drug according to need of individual patient.But today, commercialization scenario has changed much. HMP are being manufactured on commercial scalewhere manufacturer have to tackle different problems. For instance, availability of good quality of raw materi-al and its authentication, provision of quality standards, instruments for standardization of either single or polyherbal medicine, assurance of quality from vary step of propagation of herbs to packaging of finial dosage form.The scope of this review encompasses analytical techniques especially spectrophotometric and chromato-graphic fingerprint methods of analysis for standardization of HMP. Furthermore, evaluation techniques ofbioactive markers from herbal compounds through bio-chromatographic and conventional chromatographicprocedures. Such as, fingerprint and multi-constituents quantification in particular and coupling of chemicaland biological approaches such as biofingerprint and metabolic fingerprint in general are discussed here. Insummary, the analysis and standardization of HMP is in progress to better address the inherent holistic natureof HMP.

Keywords: Herbal Medicine Products (HMP), quality control, bioactive markers, chromatography fingerprint

1321

* Corresponding author: e-mail: [email protected]; phone: 0092-332-0073910

1322 IQBQL AZHAR et al.

ment of rationality between the herbal componentand conventional usage of HMP a multidiscipli-nary approach is pivotal (6). Getting beneficial factfrom samples with heterogeneous chemical con-stituents has long been a herculean task. Herbalformulations are comprised of numerous chemicalcompounds of variety of chemical structures,among them merely few, if not single, have beenrecognized by scientific screening, which areresponsible for either therapeutic and/or toxicpotential (7). Standardization of herbal productsthrough spectrophotometric and chromatographicfingerprint analysis is quite successful to resolvethis quality control riddle.

Standardization of HMP

Standardization interpreted by AmericanHerbal Product Association as ìStandardizationrefers to the body of information and controls nec-essary to produce material of reasonable consisten-cy. This is achieved through minimizing the inher-ent variation of natural product composition throughquality assurance practices applied to agriculturaland manufacturingî. Furthermore, standardizationeliminates or reduces batch to batch variation;ensure safety efficacy, quality and acceptability ofHMP. A herbal product cannot be recognized scien-tifically effectual if drug test has not been estab-lished and characterized to ascertain reproducibilityin manufacturing process (8).

The active pharmaceutical ingredients (API) ofherbal medicinal products are generally defined tobe the whole herbal preparation, e.g., the extract inits eternity (9). Individual or groups of constituentshave only been identified in selected cases to beonus for therapeutic activity. As the whole herbalproduct, e.g., extract, is regarded as the active phar-maceutical ingredient. Several extracts typesdepending on toxicological, pharmacological, phar-maceutical analytical and clinical findings, could bediscriminated into 3 categories (A, B1 and B2) (9).Extracts of Type A: this type contains either singleor groups of constituents which are solely acknowl-edged for recognized therapeutic activity.Standardization of a defined content is acceptable.In Chinese Herbal Pharmacopeia quality of 529herbs out of recorded 1203 raw materials of polyherbal formulations is based on the use of singleevaluation marker (10).

Extracts Type B1 contains well defined chem-ical constituents either single or groups which showrelevant pharmacological properties (active mark-ers). Active markers are likely to be responsible forclinical efficacy, however, evidences that they are

the only capable constituents in their respectiveHMP for clinical efficacy need evidences hitherto.The available data of efficacy, quality and safety ofan extract should be taken into consideration (9).

Extract Type B2 containing neither single norgroups of constituents responsible for efficacy, orrelevant pharmacological activity. To standardizethese types of compounds chemically defined con-stituents (markers) without known therapeutic activ-ity may be useful as a standard. These markers alsoserve to ensure good manufacturing practice or asbases for content assay of drug products (9).However, for most poly herbal medicines, the chem-ical or therapeutic components have not been fullyelucidated or easily monitored.

Fingerprint analyses fill this void to someextent, since it emphasizes more of the characteris-tic of poly herbal drug formulations. Fingerprint is acharacteristic profile or pattern which chemicallyinterpret the sample composition and in which, usu-ally, maximum information is depicted. By andlarge, fingerprints could be obtained through sever-al techniques, both chromatographic and spectro-scopic (11). Fingerprint method is widely acceptedanalytical standards by different official or non-offi-cial organizations like World Health Organization(12), British Herbal Medicine Association (13),German Commission E (14), and European Medi-cine Agency (15). Furthermore, compulsory finger-print analysis is purposed requirement for Chineseherbal products from Chinese State Food and DrugAdministration since 2004 (16).

Fingerprint analysis addresses two main issues;1. How to attain authentic and effective infor-

mation? 2. How to determine similarity with chromato-

graphic methods?Generally, chromatographic fingerprint pro-

cess is executed by determination of common andstable compound by screening out large number ofsamples by utilizing different chromatographic andseparation techniques.

Hyphenated detection methods

Detection of the target compounds is subse-quent step after sample separation. For detection,hyphenation of different techniques include ultravi-olet (UV) or diode-array detection (DAD), evapora-tive light scattering detection (ELSD), chargedaerosol detection (CAD), mass spectroscopy andenzyme-linked immunosorbent assay (MS &ELISA). A combination of multiple techniquescould provide relative analytical data for instance diode-array detection ñ evaporative light scattering

A review of emerging analytical techniques for standardization... 1323

detection (DAD- ELSD) and diode-array detection ñmass spectroscopy (DAD-MS).

Liquid chromatography ñ diode-array detection/

mass spectrometry (LC-DAD/MS)

It is a recommended technique for determina-tion of unspecified compounds by comparison withstandards. Ding and colleagues have determined thealkaloids in Corydalis yanhusuo using LC-MS/MSand LC-DAD (18). In addition, Lee et al. used LC-MS for quick identification of aristolochic acid con-tent in plant medicines (19). LC-NMR has distinctsuperiority to unambiguously identify the structuresof compounds. However, this method has somedownsides, such as relatively low sensitivity andhigh cost.

Ultraviolet (UV) or diode-array detection (DAD)

Since its inception, UV is most pervasive andmainstream analytical technology. It is ubiquitous inlaboratories, inexpensive, and easy to use. Never-theless, it has its downsides, poor absorbancedecrease sensitivity of UV due to intervention ofshort wavelength compounds. In addition, the rangeof mobile phase is narrow and option for selection ofmodifiers is also restricted. UV integrated success-fully with liquid chromatography and capillary elec-trophoresis for herbal products. DAD is now moreprevalent than UV due to its ability to measure widerange of wavelengths and capability to deliverinstant on-the-fly spectrum with the edge ofenhanced sensitivity and peak precision (20).Contemporarily, UV/DAD effectively integratedwith mass spectroscopy. UV/DADñMS techniquesfor quick qualitative and quantitative analysis ofHMP. Fan et al. determined ten major active com-ponents in Carthamus tinctorius L. to carry out aqualitative and quantitative evaluation based onHPLC-DAD. The relative standard deviation peakarea for each of the ten bioactive compounds wascalculated, respectively, to validate the precision ofthe method (21).

Evaporative light scattering detection (ELSD)

ELSD is pervasive, nonspecific analytical tech-nique that provides a steady starting line even withgradient elution (22). In addition, for enhanced dis-crimination of analyte constituents, volatile mobilephase modifiers, for example, formic acid and aceticacid are successfully used (23). An HPLCñELSDmethod has been developed for simultaneous moni-toring of 12 ginseng saponins in different parts of P.quinquefolius, (24). 14 saponins in red P. ginseng,(25) and 19 saponins in black ginseng (26).

Charged aerosol detection (CAD)

Nevertheless, ELASD is an inappropriate tech-nique for detection of non UV, weakly UV absorb-ing, and UV range absorbing samples without refer-ence standard. In such cases charged aerosol detec-tion remains method of choice. Mass is workingprinciple for either charged aerosol detection orELSD. This technique has a distinction, that neitherphysiochemical nor spectral properties of analytecould affect final result. With an identificationmethod that produce universal response factors,there is potential for a single universal standard forcalibration against which all other compounds orimpurities can be qualified (27). CAD has an edge ofincreased sensitivity over ELSD system, broaderdynamic selection, convenient operation and unifor-mity of response elements. Bai et al. successfullymaneuvered a HPLC-CAD for the simultaneousdetermination of seven different saponins in Radixet Rhizoma Notoginseng (28).

Recent advancements

Although manipulating several active ingredi-ents as evaluation markers is an effective technique,but multicomponent composition of herbal medicinerender their separation and screening substantiallydifficult, moreover many plants share the same com-pounds which further complex their standardization.This method might be inadequate to confirm theidentity of specific plants. To curb this limitationmore feasible techniques are needed. Followingadvances address these issue to certain extent.

Multiple-patterns of chromatographic finger-

prints

However, chromatographic finger print acclai-med as an appropriate and suitable analyticalmethod for quality control of HMP but usually a sin-gle chromatogram is assessed to carry out this analy-sis. By considering complex and multi componentscomposition of herbal product, a single chro-matogram could not characterize all chemical pat-terns or characteristics of sample. It is practically outof question to outline an analytical method to repre-sent all chemical features of constituents in a singlechromatogram (29, 30). To solve this problem acombination of analytical methods with multipleseparation techniques and analytical conditions areemployed. This multi-disciplinary approach is alsorecognized by FDA and Chinese State Food andDrug Administration (13, 30). It became inevitableto devise a method which could integrate multiplechromatographic fingerprint to demonstrate thecomplex multi constituent composition of herbal


formulations for determination of quality. Fan et al.carried out investigation of Danshe Dropping Pilland Panax notoginseng by multiple chromatograph-ic fingerprint for quality control. They opted for twoextraction methods to create multiple HPLC chro-matograms, then exploit retention time, UVabsorbance and MS spectra for qualitative and quan-titative analysis and finally a data level informationfusion method is manipulated to obtain integratedchemical features of binary fingerprints. The result-ant multiple fingerprints ensured lot to lot consisten-cy and avoidance of fraudulent material by usingsimilarity measures and by chemometric approach(29). Unequivocally, multiple chromatographic fin-gerprint will give detailed and precise informationabout plant material.

HPLC fingerprint integrated with multiple com-

ponent quantified assessment

In contrast with synthetic drugs, it is estab-lished fact that therapeutic potential of HMP is con-nected with synergism of their heterogeneous com-positions and different target sites on that they actupon. In chromatographic fingerprint analysis ofsuch complex compounds an analogy is drawnbetween similarity and differences of single markersof various samples which demonstrate overall viewof subjected herbal drug. However, one drawback ofthis technique is that it can only demonstrate resultby showing similarity calculated on relative magni-tude by using already known marker compound as areference standard. Little variability among alikechromatograms may not be differentiated. As a con-sequence, multiple component analysis should betaken into account for plausible illustration of HMP.Yang et al. first time reported chromatographic fin-gerprint analysis and concomitant determination ofeleven active compound in traditional Chinese med-icine Shuang-huang-lian (SHL) oral liquid formula-tion by HPLC-DAD. This novel analytical methodresolved the problem created by comparative analy-sis of single marker in sample. Moreover it provid-ed greater qualitative information than any other sin-gular evaluation and proved to be a simple, sensi-tive, accurate and reliable quality control procedurefor SHL oral liquid sample (31).

Exploitation of biochromatographic fingerprints

for bioactive markers exploration

Prevalent techniques for standardization ofherbal medicine are either compound based or pat-tern based. Compound focused technique aims par-ticular component having defined molecular struc-ture and pharmacological activity. On the other

hand, pattern oriented technique focuses alldetectable components (either they are pharmaco-logically active or not). Although pattern orientedmethods which utilize chemical markers serve thepurpose of analysis but ideal chemical markershould be a characteristic component of knownpharmacological activity to ascertain quality.Conventional way of biological screening in whichisolation of chemical compound is done first thenscreening its activity in vitro or in vivo is carried outthrough animal models, organ and tissue models,cellular models or receptor and enzyme models.These techniques proved time consuming, arduousand inadequate for recognition of synergetic activi-ties of HMP. By taking this into consideration,screening of bioactive components becomeinevitable for authentication of the therapeutic bases,exploration of leading compounds and for provisionof appropriate chemical markers for standardization.To solve this conundrum advance techniques like biofingerprinting chromatogram analysis are preferred,in which comparative assessment of fingerprint chro-matograms of the extract of HMP prior and afterwardthe interaction with biological systems is carried out.In the first step, immobilization of certain biomole-cule as solid phase is done to reflect the affinity inter-action between the analytes and non-column targets.In the second step, to interact herbal drug with asome target macro biomolecules (DNA, protein, cell,enzyme etc) and the third step is to study the metab-olism of HMP in vivo or in vitro and then analyze themetabolic fingerprint (32-35).

Isolation of each constituent of HPMs is soonerous that the aforementioned techniques arequite plausible. In addition, along with definedactive components some new compounds are likelyto be detected, this phenomenon also demonstratethat how HMP exert their synergistic or antagonisticpotential (34). For instance, a novel approach ofintegrating HPLC with microanalysis sampling inte-grated with DNA attachment for evaluation HMPextract.

In analysis of traditional Chinese medicineseven compounds were attached to calf thymusDNA from the Coptis chinesis Franch (Coptis). Butonly three from Phellodendron amurense Rupr.(Phellodendron) and none from Sophora flavescensAit to bind ct-DNA, respectively. Three of themwere identified as berberine, palmatine and jatror-rhizine and their association constant (K) to ct-DNAwere determined by microdialysis/HPL (34).

Nevertheless, limitation of these methodsshould also be taken into account during carryingout of these analyses. First, each model is not appro-

A review of emerging analytical techniques for standardization... 1325

priate for all HMP, for instance, no potential com-ponent is detected in DNA model analysis ofSophora flavescens (34). Ferulic acid, one of themain active compounds of DBD, could not appear inseveral screening methods. The parallelism of dif-ferent screening methods is not perfect, so it easilycreates mess. For example, the potential constituentsof different Chinese medicine are different in differ-ent screening strategies (35-37). This is possible dueto multi target and multi compounds characteristicof HMP. Combining with the activity verification invivo or in vitro is done in order to find those whichcan precisely predict the active components. In anutshell, bio fingerprint analyses is effective andswift approach for profiling the bioactive markers ofherbal products they also demonstrate potentialfuture for standardization of herbal products.

Quantitative analysis of multi components by sin-

gle marker (QASM)

Provision of certified standards of evaluationmarkers, is not an easy task because of shortcomingsin their separation, stability, maintenance and supplywhich render their use in assessable. To counterthese shortcoming Wang et al. designed a novelmethod named quantitative analysis of multi com-ponents by single marker (QASM), which manipu-lates a single marker to concurrent detection of mul-tiple inaccessible components because active com-ponents in plants having inherent functional andproportional relationships. The relative retentionvalues between the target compound and singlemarker were used for qualitative analysis, whiletheir relative correction factor applied to quantita-tive analysis (38).

Thin layer chromatography ñ bio ñ autographic

assay (TLC-BAA)

It is a well-developed method combining TLCisolation with biologic activity screening. It canswiftly detect and distinguish bioactive constituentsin a complex herbal formulation and has an edge ofconvenience, simplification and no need of specialequipment. From very first day of introduction ofanti-bacterial drug screening using paper chro-matography bio-autography, it is recognized andwidely acclaimed as a rapid activity-screening tool(39). Furthermore, it got acceptance for the activityscreening of pure chemical entities (40) and theactivity guided purification of bioactive naturalcompounds, for instance, antibacterial compounds(41-43), antifungal (44, 45), cholinesterase inhibi-tors (46), and free radical scavenging and antioxi-dant active ingredients (47). TLC-BBA is a swift

and simple mean to compare characteristic profileand fingerprint activity of HMP. Nevertheless, dueto its drawbacks, such as constraints in the polarityof the tested compounds and its sensitivity to bacte-ria on a TLC plate, endeavors are needed to establishTLC-BBA for broader application in quality controlof HMP.

CONCLUSION

This review briefly describes the progress andexisting standing of standardization of herbal prod-ucts, especially quality control methods and tech-nologies that have been currently introduced. Thesedevelopments stipulated that standardization andanalysis has already been entered to a new phase.This is not trivial to apply latest instruments eithersolely or in combination for quality control of cen-turies old medicinal products. But so far discussed inthis review and whatever has been done in this regardis just embankment on long expedition. Use of chro-matographic fingerprint of herbal medicine standard-ization only applied to compare the relative similari-ties and/or differences and monitoring their stability.The complicated nexus between efficacy of HMPand chromatographic fingerprints is still a grey area.Efficacy of HMP is attributed to mixture of com-pounds present in the herbs, which emphasize thatrational evaluation of their relation could not be neg-lected. HMP pose a challenge for researchers byvariation in activity of single herb and complexity ofpoly herbal formulations. Moreover, utilization ofjust chemical profiling is not enough for determina-tion of efficacy of HMP. This is area where integra-tion of interdisciplinary approaches like biochem-istry, molecular biology or cell biology might be suc-cessful. Chemical fingerprints could be linked tothese biological assays to provide a prudent solutionfor efficacy and quality issues of HMP, but hithertowork on this aspect is too little too short to meet thenecessary criteria. Thus to establish a methodologi-cal correlation between chromatographic fingerprintsand efficacy of HMP is a need of hour.

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Received: 31. 08. 2016


Malaria is the worldís greatest life bullyingparasitic infection that impacts on the productivityof community due to morbidity and mortality. Thecause of this disease is protozoan parasites of thegenus Plasmodium that infects human hosts, and isa critical world-wide health matter with about 200million cases and 1 million deceases in 2010 (1).The complicated lifespan of malaria parasite con-sists of two hosts and five tissues of host whilsthaving at least ten different morphological moves(2). Clinical symptoms of malaria are due to repeti-tion of parasites, subsequent destruction of erythro-cytes and most of medicines target asexual (human-host) stages of the lifespam. Clinically, manifesta-tions of malaria are chills, anemia, fever and pros-tration. Severity of this disease may lead to cerebralmalaria, delirium, metabolic acidosis, coma anddeath (3). Even after clearance of parasite frombloodstream relapse can occur because of the pres-

ence of some strains of malaria, particularlyPlasmodium vivax, can be in a latent liver hypno-zoite system (4). Major cause of severe malaria is P.falciparum which is a reason of majority of deathsamong five relevant species of human parasites (5).Therefore, drug development efforts are concentrat-ing on P. falciparum, generally at the expense ofother species.

New medications with exclusive structures andappliances of action are required to overcome thedrug resistance issues by continuous evolution of theparasite (6). Therefore, disease governor and treat-ment has been thorny by the advent of resistance tobroadly used antimalarial drugs reinforcing the vitalrequirement for new treatments. To overcome theproblem of drug confrontation, newer lead com-pounds and drug targets are required. Fortuitously, alarge amount of reserves have been focused on theway to antimalarial lead compounds finding in the

FUNGAL METABOLITES AND ANTIMALARIAL DRUG DISCOVERY; A REVIEW

NIGHAT FATIMA1*, SYED AUN MUHAMMAD2, MUNEER AHMED QAZI3, ZIA ULALLAH SHAH4,ABIDA K. KHAN5, ANEELA MAALIK5, HUMMERA RAFIQUE6, ABDUL MANNAN1,

MUHAMMAD DAWOOD1 and AMARA MUMTAZ5*

1Department of Pharmacy, COMSATS Institute of Information Technology, 22060 Abbottabad Pakistan2Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan

3Department of Microbiology, Shah Abdul Latif University, Khairpur, 66020 Sindh, Pakistan4Department of Pharmacy, Sarhad University of Science and Information Technology,

Peshawar, Pakistan5Department of Chemistry, COMSATS Institute of Information Technology, 22060 Abbottabad, Pakistan

6Department of Chemistry, University of Gujrat, Pakistan

Abstract: Malaria is very dangerous and destructive parasitic infection in many underdeveloped countries. Thecurrent scenario of this disease is getting eviler, mainly due to the growing battle of Plasmodium falciparum incontradiction of the pillar drug like chloroquine. There is a need of hours to discover novel drugs with uniquestructures and mechanism of action to treat drug resistant and sensitive strains of malaria. Natural products withunique structures are the major source for the lead discoveries and drug development against various diseases.This review presents recent advances and development in the field of new discoveries of antimalarial drug iso-lated from natural sources, especially fungi. This review covers investigation of fungal extracts, isolation andpurification of chemically diverse secondary metabolites (macrolides, terpenes, peptides, xanthones andcoumarines) exhibiting antimalarial activities. In sum these fungal metabolites can be used as novel scaffoldsfor drug development as antimalarial.

Keywords: antimalarial, Plasmodium falciparum, fungi, natural compounds

1327

* Corresponding authors: Nighat Fatima: e-mail: [email protected] ,phone: 092-0333 5198101;Amara Mumtaz: e-mail: ama-

[email protected] , phone: 092-0300 5316570s

1328 NIGHAT FATIMA et al.

past limited decades and fungi are considered as animportant source of antimalarial compounds.

Antimalarial metabolites from fungi

The metabolites produced by fungi represent ause of biosynthetic pathways, a wide range ofmolecular diversity, and a particular power to mod-ulate biological processes (7). They signify one-fourth of all recognized bioactive natural products.Therefore, fungi exhibit a source for the discoveryof structurally novel and diverse natural productspossessing biomedical prospective (anticancer,antileishmanial, antibacterial and antimalarial).Many scientists have been investigated fungi forantimalarial metabolites production. In current

review we covered details of isolation of fungalmetabolites and their biological activities.Following are few of important classes of reportedmetabolites.

Macrolides

Macrolides are a collection of drugs containinga great macrocyclic lactone ring with one or moredeoxy sugars. These are polyketide type of second-ary metabolites. Tanaka and his coworkers studiedantimalarial effects of fungal metabolite, radicicol(1), a macrocylic lactone antibiotic (8). In anotherstudy, Isaka and his colleagues reported resorcylicmacrolides aigialomycins A-E (2-6) purified fromAigialus parvus. Compound 5, aigialomycin D, was

Figure 1a. Fungal macrolides with antimalarial activity (1-6)

Figure 1b. Fungal macrolides with antimalarial activity (7-14)

Fungal metabolites and antimalarial drug discovery; a review 1329

found moderately active agent against P. falciparumwhich showed IC50 value of 6.6 µM/L (9).

Rukachaisirikul and his coworkers in 2004purified three new macrolides (7-9) along with sixrecognized compounds, cepharosporolides C (10) E(11) and F (12) 2-carboxymethyl-4-(3-hydroxy-butyl) furan (13) cordycepin (14) from fungusCordyceps militaris BCC 2816. The antimalarialactivity of 7-10 and 14 against P. falciparum K1 wasestimated. Only compound 14 showed noteworthyeffect with IC50 value of 4.5 µg/mL (10).

Another class of macrolides resorcylic acid lac-tones paecilomycins was also reported for antimalar-

ial activities. Six new paecilomycins A-F (15-20)and five acknowledged aigilomycin B (21), zeaenol(22), aigialomycin D (23), aigialomycin F (24) andaigialospirol were secluded from Paecilomyces sp.SC0924. Compounds 19 and 20 showed significantantimalarial activity in contradiction with P. falci-parum line 3D7 with IC50 values of 20.0 and 10.9 nM,respectively, while rest of the compounds showedmoderate activity (11). One year later, Xu and hiscolleagues isolated three paecilomycins G-I (26-28)from culture broth of the same fungus (12).

Three novel caprolactams (cyclic amide ofcaproic acid) named as pestalactams AñC (29-31),

Figure 1c. Fungal macrolides with antimalarial activity (15-28)


were purified from Pestalotiopsis sp., an endophyticfungus of Melaleuca quinquenervia. Pestalactams A(29) and B (31) showed moderate activity againstchloroquine-sensitive (IC50 16.2 and 20.7 µM, respec-tively) and chloroquine-resistant (IC50 41.3 and 36.3µM, respectively) cell lines of P. falciparum (13).

Piperazine derivatives

Piperazines are six membered ring containingorganic compounds possessing diverse pharmaco-logical activities (antifungal, antibacterial, anti-cancer and antituberculosis) because of piperazinefunctional group. Tanaka, Hatabu and their col-leagues reported that fungal metabolite gliotoxin(32) is an epipolythiodioxopiperazine toxin exhibit-ing significant plasmodicidal activity (8, 14).Gliotoxin is a sulfur containing mycotoxin producedby several species of fungi (15). In another studytwo ketopiperazines janoxepin (33) and brevicom-panine B (34) were separated from Aspergillusjanus. Janoxepin was a unique oxepin derivativepossessing a rare D-leucine integrated. Both com-pounds showed antimalarial activity against P. falci-parum 3D7 (IC50 values of 28 and 35 mg/mL,respectively) (16).

Terpene metabolites

Terpenes are a huge class of naturally occur-ring hydrocarbons consisting of multiple isopreneunits. Different classes of terpenic compoundsinclude diterpenes, triterpenes, sesquiterpenes,steroidal lanosterol have been reported from variousorganisms such as plants and microorganism.

Tanaka and his colleagues reported a sesquiter-pene antibiotic heptilidic acid (35) from fungi whichshowed antimalarial activities (8). Five trichothecenes(36-40) were isolated from Myrothecium verrucariaBCC 112. Trichothecenes are sesquiterpene mycotox-ins produced by various fungal species possessing 12,13-epoxy ring in their structure critical for biologicalpotential. These compounds showed significant anti-malarial activities with EC50 values ranging from 0.06to 0.90 ng/µL against P. falciparium (17). Later on,

two more trichothecenes (41-42) compounds werealso reported from Mangrove endophytic fungi.Compounds 41 and 42 showed potent antimalarialactivity with IC50 value below 20 nM (18). Isaka andhis colleagues in 2000 reported two unique sesquiter-penes: (+)-phomenone (43) and (+)-phaseolinone (44)from Xylaria sp. BCC 1067. Both compounds showedpromising antimalarial action with EC50 values of 0.50and 0.32 µg/mL, respectively (19).

Figure 1d. Fungal macrolides with antimalarial activity (29-31)

Figure 2. Fungal piperazine metabolites with antimalarial activity


In 2002, fungus Drechslera dematioidea wassecluded from the interior tissue of the marine redalga Liagora viscida (20). Ten new sesquiter-penoids, specifically drechslerines AñG (47ñ52),isosativenetriol (45), sativene epoxide (54) and 9-hydroxyhelminthosporol (53), However, drechs-lerins E (50) and G (52) and helminthosporol (53)

were isolated from Drechslera dematioidea. Thesecompounds exhibited antimalarial activity againstP. falciparum. Marine fungus Halorosellinia ocean-ica BCC 5149 from Thailand, produced anophiobolane sesterterpene, halorosellinic acid (54),which showed antimalarial activity (IC50 = 13µg/mL) (21). Later on, further investigation of thesame fungus led to isolation of another new

ophiobolane sesterterpene, 17-dehydroxyhaloro-sellinic acid (56) (22).

Rukachaisirikul and his colleagues isolatedtwo new sesquiterpenes, connatusins A&B (57, 58)and six known compounds (59-64) from the fungusLentinus connatus BCC 8996. Compounds (61)panepoxydone, (62) panepoxydione and (64) dihy-drohypnophilin exhibited significant antimalarialactivities with IC50 values of 3.4, 2.1 and 3.1 µg/mL,respectively (10). Chromatographic investigation ofthe extract of Ganoderma lucidum in ethyl acetateyielded seven triterpnes lanostanes (65ñ71) includ-ing three new (66, 67, 71) (Fig. 3c). These lanos-tanes exhibited moderate in vitro antiplasmodialactivity with IC50 values of 6 to 20 µM (23).

Figure 3a. Fungal terpene metabolites with antimalarial activity


Antimalarial activity of spirodihydrobenzofu-ran terpenes was stated for the first time bySawadjoon and his colleagues. They isolated twospirodihydrobenzofuran terpenic compounds (72)and 73 isolated from the fungus Stachybotrysnephrospora. Both compound showed antiplasmodi-al activity with IC50 values of 0.85 and 0.15 µg/mL,respectively (24). Dicholoromethane extract of anendophytic fungus Chalara alabamensis, secludedfrom Asterogyne martiana collected in Costa Ricadisplayed effective antimalarial activity against P.falciparum, with an EC50 value of 24 µg/mL. Furtherfractionation led to isolation of viridiol 74 (Fig. 3d),which showed EC50 value of 1.2 µg/mL against P.falciparum (25). Isaka and his colleagues reportedfive novel terpenoids sterostreins A-E (75, 76,77a/77b, 78, and 79) isolated from mushroomStereum ostrea BCC 22955. Sterostrein A unveiledpotent antimalarial activity (IC50 2.3 µg/mL) againstP. falciparum (26, 27).

Peptides

Peptides are biologically occurring smallchains of amino acids linked through peptide bonds.Peptides can act as selective signaling moleculesand are central in human physiology. Owing to theirstriking pharmacological profile signify them as anoutstanding starting point for novel design of thera-peutics. Nilanonta and his colleagues reported twocyclodepsipeptides from Paecilomyces tenuipesBCC 1614 (Fig. 4). Depsipeptide is a molecule thathas both peptide and ester linkages in proximity andplaying a vital role in early preclinical therapeuticdiscovery processes. Both compounds beauvericin(80) and beauvericin A (81) unveiled adequateantiplasmodial activities against K1 strain of P. fal-ciparum with EC50 values of 1.60 and 12.0 µg /mL,respectively (28).

Isaka and his colleagues also reported cyclo-hexadepsipeptides pullularins A-D (82-85) fromAureobasidium pullulans, isolated from a leaf of

Figure 3b. Fungal terpene metabolites with antimalarial activity


Culophyllum sp. from Thailand. Pullularin A and Bexhibited antimalarial activities with IC50 values of3.6 and 3.3 µg/mL, respectively (29). In anotherstudy Isaka and his group also reported isolation ofa new cyclohexadepsipeptide paecilodepsipeptide A(86) and its two linear analogues paecilodepsipep-tides B and C (87, 88) from Paecilomyces cinnamo-meus BCC 9616. Paecilodepsipeptide A (1) showedactivity in contradiction of P. falciparum K1 with anIC50 value of 4.9 µM (29) (Fig. 4)

Tetramic acid derivatives

Tetramic acid derivatives are nitrogenous hete-rocyclic compounds containing pyrrolidine-2,4-

dione. They are important structural units in manynatural products acquired from earthly and marinesources (30). They show a broad spectrum of bio-logical actions which includes: cytotoxic, antibiotic,antifungal, enzyme inhibitory and antiviral activities(31).

Osterhage and his coworkers reported two newunusual tetramic acid derivatives ascosalipyrrolidi-nones A (89) and B (90) as well as the new pyroneascosalipyrone (91) from marine endophytic fungusAscochyta salicorniae (Fig. 5). Ascosalipyrrolidino-ne A (89) showed antiplasmodial action in contra-diction of P. falciparum with IC50 value of 736ng/mL (32).

Figure 3c. Fungal terpene metabolites with antimalarial activity


Xanthones

Xanthones are polyphenolic compounds thatexhibit extensive biological and pharmacologicalactivities. Isaka and his colleagues reported isolationof two novel xanthone dimers, phomoxanthones Aand B (92, 93) along with compound 94 (deacetylanalogue of 92) from an endophytic fungusPhomopsis sp. BCC 1323 (33). Compounds 92 and93 demonstrated notable antimalarial actions in con-tradiction to P. falciparum K1 with IC50 values of0.11 and 0.33 µg/mL, respectively. In another study,ascherxanthone A (95), a novel tetrahydroxanthonedimer, was isolated from fungus Aschersonia sp.BCC 8401 (Fig. 6). Ascherxanthone A exhibitedactivity against P. falciparum K1 with an IC50 valueof 0.20 µg/mL (34). Pontius and his group evaluatedantiparasitic potential of extract of marineChaetomium fungus. They also reported isolationand purification of three new xanthones, chaetoxan-

thones A-C (96-98) (35). Compound 97 exposedselective activity against P. falciparum with IC50 of0.5 µg/mL.

Pyridone

Pyridone derivatives contain amide and ketogroups in it and are well known for their biologicalactivities. Four N-hydroxy- and N-methoxy-2-pyri-done compounds cordypyridones A-D (99-102)were purified from Cordyceps nipponica BCC 1389(Fig. 7). Cordypyridones A and B exhibited com-pelling antimalarial action with IC50 values of 0.066and 0.037 µg/mL, respectively (36).

Coumarins

Coumarin is a natural organic compoundbelonging to benzopyrones possessing biologicalactivities (37). Kongsaeree and his coworkers isolat-ed three novel antimalarial dihydroisocoumarin

Figure 3d. Fungal terpene metabolites with antimalarial activity


derivatives (103-105) from an endophytic fungus,Geotrichum sp. (38). Compounds 103 and 105

showed antimalarial action in contradiction of P. fal-ciparum with IC50 values of 4.7 and 2.6 µg/mL,respectively. In 2008, Sappapan and his coworkersreported isolation of a new analogue of monocerin,11-hydroxymonocerin (107) along with known 12-

hydroxymonocerin (108) and monocerin (106) froman endophytic fungus Exserohilum rostratum (Fig.8). Monocerin and 11-hydroxymonocerin revealedaction in contradiction of P. falciparum K1 strainwith IC50 values of 0.68 and 7.70 µM, respectively(39).

Quinone derivative

Quinones are oxidized derivatives of aromaticcompounds. Quinones possess many pharmacologi-cal activities such as anti-tumor, purgative, antimi-crobial and antiparasitic. A quinone derivative (7-hydroxy-8-methoxy-3,6-dimethyldibenzofuran-1,4-dione) (109) and an organohalogen natural product(2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione) (110) were isolated from the organicextract of Xylaria sp. PBR-30 an endophytic fungusisolated from Sandoricum koetjape (Fig. 9).Compounds 109 and 110 showed antimalarialagainst P. falciparum K1 strain with IC50 values of1.84 and 6.68 µM, respectively (40).

Hydronaphthalenone derivatives

Sommart and his group reported isolation ofthree new hydronaphthalenone derivatives (110-112) from the unidentified PSU-N24 endophytic

Figure 4b. Fungal peptidal metabolites with antimalarial activity

Fgure 4a. Fungal peptidal metabolites with antimalarial activity


fungus of Garcinia nigrolineata (Fig. 10).Compound 112 showed antimalarial action with theIC50 value of 7.94 µg/mL (41).

Tryptophan derivatives

Codinaeopsin 113 a newfangled tryptophan-polyketide hybrid which comprises an unusual hete-

rocyclic unit linking indole and decalin fragments,was aquired from the crude extract Codinaeopsisgonytrichoides an endophytic fungus isolated fromVochysia guatemalensis obtained from Costa Rica(Fig. 11). Codinaeopsin showed activity against 3D7strain of P. falciparum with an IC50 value of 2.3µg/mL (42).

Figure 5. Fungal tetramic acid metabolites with antimalarial activity

Figure 6. Fungal xanthone metabolites with antimalarial activity


Figure 7. Fungal pyridone metabolites with antimalarial activity

Figure 8. Fungal coumarins metabolites with antimalarial activity

Figure 9. Fungal quinones metabolites with antimalarial activity

Figure 10. Fungal hydronaphthalenone metabolites with antimalarial activity


Tropane derivatives

Tropane is a nitrogenous compound which isbicyclic amine containing piperidine and pyrrolidinerings. Tropane is the same structural part of alltropane alkaloids (43). Tropane derivatives areantiemetics, antispasmodics, anesthetics, and bron-chodilators (44). Pycnidione (114), bistropolone avastly oxygenated compound obtained from marinefungi showed antimalarial activity in contradictionof three strains of P. falciparum FCR3F86, W2 andD6 (IC50 = 0.28, 0.37 and 0.75 µM, respectively)(45). Iwatsuki and his coworkers also reported five(115-119) tropolone compounds, isolated fromPenicillium sp. FKI-4410. Compounds (115, 116)were named as puberulic acid and stipitatic acid

Figure 11. Fungal tryptophan metabolites with antimalarial activ-ity

Figure 12. Fungal tropane metabolites with antimalarial activity

Figure 13. Fungal benzaldehyde derivatives metabolites with antimalarial activity


(Fig. 12). While three compounds were new analogsof puberulic acid, named as viticolins AñC (117-

119). Among them, puberulic acid (115) unveiledeffective antimalarial activity in contradiction ofchloroquine resistant P. falciparum strain with IC50

value of 0.01 µg/mL while other compounds did notshowed significant activity (46).

Benzaldehyde derivatives

Four benzaldehyde derivatives (E)-2-(hept-1-enyl)-3-(hydroxymethyl)-5-(3-methylbut-2-enyl)benzene-1,4-diol (120), flavoglaucin (121),tetrahydroauroglaucin (122), auroglaucin (124), and2-(2í,3-epoxy-1í,3í-heptadienyl)-6-hydroxy-5-(3-methyl-2-butenyl)benzaldehyde (125) demonstratedrestrained antimalarial actions with IC50 values rang-ing from 1.1 to 3.0 µg/mL when tested in contradic-tion of chloroquine-sensitive (D6, Sierra Leone) andchloroquine-resistant (W2, Indo China) strains of P.falciparum. Antimalarial activity of auroglaucin(124) was stronger than other compounds (IC50 1.8

and 1.1 µg/mL against D6 and W2, respectively)(47).

Cytochalasins

Cytochalasins are considered an importantclass of cytotoxic fungal metabolites. Cytochalasinspossess various biological actions. Calcul and hiscolleagues reported cytochalasins (128-135). Allcytochalasins were found energetic in contradictionof P. falciparum (3D7) with IC50 values between 20-136 nM (18).

Polyamine alkaloid

Bioassay-guided fractionation of extract offungus Ramaria subaurantiaca led to isolation ofknown polyamine alkaloid, pistillarin (136) whichexhibited significant antimalarial activity with IC50

of 1.9 µM (48).

Extracts with antimalarial activity

Wiyakrutta and his colleagues reported bio-logical actions of extracts of endophytic fungi

Figure 14. Fungal cytochalasins metabolites with antimalarial activity

Figure 15. Fungal alkaloidal metabolite with antimalarial activity


secluded from Thai medicinal plants. Extracts from92 endophytes were experienced for antimalarialactivity in contradiction of P. falciparum andextracts of six of the extracts showed significantactivity with IC50 values ranging between 1.2ñ9.1µg/mL (48). Singh and Parkash in 2005 reportedantimalarial activity of culture filtrate of Beauveriabassiana fungus. Govindarajan and his group in2005 studied antimalarial activity of culture filtrateof various fungal species (49). Antiplasmodialpotential of aqueous extract of a fungus,Chlorophyllum molybdites was determined in Swissalbino mice at a dose of 200 mg/kg/day/ given oral-ly. Results showed that extract had antimalarialpotential (50). Oluba and his coworkers reportedthat all biochemical derangements that characterizedP. berghei malarial infection in mice were restored tonormal/near normal levels following treatment withaqueous extract of the fruiting bodies of G. lucidum(51). In another study, Higginbotham and his col-leagues reported isolation of 2700 fungal endo-phytes and almost 16.9% of fungal genotypes select-ed in their work were extremely active in contradic-tion of P. falciparum (52). Eighty four different fun-gal extracts were studied for their antiplasmodialactivity in contradiction of blood stage P. falciparumin human red blood cell culture. Results publicizedthat fungal endophytes belonging to diverse generashowed antiplasmodial acition such as Fusarium sp.(IC50: 1.94 µg/mL) and Nigrospora sp. (IC50: 2.88µg/mL) (53).

Future perspectives and conclusion

New bioactive metabolites are a need of timedue to ever increasing dilemma of microbial resist-ance to current therapeutic and control agents alongwith emergence of new life threatening diseases.These problems have pushed scientists to look inunconventional sources like endophytes for the newnovel compounds. The fungus capacity to synthe-size a variety of new novel bioactive metabolitesforced researchers to explore these avenues.

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Received: 12. 09. 2016


Dutasteride (N-[2,5-bis(trifluoromethyl)phen-yl]-3-oxo-4-aza-5α-androst-1-ene-17β-carboxam-ide) is an active pharmaceutical ingredient (API)which inhibits both 1 and 2 type isozymes of 5-α-reductase, the enzyme responsible for convertingtestosterone to dihydrotestosterone in the prostateand other tissues. Dihydrotestosterone is the primarycause of prostate growth and has been proven toplay a key role in the development and progressionof benign prostatic hyperplasia and prostate cancer(1-4).

The process of synthesis that was used tomanufacture dutasteride is described in the litera-ture (5, 6). According to the synthesis route, manyorganic chemical compounds were used which can

be classified as residual solvents in conformity ofthe Q3C(R5) guideline by ICH (InternationalConference on Harmonization). In this guidelineall solvents are classified into three classes withdifferent acceptable limits, mainly based on theirtoxicity.

Residual solvents are defined as organicvolatile chemical compounds that are used through-out the process of pharmaceutical substance manu-facturing. The residues of solvents remain in thefinal product despite various attempts to removethem. It is essential to estimate and control the con-centration of every individual chemical compound.Gas chromatography based methods are recom-mended to quantify and quantitate residual solvents

ANALYSIS

DEVELOPMENT AND VALIDATION OF GAS CHROMATOGRAPHY METHODS FOR THE CONTROL OF VOLATILE IMPURITIES IN THE PHARMACEUTICAL SUBSTANCE DUTASTERIDE

ALEKSANDRA GROMAN*, ELØBIETA STOLARCZYK, MARTA JATCZAK and ELØBIETA LIPIEC-ABRAMSKA

Pharmaceutical Research Institute, R and D Analytical Chemistry Department, 8 Rydygiera St., 01-793 Warszawa, Poland

Abstract: Dutasteride, N-[2,5-bis(trifluoromethyl)phenyl]-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide, isan active pharmaceutical ingredient (API) which inhibits the conversion of testosterone to dihydrotestosterone.As a 5-reductase inhibitor dutasteride is useful for the treatment of benign prostatic hyperplasia (BPH) andprostate cancer. Because of a large variety of solvents and reagents used in the synthesis, it was necessary todevelop new, sensitive and selective gas chromatography (GC) methods. The optimization of the methods con-sisted in selecting different types of the sample injection and detection as well as the optimization of experi-mental conditions that allowed to meet the appropriate range of permissible limits and suitable detection limits(LOD) of compounds. Significant differences in the volatility of these compounds forced the division intovolatile solvents (methanol, acetonitrile, dichloromethane, ethyl acetate, heptane and toluene) analyzed with theuse of the gas chromatography method with headspace (GC-HS) and less volatile compounds (pyridine,dimethylformamide, 1,4-dioxane, acetic acid, ethylene glycol, 4-dimethylaminopyridine) which were analyzedusing gas chromatography with direct injection (GC-FID). Benzene, carbon tetrachloride and 1,2-dichloroethane are potential contaminants of toluene and dichloromethane, thus the control of these solventswas a limit test procedure. Due to the low permissible limits for benzene (2 µg/mL), carbon tetrachloride (4µg/mL) and 1,2-dichloroethane (5 µg/mL) it was necessary to use gas chromatography with a mass spectrome-try detector (GC-MS). All three new methods were validated according to the requirements of the ICH(International Conference on Harmonization) validation guideline Q2R1 and the Q3C guideline for residual sol-vents. Specificity, precision, accuracy, linearity, limits of detection and quantitation as well as robustness weredetermined and the results meeting the acceptance criteria were obtained.

Keywords: GC method, GC-HS method, GC-MS method, validation, dutasteride, residual solvents

1343

* Corresponding author: e-mail: [email protected]; phone: +48 22 4563992

1344 ALEKSANDRA GROMAN et al.

indicated in the ICH and pharmacopeial guidelines.The solvents and volatile reagents used during themanufacturing process of dutasteride active pharma-ceutical ingredient are: methanol, acetonitrile,dichloromethane, ethyl acetate, heptane, toluene (asvolatile chemical compounds) and pyridine,dimethylformamide, 1,4-dioxane, acetic acid, ethyl-ene glycol, 4-dimethylaminopyridine (as lessvolatile compounds). The separation of the chemicalcompounds into two groups was induced by theneed to use two injection methods: direct liquidsample injection and gaseous sample injection withthe headspace system.

Three new gas chromatography methods weredeveloped to quantitate the residual solvents andvolatile reagents. The direct injection method wasdeveloped for less volatile organic compounds toensure the best limits of detection and quantifica-tion. The headspace technique was used to injectgaseous samples and to analyze volatile compounds.The third method (limit test according to the Q2(R1)guideline by ICH) was developed for benzene, car-bon tetrachloride and 1,2-dichloroethane as poten-tial contaminants of toluene and dichloromethane.The use of gas chromatography hyphenated with amass spectrometric detector was necessary toacquire results for very low concentrations of theanalytes (specification limit for benzene 2 µg/mL,carbon tetrachloride 4 µg/mL and 1,2-dichloroethane 5 µg/mL). All three methods werevalidated and the results meeting the acceptance cri-teria were obtained.

These are the first methods reported in the lite-rature for the organic volatile impurities determina-tion in dutasteride. The monograph related to duta-steride is presented in the European Pharmacopoeia(EP) but does not contain GC methods for thevolatile compounds determination. The develop-ment of the analytical methods and their validationare presented in this article.

EXPERIMENTAL

Chemicals and reagents

The active substance dutasteride was synthe-sized in Pharmaceutical Research Institute (Warsaw,Poland). The reagent 4-dimethylaminopyridine(DMAP) was obtained from Aldrich, (Germany).The solvents and diluents were purchased fromcommercial suppliers: dichloromethane (DCM),pyridine, N,N-dimethylformamide (DMF), 1,2-dichloroethane, ethanol from Merck (Germany),1,4-dioxane, ethylene glycol, heptane, methanol,ethyl acetate, acetonitrile, toluene, carbon tetrachlo-

ride from POCH (Poland), acetic acid from SigmaAldrich (Germany), benzene from Fluka and N,N-dimethylacetamide (DMA) from J.T.Baker (Ger-many).

Methods

Because of a large variety of solvents andreagents used in the synthesis, it was necessary todevelop three gas chromatography methods.Different detection level and significant differencesin the volatility of these compounds forced the divi-sion into less volatile compounds (pyridine, N,N-dimethylformamide, 1,4-dioxane, acetic acid, ethyl-ene glycol and 4-dimethylaminopyridine) analyzedwith the use of gas chromatography with directinjection (method I ñ GC-FID) and volatile solvents(methanol, acetonitrile, dichloromethane, ethylacetate, heptane and toluene) which were analyzedusing the gas chromatography method with head-space (method II ñ GC-HS). Benzene, carbon tetra-chloride and 1,2-dichloroethane were analyzed withthe use of gas chromatography with mass spectrumdetection (method III ñ GC-MS) because of the lowspecification limits for the solvents.

Method I (GC-FID)

Equipment and chromatographic conditions The method was performed on a Shimadzu

GC-2010 gas chromatograph with a flame ionizationdetector interacted with an auto-sampler AOC20i. ADB-WAX column (phase composition: polyethyl-ene glycol) film thickness 0.5 µm, 60 m long and0.32 mm ID, from Agilent Technologies was used.The following oven temperature program was used:the initial temperature of 80OC was raised at the rateof 5OC/min to the temperature of 180OC, thenincreased at 30OC/min to 240OC and held at this levelfor 9 min. The injection port temperature was 220OCand the detector temperature was 260OC. Nitrogenwas used as the carrier gas at 100 kPa, split 5 : 1 , 1µL of the solution was injected into the gas chro-matograph.

Sample and standard solutions preparationThe sample solution was prepared by dissolv-

ing an appropriate amount of dutasteride in DCM.The standard solution was prepared by dissolvingappropriate amounts of determined solvents andreagents in DCM and subsequent dilution to reach380 µg/mL of 1,4-dioxane, 5000 µg/mL of aceticacid, 620 µg/mL of ethylene glycol, 200 µg/mL ofpyridine, 880 µg/mL of DMF, 1500 µg/mL ofDMAP with respect to the sample preparation(100% of the permissible limit).

Development and validation of gas chromatography methods for... 1345

Method II (GC-HS)

Equipment and chromatographic conditionsThe method was performed on a Perkin Elmer

CLARUS 500 gas chromatograph with a flame ion-ization detector interfaced with a Perkin Elmerheadspace auto-sampler TURBOMATRIX 40.Chromatographic separation was performed on aDB-624 column (phase composition: 6%cyanopropylphenyl ñ 94% dimethylpolysiloxane),60 m long, 0.32 mm ID, 1.8 µm film thickness, fromAgilent Technologies.

The following oven temperature program wasused: the initial temperature of 60OC was raised atthe rate of 2OC/min to the temperature of 75OC, thenincreased at 5OC/min to 120OC, next increased at40OC/min to 240OC and held at this level for 3 min.The injection port temperature was 240OC and thedetector temperature was 260OC. Nitrogen was usedas the carrier gas at 100 kPa, split 5 : 1, attenuation-5. The vial oven temperature was set at 100OC for 30min. The needle temperature was 110OC, the trans-fer line was 120OC, inject: 0.05 min.


ing an appropriate amount of dutasteride in DMA.The standard solution was prepared by dissolvingappropriate amounts of determined residual solventsin DMA and subsequent dilution to reach 3000µg/mL of methanol, 410 µg/mL of acetonitrile, 600µg/mL of DCM, 5000 µg/mL of ethyl acetate, 5000µg/mL of heptane, 890 µg/mL of toluene withrespect to the sample preparation (100% of the per-missible limit).

Method III ñ GC-MS

Equipment and chromatographic conditions The method was conducted on a Shimadzu

GC-2010 Plus with an MS detector GCMS-QP2010Ultra, both from Shimadzu. A DB-5MS col-umn (phase composition: 5% phenyl-methylpoly-siloxane ñ 95% phenyl arylene polymer) film thick-ness 0.25 µm, 30 m long and 0.25 mm ID, fromAgilent Technologies was used.

The oven temperature program was as follows:the initial temperature of 40OC was ramped up at therate of 5OC/min to 60OC, next ramped up at the rateof 40OC/min to 280OC and maintained for 5.5 min.The injection port temperature was 260OC and thedetector temperature was 280OC. Nitrogen was usedas the carrier gas at 60 kPa, split 4 : 1, 2 µL of thesolution was injected into the gas chromatograph.Characteristic ions: benzene m/z 78, carbon tetra-chloride m/z 117 and 1,2-dichloroethane m/z 62.


ing an appropriate amount of dutasteride in DMA.The standard solution was prepared by dissolvingappropriate amounts of determined compounds inDMA and subsequent dilution to reach 2 µg/mL ofbenzene, 4 µg/mL carbon tetrachloride and 5 µg/mL1,2-dichloroethane with respect to the sample prepa-ration (100% of the permissible limit).

RESULTS AND DISCUSSION

Methods development

During the development of the GC methodwith direct injection for the determination of pyri-dine, DMF, 1,4-dioxane, acetic acid, ethylene glycoland DMAP the following columns were tested: DB-624, HP PLOT/Q, DB-5, DB-CAM. On most ofthese columns wide and tailing peaks with too lowsensitivity were obtained, or sufficient separationfrom the rest of the solvents were not obtained.Finally, the column DB-WAX was selected, and onthis column good separation of the analytes and ade-quate method sensitivity were obtained.

Appropriate diluents without interfering peaksat the retention times of the determined solventswere choosen for the preparation of the sample. Inthe course of the accuracy study of the developedmethods it has been showed that the matrix does notaffect the results of the solvents determination.

Methods validation

All the described methods were validatedaccording to the requirements of the ICH(International Conference on Harmonization) vali-dation guideline Q2R1 and the Q3C guideline forresidual solvents. The validation of the methods(quantitative test procedure) included the examina-tion of specificity, linearity, accuracy, repeatability,intermediate precision, system precision, robustnessas well as quantitation and detection limits.

Specificity

Specificity of method I The specificity of the method was evaluated

by injecting the solution containing all solvents andreagents from the synthesis route and their potentialcontaminants at 100% of the permissible limit: 1,4-dioxane (380 µg/mL), pyridine (200 µg/mL), DMF(880 µg/mL), acetic acid (5000 µg/mL), ethyleneglycol (620 µg/mL), DMAP (1500 µg/mL),methanol (3000 µg/mL), acetonitrile (410 µg/mL),ethyl acetate (5000 µg/mL), heptane (5000 µg/mL),toluene (890 µg/mL), benzene (2 µg/mL), carbon


tetrachloride (4 µg/mL), 1,2-dichloroethane (5µg/mL). In this method benzene and 1,2-dichloroethane were not found. The retention timefor all compounds was determined by analyzingindividual solvent solutions. The peaks ofmethanol, ethyl acetate and carbon tetrachloridewere in the peak of DCM (diluent of the sample).All peaks in the chromatogram of the specificitysolution were completely separated from the ana-lytes (Rs = 1.5), Rs: heptane/DCM ñ 3.70, DCM +methanol + ethyl acetate + carbon tetrachloride/benzene ñ 5.25, acetonitrile/toluene ñ 4.88,toluene/1,4-dioxane ñ 4.16, 1,4-dioxane/pyridine ñ23.43, pyridine/DMF ñ 35.37, DMF/acetic acid ñ20.82, acetic acid/ethylene glycol ñ 44.52, ethyleneglycol/DMAP ñ 27.72. Spiking the sample solution

with the analytes did not cause the peaks to split andthe retention times remained the same as for thecorresponding peaks from the sample solution. Thechromatogram of the specificity solution is present-ed in Figure 1A.

Specificity of method II The specificity of the method was evaluated by

injecting the solution containing all solvents andreagents from the synthesis route and their potentialcontaminants at 100% of the permissible limit:methanol (3000 µg/mL), acetonitrile (410 µg/mL),DCM (600 µg/mL), ethyl acetate (5000 µg/mL),heptane (5000 µg/mL), toluene (890 µg/mL), 1,4-dioxane (380 µg/mL), pyridine (200 µg/mL), DMF(880 µg/mL), acetic acid (5000 µg/mL), ethylene

Figure 1. The specificity determination: A) method I, the chromatogram of the specificity solution (1 ñ heptane; 2 ñ DCM + methanol + ethyl acetate + CCl4; 3 ñ ACN; 4 ñtoluene; 5 ñ 1,4-dioxane; 6 ñ pyridine; 7 ñ DMF; 8 ñ acetic acid; 9 ñ ethylene glycol; 10 ñ DMAP)B) method II, the chromatogram of the specificity solution (1 ñ methanol; 2 ñ ethanol; 3 ñ acetonitrile; 4 ñ DCM; 5 ñ ethyl acetate; 6 ñheptane; 7 ñ 1,4-dioxane; 8 ñ pyridine; 9 ñ toluene; 10 ñ DMA)C) method III, the chromatogram of the standard solution (1 ñ benzene m/z 78; 2 ñ 1,2-dichloroethane m/z 62; 3 ñ carbon tetrachloridem/z 117)

A

B

C


glycol (620 µg/mL), DMAP (1500 µg/mL), benzene(2 µg/mL), carbon tetrachloride (4 µg/mL), 1,2-dichloroethane (5 µg/mL). In this method aceticacid, ethylene glycol, DMF, benzene, carbon tetra-chloride and DMAP were not found, the retentiontime of 1,2-dichloroethane was the same as theretention time of the heptane impurity. The retentiontime for all compounds was determined by analyz-ing individual solvent solutions. Ethanol was used todissolve heptane in order to prepare the standardsolution. The selectivity showed no co-elution ofethanol and the solvents determined by this method.All peaks in the chromatogram of the specificitysolution were completely separated (Rs = 1.5).Spiking the sample solution with the analytes didnot cause the peaks to split and the retention timesremained the same as for the corresponding peaksfrom the sample solution. The chromatogram of thespecificity solution is presented in Figure 1B.

Specificity of method III The specificity was examined using a standard

solution (a solution consisting of the analyzed sol-vents at 100% of the specification limit: benzene (2

µg/mL), carbon tetrachloride (4 µg/mL), 1,2-dichloroetane (5 µg/mL). The use of the mass detec-tor in gas chromatography ensures the specificity ofthe method by the extraction of a specific m/z (ionmass) chromatogram from a total ions chromatogramfor all the solvents. In the said method characteristicsignals were chosen from the mass spectrum of sol-vents. Characteristic ions were selected for: benzenemean ion ñ m/z 78 and comparative ion ñ m/z 51,carbon tetrachloride mean ion ñ m/z 117 and com-parative ion ñ m/z 119 and 1,2-dichloroethane meanion ñ m/z 62 and comparative ion ñ m/z 49.

Peaks of benzene, carbon tetrachloride and 1,2-dichloroethane were not observed in the chro-matogram of the test solution. Spiking the samplewith the analyte did not cause a peak to split; noadditional peaks appeared apart from that one of theanalyte. The matrix did not affect the analyticalresults. The chromatogram of the standard solutionis presented in Figure 1C.

Linearity

It was checked that the response of the detectoris linear to the concentration of the analyte for all the

Figure 2. Results of the linearity determination (method I)


compounds. The linearity was evaluated by the lin-ear regression analysis to calculate the slope, y-intercept, and correlation coefficient (R2). An addi-tional restriction was that Student t-test should bepassed (tcr = 2.78, ta > tcr, |tb| < tcr). The linearity of themethods was evaluated by analyzing 7 solutionsranging in the concentration of the analytes fromabout 10% (or LOQ) to 120% of the specified limit(methods I and II) and from about 30% to 120% ofthe specified limit (method III). All concentrationswere prepared in triplicate and the average wasreported. The methods were linear within a widerange for the solvents included in the validation. Theacceptance criteria were confirmed. The results arepresented in Figure 2 (method I) and in Tables 1, 2(methods II and III).

Range

The range concentrations from 10% to 120%of the specification limit: pyridine 20ñ240 µg/mL,DMF 88ñ1056 µg/mL, ethylene glycol 62ñ744µg/mL, 1,4-dioxane 38ñ456 µg/mL, methanol300ñ3600 µg/mL, acetonitrile 41ñ492 µg/mL, DCM60ñ720 µg/mL, ethyl acetate 500ñ6000 µg/mL, hep-tane 500ñ6000 µg/mL, toluene 89ñ1068 µg/mL; therange concentrations from 30% to 120% of the spec-ification limit: benzene 0.6ñ2.4 µg/mL, carbon tetra-chloride 1.2ñ4.8 µg/mL, 1,2-dichloroethane 1.5ñ6.0µg/mL, DMAP 500ñ1800 µg/mL; the range concen-trations from 50% to 120% of the specificationlimit: acetic acid 2500ñ6000 µg/mL. The results ofthe analyses confirmed that the methods in theseranges are precise, linear and accurate.

Figure 3. Results of the accuracy determination (method I)


Accuracy

The accuracy of the analytical methods wasreported as the percentage of the compound added tothe sample solution in comparison to the true value.The accuracy of the method was established by

assaying 9-12 sample solutions: triplicate independ-ent preparations for 3-4 concentrations (the samplesolutions spiked with the analytes at about 10%,30%, 50%, 100%, 120% of the specification limit).The recovery for all the solutions had to be within

Table 1. Validation results of the method II (1 - methanol, 2 - ACN, 3 - DCM, 4 - ethyl acetate, 5 - heptane, 6 - toluene).

Test Acceptance criteria 1 2 3 4 5 6

Correlation Linearity coefficient > 0.990 0.999 0.999 0.998 0.998 0.998 0.997

(R2)

y-Intercept (b) -422.16 52.85 117.15 2401.50 8215.74 475.91

Slope (a) 29.08 41.75 20.66 58.47 344.89 60.97

ta ta > tcr, tcr = 2.78 58.26 65.98 42.73 41.07 45.38 37.06

tb | tb | < tcr, tcr = 2.78 0.38 0.26 0.50 0.44 0.26 0.63

AccuracyMean recovery [%] 80-120% 93.04 99.02 97.33 102.15 99.76 98.28

RSD [%] 15% 3.24 2.05 2.13 4.55 5.77 2.71

RSD [%](Solutions 10 ˜ 120%)

15% 3.19 2.02 2.04 4.85 5.12 3.03Repeatability

RSD [%](Solutions 100%)

15% 4.30 3.03 5.21 4.90 5.21 3.06

Intermediate RSD [%]precision (Solutions 100%)

15% 1.98 1.97 6.73 4.95 2.97 2.01

RSD [%]System (Solutions 100%)

10% 1.43 2.81 1.36 1.11 1.08 1.23

precision RSD [%](Solutions 10%)

10% 0.45 0.83 2.67 1.02 1.94 0.82

Table 2. Validation results of the method III.

Test Acceptance criteria BenzeneCarbon 1,2-

tetrachloride Dichloroethane

Linearity Correlation coefficient (R2) > 0.990 1.000 0.999 0.999

y-Intercept (b) -10.81 17.22 -17.45

Slope (a) 1976.8 845.8 388.7

ta ta > tcr, tcr = 2.78 126.5 64.2 85.8

tb | tb | < tcr, tcr = 2.78 0.44 0.42 0.99

AccuracyMean recovery [%] 80-120% 96.90 101.15 93.99

RSD [%] 15% 1.71 4.02 4.71

RSD [%](Solutions 30 ˜ 120%)

15% 2.08 2.97 5.26Repeatability

RSD [%](Solutions 100%)

15% 2.76 3.12 4.30

Intermediate RSD [%]precision (Solutions 100%)

15% 2.44 1.95 5.87

RSD [%]System (Solutions 100%)

10% 3.52 3.14 4.94

precision RSD [%](Solutions 30%)

10% 2.11 2.85 3.55


the range from 80 to 120%. The results of the valueof the recovery are presented in Figure 3 (method I)and in Tables 1, 2 (methods II and III). The accept-ability criteria were fulfilled.

Precision

Precision of the analytical methods was con-sidered at three levels: repeatability, intermediateprecision and system precision. Precision of an ana-lytical procedure is usually expressed as the relativestandard deviation (RSD) of a series of measure-ments.

The repeatability was performed by measuringtriplicate independent preparations for three concen-trations ñ the sample solution spiked with the ana-lytes at about 10% (or 30%, 50%), 100%, 120% ofthe specification limit and 6 independent solutions ñthe sample solution spiked with the analytes at about100% of the specification limit, then the relativeresponse (the relation of peak area to mass) was cal-culated. The intermediate precision was repeated ona different day by a different analyst by measuring 6independent solutions ñ the sample solution spikedwith the analytes at about 100% of the specificationlimit, then the relative response (the relation of peakarea to mass) was calculated. The comparison of therepeatability and intermediate precision results wasperformed using the F-Snedecor test: F = Fcr (Fcr =5.05); (n = 6) (α = 0.05, f1 = n1 ñ 1, f2 = n2 ñ 1). Theresults of the parameter F for the solvents: 1,4-diox-ane 1.60, pyridine 1.62, DMF 1.36, acetic acid 1.51,ethylene glycol 1.86, DMAP 1.45, methanol 0.25,acetonitrile 2.25, DCM 1.96, ethyl acetate 1.00, hep-

tane 2.78, toluene 2.25, benzene 1.00, carbon tetra-chloride 2.25, 1,2-dichloroethane 2.25.

The system precision was established by meas-uring the response of six replicate injections of thestandard solution with the analytes at 100% of thespecification limit) and six replicate injections of thestandard solution with the analytes at 10% or 30% ofthe specification limit). The results are expressed asthe RSD and summarized in Figure 4 (method I) andin Tables 1, 2 (methods II and III). All the criteriawere fulfilled.

Limit of Quantitation (LOQ) and Limit of

Detection (LOD)

The limit of detection (LOD) and limit ofquantification (LOQ) were estimated as signal-to-noise ratio. The LOQ and LOD were evaluatedusing the standard solutions containing known lowconcentrations of the solvents. The concentrationwhich generated the peak height of the solvent atleast 10 times as high as the noiseís height wasestablished as the LOQ and the one at least 3 timesas high as the noiseís height was stated as the LOD.The following LOD and LOQ values were obtained(with respect to the sample preparation): methanol ñ9.0 µg/mL, 19 µg/mL, acetonitrile ñ 8.0 µg/mL, 25µg/mL, DCM ñ 13.8 µg/mL, 40 µg/mL, ethyl acetateñ 7.5 µg/mL, 68 µg/mL, heptane ñ 1.2 µg/mL, 4µg/mL, toluene ñ 7.2 µg/mL, 24 µg/mL, pyridine ñ2.4 µg/mL, 6.6 µg/mL, DMF ñ 6.0 µg/mL, 20µg/mL, 1,4-dioxane ñ 3.3 µg/mL, 11 µg/mL, aceticacid ñ 22 µg/mL, 68 µg/mL, ethylene glycol ñ 17µg/mL, 44 µg/mL, DMAP ñ 180 µg/mL, 500 µg/mL,

Figure 4. Results of the precision determination (method I)


benzene ñ 0.02 µg/mL, 0.03 µg/mL, carbon tetra-chloride ñ 0.18 µg/mL, 0.44 µg/mL, 1,2-dichloroethane ñ 0.23 µg/mL, 0.83 µg/mL, respec-tively.

Robustness

The robustness of the methods was evaluatedby injecting the specificity solutions to ensure theseparation of all solvents from the synthesis routewith the use of different chromatographic condi-tions. The following parameters were tested: columntemperature ± 5OC, rate ± 1OC/min and carrier gaspressure ± 10%. The smallest resolution (Rs = 1.72)was obtained between pyridine and toluene at therate 1OC/min. in method II. Changes in the analyti-cal conditions did not influence the resolution sig-nificantly and the method was robust.

CONCLUSION

Sensitive, accurate and precise GC methods forthe determination of residual solvents (their poten-tial contaminants) and volatile reagents used in themanufacturing process of dutasteride were devel-oped. Impurities are often not removed completelyby practical manufacturing techniques and conse-quently their low levels are present in most pharma-ceuticals.

Complete validations of the quantitative testprocedures to control the presence of these com-pounds (methanol, acetonitrile, dichloromethane,ethyl acetate, heptane, toluene, pyridine, dimethyl-formamide, 1,4-dioxane, acetic acid, ethylene gly-col, 4-dimethylaminopyridine, benzene, carbontetrachloride and 1,2-dichloroethane) were per-formed. The methods turned out to be specific, accu-rate, linear and precise. The compounds were detect-ed and quantified at a µg/mL level. The validationresults clearly demonstrate that the analytical proce-dures are suitable for their intended purpose.

The developed GC methods were successfullyused for the routine quality control of differentbatches of dutasteride under GMP rules. All ana-lyzed batches were declared to be in compliancewith the in-house specifications of residual solvents.

Acknowledgments

This work has been supported by The NationalCentre for Research and Development, Poland,grant no. INNOTECH-K2/IN2/65/182982/NCBR/13.

We wish to thank Ms. Joanna Zagrodzka,Ph.D. and the employees of the ChemistryDepartment for manufacturing and delivering thesamples for testing and for their support in thisstudy.

REFERENCES

1. Avodart (Dutasteride) Drug Information: Cli-nical Pharmacology, The Internet Drug IndexRxList, http://www.rxlist.com/avodart-drug/cli-nical-pharmacology.htm

2. Schmidt L.J., Tindall D.J.: J. Steroid Biochem.Mol. Biol. 32, 125 (2011).

3. Aggarwal S., Thareja S., Verma A., BhardwajT.R., Kumar M.: Steroids 109, 75 (2010).

4. Graul A., Slivestre J., Castaner J.: Drugs Fut.24, 246 (1999).

5. Dams I., ChodyÒski M., Ostaszewska A.,Szczepek W., Krupa M. et al.: Patent 413649(2015).

6. Dams I., ChodyÒski M., Ostaszewska A., KrupaM., Lipiec-Abramska E. et al.: Przemys≥Chemiczny 94, 2229 (2015).

Received: 25. 07. 2016


Isoniazid (INH) (Fig. 1), chemically known aspyridine-4-carboxylic acid hydrazide, is an antitu-bercular drug now widely used together withrifampicin and streptomycin for the chemotherapyof tuberculosis. This has prompted many investiga-tors to devise methods for its determination in itspure form as well as in its tablet form.

The drug is official in Indian Pharmacopoeia(IP) (1), British Pharmacopoeia (BP) (2) and UnitedState Pharmacopeia (USP) (3). IP and BP describetitration of the drug with potassium bromate in thepresence of potassium bromide using methyl redindicator. USP describes HPLC method using L1column (4.6 mm ◊ 25 cm) and a mobile phase con-sisting of methanol : water (40 : 60) (pH adjusted to2.5 with H2SO4) with a flow rate of 1.5 mL/min andUV-detection at 254 nm. Apart from the above offi-cial methods, a number of methods based on sever-al techniques are found in the literature for INH andinclude: titrimetry (4-7), voltammetry (8-13), ion

selective electrode-potentiometry (14-19), amper-ometry (20, 21), spectrofluorimetry (22, 23), chemi-luminescence spectrometry (24-33), high perform-ance liquid chromatography (HPLC) (34-37), gaschromatography (GC) (38-40), LC/LC-MS (41) andcapillary electrophoresis (42-44).

Visible spectrophotometry is by far the mostwidely used technique for the assay of INH.Methods based on a variety of reaction schemessuch as, nucleophilic substitution (45), condensation(46-49), charge-transfer and ion-pair (50), derivati-zation (51-54), diazo-coupling (55, 56), oxidativecoupling (57, 58), complex formation (59-61) andredox followed by complexation (62-65). Thesemethods suffer from the disadvantages such as dras-tic experimental conditions, use of organic solvent,longer standing time, poor sensitivity, narrow linearrange, etc.

KMnO4 has been a very useful reagent for thespectrophotometric assay of several pharmaceutical

SIMPLE AND SENSITIVE SPECTROPHOTOMETRIC ASSAY OF ISONIAZIDIN PHARMACEUTICALS USING PERMANGANATE, METHYL ORANGE

AND INDIGO CARMINE AS REAGENT

KANAKAPURA BASAVAIAH1*, NAGARAJU SWAMY and UMAKANTHAPPA CHANDRASHEKAR2

1Department of Chemistry, University of Mysore, Manasagangotri, Mysuru-570 006, Karnataka, India2Department of Chemistry, UBDT College of Engineering,

Davanagere-577 004, Karnataka, India

Abstract: Two simple, sensitive and rapid spectrophotometric methods are described for the determination ofisoniazid (INH) in pharmaceuticals. The methods are based on the oxidation of INH by a measured excess ofKMnO4 in acid medium followed by the determination of residual oxidant by reacting with either methyl orangeand measuring the absorbance at 520 nm (method A) or indigo carmine, when the absorbance was measured at610 nm (method B). Experimental conditions such as KMnO4 and dye concentrations, acid medium and reac-tion time to yield better performance characteristics were carefully studied and optimized. Beerís law wasobeyed in the concentration ranges, 0.25-5.0 and 0.25-4.0 µg/mL for method A and method B, respectively, andthe corresponding molar absorptivity values were 2.5 ◊ 105 and 3.0 ◊ 105 L/M cm-1. The limits of detection(LOD) and quantification (LOQ) were 0.08 and 0.23 µg/mL (method A) and 0.16 and 0.18 µg/mL (method B).Intra-day and inter-day accuracy expressed as relative error were better than 3% and the respective precisionreported as relative standard deviation were also < 3%. Methods, when applied to tablets, yielded results whichcompared well with the label claim and also with those obtained by the reference method. No interference wasobserved from the tablet excipients.

Keywords: isoniazid, assay, spectrophotometry, KMnO4, dyes, pharmaceuticals

1353

* Corresponding author: e-mail: [email protected]; phone: +91-8212419659, fax: +91-8212516133

1354 KANAKAPURA BASAVAIAH et al.

substances (66-76). To the best of authorsí knowl-edge, there is no work in the literature describing theassay of INH using KMnO4.

The present paper deals with the application ofKMnO4 and two dyes, methyl orange and indigocarmine for the rapid and sensitive assay of INH inpharmaceuticals. In this work, INH was treated witha known excess of KMnO4 and the unreacted oxi-dant was determined by reacting with either methylorange followed by the absorbance measured at 520nm (method A) or indigo carmine, where theabsorbance was measured at 610 nm (method B).The developed methods offer the advantages of sim-plicity, speed, and sensitivity without the need forexpensive chemicals and instrumentation.

EXPERIMENTAL

Apparatus

Absorbance measurements were made usingSystronics model 166 digital spectrophotometer(Systronics, Ahmedabad, Gujarat, India) equippedwith 10-mm matched quartz cells.

Reagents and materials

All the chemicals used were of analyticalreagent grade and distilled water was used through-out the investigation. Pure INH was procured fromCipla India Ltd., Bangalore, India and used asreceived. Isonex-100, Isonex forte-300 andTubernex forte-300 tablets were purchased fromlocal commercial sources.

Potassium permanganate: An approximately 0.01M solution was prepared by dissolving about 0.4 gof KMnO4 (Merck Ltd., Mumbai, India) in water,and diluted to 250 mL in a calibrated flask, and stan-dardized using H.A Brightís procedure (77). Thestandard solution was diluted appropriately withwater to get 60 and 70 µg/mL for use in method Aand method B, respectively.

Methyl orange (MO): A standard solution equiva-lent to 500 µg/mL methyl orange was prepared bydissolving 58.8 mg of dye (S. D. Fine Chem.,Mumbai, India, 85% dye content) in water anddiluted to 100 mL in a calibrated flask; and filteredusing Whatman number 42 filter paper. It was dilut-ed to get a 50 µg/mL dye solution for use in methodA.

Indigo carmine (IC): A standard solution contain-ing 1000 µg/mL indigo carmine was prepared bydissolving 107.6 mg of indigo carmine (Loba

Chemie Pvt. Ltd., Mumbai, India, 93% dye content)in water and diluting to the mark in a 100 mL cali-brated flask. The solution was diluted with water toget a working concentration of 200 µg/mL for use inmethod B.

Sulphuric acid (5 M): Concentrated acid (S.D. FineChem, Mumbai, India, sp. gr. 1.84) was appropri-ately diluted with water to get the required concen-tration.

Standard INH solution: Ten mg of INH was dis-solved in water and made up to 100 mL in a cali-brated flask water, and then diluted to 10 µg/mL.

Assay procedures

Procedure for bulk drug

Method A (using methyl orange): Differentaliquots (0.25, 0.5, 1.0, 5.0 mL) of 10 µg/mL INHsolution were accurately transferred into a series of10 mL calibrated flasks and the total volume wasadjusted to 5.0 mL by adding adequate quantity ofwater. To each flask, 1.0 mL of 5 M H2SO4 wasadded followed by 1.0 mL of 60 µg/mL KMnO4.The flasks were stoppered, content mixed andallowed to stand for 5 min with occasional shaking.Then, 1.0 mL of methyl orange solution (50 µg/mL)was added to each flask, and after 5 min, the mixturewas diluted to the volume with water and mixedwell. The absorbance of each solution was measuredat 520 nm against the reagent blank.

Method B (using indigo carmine): Varyingaliquots (0.25, 0.5, 1.0, 4.0 mL) of 10 µg/mL INHstandard solution were transferred into a series of 10mL calibrated flasks using a micro burette and thetotal volume was brought to 4 mL by adding water.To each flask 1 mL of 5 M H2SO4 and 1.0 mL ofKMnO4 (70 µg/mL) were added. The content wasmixed well and the flasks were kept aside for 5 minwith intermittent shaking. Finally, 1.0 mL of indigocarmine solution (200 µg/mL) was added to eachflask and the volume was adjusted to the mark withwater. The absorbance of each solution was meas-ured at 610 nm against a reagent blank.

A standard graph was prepared by plottingabsorbance against concentration and the unknownconcentration was computed from the regressionequation derived using Beerís law data.

Procedure for tablets

Ten tablets of were weighed and pulverized. Anamount of tablet powder containing 10 mg INH wastransferred into a 100 mL volumetric flask. The con-

Simple and sensitive spectrophotometric assay of isoniazid in pharmaceuticals... 1355

tent was shaken well with 60 mL of water and dilut-ed to the mark with water. The insoluble residue wasfiltered off using Whatman No. 42 filter paper. First10 mL portion of the filtrate was discarded and a sub-sequent portion was diluted to get a working concen-tration of 10 µg/mL and subjected to analysis follow-ing the general procedure described earlier.

Procedure for placebo blank and synthetic mixture

A placebo blank of the composition acacia (15mg), hydroxyl cellulose (10 mg), magnesiumstearate (15 mg), starch (10 mg), sodium citrate (10mg), sodium alginate (10 mg) and talc (20 mg) wasprepared by thorough mixing. Ten mg of the place-bo was taken and its solution prepared as describedunder ìProcedure for tabletsî and then analyzedusing the procedures described above. To 10 mg ofthe placebo blank, 5 mg of INH was added andhomogenized, transferred to 50 mL volumetric flaskand the solution was prepared as described underìProcedure for tabletsî. Synthetic mixture solution(100 µg/mL in INH) was diluted to 10 µg/mL leveland 2 mL aliquots were taken in five replicates andassayed following the general procedures.


The present work involves the oxidation ofINH by a measured excess of KMnO4 in H2SO4

medium followed by determination of surplusKMnO4 after the reaction is ensured to be complete.The unreacted KMnO4 is determined by reactingwith a fixed amount of either MO and measuring theabsorbance at 520 nm or IC and measuring theabsorbance at 610 nm. In both the methods, theamount of KMnO4 reacted is related to the amountof INH. The methods make use of the bleachingaction of KMnO4 on the dyes, where the discol-

oration is caused by oxidative destruction of the dye.Possible reaction pathway is shown in Scheme 1.

Many dyes are irreversibly destroyed to color-less species by oxidizing agents in acid medium (78)and this observation has been exploited for the indi-rect spectrophotometric determination of some phar-maceutical compounds (79-85). In the proposedmethods, the ability of KMnO4 to effect the oxida-tion of INH and irreversibly destroy MO and IC tocolourless products in acid medium has been capi-talized. In either method, the absorbance increasedlinearly with increasing concentration of drug. INHwhen added in increasing concentrations to a fixedconcentration of KMnO4 consumes the latter andthere will be a concomitant decrease in the concen-tration of KMnO4. When a fixed concentration ofeither dye is added to decreasing concentrations ofKMnO4, a concomitant increase in the concentrationof dye is obtained. This is observed as a proportion-al increase in the absorbance at the respective wave-lengths of maximum absorption with increasingconcentration of INH (Figs. 2, 3).

Optimization of KMnO4 and dye concentrations

Preliminary experiments were performed to fixthe upper limits of the methyl orange (MO) and indi-go carmine (IC) that would produce a reasonablyhigh absorbance, and these were found to be 5µg/mL MO in method A and 20 µg/mL IC in methodB. To fix the optimum concentration of KMnO4, dif-ferent concentrations of KMnO4 were reacted with afixed concentration of MO (5 µg/mL) or IC (20µg/mL) in H2SO4 medium, and the absorbance wasmeasured at 520 or 610 nm. A constant and mini-mum absorbance resulted with 6.0 and 7.0 µg/mLKMnO4 for method A and method B, respectively.Different concentrations of INH were reacted with 1mL of 60 µg/mL KMnO4 in method A and 70 µg/mL

Scheme 1. Possible reaction pathway for INH determination by the proposed methods


in method B in H2SO4 medium before determiningthe residual KMnO4. This facilitated the optimizationof the linear dynamic range over which each methodcould be applied for the assay of INH.

Effect of reaction medium

The reaction between INH and KMnO4 wasperformed in different acid media viz. sulphuricacid, hydrochloric acid and perchloric acid.

Sulphuric acid was found to be the ideal medium forthe oxidation of INH by KMnO4 as well as the lat-terís determination employing either dye. The effectof acid concentration on the reaction between INHand KMnO4 was studied by varying the concentra-tion of H2SO4 keeping the concentrations of KMnO4

and drug fixed. Higher acid concentrations showedlower sensitivity, hence, 1 mL of 5 M H2SO4 in atotal volume of 10 mL was fixed as optimum.

Study of reaction time and stability

Under the described experimental conditions,for a quantitative reaction between INH andKMnO4, contact time of 5 min was found necessaryin both methods at room temperature. After additionof dye, the reaction between KMnO4 and dye wasinstantaneous and absorbance of the unreacted dyewas stable at least for 45 min in method A and 60min in method B.

Method validation

Linearity and sensitivity

The proposed methods were validated for lin-earity, selectivity, precision, accuracy, robustnessFigure 1. Structure of INH

Figure 2. Absorption spectra of 6 µg/mL KMnO4 after reacting with: a. 2; b. 3; c. 4 and d. 5 µg/mL INH


and ruggedness, and recovery. In both the methods,a linear correlation (Fig. 4) was found betweenabsorbance at λmax and concentration of INH in the

ranges given in Table 1. Regression analysis of theBeerís law data using the method of least squareswas made to evaluate the slope (b), intercept (a) and

Figure 3. Absorption spectra of 7 µg/mL KMnO4 after reacting with: a. 1; b. 2; c. 3 and d. 4 µg/mL INH

Table 1. Sensitivity and regression parameters.

Parameter Method A Method B

λmax, nm 520 610

Beerís law limits, µg/mL 0.25 ñ 5.0 0.25 ñ 4.0

Molar absorptivity, L/M cm-12.5 ◊ 105 3.0 ◊ 105

Sandell sensitivity*, µg cm-2 0.0055 0.0045

Limit of detection, mg/mL 0.08 0.06

Limit of quantification, mg/mL 0.23 0.18

Regression equation, Y**

Intercept, (a) 0.0192 0.0102

Slope, (b) 0.1666 0.2167

Standard deviation of intercept (Sa) 0.0045 0.0021

Standard deviation of slope (Sb) 0.0109 0.0226

Regression coefficient (r) 0.9989 0.9990

a Limit of determination as the weight in µg/mL of solution, which corresponds to an absorbance of A = 0.001measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm.b Y = a + bX, where Y is the absorbance, X con-centration in µg/mL.


correlation coefficient (r) for each system and thevalues obtained from this investigations are also pre-sented in Table 1. Sensitivity parameters such as

apparent molar absorptivity and Sandell sensitivityand the limits of detection and quantification are cal-culated as per the current ICH guidelines and are

Figure 4. Calibration curves for: a) method A; b) method B

Table 2. Results of intra-day and inter-day accuracy and precision study.

Intra-day accuracy and Inter-day accuracy and

Method INH taken* precision (n = 7) precision (n = 5)

INH found* %RE %RSD INH found* %RE %RSD

1.0 1.02 2.04 2.18 1.03 2.89 2.28

A 2.0 1.96 2.27 1.34 1.95 2.30 2.45

3.0 3.04 1.31 1.67 3.06 1.96 2.85

2.0 1.98 1.01 1.72 1.96 2.02 2.03

B 3.0 3.04 1.36 2.16 3.05 1.65 2.72

4.0 4.05 1.27 1.49 4.07 1.78 2.98

RE - Relative error and RSD - Relative standard deviation; INH taken and found are in µg/mL; Mean value of n determinations.

Table 3. Results of robustness and ruggedness expressed as intermediate precision (% RSD).

Robustness Ruggedness

Method Nominal Reaction times* Volume of acid$ Inter- analysts Inter- instruments concentration (n=3) (n=3) (n=3) (n=3)

1.0 2.65 2.13 1.04 2.42

A 2.0 3.14 2.86 1.28 2.16

3.5 3.38 2.73 0.92 1.85

2.0 1.85 1.05 0.73 2.16

B 3.0 2.16 1.27 1.12 2.72

4.0 2.08 1.97 1.08 3.04

* Reaction time was 4, 5 and 6 min.; $ Volume of H2SO4, 0.9, 1.0 and 1.1 mL.

a) b)


compiled in Table 1 and speak of the excellent sen-sitivity of the proposed methods.

Accuracy and precision

In order to assess the precision and accuracy ofthe proposed methods, pure INH at three concentra-tion levels within the linearity range were analyzed,each determination being repeated seven times(intra-day precision) on the same day and one timeeach for five successive days (inter-day precision).The percent relative standard deviation (% RSD)was = 1.34% (intra-day) and = 2.10% (inter-day). Inaddition, the accuracy of the proposed method wasevaluated by calculating the percentage relativeerror (% RE), which varied between 1.01% and ≈ 3%. The results of this study compiled in Table 2indicate the high accuracy and precision of the pro-posed methods.

Selectivity

Selectivity was evaluated by both placeboblank and synthetic mixture analyses. The placebo

blank, whose composition was given earlier wasprepared and analyzed as described under the rec-ommended procedures. The resulting absorbancereadings for the methods were the same as thereagent blanks, inferring no interference from theplacebo. The selectivity of the methods was furtherconfirmed by carrying out recovery study from syn-thetic mixture. The percent recoveries of INH were98.7 ± 1.18 and 101.4 ± 1.63 for method A andmethod B, respectively. This confirms the selectivi-ty of the proposed methods in the presence of thecommonly employed tablet excipients.

Robustness and ruggedness

To evaluate the robustness of the methods, twoimportant experimental variables, viz. the amount ofacid and reaction time, were slightly varied, and thecapacity of the methods was found to remain unaf-fected by small deliberate variations. The results ofthis study are presented in Table 3 and indicate thatthe proposed methods are robust. Method rugged-ness is expressed as %RSD of the same procedure

Table 4. Results of analysis of tablets by the proposed methods and statistical comparison with the official method.

Founda (Percent of label claim ± SD)Tablet Label claim*

Reference Proposed methodsstudied mg/tablet

method Method A Method B

98.84 ± 1.42 98.75 ± 1.38Isonex1 100 99.36 ± 1.65 t = 2.72 t = 2.82

F = 1.35 F = 1.42

99.07 ± 1.22 98.63 ± 1.09Isonex forte1 300 97.99 ± 1.03 t = 1.51 t = 0.95

F = 1.4 F = 1.12

101.2 ± 1.29 100.9 ± 1.15Tubernex forte2 300 100.5 ± 0.81 t = 1.02 t = 0.64

F = 2.54 F = 2.02

a Mean value of five determinations; the value of t and F (tabulated) at 95% confidence level and for four degrees of freedom are 2.77 and6.39, respectively.*Marketed by: 1 Pfizer Limited (Pharmacia India Pvt. Ltd.); 2 Radicura Pharmaceuticals Pvt. Ltd. India.

Table 5. Results of recovery study via standard addition method.

INH in Pure INH Total Pure INH Method

Tablettablet added found recovered*studiedµg/mL µg/mL µg/mL Percent ± SD

1.48 0.75 2.18 97.85 ± 1.19

Method A Isonex-100 1.48 1.50 3.02 101.5 ± 0.79

1.48 2.25 3.82 102.5 ± 1.76

1.48 0.75 2.19 98.55 ± 0.81

Method B Isonex-100 1.48 1.50 2.95 99.26 ± 0.58

1.48 2.25 2.75 100.9 ± 0.84

*Mean value of three determinations.

1360 KANAKAPURA BASAVAIAH et al.T

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applied by three analysts using the same instrumentand also by a single analyst using three differentspectrophotometers. The inter-analystsí and inter-instrumentsí RSD values were = 3.04% indicatingruggedness of the proposed methods. The results ofthis study are presented in Table 3.

Application to tablets

The results presented in Table 4 showed thatthere was a close agreement between the resultsobtained by the proposed methods and the labelclaim. The results were also compared with those ofthe reference method (2) statistically by Studentís t-test for accuracy and variance ratio F- test for preci-sion at 95% confidence level. The calculated t- andF-values indicated that there was no significant dif-ference between the proposed methods and the ref-erence method with respect to accuracy and preci-sion.

Accuracy by recovery study

To further ascertain the accuracy of the pro-posed methods, a standard addition procedure wasfollowed. A fixed amount of pre-analyzed tabletpowder was taken and pure drug at three differentlevels (50, 100 and 150% of that in tablet powder)was added. The total was determined by the pro-posed methods. The determination at each level wasrepeated three times and the percent recovery of theadded standard was calculated. Results of this studyare presented in Table 5 and are reflective of thehigh accuracy, selectivity and reliability of the pro-posed methods for routine use.

CONCLUSIONS

Two rapid, simple, and selective spectrophoto-metric methods for the determination of isoniazid inpharmaceuticals are described. The methods are freefrom rigid experimental conditions such as criticalpH control, heating, extraction, etc. They employinexpensive and easily available chemicals andinstrument compared to most methods reported pre-viously (Table 6), making them cost-effective. Themethods are applicable over wide linear dynamicranges and highly sensitive (∈ = 105) compared tothe existing spectrophotometric methods. The meth-ods can be used as alternatives to highly sophisticat-ed instrumental methods reported for isoniazid.

Acknowledgments

Authors express their gratitude to the qualitycontrol manager, Cipla Ltd., Bangalore, India, for

gift sample of pure isoniazid and the authorities ofthe University of Mysore, Mysore, for permissionand facilities. Prof. K. Basavaiah thanks UniversityGrants Commission New Delhi, India, for the awardof UGC-BSR faculty fellowship.

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Received: 25. 08. 2016


Quantitative structureñretention relationships(QSRRs) describe relationships between moleculardescriptors and chromatographic properties of mol-ecules. The QSRR approach can be applied: (1) forprediction of retention; (2) for identification of themost revealing structural descriptors (regardingproperties); (3) to gain insight into the retentionmechanism of separation; (4) for the evaluation ofcomplex physicochemical properties (other thanchromatographic) of analytes, e.g., lipophilicity; and(5) for the prediction of relative biological activity(1-4).

Micellar liquid chromatography (MLC) is areversed-phase liquid chromatographic (RPLC)mode with a mobile phase consisting of an aqueoussolution of surfactant above its critical micellar con-centration (CMC) (5). In such system, solute reten-

tion is governed by three different equilibria. Thoseare solute distribution between the micelles and thebulk phase, solute partition between the stationaryphase and the bulk phase and direct transfer ofsolute molecules between surfactant-modified sur-face to the micelles (6). In recent years there were afew articles where MLC was successfully appliedfor lipophilicity prediction (6, 7).

Chromatographic parameters can be useful notonly for lipophilicity determination, but also forQuantitative Structure-Activity Relationships(QSAR) study. In the literature, there are alreadysome QSAR models based on TLC retention param-eters (8, 9).

The primary goal of the study was to determinemolecular descriptors which affect retention ofmacrolide antibiotics in selected MLC systems.

QUANTITATIVE STRUCTUREñRETENTION RELATIONSHIPS AND QUANTITATIVE STRUCTUREñACTIVITY RELATIONSHIPS STUDY

OF MACROLIDE ANTIBIOTICS ON MICELLAR THIN LAYER CHROMATOGRAPHY

KRZESIMIR CIURA1*, JOANNA NOWAKOWSKA1, PIOTR KAWCZAK2, KATARZYNA EWA GREBER1 and MICHA£ JAN MARKUSZEWSKI3

1Medical University of GdaÒsk, Faculty of Pharmacy, Department of Physical Chemistry, Al. Gen. J. Hallera 107, 80-416, GdaÒsk, Poland

2Medical University of GdaÒsk, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Al. Gen. J. Hallera 107, 80-416 GdaÒsk, Poland

3Medical University of GdaÒsk, Faculty of Pharmacy, Department of Biopharmaceutics andPharmacodynamics, Al. Gen. J. Hallera 107, 80-416, GdaÒsk, Poland

Abstract: Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatographic (RPLC) modewith a mobile phase consisting of an aqueous solution of surfactant above its critical micellar concentration(CMC). Chromatographic properties of nine macrolide antibiotics have been studied on cyano-bonded silicastationary phase. Four surfactants, with different chemical character: anionic (SDS) cationic (CTAB and SC)and nonionic (triton X-100), were tested as modifiers of mobile phase. The influence of physicochemicalparameters of tested macrolide antibiotics on the retention were investigated utilizing quantitativestructureñretention relationships (QSRR) approach. Molecular descriptors were calculated from the optimizedstructures using HyperChem and ChemAxon software. Additionally, the quantitative structureñactivity rela-tionships (QSAR) equations, based on chromatographic parameters, were proposed. The QSAR models can beused for predictions of antimicrobial activity of Staphylococcus aureus, Steptococcus pneumoniae and Listeriamonocytogenes against.

Keywords: micellar liquid chromatography (MLC), Quantitative Structure-Retention Relationships (QSRR),Quantitative Structure-Activity Relationships (QSAR), macrolide antibiotics

1365

* Corresponding author: e-mail: [email protected]

1366 KRZESIMIR CIURA et al.

Additionally, the utilization of obtained chromato-graphic parameters in QSAR analysis were exam-ined.

EXPERIMENTAL

Analytes

The 9 reference standards (dirithromycin,josamycin, erythromycin, azithromycin, clarithromy-cin, roxithromycin, spiramycin, troleandomycin andtylosin) used during the study were purchased fromSigma-Aldrich (Steinheim, Germany).

Mobile and stationary phases

Chromatographic analyses were performed onsilica gel 60 CN F254s HPTLC plates manufacturedby Merck (Darmstadt, Germany). The organic sol-vents (HPLC-grade: acetone, acetonitrile, dioxaneand methanol) were supplied by Sigma-Aldrich(Gillingham, Dorset, UK). Water was purified usingMillipore Direct-Q 3 UV Water Purification System(Millipore Corporation, Bedford, MA, USA). Thesurfactants: sodium dodecyl sulfate (SDS), cetrimo-nium bromide (CTAB), Triton X-100 and sodiumcholate (SC) were purchased from Sigma-Aldrich(Steinheim, Germany). The mobile phases were pre-pared by mixing appropriate quantities of pureorganic solvents and aqueous solution of surfactantin proportion listed bellow: a) 50, 65, 80, 95, 110 mM aqueous solution of SDS

with 15 % (v/v) addition of acetonitrileb) 85, 95, 110, 125 mM aqueous solution of CTAB

with 15 % (v/v) addition of acetonitrilec) 30, 40, 50, 60, 70 mM aqueous solution of SC

with 15 % (v/v) addition of acetonitrile

Chromatographic analysis

Macrolide antibiotics were dissolved inmethanol at a concentration of 5 mg/mL and 1 µl of

the obtained solutions were spotted with the used ofmicropipettes on the plates. 65 mm wide and 105mm high cylindrical glass chambers (Sigma-Aldrich; Steinheim, Germany) were used to developthe plates. The chromatographic chamber was satu-rated 20 minutes before every analysis. Chromato-grams were developed by the ascending technique,at room temperature (20 ± 2OC) to the distance of 8cm. The studied substances were visualized byspraying the plates with the mixture of concentratedsulfuric acid and methanol (1 : 4, v/v). After spray-ing, the plates were heated at 121OC for 10 min(Camag TLC Plate Heater III). Subsequently, theeluted compounds were visible in UV light (254 and366 nm wavelength) as colored spots. The chro-matographic analyses were performed in triplicateand mean retardation factor (RF) values were calcu-lated.

Chromatographic parameters

The RM parameters were calculated with use ofBate-Smith and Westall formula:

1Rm = log (ñññ ñ 1) (1)

Rf

The correlation between chromatographic parameterk and surfactant concentration in the mobile phase ispresented with Foley formula (10):

1 1 KAMññ = ññññ + ññññ [M] (2)k km km

where [M] is the total concentration of surfactant inthe mobile phase, KAM is the constant describingsoluteñmicelle binding and km is the solute retentionparameter at zero micellar concentration, i.e. at sur-factant monomer concentration equal to the CMC(6). The Foley formula can be applied for aqueoussolutions of surfactant or mobile phases with theequal. The parameters KAM and km can be calculatedfrom the slope and intercept of experimental 1/k ver-sus [M] relationships (6, 10, 11).

Table 1. The physicochemical parameters of the tested macrolide antibiotics.

log D log DpH5.5 ACD pH7.4 ACD

H-Ac EHOMO σmin µ EnergyDreiding

Azithromycin -0.74 1.40 14 -42.00 -0.35 5.57 218.34

Clarithromycin 0.71 2.41 14 -41.89 -0.31 12.69 223.45

Dirithromycin -0.79 1.99 16 -42.02 -0.35 6.05 277.79

Erythromycin 0.38 2.08 14 -41.50 -0.35 10.64 242.31

Josamycin 2.09 3.06 16 -42.37 -0.37 7.41 189.70

Roxithromycin 1.28 2.98 17 -41.87 -0.35 10.06 224.17

Spiramycin -0.92 1.58 16 -42.03 -0.38 3.01 245.26

Troleandomycin 1.09 2.79 16 -42.69 -0.32 9.82 324.64

Tylosin 1.49 3.02 18 -42.21 -0.33 7.95 276.56

Quantitative structure-retention relationships and quantitative... 1367

Molecular descriptors

The structures of tested compounds were con-structed using the Open Babel 2.3.3 software (12).HyperChem 8.08 software with ChemPlusExtension (Hypercube, Waterloo, Canada) was usedfor the calculation of molecular bulkiness-relateddescriptors and molecular polarity-related (electron-ic) descriptors. In the first step, the structures wereoptimized using the molecular mechanic calcula-tions (MM+). In the next step, semi-empirical calcu-lation method Austin Model 1 (AM1) was applied.The number of hydrogen donors and acceptors weretaken from PubChem database (13). Geometricaland topological descriptors were obtained with theuse of ChemAxon software (14). All descriptorsused in this study are presented in Table 1.

Determination of critical micelle concentrations

Critical micelle concentrations of CTAB and SCwere determined by the surface tension measure-ments on an Easy Dyne (Kr¸ss, Germany) tensiome-ter. Solutions of known concentration were progres-sively diluted and examined with the use of Wilhelmyplate method. Temperature was kept at 25 ± 0.2OCusing a thermostated circulating water bath. Beforeeach measurement, the Wilhelmy plate was washedwith pure water and then heated to redness using aBunsen burner. All samples were prepared in 15%solution of ACN in water. The CMCs values werecalculated by plotting the surface tension against thelog of the concentration of surfactants. The accuracyof the measurement was within ± 0.1 mN/m.

Data analysis

All statistical calculations were performedusing STATISTICA 9.1 (StatSoft, Tulsa, Okla-homa, USA). Correlations between moleculardescriptors and retention or biological activitieswere presented as QSRR/QSAR equations. In orderto establish the QSRR/QSAR equations, the multi-ple linear regression (MLR) was derived, using step-wise regression. During calculations, the retentiondata were used as a dependent variables and struc-tural parameters as the independent ones.Coefficients of correlation (r) and determination(R2), value of F-test and standard estimation errorwere used as the basis for testing the linearity ofregression plots. All presented QSRR/QSAR equa-tions were performed at a significant level of ≤ 5 %.

RESULTS AND DISCUSSIONS

In our research, a group of 9 selected macrolideantibiotics were used. All studied compounds,

except for tylosin, belong to the same macrolideclass in the Anatomical Therapeutic Chemical(ATC) classification system (J01F Macrolides, lin-cosamides and streptogramins). Tylosin is struc-turally similar to each other and it is used as a vet-erinary drug. The size of the tested group was limit-ed but numbers of widely available analytical stan-dards of macrolide derivatives were not much larg-er.

Chromatographic analysis

CN plates were chosen as stationary phase,because analysis on those plates is noticeably short-er in comparison to analysis on C18 or C8 bonded sil-ica gel (6). Four surfactants were explored duringthis study. The surfactants have different chemicalcharacter and belong to various class: anionic (SDS)cationic (CTAB and SC) and nonionic (triton X-100). Preliminary chromatographic study showedthat the addition of small amount of organic solventto the aqueous solution of surfactant is necessary.When only aqueous solution of surfactants wasused, the solutes remained at the starting point. Thismight be the consequence of high molecular massand size of tested compounds. Four organic sol-vents, at different concentrations were tested: ace-tone, acetonitrile, dioxane and methanol.

Unfortunately, when triton X-100 was used asa modifier of mobile phase, the solutes remained onthe starting line, although various proportion of tri-ton X-100 and different organic modifiers wereexamined. Due to this fact, this chromatographicsystem was excluded from further analysis.

For further analyses of other chromatographicsystems, the addition of 15 % (v/v) of acetonitrile inthe mobile phase was selected, since it causes sig-nificant increase in Rf values. Additionally, acetoni-trile significantly reduced the time of the analysis.On the other hand, the short-chain alcohol, as wellas acetonitrile, in the mobile phase can lead to disin-tegration of micelles. For this reason, the concentra-tion of surfactants was considerably higher thanCMC in pure water. In the literature there is infor-mation that SDS micelles are stabile in 20% solutionof ACN (15) but there are no information aboutinfluences of ACN on CMC in case of using SC andCTAB. Therefore, the self-assembly abilities ofCTAB and SC in 15% ACN have been investigated.Interesting might be the fact that ACN have onlyslight influence on CMC value in case of SC. Theobtained CMC value is 11.5 mM, showing smallincrease in comparison to pure water (CMC valuerange between 2 ñ 6 mM). On the contrary, whenCTAB was used, the CMC value increased extreme-


ly to the value of 83.6 mM in comparison to purewater (0.92 mM).

The obtained chromatographic parameters cal-culated according to equation 2 are listed in Table 2.Generally, the correlation coefficient was close to0.95 confirming the applicability of the presentedequation. Significantly high correlation wasobserved when SDS was used. When mobile phaseincluded CTAB, the correlation coefficient waslower than 0.9 for josamycin (r = 0.866) and rox-ithromycin (r = 0.878). Moreover, clarithromycinhas near linear behavior (r = 0.891) when mobilephase contained SC. The obtained results may sug-gest that for the cationic and the anionic surfactantsthe equation 2 represents the chromatographicbehavior very well. The additional studies, based on

larger number of different surfactants should be per-formed to confirm the hypothesis.

Since parameters KAM/km and 1/km are analogi-cal for RM

0 and m in classical reserved phase thinlayer chromatography (RP-TLC), the inter-correla-tions were examined. The results of the analysis areshowed in Table 3 and Figure 1. Generally, linearcorrelations of good statistical quality wereobserved, what is confirmed by high value of r, val-ues of F- Snedecorís test and small standard estima-tion error. This proves that, from the chromato-graphic point of view, the macrolide antibiotics canbe regarded as one group of structurally similar ana-lytes, and also suggests, that the mechanisms ofchromatographic retention for the analyzed com-pounds are similar within their groups (9).

Table 2. Parameters of Eq. (2) calculated for tested macrolide antibiotics with statistical parameters.

Analyte KAM/km σ KAM/km 1/km σ 1/km r R2 F s

Azithromycin -0.16 0.085 0.52 0.114 0.956 0.914 21.27 0.038

Clarithromycin -0.04 0.025 0.25 0.033 0.983 0.966 56.63 0.011

Dirithromycin -0.12 0.018 0.42 0.024 0.997 0.993 300.38 0.008

Erythromycin -0.07 0.038 0.35 0.052 0.979 0.959 46.43 0.017

SDS Josamycin 0.00 0.014 0.06 0.018 0.928 0.861 12.36 0.006

Roxithromycin -0.05 0.017 0.25 0.023 0.991 0.983 115.91 0.008

Spiramycin -0.17 0.027 0.53 0.037 0.995 0.991 211.02 0.012

Troleandomycin -0.06 0.017 0.22 0.023 0.989 0.979 93.102 0.008

Tylosin -0.05 0.029 0.30 0.039 0.983 0.966 57.173 0.013

Azithromycin 0.30 0.047 0.26 0.064 0.947 0.897 17.81 0.020

Clarithromycin 0.16 0.050 0.40 0.067 0.975 0.951 32.38 0.021

Dirithromycin 0.27 0.029 0.29 0.040 0.983 0.966 54.29 0.013

Erythromycin 0.16 0.068 0.40 0.091 0.954 0.910 21.48 0.029

CTAB Josamycin 0.34 0.019 0.06 0.025 0.866 0.750 9.78 0.008

Roxithromycin 0.35 0.043 0.11 0.059 0.878 0.771 6.22 0.019

Spiramycin 0.45 0.032 0.04 0.004 0.930 0.865 12.82 0.019

Troleandomycin 0.43 0.016 -0.03 0.022 0.967 0.935 41.571 0.007

Tylosin 0.30 0.043 0.21 0.059 0.909 0.826 10.385 0.029

Azithromycin 0.89 0.098 -0.65 0.153 0.978 0.957 44.27 0.024

Clarithromycin 0.74 0.049 -0.32 0.094 0.891 0.794 11.58 0.030

Dirithromycin 0.90 0.004 -0.64 0.007 0.999 0.999 8317.75 0.002

Erythromycin 0.78 0.047 -0.42 0.090 0.937 0.879 21.76 0.028

SC Josamycin 0.79 0.032 -0.41 0.062 0.967 0.936 43.54 0.020

Roxithromycin 0.83 0.041 -0.61 0.079 0.976 0.952 59.49 0.025

Spiramycin 0.93 0.037 -0.77 0.071 0.988 0.976 119.57 0.022

Troleandomycin 0.89 0.059 -0.70 0.105 0.978 0.957 44.266 0.024

Tylosin 0.91 0.062 -0.73 0.118 0.942 0.887 39.437 0.038

r - the coefficient of correlation; R2- the coefficient of determination; F - the value of Snedecor F-test; s - the standard estimation error; p-value < 0.05.


QSRR analysis

In QSRR study, the predictor variable is theretention constant, in the case of TLC ñ it is RM

0

parameter, and the independent variables are molec-ular descriptors. In order to establish QSRR modelsMLR was applied. Since the descriptors in finalMLR models must be independent, the inter-corre-lations were tested. According to the number ofcompounds, examined models contained maximumtwo independent variables. The best one and twoparameters equations are summarized in Table 4.

Positive values of regression coefficient indi-cate that observed descriptor contributes positivelyto the value of KAM/km. Whereas negative valuesindicate that the greater value of descriptor, thelower value of KAM/km.

In case of two tested cationic surfactants stronginfluences of the electronic descriptors on the reten-tion are noticeable. In order to show moleculardescriptors, govern retention in chromatographicsystem, included CTAB, two parameters modelshave been applied. It is based on two electronicdescriptors, but they are not correlated (r = 0.121).Moreover, two parameters model also explains bet-ter retention mechanism, when SC as modifier ofmobile phase was used. Obtained models presentthat dipol moment and Dreiding energy plays a cru-cial role, affecting the retention of macrolide antibi-otics in this chromatographic systems.

QSRR analysis showed that only SDS as mod-ifier of mobile phase is useful for the prediction of

lipophilicity properties of macrolide antibiotics. Theuniversal scale of lipophilicity is represented by thelogarithms of the partition coefficients (log P)between two phases, n-octanol and water in the caseof neutral species, or the distribution ratio (log D)for ionizable compound. The traditional directmethod for lipophilicity determination (ìshakeflaskî method based on n-octanol-water partition-ing) is labor-intensive and time-consuming, so thatit is not commonly used nowadays. For this reason,current separation techniques are increasingly usedfor lipophilicity determination (3). Chromatographicapproach has many advantages in comparison to tra-ditional method, it is more convenient, reproducible,faster and inexpensive (4). Additionally, the pro-posed method for lipophilicity estimation ofmacrolide antibiotics is attractive from ìgreenchemistryî point of view, because micellar mobilephases are less toxic, nonflammable and have lowerenvironmental impact compared to conventional LCmethods. Furthermore, MLC method is faster thanpreviously proposed SOTLC method (17).Although, in SOTLC vapor pressure is lower,because an aqueous solutions of inorganic salts areused as mobile phases.

QSAR analysis

The emergence and spread of multiple drugresistance (MDR) bacteria strains, causes that adevelopment of a new antibiotics is nowadays oneof the most important challenges of medicinal chem-

Table 3. Correlations between and obtained for tested chromatographic systems.

Surfactant Equation r F s

SDS KAM/km = -0.37 (± 0.038) 1/km + 0.037 (± 0.014) 0.964 91.563 0.0164

CTAB KAM/km = -0.64 (± 0.082) 1/km + 0.41 (± 0.018) 0.948 62.0143 0.0341

SC KAM/km = - 0.41 (± 0.039)1/km - 0.61 (± 0.023) 0.969 107.526 0.0174

r - the coefficient of correlation; F - the value of Snedecor F-test; s - the standard estimation error; p-value < 0.05; n = 9.

Table 4. One and two parameters obtained QSRR equations with statistical parameters.

r R2 F s

KAM/km SDS = 0.05(± 0.006) log D pH5.5 ACD ñ 0.11(± 0.007) 0.942 0.888 55.592 0.021

KAM/km SDS = 0.11(± 0.008) log D pH7.4 ACD ñ 0.02(± 0.003) H-Ac ñ 0.02(± 0.004) 0.984 0.968 93.106 0.012

KAM/km CTAB = ñ 0.22(± 0.064) EHOMO ñ 2.63(± 1.051) σmin ñ 9.99(± 2.784) 0.855 0.731 8.175 0.061

KAM/km SC = -0.02(± 0.006) µ + 0.99(± 0.047) 0.762 0.580 9.669 0.047

KAM/km SC = 0.0009(± 0.0001) Energy Dreiding ñ 0.02(± 0.002) µ + 0.76(± 0.059) 0.948 0.898 26.312 0.025

r - the coefficient of correlation; R2 - the coefficient of determination; F - the value of Snedecor F-test; s - the standard estimation error; p-value < 0.05 n = 9.


Figure 1. Relationship between 1/KM and KAM/km for tested chromatographic systems a (CTAB), b (SDS) andc (SC)

a

b

c


istry. Macrolide are broad spectrum antibiotics fre-quently used to treat infections caused by Gram-pos-itive bacteria, such as respiratory tract and soft-tis-sue infections. They act by inhibiting protein syn-thesis by blocking the 50S ribosomal subunit (16).In the early stages of the development of drug can-didates, same physicochemical properties of drugcandidates such as solubility, lipophilicity, stabilityand acidñbase character should be determined.Since, they can effect on oral absorption, proteinbinding and metabolism, as well as a pharmacologi-cal effect including antimicrobial activity (1). In ourprevious study, SOTLC was applied in order to findthe best SOTLC chromatographic system to predictlipophilicity and biological activity for this class ofchemical compounds (17). The antimicrobial activi-ty of antibiotic frequently depends on its lipophilic-ity (18). However, other chromatographic data, notonly chromatographic estimated lipophilicity, canbe useful for QSAR study. The data on in vitro anti-bacterial activity for seven of tested compounds waswidely presented in the literature (19). Tylosin isused as veterinary drug, for this reason there areinformation about its activity for typical bacteriacausing animal diseases; Pasteurella multocida,Actinomyces pyogenes, Fusobacterium necropho-rum, Brachyspira hyodysenteriae or Mycoplasmahyosynoviae. Furthermore, in the literature are noavailable data on antibacterial activity of trolean-domycin. It is not popular, since this drug is soldonly in two countries: Italy and Turkey.

Finally, the minimal inhibitory concentrations(MIC) 50 and 90 of the following bacteria:Staphylococcus aureus, Streptococcus pneumonia,Listeria monocytogenes, were successfully used forQSAR analysis. Establish QSAR equations arenoticed bellow. Staphylococcus aureusMIC 50 = 0.25 (± 0.068) KAM/km CTAB + 0.16 (±0.042) r = 0.853, R2 = 0.727, F = 13.324, p < 0.014, s =0.060 n = 7MIC 50 = -0.39 (± 0.077) 1/km CTAB + 0.43 (±0.047) r = 0.914, R2 = 0.837, F = 25.619, p < 0.0039 s =0.068 n = 7MIC 90 = -0.11 (± 0.033) 1/km CTAB + 0.50 (±0.089) r = 0.832, R2 = 0.692, F = 11.228, p < 0.02031 s =0.0933 n = 7Steptococcus pneumoniaeMIC 50 = -1.89 (± 0.536) + 0.51 (± 0.062) 1/km SDS r = 0.844, R2 = 0.713, F = 12.43, p < 0.01682, s =0.098 n = 7

Listeria monocytogenesMIC 90 = 0.06 (± 0.020) KAM/km CTAB + 0.18 (±0.044) r = 0.809, R2 = 0.655, F = 9.503, p < 0.0274, s =0.067 n = 7MIC 90 = 0.04 (± 0.012) + 0.75 (± 0.027) KAM/km SCr = 0.851, R2 = 0.725, F = 13.155, p < 0.01511, s =0.041 n = 7

The results showed, that it is possible to applychromatographic parameters in order to predictantimicrobial activity of macrolide antibiotics.Similar conclusions can be drawn when we compareobtained results with our previous analyses based onSOTLC. Both SOTLC and MLC chromatographicparameters affect MIC value of Steptococcus pneu-moniae, although in case of SOTLC stronger corre-lations were find. Important might be the fact that inboth chromatographic methods retention was highlycorrelated with lipophilicity.

The significant correlations were foundbetween both MIC values against Staphylococcusaureus and chromatographic parameters obtained onchromatographic system included CTAB in mobilephase. Retention parameters obtained in this chro-matographic system were not correlated withlipophilicity of macrolide. This could suggest thatantimicrobial activity against Staphylococcus aureusdepends on properties of macrolides other thanlipophilicity.

Furthermore, chromatographic systems,including CTAB and SC as modifiers of mobilephases, can be used for estimation of antimicrobialactivities against L. monocytogenes. The correlationcoefficients were close to those observed in our pre-vious study (17).

CONCLUSION

Obtained QSRR equations explain the mecha-nism of retention and illustrated differences betweentested MLC systems. This analysis also confirmedthat regression models, which are based on calculat-ed molecular descriptors, can be successfully usedfor the prediction of chromatographic parameters.Additionally, the study presents the usefulness ofchromatographic parameters obtained on differentmicellar TLC systems on QSAR analysis ofmacrolide antibiotics. The high correlation coeffi-cients (r), values of F and small values of the stan-dard deviation indicate that the obtained equationscan be used for prediction of antimicrobial activityagainst Staphylococcus aureus, Steptococcus pneu-moniae and Listeria monocytogenes. Important isthe fact that micellar thin layer chromatography


reduces the time of analysis in comparison toSOTLC. Furthermore, chromatographic systemincluding aqueous solution of SDS may be usefulfor lipophilicity estimation of those classes of com-pounds.

REFERENCES

1. Kaliszan R.: Chem. Rev. 107, 3212 (2007).2. Kaliszan R.: Structure and Retention in

Chromatography. A Chemometric Approach;Harwood Academic Publishers, Amsterdam1997.

3. Kaliszan R.: Quantitative StructureChromatographic Retention Relationshipsî;John Wiley & Sons, New York 1987.

4. HÈberger K.: J. Chromatogr. A 1158, 273(2007).

5. Ruiz-Angel M.J., Carda-Broch S., Torres-LapasiÛ J.R., GarcÌa-Alvarez-Coque C.: J.Chromatogr A 1216, 1798 (2009).

6. Janicka M., StÍpnik K., Pachuta-Stec A.:Chromatographia 75, 449 (2012).

7. Medina-Hernandez M.J., Sagrado S.: J.Chromatogr A. 718, 273 (1995).

8. Odovic J., Markovic B., Vladimirov S.,Karljikovic-Rajica K.: J. Chromatogr. B 102,953 (2014).

9. EriË S., PavloviË M, PopoviË G., Agbaba D.: J.Chromatogr. Sci. 45, 140 (2007).

10. Foley J.P.: Anal. Chim. Acta 231, 237 (1990).11. Garcia-Alvarez-Coque M.C., Torres-Lapasio

J.R., Baeza-Baeza J.J.: J. Chromatogr. A 780,129 (1997).

12. http://openbabel.org/wiki/Main_Page/ (acces-sed on 01.06.2016).

13. https://pubchem.ncbi.nlm.nih.gov/ (accessed on01.06.2016).

14. https://www.chemaxon.com/ (accessed on01.06.2016).

15. Berthod A., Garcia-Alvarez-Coque C.: MicellarLiquid Chromatography, CRC Press, BocaRaton, Ann Arbor, London, Tokyo 2000.

16. Brayfield A.: Martindale: The complete drugreference, Pharmaceutical Press, London 2014.

17. Ciura K., Nowakowska J., Rudnicka-Litka K.,Kawczak P., Bπczek T., Markuszewski M.J.:Monatsh. Chem. 147: 301 (2016).

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19. Zhanel G.G., Dueck M., Hoban D.J., VercaigneL.M., Embil J.M. et al.: Drugs 61, 443 (2001).

Received: 12. 09. 2016


Peloid is described as a maturated mud ormuddy dispersion with healing and/or cosmeticproperties, composed of a complex mixture of fine-grained natural materials of geologic and/or biolog-ic origins, mineral water or sea water, and commonorganic compounds of biological metabolic activity(1). The peloids also named as therapeutic muds,thermal muds, clay or ancient peat are known fromprehistoric times and were used for healing purpos-es by Egyptians and Romans (2, 3). Currently,peloids are used in cosmetics and medical treatmentin spa resorts. The application of peloids ñ calledpelotherapy is becoming increasingly popular (4).The use of healing muds in pharmaceutical formula-tions as gastrointestinal protectors, oral laxatives,anti-diarrheas, dermatological protectors and inesthetic medicine, became more common during lastfew years due to increasing interest in natural medi-cine (5, 6). While being selected on empirical bases,all natural materials are not free of possible side-effects (7). Various components of mud, particular-ly fulvic acid, trace elements may be absorbedthrough the skin or any skin abrasions, cuts, or other

breaches in the integrity of the skin (8-13).Furthermore, after applying the mud directly on thebody when it dries, released particles may be inhaledimmediately, or they may be incorporated intohouse dust and enter the respiratory tract at a latertime. Accidental mud swallowing by children or atransfer of the material from their hands to theirmouths should also be considered. However, therehas been no assessment of the potential health risk toconsumers posed by toxic elements possibly presentin the peloid itself and their toxicity are still lacking(6, 14). In the U.S., the Food and Drug Admini-stration has limited authority over the cosmeticsindustry and rarely does any routine testing for toxicmetals in products used for cosmetic purposes (15,16). However, FDA regulations state that heavymetal concentrations in cosmetic products shouldnot exceed 10 ppm in the case of Pb. According toEuropean directives, lead is not allowed in cosmeticmaterials, including clays and peats for pelotherapy(85/391/CEE, 86/179/CEE and 86/199/CEE) (7).Potential toxicity associated with elemental compo-sition of cosmetics is well documented in e.g., eye

VOLTAMMETRIC DETERMINATION OF TRACE ELEMENTS (Cu, Pb, Zn) IN PELOID-BASED PHARMACEUTICALS

MAREK SZL”SARCZYK1, IWONA NOWAKOWSKA1, ROBERT PIECH2, W£ODZIMIERZ OPOKA1,and JAN KRZEK1

1Department of Inorganic and Analytical Chemistry, Jagiellonian University, Collegium Medicum, 30ñ688 KrakÛw, Medyczna 9, Poland

2Faculty of Materials Science and Ceramics, AGH University of Science and Technology, 30ñ059 KrakÛw, al. Mickiewicza 30, Poland

Abstract: The research on the potential health risk posed to consumers by toxic elements that can be found inpeloids is still lacking. Moreover, in Polish law no clinical or pharmacological tests are required to identifyhealing properties of peloids. The objective of this work was to determine some mineral content in selectedpeloids used in medical treatment. Anodic stripping voltammetry with differential pulse step was used for zinc,copper and lead determination. Decomposition of organic matrix was conducted by a simple wet digestion pro-cedure using acid digestion vessel. Obtained results showed that proposed methods were suitable for the deter-mination of investigated metallic elements. Lead content varied between 0.18 mg/kg (in MaúÊ Borowinowa)and reached up to 15.5 mg/kg of dry weight for Chokrak peloid. Zinc content ranged from 0.64 to 66.87 mg/kgand copper content was between 0.57 and 7.50 mg/kg. The proposed method was validated, the recovery forpeloid samples were 94 ñ 102%; 92 ñ 97%; 96 ñ 106% for copper, zinc and lead, respectively.

Keywords: trace element, muds, peloids, medical muds, therapeutic mud

1373

* Corresponding author: e-mail: [email protected]; phone/fax: +48 12 6205480

1374 MAREK SZL”SARCZYK et al.

highlighters (17, 18) muds and clays (6, 10, 19) andother products (20, 21). Moreover, Polish MinistryRegulation in this case defines that peloids shouldbe physico-chemically and microbiologically testedto determine their healing properties, but no clinicalor pharmacological tests are required (Dz. U. 2006Nr 80 No. 565).

Available databases show that spectroscopicmethods (ICP-MS, ICP-AES, AAS, XRF) wereused for determination of copper, zinc and lead inthe majority of available research works (6, 22, 23).All the methods described above, are not free frominterferences. The direct determination of metals insamples with high organic matter content by atomicabsorption spectrometry is not always possible dueto matrix composition. In that case liquid-liquidextraction, flow injection system and solid phasepreconcentration techniques were applied (24, 25).The sequential voltammetric procedure allowingdetermination of zinc, copper and lead was rarelyused in biological and environmental samples.Therefore, the application of simple electroanalyti-cal methods should improve analytical performance.The aim of this research was to determine content ofzinc, copper and lead in peloids and its products byanodic stripping voltammetry.

MATERIAL AND METHODS

Material

Artificial clays (peloids) were purchased indrugstore or directly in spa resort. All raw materialswere conditioned in room temperature before use.Pasta borowinowa lecznicza ñ BiochemÆ,Borowinowa Kostka IwonickaÆ from Iwonicz Spa(Poland), MaúÊ borowinowa, Sulphur ZdrÛjÆ (BuskoZdrÛj, Poland), Lecznicza pasta borowinowaÆ,KamieÒ Pomorski Spa (Poland) were purchased indrugstore. Chokrak peloid sample was collectedfrom its environment in the Chokrak lake area(Ukraine) in April and May, 2013.

Apparatus

A Multipurpose Electrochemical AnalyserM161 with a M164 electrode stand (both mtm-anko,

Poland) were used for all voltammetric measure-ments. The standard three-electrode cell consisted ofa controlled growth mercury drop electrode(CGMDE) as a working electrode, Ag/AgCl in 3 MKCl with a double junction filled with 3 M KCl(Mineral, Poland) as reference and platinum wire asan auxiliary electrode. Voltammograms wererecorded, interpreted and stored by EAGRAPH 6.0(mtm-anko, Poland) software.

Reagents

A standard solutions of Zn(NO3)2, Cu(NO3)2,and Pb(NO3)2 were prepared by proper dilution of 1g/L stock standard solution (OUM, £Ûdü, Poland).Electrolyte used as ionic medium was prepared bydissolving KNO3 (Merck, SuprapurÆ). For digestionprocedures HNO3 (Merck, SuprapurÆ) and 30%H2O2 (Cheman, Poland) was used. All the solutionswere prepared with double-distilled water fromquartz distiller (SZ-97A, Chemland, Poland) and allother reagents were of analytical grade.

Sample preparation

Samples were homogenized in agate mortar andthen dried at over 70OC for 4 hours. Approximately250 ñ 500 mg of sample material was accuratelyweighed and inserted in a TeflonÆ container of an aciddigestion vessel (4748, Parr Instruments Co., USA).Next, the sample was treated with 5 ñ 6 mL of nitricacid and 1 mL of perhydrol, tightly sealed and wasplaced in drying oven (Binder, Germany). The diges-tion of the sample was carried 24 h in 160OC and afterit cooled to the room temperature the vessel wasunsealed and the sample was quantitatively trans-ferred to an evaporation dish. The digested samplewas placed at the heated plate for evaporation andremoving the nitrates. The sample solutions were thencooled to room temperature, transferred quantitative-ly into volumetric flasks (10 mL) and filled up to themark with double distilled water. All the procedureswere carried out in triplicate for each sample.

Analytical procedure

The voltammetric procedure for determinationof copper, zinc and lead level was in accordance

Table 1. Analytical parameters of voltammetric analysis of selected trace elements.

Element Eacc [mV] tacc [s] Method dE [mV] Working electrode Medium

Zn(II) -1200 20 DP ASV -20 CGMDE 0.2 M KNO3

Cu(II) -400 40 DP ASV -20 CGMDE 0.04 M KNO3

Pb(II) -600 40 DP ASV -20 CGMDE 0.04 M KNO3

Eacc - accumulation potential, tacc - accumulation time, dE - pulse amplitude.

Voltammetric determination of trace elements (Cu, Pb, Zn) in... 1375

with a method that was previously used (26). Beforeeach measurement the solution in the voltammetriccell was deaerated by high purity argon for 5 minand then, after redirecting the argon flow over thesolution surface, measurements were performed.Zinc, copper, lead were determined using differen-tial pulse anodic stripping voltammetry with a con-trolled growth mercury drop electrode (CGMDE)with a differential pulse stripping step (27). Themeasurements were performed according to thestandard addition method, voltammograms corre-sponding to individual additions were registeredthree times

All experiments were carried out at room tem-perature. All instrumental parameters of performedmeasurements were arranged in Table 1.


The proposed voltammetric method usingCGMDE after digestion procedure allows determi-nation of zinc (20 s deposition time) with LOD =0.49 µg/L and LOQ = 1.49 µg/L. The slope forregression line is -2.809 ± 0.036 (nA/µg/L) with acorrelation coefficients r = 0.9989. The recovery ofselected peloid samples ranged from 92 to 97% forzinc. The proposed method also allows the determi-nation of copper (40 s deposition time) with LOD =1.71 µg/L and LOQ = 5.15 µg/L. The slope forregression line is -2.399 ± 0.019 (nA/µg/L) withcorrelation coefficients r = 0.9967. The recovery ofselected samples ranged from 94 to 102% for cop-per. Furthermore, the same method also allowsdetermination of lead (40 s deposition time) withLOD = 0.15 µg/L and LOQ = 0.47 µg/L. The slopefor regression line is -1.949 ± 0.036 (nA/µg/L) withcorrelation coefficients r = 0.9995. The recovery of

selected peloids ranged from 96 to 106% for lead.For cadmium only LOD and LOQ were estimated atthe level 0.09 µg/L and 0.27 µg/L, respectively. Theinvestigated samples showed cadmium level belowdetection limit and no interpretable signal indicatedcadmium presence. During zinc and copper analysisa 1000-fold excess of Fe (III), and 100-fold excessof Pb (II), Cd (II), Mn (II) did not interfere. The sur-face-active compounds are usually a source ofstrong interferences in voltammetric methods andshould be thoroughly destroyed by digestion prior toanalysis.

Zinc showed the highest concentration levelsin natural peloid samples, whereas the lowest levelvalues were observed for Pb (II) (Table 2). Thehighest zinc content was determined in natural sam-ples from Chokrak lake 66.9 mg/kg and the lowestwas in pharmaceutical product Borowinowa Kostkalecznicza 0.8 mg/kg. The highest value of copperconcentration was determined also in Chokrak lakepeloid (4.6 mg/kg) and the lowest was in pharma-ceutical ointment MaúÊ borowinowa (0.6 mg/kg).The highest lead contamination was determined inpeloid from Chokrak lake 15.5 mg/kg with thesmallest moisture. Basing on obtained results posi-tive correlations were observed between the investi-gated trace elements contents: very high betweenzinc and lead (r = 0.86) and high in pairs Cu-Pb (r =0.61) and Zn-Cu (r = 0.53).

The investigated samples were different in ori-gin and a direct comparison is difficult. Amongthem the Chokrak mud was interesting because itprovided a reliable test for the effectiveness of ana-lytical method. Additionally, there are no availabledata of voltammetric analysis of trace element inpeloids. When comparing the results obtained in thisstudy, to the ones from spectroscopic analysis it can

Table 2. Trace element and water content of investigated peloid products.

Product name Trace element content (mg/kg d.w.)

Zn(II) Cu(II) Pb(II) Water content [%]

Pasta borowinowa lecznicza - BiochemÆ 17.6 ± 1.5 1.6 ± 0.2 11.6 ± 3.2 92.31

Borowinowa Kostka IwonickaÆ

from Iwonicz Spa (Poland)0.8 ± 0.2 0.7 ± 0.1 0.55 ± 0.17 80.26

Peloid from Chokrak lake (Ukraine)

66.9 ± 4.7 4.6 ± 0.4 15.5 ± 3.4 52.27

MaúÊ borowinowa. Sulphur ZdrÛjÆ

(Busko ZdrÛj. Poland)1.0 ± 0.1 0.6 ± 0.1 0.18 ± 0.05 -*

Lecznicza pasta borowinowaÆ

KamieÒ Pomorski Spa (Poland)20.5 ± 7 7.5 ± 1.2 9.6 ± 1.0 80.67

*40 g water extract of peloid/100 g ointment.

1376 MAREK SZL”SARCZYK et al.

be observed that both methods give the same rangeof concentrations. Chokrak mud zinc and lead con-tent were in the range showed by other works con-ducted over Portuguese mud where approximately50 to 150 mg/kg of zinc and 5 to 38 mg/kg of leadwere found (6). Moreover, Spanish peloids showedsimilar zinc and lead concentration with a rangebetween 33.1 - 89.8 mg/kg and 10.9 - 37.5 mg/kg,respectively (23). Peculiarly, according to priorresearch some peloids can show zinc content up to160 mg/kg with elevated levels of lead and copper.In comparison, copper level in Chokrak peloid wastwo times lower than the lowest of previouslyreported content and in average five to ten timeslower than the highest value showed in earlierresearch (23). However, the distribution of elementcontent was similar showing the following order:Zn > Cu > Pb.

The two types of paste, first from Spa resortand second from market, had similar zinc and leadcontents but differed in copper. Correlation betweenwater content and element content were quite low (r= 0.42) mostly due to lead and copper contents. Forzinc and water content solely, correlation coefficientshowed high value (r = 0.86) but was statisticallyinsignificant. This fact indicates that all the peloidspastes have similar zinc concentration taking intoaccount their water content. According to regula-tions, the investigated samples can be consideredfree from heavy metal contamination excluding thepeloid from Chokrak lake and Pasta borowinowalecznicza (Biochem). I this case the lead content wasabove 10 mg/kg and exceeded the value approvedby Polish Pharmacopoeia. However, the limits wereexceeded only when calculated for dry sample mass.When water is taken into consideration the contentsof lead in the peloid from Chokrak lake and Pastaborowinowa lecznicza ñ Biochem were 7.39 mg/kgand 0.89 mg/kg, respectively. Moreover, the peloidpaste or peloid mud in medical treatment are used inhydrated form directly on the body, so the exceededlimits should not be considered in this case.

CONCLUSIONS

It is necessary to expand study on more samples,but at Polish market there is only a limited number ofpeloid-based products. However, this study gives anoverview on products that are available in Poland anda single, less known peloid from its natural source.The obtained results showed that voltammetric meth-ods used in this study were suitable for determinationof copper, zinc and lead both in peloids and derivedproducts. The proposed method is characterized by

low instrumental and single analysis costs, ten timeslower than the usually applied methods, achievingcomparable results with spectroscopic analysis.According to FDA and EC regulations, a trace ele-ment content study and analytical quantification forselected elements seem to be necessary, especiallywhen concerning their life important functions and/ortheir toxicological hazard.

Acknowledgment

The study was financed as an R&D project by the Polish Ministry of Science of Education from research funds for the years 2013ñ2015K/DSC/000796.

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Received: 15. 09. 2016


Mushrooms collected in wild are importantfood item in certain regions of the World and certainspecies are used also because of medicinal value (1,2). Demand for edible wild-growing and cultivatedfungi in recent decades of years is growing world-wide (3). Sclerotia is a dense mass of myceliumdeveloped by some saprophytic fungi, which islocated under the ground and is collected in someregions of the World as traditional medicine andingredient in meals (4). Sclerotia of Wolfiporiaextensa (Peck) Ginns fungus is popular in theTraditional Chinese Medicine ñ is collected in wildand is cultivated in field condition (2, 5). Databaseavailable on biologically active compounds in scle-rotia of W. extansa is relatively large and manyorganic constituents have been identified (5, 6),while there is a gap on information about inorganicconstituents (7, 8).

Unprocessed fruiting bodies of edible fungi(mushrooms) collected in wild could be rich in anessential macro- and trace inorganic constituents butalso at elevated level in certain harmful metallic ele-ments (9). Cadmium (Cd), mercury (Hg) and lead(Pb) are usually considered but some other harmfulelements could also matter (10-14).

The province of Yunnan in China is one of theregions in Asia where foraging for sclerotia of W.extensa is tradition while cultivation in field condi-tion becomes popular too (5). The Yunnan land,because of the occurrence of the Circum-PacificMercuriferous Belt, is one of a specific regions inthe World with polymetallic soils that are enrichedin Hg, antimony (Sb), barium (Ba), strontium (Sr)and certain other metallic elements (15-17). Thiscan have a consequence in elevated or highly ele-vated content of Hg in fruiting bodies of certain

OCCURRENCE, VARIABILITY AND ASSOCIATIONS OF TRACE METALLICELEMENTS AND ARSENIC IN SCLEROTIA OF MEDICINAL WOLFIPORIA

EXTENSA FROM POLYMETALLIC SOILS IN YUNNAN, CHINA

JERZY FALANDYSZ1*, MARIA CHUDZI—SKA2, DANUTA BARA£KIEWICZ2, MARTYNA SABA1,YUAN-ZHONG WANG3, 4* and JI ZHANG3, 4

1Laboratory of Environmental Chemistry and Ecotoxicology, GdaÒsk University, GdaÒsk, Poland2Department of Trace Element Analysis by Spectroscopy Method, Adam Mickiewicz University,

PoznaÒ, Poland3Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan, China

4Yunnan Technical Center for Quality of Chinese Materia Medica, Kunming, Yunnan, China

Abstract: Dried sclerotia of Wolfiporia extensa has a long history of a wide medicinal uses in Asia because ofanti-inflammatory effect. There is deficit of information on inorganic constituents of sclerotia. This study aimedat providing and evaluating data on some trace metals (Ag, As, Ba, Cd, Co, Cr, Cs, Cu, Li, Mn, Ni, Pb, Rb, Sr,Tl, U, V, Zn) contents of sclerotia collected across Yunnan land in China. Sclerotia, when compared to fruitingbodies of fungi collected in wild, were low in toxic Ag: range (< 0.005-0.21) mg/kg dry biomass (db); As: (<0.004-0.041) mg/kg db; Cd: (< 0.002-0.034) mg/kg db; Pb: (0.020-0.24) mg/kg db and Tl: (< 0.001-0.015)mg/kg db. Sclerotia contained detectable amounts of essential trace metals such as Co: (0.011-0.21) mg/kg db;Cu: (0.44-3.8) mg/kg db; Li: (0.005-0.092) mg/kg db; Mn: (2.2-48) mg/kg db and Zn: (1.8-9.0) mg/kg db; werelow in Rb: (0.11-2.8) mg/kg db and stable Cs (133Cs): (< 0.002-0.043) mg/kg db while relatively rich in Ba:(0.78-22) mg/kg db and Sr: (0.15-12) mg/kg db. The elements such as Cr, Ni and V occurred in sclerotia in dif-ferent concentrations, Cr: (0.24-3.3) mg/kg db; Ni: (0.037-14) mg/kg db; V: (0.009-0.25) mg/kg db; and U: (<0.001-0.015) mg/kg db. The sclerotia of W. extensa from Yunnan were specifically enriched in Ba and Sr, andcontent of Mn was similar as showed for fruiting bodies of fungi foraged in Europe. The relatively high con-tents of Ba and Sr in the sclerotia examined could be attributed to a specific or anomalous geochemistry of soilsin the regions of Yunnan.

Keywords: medicinal fungi, wild foods, bioconcentration, heavy metal

1379

* Corresponding authors: e-mail: [email protected]; e-mail: [email protected]

1380 JERZY FALANDYSZ et al.

fungi (mushrooms) foraged in some regions of theYunnan Province (18-20). Some fungi, e.g. from thegenus Agaricus, Boletus, Macrolepiota and others,because of specific genetic features could ñ evenwhen grown-up at the background areas (withoutgeochemical anomaly and unpolluted) ñ efficientlyaccumulate in fruiting bodies harmful Cd, Hg, Ag,and examples of species efficient in accumulationare given by BoroviËka et al. (21), Falandysz et al.(22), GarcÌa et al. (23), Gucia et al. (24), KrasiÒskaand Falandysz (25), and Melgar et al. (26).

The fungus Wolfiporia extensa from the familyof Polyporaceae grows on the roots of an old, deadpine trees. Sclerotia of W. extensa has attracted con-siderable attention due to the various medicinalproperties, such as anti-inflammatory, sedative,stomachic and diuretic in the Traditional ChineseMedicine for millennia (5, 6). Dried sclerotia iswidely used in the form of the decoctions (usually 9to 18 g of dried powder per day, up to 45 g) and incombination with some other herbs. A traditionalsnack in Beijing that is called ìfuling jiabingî alsocontains dried sclerotia.

This study is aimed at investigating the occur-rence and variability and evaluate data on Ag, As,Ba, Cd, Co, Cr, Cs, Cu, Li, Mn, Ni, Pb, Rb, Sr, Tl,U, V, and Zn in sclerotia of W. extensa from poly-metallic soils of Yunnan in China. Elements weredetermined with inductively coupled plasma ñ mass

spectroscopy and collision cell, what enabled for anefficient elimination of interferences arising fromthe argon plasma etc. in metal ion detection, and todetermine possible variability, associations betweenthe elements and to evaluate the levels.

MATERIALS AND METHODS

Sample collection and initial preparation

Sclerotia of Wolfiporia extensa (Peck) Ginnswere collected from 19 places across of the YunnanProvince in China in July 2012 (Fig. 1). The local-izations were selected at an altitude above sea levelbetween 1371 and 2061 m. No specific permits wererequired for the described field studies. No endan-gered or protected species were sampled, and thelocalities where the samples came from are not pro-tected in any way. The inner part of the sclerotia ìfu-lingî which is white in color and free of skin ìfu-ling piî or pine roots ìfu-shenî were separated for astudy. From each sclerotia 6 sub-samples of theinner part were taken (each ca. 300 g) and pooled tomake a bulk sample. Further, the sclerotia weresliced into small pieces, dried at 105OC and pow-dered using porcelain mortar. The bulk samples ofdry powdered sclerotia (each ca. 600 g) were placedinto sealed new polyethylene bags and kept in dryand clean conditions in a herbal materials deposito-ry room prior to further analyses.

Figure 1. Localization of the sampling places of W. extensa in Yunnan

Occurrence, variability and associations of trace metallic elements and... 1381

Sample preparation for analyses

Before digestion, the samples were dried at105OC for 12 h using an electrically heated laborato-ry oven. Then, subsamples of dried and powderedmushrooms (~0.5000 g) were digested with 5 mL of65% HNO3 (Suprapure, Merck, Germany) underpressure into the microwave oven model Ethos One(Milestone Srl, Italy). The heating program was per-formed in one step: the power of the process was1500 W, ramp time 20 min, temperature 200OC andhold time 30 min. Reagent blank solutions were pre-pared in the same way. For every set of 10 mush-room samples digested, two blank samples were run.The digest was diluted to 10 mL using deionizedwater (TKA Smart2Pure, Niederelbert, Germany).

Analytical procedure

Elemental analysis of Li, V, Cr, Mn, Co, Ni,Cu, Zn, As, Rb, Sr, Ag, Cd, Ba, Pb, Tl, U was car-ried out using the ELAN DRC II ICP-MSInductively Coupled Plasma Mass Spectrometer(PerkinElmer, SCIEX, Canada) equipped with aMeinhard concentric nebulizer, cyclonic spraychamber, collision reaction cell, Pt cones andquadruple mass analyzer. Typical instrument operat-

ing conditions for ICP-MS spectrometer were: RFpower ñ 1100 W; plasma Ar flow rate ñ 15 L/min;nebulizer Ar flow rate ñ 0.87 L/min and auxiliary Arflow rate ñ 1.2 L/min and lens voltage ñ (7.5-9.0) V.For calibration curves construction, a mixed stan-dard solution with concentration of 10 mg/L wasused (Multielement Calibration Standard 3, AtomicSpectroscopy Standard, PerkinElmer Pure).Moreover, the isotopes of 45Sc, 74Ge, 103Rh and 159Tbprepared from individual solutions with concentra-tion of 1000 mg/L were applied as an internal stan-dards in order to effectively correct temporal varia-tions in signal intensity (ICP Standard CertiPUR,Merck, Germany). Calibration curves for elementswere constructed in the range of 0.1 to 50 µg/L.Argon with a purity of 99.999% was used as a neb-ulizer, auxiliary and plasma gas in ICP-MS (Messer,ChorzÛw, Poland).

Analytical performance

The methods of trace element measurementwas validated and controlled by preparation of stan-dard solutions, calibration of instrument and dailyrun of blank samples and duplicates and replicateswith each analytical cycle. All samples were ana-

Table 1. Results of the measurements of accuracy of the analytical data using certificate reference materials ìfungal powdered fruiting bod-ies of Leccinum scabrumî IC-CS-M-4 (n = 5) and Oriental Basma Tobacco Leaves, INCT-OBTL-5 (n = 5).

Analyte Measured value (mg/kg) Certified value (mg/kg) Recovery (%)

Ag2 0.062 ± 0.003 0.053 ± 0.011 116

As1 0.416 ± 0.018 0.375 ± 0.051 111

Ba2 63.2 ± 2.2 67.4 ± 3.8 94

Cd1 1.43 ± 0.13 1.33 ± 0.09 107

Co2 0.949 ± 0.043 0.981 ± 0.067 97

Cr1 1.05 ± 0.11 1.09 ± 0.21 96

Cs2 0.261 ± 0.01 0.288 ± 0.02 91

Cu1 28.18 ± 0.98 26.37 ± 1.74 107

Li2 18.8 ± 0.65 19.3* 97

Mn2 174 ± 5 180 ± 6 97

Ni2 8.94 ± 0.49 8.5 ± 0.49 105

Pb1 1.032 ± 0.06 0.998 ± 0.072 103

Rb2 18.5 ± 0.6 19.1 ± 1 97

Sr2 101 ± 1 105 ± 5 96

Tl2 0.044 ± 0.001 0.051* 86

U2 0.100 ± 0.002 0.113* 88

V2 3.98 ± 0.10 4.12 ± 0.55 97

Zn1 127.8 ± 5.2 120.7 ± 5.9 106

1 IC-CS-M-4. 2 INCT-OBTL-5. * Information values.


lyzed in batches with certified references materialand blanks. Duplicates and blanks were measuredwith every set of 10 mushroom samples. Evaluationof the accuracy of the analytical method was basedon analysis of the certified reference material mush-rooms powder CS-M-4, Institute of NuclearTechnology and Chemistry in Warsaw, ICHTJ,Poland (Table 1). Precision was calculated as thecoefficient of variations (CV) of duplicates. As aresult of the analysis, the following measurementprecision values were in the range from 1 to 8% forall investigated elements. Finally, the limits ofdetection, calculated as three standard deviations of7 independent replicates of the reagent blank, wererespectively (in µg/L) for 0.003 for Ag; 0.03 for As;0.2 for Ba; 0.008 for Cd; 0.001 for Co; 0.2 for Cr;0.15 for Cu; 0.02 for Li; 0.4 for Mn; 0.05 for Ni;0.07 for Pb; 0.03 for Rb; 0.06 for Sr; 0.001 for TL;0.004 for U; 0.09 for V and 4 for Zn.

Statistical analyses

The computer software Statistica, version 10.0(Statsoft Polska, KrakÛw, Poland), was used for sta-tistical analysis of data.


The data on metallic elements and arsenic con-tents of sclerotia are summarized in Table 2, and theresults from the multivariate examination in Table 3and in Figure 2. The results presented in this studyshowed on a highly varying contents of certain ele-ments in sclerotia with the data differing betweenthe places for Ag, Ba, Ni and Sr, low and roughlyconsistent for Ag, Co, Cs, Li, U and V, and greaterand also roughly consistent for Ba, Cr, Cu, Mn, Pb,Rb, Sr and Zn.

Cu, Mn, Zn

Copper, manganese and zinc are essential tracemetallic elements both in fungi and fauna, becausethey are key cofactors in many enzymes. Ediblemushrooms foraged in Europe could be consideredas good in Cu, Mn and Zn (9). The median value ofCu in examined sclerotia was at 1.3 mg/kg dry bio-mass (db) and range at 0.085-3.9 mg/kg db. Thosevalues are substantially lower if compared to sclero-tia of the fungus named Pleurotus tuber-regium (Fr.)Singer [current name is Lentinus tuber-regium (Fr.)

Figure 2. Principal component analysis of the trace metallic elements and arsenic in the Yunnanís sclerotia of W. extensa

A B

C D


Tab

le 2

. Tra

ce m

etal

lic e

lem

ents

and

ars

enic

con

tent

(m

g/kg

db)

of

scle

rotia

of

the

fung

us W

. ext

ensa

from

Yun

nan.

Plac

e of

col

lect

ion

Li

VC

rM

nC

oN

iC

uZ

nA

sR

bSr

Ag

Cd

Ba

PbC

sT

lU

Zhe

nyua

n, P

u'er

0.01

00.

016

0.24

9.9

0.02

20.

203.

45.

5<

0.0

020.

350.

52<

0.0

01<

0.0

002

0.91

0.02

10.

006

< 0

.001

< 0

.001

Wen

shan

cou

nty,

Wen

shan

0.09

20.

231.

85.

10.

110.

370.

441.

80.

024

0.43

6.7

< 0.

001

0.01

52.

70.

240.

029

< 0.

001

0.00

4

Ten

gcho

ng, B

aosh

an0.

022

0.04

70.

6516

0.12

0.10

1.2

6.5

< 0.

002

2.1

0.25

< 0.

001

< 0.

0002

3.6

0.04

00.

012

< 0.

001

< 0.

001

Jing

dong

, Pu'

er0.

003

0.04

13.

333

0.02

50.

211.

52.

2<

0.00

20.

1112

< 0.

001

0.03

422

0.17

0.00

4<

0.00

1<

0.00

1

Xin

ping

, Yux

i0.

010

0.01

80.

2710

0.02

60.

044

0.90

6.1

< 0.

002

2.0

0.15

0.01

10.

009

0.84

0.02

40.

003

< 0.

001

< 0.

001

Yua

njia

ng,Y

uxi

0.00

80.

030

0.36

7.9

0.01

60.

098

0.80

5.6

< 0.

002

0.21

0.25

< 0.

001

< 0.

0002

0.89

0.03

8<

0.0

01<

0.00

1<

0.00

1

Kai

yuan

, Hon

ghe

0.01

20.

038

0.54

310.

064

0.09

22.

48.

10.

015

2.8

0.41

0.08

00.

016

1.6

0.07

30.

007

< 0.

001

< 0.

001

Ship

ing,

Hon

ghe

0.01

00.

020

0.34

9.0

0.03

30.

065

0.08

55.

3<

0.00

20.

390.

190.

20<

0.00

020.

670.

035

< 0.

001

< 0.

001

< 0.

001

Shua

ngba

i, C

huxi

ong

0.00

50.

038

0.26

5.3

0.01

80.

089

3.4

3.0

< 0.

002

0.30

1.8

< 0.

001

0.01

00.

920.

054

< 0.

001

< 0.

001

< 0.

001

Esh

an, Y

uxi

0.02

10.

180.

6716

0.03

50.

310.

612.

70.

012

0.31

10<

0.00

1<

0.00

022.

40.

130.

011

< 0.

001

< 0.

001

Jian

shui

, Hon

ghe

0.00

50.

010

0.30

2.9

0.01

10.

027

2.0

6.2

< 0.

002

0.13

0.26

< 0.

001

< 0.

0002

0.81

0.03

0<

0.00

1<

0.00

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Lux

i, H

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680.

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Mile

, Hon

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0.04

70.

111.

18.

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140.

703.

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4.2

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83.

20.

120.

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< 0.

001

< 0.

001

Xin

cun

tow

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0.76

9.4

0.10

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200.

97<

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0.04

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0.04

< 0.

001

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< 0.

001

Nin

glan

g, L

ijian

g0.

080

0.25

1.8

480.

250.

383.

97.

10.

041

1.3

0.27

0.01

60.

022

2.1

0.09

70.

043

0.00

50.

005

Mea

n0.

022

0.06

20.

7914

0.05

40.

801.

75.

10.

010

0.81

2.6

0.01

80.

018

2.8

0.07

70.

008

< 0.

001

< 0.

001

SD0.

027

0.07

50.

7912

0.03

72.

91.

12.

10.

012

0.84

4.5

0.04

90.

010

4.4

0.06

00.

011

NA

NA

Med

ian

0.01

00.

038

0.52

9.9

0.03

20.

101.

35.

1<

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20.

370.

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1.5

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Fr.] (27) in the south-eastern Nigeria (median at 4.7mg/kg db and range (1.2-20) mg/kg db) (4).

Manganese in sclerotia of W. extensa was at 9.9mg/kg db (range 2.9-48) and followed by zinc at 5.1mg/kg db (range 1.8-9.0) (Table 2). The Mn and Zncontents of the sclerotia of W. extensa are much lessof L. tuber-regium with the manganese median val-ues at 18 mg/kg db and zinc median value at 20mg/kg db (4).

Fungus W. extansa is wood decaying species.Hence, its feeding habit could, to some degree,explain difference in content of Mn and Zn betweenthe sclerotia of W. extensa and L. tuber-regium. TheL. tuber-regium is also saprophytic fungus, which isabsorbing minerals from a decaying biomassdegrading in soils and from soil solution, while usu-ally greater content of Mn and Zn in soils than woodsubstrata could also matter.

From data published on the trace element com-position of mushrooms that are popular in Yunnancould be suggested, that the species substantiallyvary in contents of Cu and Zn. For example, Boletustomentipes (Earle) Murrill was poor in both ele-ments: Cu was at 11-16 mg/kg db and Zn at 19-23mg/kg db (28), while much richer was Boletusedulis foraged in the central region of Yunnan:

range of cooper means value in caps from severalplaces at (17 ± 6) mg/kg db to (880 ± 62) mg/kg dband zinc range was from (54 ± 17) mg/kg db to (530± 120) mg/kg db (29).

Cr, Co, Li

Chromium (trivalent ion), cobalt and lithiumplay the statutory roles in human body and areessential, while nutritional requirements for theseelements is much lower than for Cu, Mn and Zn.Sclerotia contained Cr at 0.52 mg/kg db (median value), Co at 0.032 mg/kgdb and Li at 0.010 mg/kg db. There is no earlier datapublished on Cr or Li composition of fungal sclero-tia. The results for Co in this study are within arange reported for L. tuber-regium ñ with cobaltmean content value at (0.049 ± 0.012) mg/kg db (4).

Cs, Rb

Sclerotia contained Cs (133Cs) in range from <0.001 mg/kg db to 0.043 mg/kg db. Rubidium was at0.37 mg/kg db (median value), while its minimumwas 0.12 mg/kg db and 2.8 mg/kg db was maximumcontent. No previous data are available on Cs an Rbin sclerotia of W. extensa. The sampled sclerotia ofW. extensa from Yunnan showed very low activity

Table 3. Factor loadings obtained with unrotated factor matrix.

Eigenvalues 6.82 3.63 2.25 1.48 1.21

Total variance (%) 37.91 20.16 12.53 8.25 6.75

Variables PC1 PC2 PC3 PC4 PC5

Li 0.92 0.01 0.15 -0.10 -0.14

V 0.90 0.20 0.23 0.06 -0.06

Cr 0.35 0.88 0.13 0.13 -0.02

Mn 0.41 0.55 -0.50 0.40 0.08

Co 0.88 0.06 -0.23 0.22 0.08

Ni 0.09 0.04 0.14 -0.11 -0.96

Cu 0.05 -0.05 -0.19 0.88 0.07

Zn -0.09 -0.36 -0.86 0.13 0.08

As 0.83 0.08 0.05 0.19 -0.37

Rb 0.06 -0.05 -0.87 0.05 0.03

Sr 0.08 0.77 0.47 -0.24 -0.02

Ag -0.10 -0.08 -0.55 -0.35 0.05

Cd 0.23 0.73 -0.17 0.26 -0.50

Ba -0.16 0.93 0.14 -0.03 0.06

Pb 0.52 0.62 0.45 -0.13 -0.11

Cs 0.96 0.14 -0.04 0.10 -0.04

Tl 0.14 0.05 0.16 0.78 0.00

U 0.94 0.10 -0.04 0.08 0.11


level from radioactive 137Cs for which the medianvalue was at 3.4 Bq/kg db (7). Sclerotia of W. exten-sa are remarkably lower in Rb than sclerotia of L.tuber-regium, for which the mean value was at 15 ±14 (0.05-46) mg/kg db (4).

The elements Rb is common constituentsinfruiting bodies. For example in caps of Amanitamuscaria Rb was at 58-310 mg/kg db and inPaxillus involutus at 240-480 mg/kg db (30, 31). Inthe light of data presented, sclerotia of W. extensafrom Yunnan are poor in Rb.

Ba, Sr

The median value of Ba in sclerotia of W.extensa was at 1.5 mg/kg db and of Sr at 0.68 mg/kgdb while for barium range were wide, i.e., from(0.62 to 22) mg/kg db and strontium from (0.15 to12) mg/kg db. Those values may suggest on a highdiversity in composition of the geochemical back-ground between the sites sampled. Sclerotia of L.tuber-regium from Nigeria contained Ba at 2.4(0.045-17) mg/kg db and Sr at 4.3 (0.13-20) mg/kgdb (4), which are roughly similar levels.

Ni, V, U

The elements Ni and V were at detectable levelin all sclerotia sampled. The median value for Niwas at 0.10 mg/kg db, while maximum content at asingle place was as high as 14 mg/kg db (Table 3).Sclerotia of W. extensa are on the average poorer inNi than sclerotia of L. tuber-regium, for which medi-an content was at 0.39 mg/kg db, while maximumwas 0.86 mg/kg db (4). Vanadium also could bedetected in all samples of sclerotia but at low level,i.e., median value was at 0.038 mg/kg db, while ura-nium could not be detected (< 0.001 mg/kg db) withexception of sclerotia from two places (Table 2).There is no data available on occurrence of V and Uin sclerotia of fungi other than this study.

Ag, As, Cd, Pb, Tl

The elements Ag, As, Cd and Tl could bedetected respectively in one to a few samples ofsclerotia but at very low level, while element Pb wasfound in all sclerotia sampled. The median value ofPb in sclerotia was at 0.054 mg/kg db, which is anorder of magnitude less when compared to sclerotiaof L. tuber-regium ñ also richer in Ag and Cd (4).Nevertheless, the contents of Ag, Cd and Pb in scle-rotia of both species mentioned were, on the aver-age, from two to three orders of magnitude lowerwhen compared to fruiting bodies of popular ediblewild-growing fungi (32-34). No data on As and Tl insclerotia could be obtained from the scientific liter-

ature. Altogether, Ag, As, Cd, Pb, Rband Tl are verylow in sclerotia and without toxicological signifi-cance.

Because of the polymetallic character of aweathered, the red and yellow lateritic soils ofYunnan, any further study aiming to provide moredata on mineral constituents of sclerotia could focuson a specific areas of Yunann and on associations offungus with soil geochemistry there. Transfer of theelements from a substrate (sclerotia) to edible or ofa medicinal use product (decoct etc.) is also worth aconcern.

Multivariate analysis of data

To examine if any association exits betweenelements determined in sclerotia has been used amultivariate approach and applying the principalcomponent (PC) analysis (35). We examined thecorrelation matrix obtained from a possible 19 x 18data matrix and the results are given in Table 3. Themodel could explain up to 85.6% variability in thedata matrix by five factors for which an eigenvaluewas greater than 1. Absolute values of the correla-tion coefficient were above 0.73 (significance at p <0.05) for several elements.

The first PC (PC1) was under influence by vari-ables associated with positively correlated As, Co,Cs, Li, U and V. The second PC (PC2) was stronglyinfluenced by positively correlated Ba, Cd, Cr and Sr.The third PC (PC3) was influenced by negatively cor-related variables describing Rb and Zn, the fourth(PC4) by positively correlated Cu and Tl, and the fifth(PC5) by negatively correlated for Ni. Figure 2 showsgraphically the associations among the elements andsampling sites in the factor space as a PCA.

The trace elements in sclerotia may originatefrom the wood substrate which provides organic andinorganic compounds to fungus as well as from soilsolution which provides water and hence also watersoluble compounds ñ largely inorganic ions but alsosoluble organic molecules. As can be read fromFigure 2A, certain elements tend to cluster together,i.e. Cs, U, Li, V, Co and As (associated with PC1).This configuration of cluster interrelations could beexplained by considering that the major source ofelements to W. extensa and its host trees in theforested regions of Yunnan is characteristic geo-chemical composition of soil bedrock and propertiesof soils of southwestern China but not anthro-pogenic sources. Those elements at elevated contentwere specifically in sclerotia from the region ofWenshan in Wenshan country (site 2) and of theNinglang in the Lijiang area (site 19; Fig. 2C), whatstrongly suggests an impact from the soil bedrock.


This has been observed recently for Hg and mush-rooms foraged in the Yunnan land (18-20).

The Ba and Sr, which were positively associat-ed with PC2 usually inter-correlate in fruiting bod-ies of fungi and this could be related to their chemi-cal analogy with calcium (Ca) that was not meas-ured in this study (36-38). Sclerotia from the regionof Jingdong in the Puíer prefecture (site 4) werespecifically enriched in the elements Ba, Sr, Ce andCd (Fig 2C). The Kaiyuan, Honghe (site 7) andHuize, Qujing (site 18) were associated with PC3due to Rb and Zn (Fig 2D). With PC4 were associ-ated the Bajiao in the Chuxiong area (site 15) andthe Ninglang in the Lijiang (site 19) area due to Cuand Tl (Fig 2D), and with PC5, the Mile in theHonghe area (site 13) due to Ni (Fig. 2D).

CONCLUSION

Dried sclerotia of the fungus W. extensa for-aged somewhere in the Yunnan Province of Chinacould be enriched in the metallic elements such asBa, Cr, Sr and Ni. Manganese content of sclerotia isrelatively great and similar to levels noted in ediblefruiting bodies of wild-growing mushrooms foragedin Europe. The metallic elements such as Co, Cs,Cu, Li, Rb, U, V and Zn could be found in sclerotiabut contents are smaller than in fruiting bodies ofwild growing mushrooms. The toxic compoundssuch as Ag, As, Cd, Pb, Rb and Tl are at very lowlevel in sclerotia and without toxicological signifi-cance. In future, because of the polymetallic charac-ter of a weathered, the red and yellow lateritic soilsof Yunnan, any study aiming to provide more dataon mineral constituents of sclerotia could focus on aspecific areas in this vast in space and rich in forestsland, and on associations of fungus with soil geo-chemistry. Transfer of the elements from a substrate(sclerotia) to edible or of a medicinal use product(decoct etc.) could be also a matter of concern.

Acknowledgment

This study in part was supported by theNational Natural Science Foundation of China (No.31460538).

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Received: 25. 05. 2016


Cancer is uncontrolled proliferation of abnor-mal cells associated with genetic mutation. Despiteof all considerable clinical interventions, cancerremains a devastating disease and World HealthOrganization (WHO) has reported death of 7.6 mil-lion people in 2008 (1) and 8.2 million in 2012 (2).Most common cancers include; lungs, breast, liver,stomach and kidneys (3). Incidence rates for canceris twice as high in developed countries than lessdeveloped countries (4). However, mortality rate ishigh in less developed countries due to lack ofhealth care facilities, poor prognosis and high cost ofavailable treatments. Current therapeutic options forcancer are mainly chemotherapy, radiotherapy, sur-gery, DNA-interactive agents and hormones (5).However, these treatments are costly, painful andhave severe adverse effects. Therefore, there is per-sistent requirement for new, affordable and effectivetherapeutic opportunity against cancers. Contempo-rary health management systems are now preferringmedicinal plants for treatment of different ailments(6). In most of the developing countries indigenousmedicinal plants are used as folk medicine.

Pharmacological evaluation of plants proves them agood source for the medicine development againstdifferent infections.

More than 50% of drugs available for cancertherapy are now based on plant derived natural prod-ucts (7). This is a part of an effort to search new phy-tochemical based anti-cancer agent with more effi-cacy, less toxicity and cost effectiveness (8).Moreover, during various studies, phytochemicalshave proved to be effective against cell prolifera-tion, apoptosis, metastasis and angiogenic effects (9,10). However, presently most of them are underclinical trials and only limited number of such natu-ral product based drugs are available for cancerpatients (11). In 2005, Chinese Pharmacopoeia list-ed that petroleum ether, ethyl acetate and methanolextracts of B. chinensis root exhibited significantantitumor activity in PC3, Bcap-37 and BGC-823cells lines (12).

Ornamental plants are mainly grown for theiresthetic value but many of them possess lignin,flavonoids and other medicinally important phyto-chemicals. Some ornamental plants are reported to

DRUG BIOCHEMISTRY

IN VITRO CYTOTOXICITY OF SANCHEZIA SPECIOSA EXTRACTS ONHUMAN EPITHELIAL CERVICAL CANCER (HELA) CELL LINE

NUSRAT SHAHEEN1*, MUHAMMAD UZAIR, BASHIR AHMAD CH and ALAMGEER2*

1Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan2Department of Pharmacology, Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan

Abstract: Cancer is one of the most distressing, life threatening disease regarding morbidity and mortalityworldwide. Many folk medicinal and ornamental plants possess chemotherapeutic and chemo preventive poten-tial. In present study, dichloromethane extract of wood, bark and root and methanol extracts of bark and root ofSanchezia speciosa were prepared. Sanchezia speciosa, an ornamental plant was evaluated for phytochemicalmanifestation, in vitro cytotoxic activity (brine shrimp lethality bioassay) and anti-cancer activity on humanepithelial cervical cancer cell line (MTT assay). Preliminary phytochemical evaluation revealed the presence ofalkaloids, glycosides, steroids, terpenoids and tannins. Sanchezia speciosa root extract in dichloromethane(DCM) showed substantial cytotoxic activity (IC50 2.528 ± 0.31 µg/mL) in brine shrimp assay and in MTT cellproliferation inhibition assay on HeLa cell line, it showed IC50 value of 26.7 ± 0.72 µg/mL. Effect of differentconcentrations of S. speciosa roots showed optimum cell proliferation inhibition at 100 µg/mL. Thus from theresults it can be deduced that Sanchezia speciosa extract root (DCM) possess significant cytotoxic potential inbrine shrimp and MTT bioassay.

Keywords: Sanchezia speciosa, HeLa cell line, MTT assay, anticancer, cytotoxic activity

1389

* Corresponding author: e-mail: [email protected]; e-mail: [email protected]

1390 NUSRAT SHAHEEN et al.

have biological activities in various in-vitro and in-vivo studies (13). Sanchezia speciosa (Family:Acanthaceae), is planted as an ornamental plant.Previous reports revealed that methanol extract ofSanchezia speciosa leaves has shown anti-oxidanteffects and significant cell growth inhibition onMCF-7 cell line (14). Ethanolic extracts of this plantpossess in-vitro cytotoxic, anti-bacterial, anti-fungaland insecticidal activities (15). The aim of presentstudy was to screen phytochemicals and analyzecytotoxic potential of Sanchezia speciosa usingBrine shrimp lethality bioassay and MTT cell prolif-eration assay.


Plant material

Sanchezia speciosa fresh, mature plants of183-244 cm height, were collected in July 2015from Multan and recognized by Dr. Zafar Ullah,Professor of Taxonomy, Department of Botany,Bahauddin Zakariya University, Multan (Voucherspecimen: KEW Royal Botanic GardensK00882617).

Preparation of plant extract

Sanchezia speciosa leaves, bark, wood androots were shade dried and ground in mill. The pul-verized plant material (700 g) was soaked indichloromethane for 24 h; followed by methanolextraction. Extracts were concentrated by rotaryevaporator under reduced pressure. Plant extractsfor wood, bark and root in dichloromethane (DCM)were labeled as SSWD, SSBD and SSRD. Whereas,methanol extracts for wood, bark and root werelabeled as SSWM, SSBM and SSRM, respectively.

Primary phytochemical screening

For phytochemical analysis, Sanchezia spe-ciosa was subjected to recommended tests for alka-

loids, anthraquinones, glycosides, saponins, flavo-noids, tannins, terpenes and steroid as described pre-viously (16-18).

Cytotoxicity analysis

Brine shrimp lethality bioassay

Brine shrimp larvae Artemia salina (Leech) areoften sensitive to toxic bioactive compounds.Artificial sea water was managed by adding 3.8 gsea salt in 1000 mL water and then filtered. Seawater was poured in a tank, in larger compartmentshrimp eggs were added and covered with aluminumfoil to darken the surrounding. Brine shrimp larvaehad matured in two days. Thereafter, vials were pre-pared with 5 mL of sea water in each and by usingpasture pipette 10 shrimps were placed in respectivevial (30 shrimps/dilution) and sustained in illumina-tion. Number of survived shrimps was examined by3× magnifying glass, results was documented (19)and recorded as IC50 value.

Hela (human epithelial cervical cancer) cell line

culture

The Hela cell line was cultured in 10% fetalcalf serum with 50 U/mL penicillin and 50 µg/mLstreptomycin, placed in humidified incubator with5% carbon dioxide and at 37OC temperature main-tained.

MTT assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-yltetrazolium bromide) tetrazolium) assay was per-formed to access reduction of tetrazolium to for-mazan by production of dehydrogenase enzymes inmitochondria of proliferation cells. Hela cells wereseeded (on 96 well microtiter plate) with 5 ◊ 104 ñ105 mL-1 concentration, 200 µL per well total vol-ume maintained and incubated at 37oC. After 24 h,fresh medium was added with different concentra-tions of plant extracts and control with standard anti-

Table 1. Preliminary phytochemical screening of Sanchezia speciosa extract.

Phytochemicals Qualitative Screening Result

Alkaloids Dragendorff/ Mayer/ Wagner +

Anthraquinone Borntrage/ Modified Borntrager -

Glycosides Keller Kelliani +

Flavonoids Ferric Chloride/ Alkaline -

Steroids and Terpenoids Salkowski/ Libermann-Burchard +

Saponins Frothing -

Tannins Lead acetate /Ferric chloride +

(+) Phytoconstituent present; (-) Phytoconstituent absent

In vitro cytotoxicity of Sanchezia speciosa extracts on... 1391

cancer drug followed incubation for 72 h at 37OC.After incubation, 15 µL of medium was replacedwith 150 µL of fresh medium and MTT, respective-ly, and incubated for 4 h. After that, insoluble for-mazan was dissolved in 50 µL DMSO andabsorbance was taken at 540 nm. IC50 value was cal-culated for potent cytotoxic plant extracts againstHela cell line. Dose response of potent extract wasevaluated at various concentration (6.25 µg/mL,

12.5 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL),plates were incubated for 72 h, absorbance wasmeasured at 540 nm by ELISA plate reader andresults were recorded (20, 21).

Statistical analysis

Results were expressed as the mean ± SEM.One way Anova and T test were applied in differentexperiments using Graph Pad Prism 6 software.

Figure 1. Cytotoxic effect of Sanchezia speciosa crude extracts on Brine shrimps

Figure 2. Hela cells survival rate after treatment with Sanchezia speciosa extracts

Brine Shrimp Lethality Assay

Etoposide SSRM SSRD SSBM SSBD

IC50

valu

es (

mg

/mL

)500

400

300

200

100

0

Effect of Sanchezia speciosa on HeLacell line (MTT assay)

Sanchezia speciosa extracts

Cel

l sur

viva

l (LD

50µg

/mL)

SSWD SSBD SSBM SSRD SSRM Doxorubicin


RESULTS

Qualitative screening of Sanchezia speciosa forphytochemical evaluation was performed as per rec-ommended protocols. Results illustrate the presenceof alkaloids, glycosides, steroids terpenoids and tan-nins. On the other hand, anthraquinones, flavonoidsand saponins were not detected in Sanchezia spe-ciosa extract (Table 1). The cytotoxic effect of DCMand methanol extracts of Sanchezia speciosa barkand root were evaluated by brine shrimp lethalityassay (BSLA). According to present investigation,varying degrees of lethality were observed whenexposed to different doses of test samples. Moreover,lethality was found to be directly proportional to con-centration of extracts tested (Fig 1). The DCMextract of S. speciosa root appeared to have signifi-cant cytotoxic potential with IC50 value of 2.52µg/mL comparable to standard anti-cancer drug,etoposide IC50 value of 7.46 µg/mL.

To evaluate the effect of S. speciosa on cellproliferation assay, growth prospect of cancer cellswas considered in MTT assay, with bark and rootextract of S. speciosa in DCM and methanol solventrespectively. In-vitro anti-cancer effect was assessedon Hela cells in 96-well plates and then incubated at37OC at different daily intervals (n = 3) againstabsorbance of formazan at 540 nm. The number ofviable cells were determined by taking doxorubicinas control with IC50 value calculated as 0.1 ± 0.02µg/mL. Whereas, SSRD found to have chemothera-peutic effect with IC50 value 46.7 ± 1.7 µg/mL (Fig

2). Similarly, dose response MTT assay for SSRDwas performed at different concentrations indicatingmaximum chemotherapeutic effect at 100 µg/mL(Fig 3).

DISCUSSION

Natural products are major source of 40% ofdrugs available in modern medicine system (20).Phytochemical evaluation is foremost step prior tobioactivity investigation, which leads to discovery ofnovel therapeutic compound. Most of the people inPakistan rely on folk medicine from diverse flora ofcountry. Plants contain phytochemicals which playvital role in treatment of various infections (22).Phytoconstituents such as triterpenoids, flavonoids,alkaloids and glycosides are reported to be effectivein inflammation, pain, infection and cancers. In pres-ent study, phytochemical evaluation of Sancheziaspeciosa revealed presence of alkaloids, glycosides,terpenoids, steroids and saponins (Table 1) while,alkaloids possess cytotoxic effect which is utilizedfor medicinal purpose (23). Previously, Sancheziaspeciosa was explored to have significant anti-bacte-rial, anti-fungal and moderate insecticidal property(24). Moreover, brine shrimp lethality assay is con-sidered as effective bioassay to assess cytotoxiceffect of plant material (25, 26). In addition, this sig-nificant lethality is accredited to the occurrence ofeffective cytotoxic phytoconstituents (27). Anextract having IC50 below 30 µg/mL is generally con-sidered as a potent bioactive extract (23). Plant

Figure 3. Hela cells survival rate at different concentrations of Sanchezia speciosa root (SSRD)

Cel

l sur

viva

l (µg

/mL)

Control 6.25 12.5 25 50 100

Sanchezia speciosa concentration (µg/mL)

Dose response assay

10000

8000

6000

4000

2000

0

In vitro cytotoxicity of Sanchezia speciosa extracts on... 1393

extracts obtained from root of Sanchezia speciosa(SSRD) were found to have potent cytotoxic activity(IC50 value 2.52) as compared to standard anti-cancerdrug etoposide (IC50 value 7.46). This inhibitoryeffect of extract might be due to cytotoxic com-pounds present in extract.

MTT rapid colorimetric assay was employed todetermine the effect of Sanchezia speciosa wood(SSWD), root (SSRM) (SSRD) and bark (SSBM)(SSBD) on Hela cells proliferation. Meanwhile, for-mazan product is only formed in viable respiringmitochondria of cells. To recognize in-vitro cell sur-vival of Hela cells, graph was plotted between cellssurvived and extracts (Fig 3). The results illustratethat SSRD possess chemotherapeutic effect in theperiod of administration. In a previous study,methanol leaves extract of Sanchezia speciosa haveshown anti-oxidant and anti-cancer effects (14).Whereas, no earlier study on wood, root and bark ofSanchezia speciosa was found. In order to investigatethe effect of SSRD extract on Hela cell, anotherexperiment was performed with serial dilutions ofSSRD extract. The cell viability was significantlyreduced, especially at concentration of 100 µg/mL(Fig 3). These findings suggest that Sanchezia spe-ciosa root possesses cytotoxic ptroperties due to itssecondary metabolites. However, further fractiona-tion, isolation and bioassay guided studies are indis-pensable to obtain pure potent anti-cancer compound.

CONCLUSION

Preliminary screening of Sanchezia speciosarevealed the presence of different phytochemicals.Proliferation and survival rate of Hela cells wasreduced by Sanchezia speciosa root extract duringMTT assay. Moreover, 100 µg/mL concentration ofSSRD was found to be most effective in Hela cells.Therefore, Sanchezia speciosa root extract may pos-sess novel potent anti-cancer compound withenhanced efficacy and limited adverse effects.Further, rigorous phytochemical mechanistic andbioassay guided studies are indispensable.

Acknowledgments

The authors highly acknowledge Faculty ofPharmacy Bahauddin Zakariya University, Multan,Pakistan for providing research facilities.

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4. Torre LA., Bray F., Siegel R.L., Ferlay J.,Lortet-Tieulent J., Jemal A.: Cancer J. Clin. 65,87 (2015).

5. Nussbaumer S., Bonnabry P., Veuthey J.L.,Fleury-Souverain S.: Talanta 85, 2265 (2011).

6. Ullah M., Khan M.U., Mahmood A., MalikR.N., Hussain M. et al.: J. Ethnopharmacol.150, 918 (2013).

7. Solowey E., Lichtenstein M., Sallon S.,Paavilainen H., Solowey E., Lorberboum-Galski H.: Sci. World J. (2014).

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9. Hadjzadeh M., Tavakol Afshari J., Ghorbani A.,Shakeri M.T.: J. Med. Plants 2, 41 (2006).

10. Sadeghnia H.R., Ghorbani Hesari T., Morta-zavian S.M., Mousavi S.H., Tayarani-NajaranZ., Ghorbani A.: BioMed. Res. Int. (2014).

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14. Paydar M., Wong Y.L., Moharam B.A., WongW.F., Looi C.Y.: Pak. J. Biol. Sci. 16, 1212(2013).

15. Parvin S., Rafshanjani M.A.S., Kader M.A.,Sharmin T.: Int. J. Adv. Sci. Res. 1, 145 (2015).

16. Brain K.R., Turner T.D.: Practical evaluation ofphytopharmaceuticals, 1st edn., Wright-Scientechnica, Bristol 1975.

17. Evans W.C.: Trease and Evansí pharmacog-nosy, 16 edn., Elsevier Health Sciences 2009.

18. Raaman N.: Phytochemical techniques. 1st edn.New India Publishing agency, New Delhi 2006.

19. Choudhary M.I., Thomsen W.J.: Taylor &Francis, (2001).

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21. Huebers H.A., Finch C.A.: Physiol. Rev. 67,520 (1987).

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Received: 14. 08. 2016


Diabetes mellitus is still one of the most lead-ing causes of mortality and chronic morbidity in theworld. Generally, diabetes is classified into twomain types according to the etiological definitions.Type 1 diabetes is caused by destruction ofLangerhans islet beta cells, while type 2 diabetes isdue to decreased insulin sensitivity which in latestages is also associated with inadequate mass offunctional islet cells (1, 2). Although insulin andoral antidiabetic agents are the mainstay of control-ling hyperglycemia and management of early onsetcomplications of diabetes, serious late onset compli-cations appear in a large number of patients (3, 4). Amore promising approach to the management of dia-

betes in insulin-dependent patients seems to betransplantation of Langerhans islet cells (5, 6).However, the scarcity of islets donors, graft failure,and issues of ethics are major problems of thisapproach.

In the last decade several attempts have beenmade to use stem cells for generating a renewablesource of insulin-producing cells (IPCs) (7). From apractical view, an ideal technique for differentiationof stem cells into IPCs should be simple and repeat-able in different labs, and should lead to generationof cells that are responsive to both physiological andpharmacological interventions. Recently, it has beenreported that a simple procedure comprising manip-

EXPOSURE TO FORSKOLIN IMPROVES TRANS-DIFFERENTIATION OFBONE MARROW STROMAL CELLS INTO INSULIN PRODUCING CELLS;GENERATING CELLS WITH BETTER RESPONSES TO PHYSIOLOGICAL

AND PHARMACOLOGICAL AGENTS

SEYYED ABBAS ZOJAJI1, AHMAD GHORBANI2, FAEZEH GHASEMI3, and REZA SHAFIEE-NICK1, 2*

1Deptartment of Pharmacology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2Pharmacological Research Center of Medicinal Plants, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

3Medical Biotechnology, Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences Mashhad, Iran

Abstract: Several attempts have been made to differentiate stem cells into insulin-producing cells (IPCs) forthe treatment of diabetes. Ideally, the differentiation procedures should be simple, repeatable, and must gener-ate cells responsive to physiological and pharmacological stimuli. In this study, we first tested repeatability ofa differentiation method for generating IPCs and then tried to improve the method through pharmacologicalapproach to increase capability of IPCs for glucose- and drug-induced insulin secretion. Rat bone marrow stro-mal cells were trans-differentiated into IPCs by manipulation of the level of glucose, serum and dimethyl sul-foxide (GSD) in culture medium. The effects of addition of nicotinamide (GSD-NA method) and forskolin(GSD-FK method) to differentiation medium on responses of generated IPCs to physiological and pharmaco-logical stimuli were examined. Results demonstrated that the generated IPCs expressed insulin gene and couldreduce blood glucose of diabetic mice following intraperitoneal transplantation. When stimulated with glucoseas a physiological cue, the IPCs of GSD and GSD-FK methods expressed and secreted insulin in a regulatedmanner. The expression of insulin gene was also increased in IPCs of GSD-FK method following treatmentwith glyburide, 3-isobutyl-1-methylxanthine and forskolin. In conclusion, the GSD method is a simple andrepeatable procedure for generating IPCs and modification of the method by increasing cell cAMP usingforskolin enhances responsiveness of them to physiological and pharmacological agents.

Keywords: differentiation, forskolin, glucose, insulin, nicotinamide, stem cells

1395

* Corresponding author: e-mail: [email protected]; phone: +985138002253; fax: +985138828567

1396 SEYYED ABBAS ZOJAJI et al.

ulation of the level of glucose, serum and dimethylsulfoxide (GSD method) in culture medium success-fully induces trans-differentiation of bone marrow(BM) derived cells into IPCs that secret insulin andreduce blood glucose after transplantation intohyperglycemic mice (8). However, the authors ofthis report, like others, have not investigated theresponsiveness of differentiated insulin producingcells to pharmacological agents.

In this study, we tested the repeatability ofGSD method and investigated the capability of IPCsfor initiation of insulin secretion in response tophysiological (glucose) or pharmacological (gly-buride) stimuli and potentiation of glucose-inducedinsulin secretion using 3-isobutyl-1-methylxanthine(IBMX, a non-selective phosphodiesterase inhibi-tor) or forskolin, a known adenylyl cyclase activa-tor. Also, we tried to improve this technique by theaddition of nicotinamide or forskolin to the differen-tiation medium. Nicotinamide, the amide derivativeof nicotinic acid, is poly (ADP-ribose) polymeraseinhibitor. It has been shown that nicotinamide caninduce beta cell outgrowth from undifferentiatedfetal pancreatic cells (9), stimulates differentiationof embryonic stem cells into insulin-secreting cells(10-12) and enhances insulin production in fetal pigIPCs (13). Forskolin is known to alter the lineagecommitment of BM stromal cells through activationof cAMP signaling pathway (14). The cAMP playsan important role in cell proliferation and differenti-ation of progenitor cells and has been suggested topromote beta cell survival (15-17). It was found herethat the addition of forskolin to the differentiationmedia generates IPCs which are more responsive tothe pharmacological interventions.

EXPERIMENTAL

Chemical compounds

Low glucose (5.5 mM) and high glucose (25mM) Dulbeccoís Modified Eagles Medium(DMEM), trypsin, and fetal bovine serum (FBS)were purchased from Gibco (Grand Island, NY,USA). Forskolin, 3-isobutyl-1-methylxanthine(IBMX), penicillin-streptomycin solution, and di-methyl sulfoxide were obtained from Sigma-Aldrich(St. Louis, MO, USA). Streptozotocin was pur-chased from Enzo Life Sciences (Farmingdale, NY,USA). Rat insulin RIA kit was obtained fromIzotope (Budapest, Hungary). Total RNA purifica-tion kit was purchased from Jena Bioscience(Germany). The RevertAid first strand cDNA syn-thesis kit was supplied from Thermo Scientific(Pittsburgh, PA, USA). Taq polymerase, dNTP, 10x

buffer were obtained from Cinagen Company(Tehran, Iran).

Animals

Male Wistar rats (180-200 g, 10-12 weeks old)and male Bulb/C mice (26-30 g, 10-12 weeks old)were obtained from Laboratory Animals ResearchCenter, Mashhad University of Medical Sciences.The animals were maintained in the laboratory inroom temperature (22 ± 2OC) under a 12 h light/darkcycle. They had free access to normal laboratorychow and tap water ad libitum. All procedures wereperformed in accordance with the NIH Guide for theCare and Use of Laboratory Animals and wereapproved by the Animal Ethics Committee ofMashhad University of Medical Sciences.

Preparation of collagen-coated coverslips

Tail tendon fibers were taken from male ratsand sterilized under UV light for 24 h. Then, thefibers were suspended in 0.1% acetic acid andstirred at 4OC for 48 h. The mixture was left at 4OCfor 24 h without agitation allowing the undissolvedfibers to sediment. Upper clear solution was sepa-rated as collagen solution. The slide coverslips (22 ◊22 cm2) were sterilized by autoclave and immersedin 0.3% collagen solution for 48 h at 4OC. At the endof this period, the coverslips were dried under lami-nar hood and then deacidified by a solution of phe-nol red in phosphate buffer). The collagen-coatedcoverslips were placed in wells of six-well tissueculture plates and sterilized under UV light for 24 h.

Preparation of BM cells

The BM stromal cells were isolated fromfemur and tibia of rats. The animals were sacrificedwith high ether anesthesia and with aseptic tech-nique the bones of two hind limbs were removed.The proximal and distal heads of bones were cut,and the marrows were harvested by flushingmedullary cavities with low glucose (5.5 mM)DMEM. The BM cells were filtered through a bloodset filter (pore size 200 µm) to remove debris andbone fragments.

Differentiation of BM cells into IPCs

In GSD method, the BM stromal cells wereseeded onto collagen-coated coverslips in six-wellculture plates (1.5 ◊ 105 cells/well) containingserum-free low glucose (5.5 mM) DMEM supple-mented with 1% DMSO. After 3 days culture, themedium was changed by high glucose (25 mM)DMEM containing 10% FBS. The cells were incu-bated in this medium for 7 days, while the medium

Exposure to forskolin improves trans-differentiation of bone marrow stromal cells into insulin... 1397

was replaced every 3 days. In separate experiments,after 3 days culture in the above mentioned medium,the cells were incubated for 7 days in high glucoseDMEM containing 10% FBS plus 10 mM nicoti-namide (GSD-NA method) or 1 µM forskolin (GSD-FK method).

PCR test

In order to approve that the IPCs have trans-differentiated into endocrine hormone-producingcells, the expression of insulin-1, somatostatin andglucagon genes was evaluated by RT-PCR. TotalRNA was extracted using RNA purification kit andcDNA was synthesized according to the manufac-turerís instruction. Then, PCR was performed usingspecific primers mentioned in Table 1. For the men-tioned genes, PCR amplification was carried out in20 µL reaction volumes containing 40 ng cDNA,0.25 mM dNTPs, 2.5 mM MgCl2, 1X reactionbuffer, 0.5 µM of each primers, and 0.4 units of Taqpolymerase. PCR experiment was performed underthe following conditions for all three genes: 95OCfor 5 min, followed by 35 cycles at 95OC for 45 sec-onds, 58OC for 40 seconds, 72OC for 1 min, andfinally a 7 min extension at 72OC. Finally, the ampli-fied products were carried out using VeritiÆThermal Cycler ñ Applied Biosystems (USA) andall PCR products were analyzed using 2% agarosegel. Results were visualized under UV light by aGelDoc system.

Transplantation of IPCs

The repeatability of GSD method for control-ling blood glucose following transplantation of IPCswas investigated in diabetic mice. Diabetes wasinduced by intraperitoneal injection of 160 mg/kgstreptozotocin. At day 10 after streptozotocin injec-tion, mice were randomly divided into two groups (n= 4): diabetic transplant group that received an

intraperitoneally transplant of 100 µL IPCs (105

cells/100 µL) suspension in saline, and diabetic con-trol group that received 100 µL saline solutions asvehicle. The animals were considered to be diabeticif they had fasting blood glucose level of 300 mg/dLor higher (18) at day of transplantation. Blood glu-cose was measured using a standard blood glucosemeter (Roche Diagnostics).

Evaluation of physiological responses of IPCs

Physiological responses to different concentra-tions of glucose were investigated in IPCs trans-dif-ferentiated with GSD, GSD-NA, and GSD-FKmethods. The IPCs were pre-incubated for 24 h inlow glucose DMEM containing 10% FBS. Then, tospecifically measure insulin release without interfer-ence from the FBS, the IPCs were incubated inserum-free media containing 5.5, 10 or 20 mM glu-cose. After 2 h of incubation, the conditioned mediawere collected and stored at -20OC until insulinassay. Insulin-1 concentration in the culture mediawas measured by an immunoradiometric assay. Toevaluate the effect of glucose on the level of insulinexpression, similarly the IPCs were incubatedovernight in the media containing 5.5, 10 or 20 mMglucose.

Evaluation of pharmacological responses of IPCs

Pharmacological responses to IBMX, forskolinand glyburide were investigated in IPCs trans-differ-entiated with GSD, GSD-NA, and GSD-FK meth-ods. The IPCs were pre-incubated for 24 h in lowglucose DMEM containing 10% FBS. Then, theIPCs were incubated in serum-free media containing10 mM glucose in the absence or presence of 100 µMIBMX, 10 µM forskolin or 10 µM glyburide. After 2h of incubation, the conditioned media were collect-ed for insulin assay. To evaluate the effect of IBMX,forskolin and glyburide on the level of insulin-1

Table 1. Sequence of primers.

Gene Primer sequencePCR product length (bp)

Insulin-1Forward: 5-AGCCTCTGCTTCTGCTTCTG-3

250Reverse: 5-GAGAAGGTCTCGGAGCTGTG-3

GlucagonForward: 5-GAGGAACCGGAACAACATTGCCAA -3

261Reverse: 5-ACGGTGGTCTCTGAAGTAGTT-3

SomatostatinForward: 5-ATGCTGTCCTGCCGTCTCCA-3

321Reverse: 5-GCGTTTCGACCGACGTTCTTG-3

GAPDHForward: 5-ATGGTGAAGGTCGGTGTGA-3

250Reverse: 5-GACCAGCTTCCCATTCTCAG-3


expression, the IPCs were incubated overnight in themedia containing 10 mM glucose and test drugs.

Quantitative real-time PCR

The mRNA expression level of insulin-1 wasevaluated by Real time PCR. After total RNA

extraction and cDNA synthesis, PCR was performedusing specific primers mentioned in Table 1. Realtime PCR was carried out by SYBR green method.All reactions were performed in triplicate and glyc-eraldehyde phosphodehydrogenase (GAPDH) wasused as reference gene. Expression level changeswere estimated by ∆∆CT method.

Figure 1. Morphological changes of BM stromal cells trans-differentiated into IPCs with GSD method. A: Undifferentiated cells cultured10 days in control medium; B: the BM cells underwent morphological changes into more rounded shapes and generated cell clusters underdifferentiated medium. Scale bars, 50 µm

Figure 2. Expression of genes related to pancreatic islets cells trans-differentiated from BM stromal cells with GSD method. RT-PCR wasperformed to detect insulin-1, glucagon and somatostatin

Insulin Glucagon Somatostatin


Figure 3. Glucose change of diabetic mice after intraperitoneal transplantation of IPCs trans-differentiated from BM stromal cells. Diabeteswas induced by intraperitoneal injection of streptozotocin at day 1. #p < 0.05, ###p < 0.001 |compared with the values of day 1 in the cor-responding group. | p < 0.05, | | p < 0.05, | | | p < 0.001 compared with the values of day 10 (transplantation day) in the corresponding group.*p < 0.05, **p < 0.01 compared with diabetic transplant group at the corresponding day. Each value represents mean ± SEM (n = 4)

Figure 4. Effect of different concentrations of glucose on the release of insulin by IPCs. Rat BM stromal cells were trans-differentiated intoIPCs by manipulation of the level of glucose, serum and dimethyl sulfoxide (GSD) in culture medium, in the absence or presence of nicotin-amide (GSD-NA method) or forskolin (GSD-FK method). The IPCs were incubated in serum-free media containing 5.5, 10 or 20 mM glu-cose for 2 h. *p < 0.05, **p < 0.01 compared with glucose of 5.5 mM in the corresponding group. ##p < 0.01 compared with glucose of5.5 mM in GSD group. Each value represents mean ± SEM (n = 4)

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Statistical analyses were performed usingStudentís t-test, when there were two groups to becompared and one-way analysis of variance fol-lowed by Tukeyís post hoc test, when there weremore than two groups. All experiments were repeat-ed at least four times. Results are expressed as themean ± standard error of the mean (SEM) and the p-value less than 0.05 was considered to be statistical-ly significant.

RESULTS

Morphological changes and gene expression of

BM-derived IPCs

Figure 1A shows BM-derived stromal cellsafter day 10 culture in control condition. The cellswere spindle shaped with long projection and couldproliferate in vitro. On the other hand, the cells cul-tured for 10 days in differentiation medium withGSD protocol underwent morphological changesinto more rounded shapes and generated cell clusterslook like islet structure (Fig. 1B). The islet-like clus-ters are stable for at least 4 weeks in culture medi-um.

To confirm BM cells have undergone pancre-atic differentiation, gene expression profiles related

to pancreatic islet cells were assessed using RT-PCRanalysis. Figure 2 shows the expressions of insulin-1, glucagon, and somatostatin in cells trans-differen-tiated with GSD method. While all of the threegenes were expressed in the differentiated cells, noexpressions were detected in control (undifferentiat-ed) cells.

Glycemic status in diabetic mice following trans-

plantation of IPCs

In diabetic control rats, the level of fastingblood glucose continually increased from day 1 to30 post-diabetes induction (Fig. 3). The animalsreceiving intraperitoneal transplant of IPCs began todecrease their blood glucose within one week. Atday 7 and 20 after cell therapy, the level of bloodglucose in treated group was significantly lowerthan that in control group (p < 0.05).

Physiological responses of IPCs to glucose level

In IPCs trans-differentiated with GSD method,the release of insulin in the presence of 5.5 mM, 10mM and 20 mM glucose was 36 ± 1.5 pg/ml, 69 ± 4pg/mL (p < 0.01 vs. 5.5 mM) and 92 ± 10 pg/mL (p< 0.01 vs. 5.5 mM), respectively (Fig. 4). Similarly,the release of insulin was significantly increased byenhancing glucose concentration of incubation

Figure 5. Effect of different concentrations of glucose on the expression of insulin gene in IPCs. Rat BM stromal cells were trans-differ-entiated into IPCs by manipulation of the level of glucose, serum and dimethyl sulfoxide (GSD) in culture medium, in the absence or pres-ence of nicotinamide (GSD-NA method) or forskolin (GSD-FK method). The IPCs were incubated overnight in serum-free media con-taining 10 or 20 mM glucose. *p < 0.05 compared with 10 mM glucose in GSD method; ***p < 0.001 compared with 20 mM glucose GSDand GSD-NA groups. #p < 0.05, ##p < 0.01 compared with 10 mM glucose in the corresponding group. Each value represents mean ±SEM (n = 4)

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media in IPCs trans-differentiated with GSD-FKtechnique. In IPCs of GSD-NA method, the increaseof insulin secretion in response to 10 mM glucosewas comparable to those of GSD and GSD-FK tech-niques (p < 0.01 vs. 5.5 mM); however, no furtherincrease was observed in the presence of 20 mMglucose. Comparison between methods showed thatthe level of basal insulin release (in the presence of5.5 mM glucose) by IPCs trans-differentiated withGSD-FK and GSD-NA methods is higher than GSDtechnique, while their response to 10 mM and 20mM glucose was not significantly different amongthe three methods.

The data of quantitative real-time PCR demon-strated that, in glucose concentration of 10 mM,insulin-1 gene expression was significantly (p <0.05) higher in IPCs generated by GSD-NA methodthan those of GSD method (Fig. 5). Increase of glu-cose concentration in the culture medium from 10mM to 20 mM led to a significant increase inexpression of insulin gene in IPCs of GSD (p < 0.05)and GSD-FK (p < 0.01) methods, but not in IPCs ofGSD-NA method. The IPCs generated by GSD-FKmethod showed the highest insulin gene expressionin the presence of 20 mM glucose.

Pharmacological responses of IPCs to IBMX,

forskolin and glyburide

As shown in Figure 6, 2 h incubation with 10µM glyburide led to a nonsignificant increase ininsulin secretion by IPCs of all GSD, GSD-FK andGSD-NA methods. Similarly, incubation with 10 µMforskolin increased the secretion of insulin by IPCstrans-differentiated with either the three methods,however the level of increase was not statisticallysignificant. Regarding IBMX, the IPCs trans-differ-

entiated with GSD-FK were the only IPCs thatshowed significant pharmacological responses. Inthe presence of 100 µM IBMX, insulin released tothe culture media increased from 74 ± 3.6 pg/mL to96 ± 6 pg/mL (p < 0.05).

Quantitative real-time PCR demonstrated thatthe expression of insulin-1 was not significantlyaltered in IPCs of GSD method as a result of gly-buride or forskolin treatment (Fig. 7). Only IBMXwas able to enhance (p < 0.05) insulin gene expres-sion in IPCs trans-differentiated with GSD method.In IPCs generated by GSD-NA method, forskolinsignificantly decreased insulin expression (p < 0.05)while glyburide and IBMX were ineffective. On theother hand, the level of transcript insulin wasincreased significantly in the IPCs of GSD-FKmethod as a result of glyburide (p < 0.05), forskolin(p < 0.05) or IBMX (p < 0.01) treatment.

DISCUSSION AND CONCLUSION

Utilization of stem cells for generating arenewable source of pancreatic islet cells for trans-plantation to diabetic patients has become the sub-ject of intense research over the past years (8, 19-21). Stem cells can be obtained from embryos, fetus-es, and adults (usually from BM, adipose tissue,teeth, skin, and umbilical cord blood) (22-24). Thereare reports that stem cells harvested from BM, adi-pose tissue, pancreas, skin, and embryos have thepotential to differentiate into endocrine cells capableof releasing islet hormones (19, 20, 25-27). From apractical view, differentiation techniques for gener-ating islet cells should be simple, repeatable and eth-ically justifiable, and the generated IPCs should pos-sess physiological secretory machinery in terms of

Figure 6. Effect of glyburide, forskolin and IBMX on the release of insulin by IPCs. Rat BM stromal cells were trans-differentiated intoIPCs by manipulation of the level of glucose, serum and dimethyl sulfoxide (GSD) in culture medium, in the absence or presence of nicoti-namide (GSD-NA method) or forskolin (GSD-FK method). The IPCs were incubated in serum-free media containing 10 mM glucose with-out or with 10 µM glyburide (A), 10 µM forskolin (B) or 100 µM IBMX (C) for 2 h. *p < 0.05 compared with control in the correspon-ding group. Each value represents mean ± SEM (n = 4)

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glucose-induced insulin release which responds toinsulinotropic agents. From the legal and ethicalpoint of view, the use of adult sources for cell ther-apy appears to raise no new issues and is preferableto the use of embryos or fetuses sources (27). Afteran extensive review of literature on techniques fordifferentiation of adult stem cells into IPCs, it wasfound that the method of GSD described by Oh et al.is a simple, rapid, and inexpensive technique (8). Inthe present work, our results on gene expression,insulin release, and transplantation are in accor-dance with those of Oh et al. and therefore confirmthe repeatability of GSD method. Then, we tried toimprove the method of Oh and coworkers by theaddition of some factors, which are reported to influ-ence beta cell mass, to the differentiation medium.

Until now, several factors have been identifiedthat influence beta cell mass in the pancreas. Theseinclude hormones, glycemic status, growth factors,and phytochemical ingredients (28-31). In the pastyears, some attempts have been made to use thesefactors for increasing islet mass in vivo or improvingmethods of beta cells differentiation in vitro. Forexample, nicotinamide has been reported to stimu-late differentiation of embryonic stem cells intoIPCs (9-12). Also, glucagon-like peptide-1 (GLP-1)and exendin-4, a long-acting GLP-1 receptor ago-nist, have been shown to enhance regeneration ofbeta cell mass in diabetic animals (32). Incubation offetal human islet cells with GLP-1 caused matura-tion of the beta cells (33). In neonatal rat islets, athree-fold increase in the number of beta cells wasseen following 24 h incubation with GLP-1 (31). Inour study, it was found that supplementation of dif-ferentiation medium of GSD method with nicotin-amide improves glucose sensitivity of IPCs, andsupplementation with forskolin generates IPCs

which are more responsive to both glucose and phar-macological insulinotropic agents.

Insulin secretion from beta cells is initiated bymembrane depolarization which is regulated byATP-dependent potassium (KATP) channels and aug-mented by protein kinase A and protein kinase Cdependent signaling pathways. Glucose increasesinsulin secretion through increasing the ratio ofATP/ADP and closing the KATP channels, and itsextracellular concentration is the major physiologiccontroller of insulin secretion (34). Forskolin andIBMX increase intracellular cAMP by stimulatingadenylyl cyclase and inhibiting cyclic nucleotidephosphodiesterases (PDEs), respectively, and there-fore potentiate glucose-induced insulin secretion(35, 36). On the other hand, glyburide, a sulfonyl-urea hypoglycemic agent, elicits insulin release byclosing KATP channels (34). In our study, to investi-gate the physiological capability of IPCs, weassessed their glucose-induced insulin release. Itwas found that insulin release by IPCs of all differ-entiation methods was significantly increased inresponse to glucose enhancement in a concentration-dependent manner. It seems that forskolin andnicotinamide improves the sensitivity of the IPCs toglucose as evidenced by increasing insulin release in5.5 and 10 mM glucose while the maximum of glu-cose-induced insulin secretion was not changed. Wemay conclude that in the modified methods glucoseresponse curve was shifted to the left resulting inreduction of glucose EC50. This finding is in agree-ment with previous evidence which showed cAMP-dependent signal transduction has an important rolein susceptibility of beta cells to glucose (35, 36).

Glucose controls insulin gene expressionthrough recruitment of several transcription factorsincluding mammalian homologue of avian MafA/L-

Figure 7. Effect of glyburide, forskolin and IBMX on the expression of insulin gene in IPCs. Rat BM stromal cells were trans-differenti-ated into IPCs by manipulation of the level of glucose, serum and dimethyl sulfoxide (GSD) in culture medium, in the absence or presenceof nicotinamide (GSD-NA method) or forskolin (GSD-FK method). The IPCs were incubated in serum-free media containing 10 mM glu-cose without or with10 µM glyburide (A), 10 µM forskolin (B) or 100 µM IBMX (C) for 2 h. *p < 0.05, **p < 0.01 compared with thecorresponding value in the GSD group. #p < 0.05, ##p < 0.01 compared with control in the corresponding group. Each value representsmean ± SEM (n = 4)

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Maf, pancreatic/duodenal homeobox-1, andBeta2/Neuro D (37, 38). Accordingly, our resultsshowed that the expression of insulin gene increasedin the IPCs of GSD and GSD-FK methods followingglucose increase. This effect was especially obviousin IPCs of GSD-FK method. On the other hand, theIPCs of GSD-NA method showed the highest levelof insulin gene expression in glucose level of 10mM.

It has been shown that both cAMP and nicotin-amide augment the glucose-activated insulin genetranscription, but with different mechanism. While,cAMP stimulates insulin gene transcription throughcAMP response elements, nicotinamide enhancesinsulin biosynthesis, in part, through increasedMafA gene transcription (39, 40). These data indi-cate that continuous stimulation of MafA andcAMP-dependent pathway during differentiation ofprogenitor cells leads to IPCs that have betterresponse to physiological and pharmacological cues.

Regarding response to pharmacologicalinsulinotropic agents, the IPCs of GSD-FK methodwere the only IPCs that showed increased insulinrelease in response to IBMX, and demonstratedincreased insulin expression in response to all gly-buride, forskolin and IBMX. These data demon-strate that stimulation of cAMP pathway during dif-ferentiation leads to IPCs that also have betterresponse to pharmacological cues. It has beenshown that elevation of intracellular cAMP contentstimulates the transcription of cyclic nucleotidephosphodiesterases (PDEs) (40). A previous studyshowed that PDEs have important role in secretionof insulin from isolated pancreatic islets (41). Thebetter insulin secretion in IPCs of GSD-FK methodmay be the result of higher activity of PDEs whichimplicates an active role for PDEs in glucose-induced insulin release.

In conclusion, our data indicate that the GSDmethod is a simple and repeatable procedure forgenerating IPCs and use of forskolin improves thismethod so that enhances glucose-induced insulinsecretion and responsiveness of these cells to phar-macological initiators of insulin secretion (such asglyburide) and to potentiator of glucose-inducedinsulin secretion (IBMX, and forskolin). Keeping inmind the growing demand for IPCs transplants, fur-ther studies are warranted for optimizing protocolsof generating these cells from stem cells.

Acknowledgments

This work was a part of the Ph.D. thesis of oneof the authors (Abbas Zojaji) and supported by a

grant from Research Council of Mashhad Universityof Medical Sciences, Iran. The authors declare thatthey have no conflict of interest.

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Received: 28. 10. 2016


Cancer, diabetes and hemophilia are caused bythe over or under production of proteins and mutat-ed proteins (1). The genetic material that codes forproteins production is DNA, so extensive research isbeing carried out in order to understand the interac-tions of drug molecules with DNA (2). Many fea-tures of the rich literature insighting drug binding toDNA have been studied in recent articles, symposiaand books (3-5). There are different modes of inter-action of drug with DNA but the molecules whichare planar and hydrophobic in nature tend to haveability to bind with DNA via intercalative mode ofinteraction. This mode of binding is also confirmedby the theoretical studies and molecular dockingshows this mode clearly in the different conformerfor the ligand (6).

Heterocyclic compounds play an importantrole in an untiring effort aimed at developing newchemotherapeutic agents with new mechanism ofaction (7). Among them, conjugated heterocyclesderived from 1,2,4-triazoles have received consider-able attention due to the synthetic and effective bio-logical prominence. A large number of ring systemscontaining 1,2,4-triazoles have been incorporatedinto a wide variety of therapeutically interestingdrug candidates (8, 9). 4-Amino-3-substituted-5-mercapto-1,2,4-triazoles by virtue of their ambidentnucleophilic centers are good starting materials forthe synthesis of several interesting fused heterocy-cles. A variety of biological activities have beenreported in mercapto- and thione-substituted-1,2,4-triazole ring systems (10).

DRUG SYNTHESIS

TRIAZOLOTHIADIAZINES AS A NEW CLASS OF EFFICIENT DNA INTERCALATING AGENTS

ALIYA IBRAR1*, SHAMYLA NAWAZISH2, IMTIAZ KHAN3, TAYYABA NOOR4, BUSHRA UZAIR5,SYED MAJID BUKHARI1*, FARHAN A. KHAN1 and ASMA ZAIDI1

1Department of Chemistry, 2Department of Environmental Sciences, COMSATS Institute of InformationTechnology, Abbottabad-22060, Pakistan

3Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan4School of Chemical and Materials Engineering, National University of Science and Technology,

Islamabad-44000, Pakistan5Department of Bioinformatics and Biotechnology, International Islamic University,

Islamabad-44000, Pakistan

Abstract: A new series of N-bridged heterocycles 1,2,4-triazolo[3,4-b][1,3,4]thiadiazine derivatives (6a-e)have been synthesized by the condensation of 4-amino-5-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thiol and var-ious substituted phenacyl bromides in absolute ethanol in high yields. Cyclic voltammetric and molecular dock-ing methods have been used to investigate the interaction of potential binding affinities of 6a-e with DNA. Thechange in peak potential and peak current is used to probe the mode of interaction, formation constant Kf, freebinding energy and irreversible nature of system. The DNA-binding potency of 6a (Kf = 5.00 ◊ 105 M-1) and 6c

(Kf = 4.80 ◊ 105 M-1) are found to be greater than epirubicin (Kf = 4.1 ◊ 105 M-1), the clinically used anticancerdrug. The positive shift in peak potential revealed the intercalative mode of interactions. The high negativevalue of ∆G signifies the spontaneity and high conformational stability of compound-DNA adduct. A molecu-lar docking study of all the compounds using Auto-Dock 4.2 confirmed additional specificity and insight of theinteraction with the DNA. Free energies by theoretical studies had much resemblance and almost completelysatisfied the experimental results. From results investigation, it is revealed that the examined compounds exhib-it intercalation mode.

Keywords: conjugated heterocycles, free binding energy, intercalation, molecular docking

1405

* Corresponding authors: e-mail: [email protected] (A. Ibrar); e-mail: [email protected] (S.M. Bukhari)

1406 ALIYA IBRAR et al.

The need for privileged scaffolds in medicinalresearch gives an impetus for the synthesis of hybridstructures with remarkable activity profile and in acontinuation of an ongoing program aiming at find-ing new structural leads with potential chemothera-peutic applications (11-15), it was rationalized toemphasize on a structural library of triazolothiadi-azines in combination with DNA binding affinitiescalculated by the voltammetric measurements (16-18). The computer-aided molecular docking studywas also performed in this work to evaluate thebinding properties which affords valuable informa-tion of drug binding mode in the active site of DNAand may lead to the rational design of new class ofanticancer drugs (6, 19).


Synthetic pathway adopted for the synthesis of1,2,4-triazolo[3,4-b][1,3,4]thiadiazine derivatives(6a-e) is sketched in Scheme 1. Ethyl 4-methoxy-benzoate (2) was synthesized by the acid-catalyzedesterification of 4-methoxybenzoic acid (1) inabsolute ethanol. The ester (2) was converted intocorresponding aromatic acid hydrazide (3) byrefluxing with hydrazine hydrate (80%) in ethanol.

The 4-methoxybenzohydrazide (3), on reaction withcarbon disulfide in ethanolic potassium hydroxideafforded corresponding dithiocarbazinate (4) ingood yield and was directly used for the next stepwithout further purification. 4-Amino-5-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thiol (5) wassynthesized by refluxing 4 with hydrazine hydrate(80%). Condensation of triazole (5) with variousphenacyl bromides in absolute ethanol under refluxconditions afforded 1,2,4-triazolo[3,4-b][1,3,4]thia-diazines (6a-e) in good yield. The spectro-analyticaldata of these compounds are reported in our recent-ly published report (20).

Voltammetric studies of the interaction of com-

pounds (6a-e) with DNA

The cyclic voltammetric measurements werecarried out in order to understand the redox behaviorand the DNA binding affinities of the newly pre-pared compounds (6a-e). The cyclic voltammo-grams of 6a-e showed only one broad cathodic peakthat is reduction peak, there is no oxidation peakwhich reflects the irreversible nature of the system.The absence of oxidation peak during reverse scanrevealed the unwavering reduced form of com-pounds 6a-e.

Scheme 1. Synthetic scheme for triazolothiadiazines (6a-e)

Table 1. Formation constant (Kf), free binding energy (∆G) for (6a-e) revealed by the voltammetric measurements and docking studies.

Entry ∆E/VFormation constant ∆G (kJ/M) ∆G (kJ/M)

Kf (M-1)/105 Experimental Theoretical

6a 0.0288 5.00 -32.51 -34.31

6b 0.0671 2.50 -30.79 -32.75

6c 0.0198 4.80 -32.41 -33.87

6d 0.0158 1.18 -28.93 -31.33

6e 0.0231 3.25 -31.44 -33.72

Triazolothiadiazines as a new class of efficient dna intercalating agents 1407

For irreversible system, the number of electrontransfer in the electrochemical process (21) can bedetermined by the following equation:

|Epñ E1/2| = 47.7/αn mV (1)where α is the transfer coefficient and n is the num-ber of electron transfer.

For every compound it had been calculated thatthe number of electron transfer in each system was~~ 1. The addition of 0.77, 1.12, 1.54 and 2.7 µMDNA to 1 mM concentration of the compounds (Fig.2), showed a decrease in cathodic peak current andpositive shift in peak potential. The variation in peakcurrent and shift in potential of compounds (6a-e)was used to calculate formation constant Kf andmode of interaction with DNA. The positive shift inpeak potential is attributed to the intercalative modeof the 6a-e into the double helix strands of DNA(22) whereas the decrease in peak current can beexplained as, there is formation of drug-DNAadduct due to which free drug concentrationdecreases as a result peak current decreases. Thisgradual decrease in peak current of drugs by addi-

tion of increasing concentration of DNA was probedto calculate the formation constant Kf of drug-DNAadduct (23) by using equation 2 and is listed inTable 1.

1Ip

2 = ñññññññññ (Ipo2ñ Ip

2) + Ipo2 (2)

Kf [DNA]where Ip2 and Ipo

2 is peak current with and withoutDNA, Kf is the formation constant and [DNA] is theconcentration of DNA used. By plotting Ip2 vs.(Ipo

2ñIp2)/[DNA], we get the slope value and the

inverse of slope gives the value of formation con-stant Kf.

From the data listed in Table 1, the values offormation constant Kf obtained for all compoundsare impressive having order of 105. The greaterbinding affinity is revealed by compounds 6a and6c substituted with chloro and fluoro groups where-as compound 6d possessing a naphthyl group showsthe least binding potency. The increase in sterichindrance may well be the responsible factor for thedecrease in binding affinity and it can also be justi-fied by the introduction of another chloro sub-

Figure 1. Cyclic voltammograms of 20% aqueous DMSO buffer at pH 6.0 (no peak); supporting electrolyte 0.1 M TBAP and 0.1M 6a,showing strong reduction peak in selected potential window

Table 2. Data from PyRx for ligand 6a (6 different conformers and their respective free energy and slandereddeviation values).

Ligand-DNA Binding affinity RMSD/upper RMSD/lowerkJ/M bound bound

DNA-6a (1) -34.317 0 0

DNA-6a (2) -34.317 12.723 8.397

DNA-6a (3) -33.0615 15.681 12.55

DNA-6a (4) -32.2245 7.264 4.965

DNA-6a (5) -33.0615 15.806 11.558

DNA-6a (6) -31.0615 14.860 11.509

E/V vs. SCE


stituent (6e), the value of formation constantdecreases which may also be due to the same reasoni.e., steric hindrance. But having values of forma-tion constant Kf in order of 105 make us to think thatthese compounds prove to have value as DNAbinder.

The DNA binding potency of (6a and 6c) aregreater than epirubicin (Kf = 4.1 ◊ 105 M-1), the clin-ically used anticancer drug (24). The binding freeenergy change (∆G = -RTlnK) of synthesized com-

pounds in KJ/M at 25OC signifies the spontaneity ofdrug-DNA interaction and the high value of ∆G alsojustifies the high conformational stability of com-pound-DNA complex (25).

Docking studies

Theoretically different software like autodockvina and PyRx are very useful and give very inter-esting information about the drug-DNA interaction.By using these computational studies, we can pre-

Figure 2. Cyclic voltammograms of a) 6a; b) 6b; c) 6c; d) 6d; e) 6e; 1 mM each recorded at 100 mV/s potential sweep rate on glassy car-bon electrode at 298 K in the absence and presence of (0.77, 1.12, 1.54 and 2.7 µM) DNA concentration in 20% aqueous DMSO buffer atpH 6.0; supporting electrolyte 0.1 M TBAP


dict and design some new drugs without wet lab (19,26). The behavior for these compounds shows somestrong interaction and from different conformers, weobserve the mechanism which is mostly intercala-tion (Fig. 3). Different conformers after resultsanalysis in PyRx that is supported by vina and autodock show mechanism of binding and also tabulatedresults for free energy.

With the same structure for DNA ligand hasdifferent probabilities to bind with intercalationmode. Free energy values from dockings areapproximately equal to the experimental values andfully support the experimental data as representedby compound 6a in Table 2. We use the minimumslandered deviation values to compare and in Table2, itís the first one. Furthermore, docking studies ofthe synthesized compounds confirmed both thequalitative as well as quantitative results.

EXPERIMENTAL

Synthesis

The compounds were synthesized according toour recently published report (20).DNA binding studies

Sodium salt of DNA (Acros) was used asreceived. The concentration of DNA stock solutionwas calculated by measuring UV absorbance at 260nm using molar extinction coefficient (ε) value 6600M-1/cm whereas the ratio of absorbance at 260 and280 nm indicated that the DNA is free from protein(27). Cyclic voltammetric (CV) measurements wereperformed with the objective of getting insight intoredox behavior and DNA binding potency of com-pounds (6a-e) in a single compartment cell with athree electrode configuration by using Eco ChemieAuto lab PGSTAT 12 potentiostat/galvanostat

Figure 3. Different conformers of 6a by molecular docking


(Utrecht, The Netharlands) with the electrochemicalsoftware package GPES 4.9. The three electrodesystem consisted of reference electrode; StandardCalomel Electrode SCE (Fisher Scientific Companycat # 136395), a Beckman platinum wire of thick-ness 0.5 mm with an exposed end of 10 mm as thecounter electrode and a bare glassy carbon electrode(surface area of 0.071 cm2) as working electrode.Prior to experiments, the voltammogram of a knownvolume of the solvent system was recorded in theabsence of compound and DNA after flushing outoxygen via purging argon gas for 10 min. The pro-cedure was then repeated for systems with constantconcentration of the drug and varying concentrationof DNA. The working electrode was cleaned afterevery electrochemical assay.

Molecular docking

Molecular structures for the ligands weredrawn on the chemsk11 and then geometrically opti-mized by the HyperChem8. Autodock4 and auto-grid4 run in PyRx-0.8 docking software which auto-matically generates the PDBQT files for both ligandand macromolecule. Selection of the appropriategrid PyRx run its calculation and gives the results intabulated form.

CONCLUSIONS

In summary, this work demonstrates the suc-cessful synthesis of a series of condensed heterocy-cles; 3,6-disubstituted-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine derivatives which show higher DNAbinding affinities calculated from the voltammetricmeasurements. These results unfailingly open up thebinding mode and interaction strength as obligatoryfor the design of targeted DNA binders. The molec-ular docking studies were also performed for thenewly synthesized hybrid structures and the resultsobtained strongly supported the theoretical calcula-tions. Free energy values from dockings are found tobe in good agreement with those of experimentalvalues. The mechanism of interaction of these com-pounds from different conformers has found to bethe intercalation. Furthermore, docking studies ofthe synthesized compounds confirmed both thequalitative as well as quantitative results.

Acknowledgment

A. Ibrar gratefully acknowledges the HigherEducation Commission of Pakistan for the supportof this work.

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Received: 9. 08. 2016


Inflammation is associated with numerous dis-eases like atherosclerosis, asthma, Alzheimerís dis-ease, gout, multiple sclerosis, osteoarthritis,rheumatoid arthritis, diabetes mellitus, carcinoma,bacterial or viral infections, etc. which result inchronic inflammation (1). Nonsteroidal anti-inflam-matory drugs (NSAIDs) are the first choice of drugsfor the treatment of various inflammatory diseases(2). They block prostaglandin synthesis bycyclooxygenases. The major side effect with chron-ic use of NSAIDs is gastrointestinal ulcerations dueto inhibition of cycloxygenase in tissues. The twoCOX isoforms, constitutively expressed COX-1 inmost tissues, while COX-2 is induced at the site ofinflammation (3), led to the development of selec-tive COX-2 inhibitors that significantly reduce gas-tric ulceration associated with chronic use ofNSAIDs. However, some selective COX-2inhibitors are withdrawn from the market because ofcardiovascular toxicity (4). So a real need exists todevelop new anti-inflammatory and analgesic agentswith enhanced efficacy, less toxicity and gastriculceration.

Benzimidazole is an important pharmacophorepossessing wide range of biological activities suchas anti-allergic (5), antimicrobial (6) antioxidant (7),PARP (Poly ADP ribose polymerase) inhibitors- asanticancer (8), cytomegalovirus (HCMV) inhibitors(9), antiulcer (10), anti-inflammatory (11) and asantihistaminics (12). Substitution of benzimidazoleat 1, 2, 5 and 6 positions accomplish the minimumstructural requirements for anti-inflammatory andanalgesic activity (13, 14) along with this benzimi-dazole nucleus substituted with an appropriate groupat 2- position is an essential structural feature forgastric safety of the molecule (15). These character-istic features create benzimidazole an importanttherapeutic target for drug development.

Mannich bases are the end products ofMannich reaction and are known as beta aminoketone carrying compounds (16). In Mannich reac-tion, formaldehyde is condensed with primary orsecondary amine and a compound containing activehydrogen. Introduction of a basic functional grouprendering the molecule in aqueous solvent, makesMannich base very reactive and are easily trans-

DESIGN AND SYNTHESIS OF N-(BENZIMIDAZOL-1-YL METHYL)-BENZAMIDE DERIVATIVES AS ANTI-INFLAMMATORY

AND ANALGESIC AGENTS

RITCHU SETHI1, SANDEEP ARORA1, DEEPIKA SAINI2, THAKUR GURJEET SINGH1

and SANDEEP JAIN2*

1Chitkara College of Pharmacy, Chitkara University, Rajpura, Distt. Patiala-140401, India2Department of Pharmaceutical Sciences, Guru Jambheshwar University, Hisar-125001, India

Abstract: A series of N-benzimidazol-1-yl methyl-benzamide derivatives were synthesized by Mannich reac-tion and evaluated for anti-inflammatory, analgesic and ulcerogenic activity. The newly synthesized com-pounds were designed using docking simulations with the help of AutoDock Vina. The compounds were fur-ther characterized by spectral and analytical techniques. The results revealed that out of thirteen compounds,five compounds showed significant analgesic and anti-inflammatory effect at a dose of 100 mg/kg p.o. alongwith less gastric toxicity profile and the experimental data are statistically significant at p < 0.05 level.Compound 3a N-(2-chloromethyl-benzimidazol-1-yl methyl)-benzamide, 3e N-[2-(2-chloro-phenyl) -benzimi-dazol-1-yl methyl] -benzamide, 3g N-[2-(4-chloro-phenyl)-benzimidazol-1-yl methyl]-benzamide, 3h N-[2-(2-Bromo-phenyl)-benzimidazol-1-yl methyl]-benzamide, 3j N-[2-(4-bromo-phenyl)-benzimidazol-1-yl methyl]-benzamide were found to be the most effective compounds. Compounds were found to have good correlationwith in-silico study.

Keywords: Mannich bases, benzimidazole, docking studies, anti-inflammatory activity, analgesic activity

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* Corresponding author: e-mail: [email protected]; phone: +918295968173

1414 RITCHU SETHI et al.

Scheme 1a. Synthesis of 2-substituted benzimidazole using o-phenylene diamine dihydrochloride

Compound Ar Compound Ar

2b -C6H5 2h 2-Br-C6H4-

2c 3-C5H4N- 2i 3-Br-C6H4-

2d 2-OH-C6H4- 2j 4-Br-C6H4-

2e 2-Cl-C6H4- 2k 4-NO2-C6H4-

2f 3-Cl-C6H4- 2l 4-F-C6H4-

2g 4-Cl-C6H4- 2m 2-NH2-C6H4-

Scheme1b. Synthesis of 2-substituted benzimidazoles using o-phenylene diamine

Compound R Compound R

3a -CH2Cl 3h 2-Br-C6H4-

3b -C6H5 3i 3-Br-C6H4-

3c 3-C5H4N- 3j 4-Br-C6H4-

3d 2-OH-C6H4- 3k 4-NO2-C6H4-

3e 2-Cl-C6H4- 3l 4-F-C6H4-

3f 3-Cl-C6H4- 3m 2-NH2-C6H4-

3g 4-Cl-C6H4-

Scheme 2. Synthesis of Mannich bases from 2-substituted benzimidazoles

Design and synthesis of N-(benzimidazol-1-yl methyl)-benzamide... 1415

formed into numerous other compounds (17). Thereactivity of these bases accounts for their severalpharmacological properties such as analgesic (18),anti-inflammatory (19, 20), anticancer (21, 22), anti-convulsant (23), antiviral, anthelmintic, antimalari-al, antibacterial (24) antifungal (25, 26) and severalother activities.

Looking into these findings together withdiverse biological activities exhibited by benzimida-zoles and less GI toxicity with 2-substituted benz-imidazoles, also, increased activity by the formationof Mannich bases inspired us for the development ofnew Mannich bases of 2-substituted benzimidazolederivatives and evaluated for analgesic, anti-inflam-matory and ulcerogenic activity. In silico methodwas used for the screening of novel compoundsderived from mannich reaction. The target com-pounds after screening were further synthesized andthe structures of all the compounds were confirmedby elemental and spectral analysis.


Docking studies

Molecular docking studies were performedusing Autodock Vina with pdb id: 1CX2. All the lig-ands employed in this study, were drawn usingMarvin Sketch application of ChemAxon. The 3Dstructures of the receptor were obtained fromProtein Data Bank (www.rcsb.org). Here, moleculardocking simulations were performed for anti-inflammatory (PDB id: 1CX2) using Autodock toolsof mgl lab for further pre-processing of our ligandsand receptor proteins (27). In the pre-processingstep, gasteiger charges were added both to the ligandas well as receptor. All the non-polar hydrogenswere merged and water of crystallization wasremoved using the same graphical interface.Autodock tool uses the hybrid global-local searchalgorithm which is a big improvement on the genet-ic algorithm for the best confirmations of ligands.Autodock Vina requires 3 calculated grid maps formaking our docking calculations extremely fast. TheAutodock Vina uses a hybrid scoring function

(empirical + knowledge based function) for evaluat-ing the binding affinity of ligands with the receptor(28).

A set of 24 compounds was screened for anti-inflammatory activity by molecular docking simula-tions using pdb id: 1CX2. The screening resulted in13 hit compounds which were further synthesizedand evaluated for anti-inflammatory, analgesic andulcerogenic activity.

Chemistry

Melting points were determined in open capil-lary tubes and are uncorrected. The purity of thesynthesized compounds was checked on precoatedaluminium silica gel G plates using UV/ iodinechamber as visualizing agent. Infrared spectra wererecorded on Perkin Elmer Spectrum FTIR spec-trophotometer using the potassium bromide discmethod. Proton NMR spectra were recorded onBruker Avance II (400 MHz) NMR spectrometerusing DMSO-d6 as a solvent and tetramethyl silane(TMS) as internal standard. Chemical shift valueswere expressed in delta parts per million (δ ppm).Elemental analysis was done using Eager XperienceCHN analyzer. Mass spectra were recorded onWATERS, Q-TOF MICROMASS spectrometer. 2-substituted benzimidazole Mannich bases were syn-thesized by the reaction of 2-substituted benzimida-zole (secondary amine), formalin and benzamide(active hydrogen compound) (3a-m). 2-substitutedbenzimidazoles (2a-m) were synthesized by thereaction of ortho-phenylenediamine with substitutedcarboxylic acid (aliphatic moiety) and with substi-tuted aromatic aldehyde (aromatic moiety), respec-tively. The compounds were characterized by spec-tral and analytical techniques.

General method

The title compounds were prepared by the followingsteps:

Synthesis of 2-substituted benzimidazole fromortho-phenylenediamine dihydrochloride [2a]

2-Substituted benzimidazole was synthesizedby the reaction of o-phenylenediamine dihydrochlo-ride with substituted carboxylic acid by the methoddescribed in the literature (29).

Synthesis of 2-substituted benzimidazoles fromortho-phenylenediamine [2(b-m)]

2-Substituted benzimidazoles were synthesizedby the reaction of o-phenylenediamine and substi-tuted benzaldehyde by the procedure reported in theliterature (30).

Table 1. X, y, z coordinates of grid box (PDB ID: 1CX2).

Center_x 42.332

Center_y 33.59

Center_z 36.01

Size_x 40

Size_y 40

Size_z 40


Synthesis of Mannich base of 2-substituted benz-imidazoles with benzamide [3(a-m)]

2-Substituted benzimidazole (0.01 M) wasadded to the ethanolic solution of benzamide (0.01M). Formaldehyde (37-41% w/v) (0.01 M) wasadded and the reaction mixture was then adjusted tothe pH of 3.5 with concentrated HCl. The mixturewas kept at efficient ice cooling for half an hour.Then, it was refluxed with stirring at 80OC for 10-12h. Formaldehyde (37-41% w/v) was added to thereaction mixture in portions for completion of thereaction. End of reaction was monitored by TLC.Reaction mixture was kept in refrigerator overnight.Solvent was evaporated under reduced pressure andproduct was collected, washed with water andrecrystallized from ethanol.

By adopting the same procedures and takingequimolar quantities of reactants, 13 new deriva-tives were synthesized. Synthetic method for prepa-

ration of novel compounds [3(a-m)] is shown ingeneral schemes.

Spectral data:

N-(2-chloromethyl-benzimidazol-1-yl methyl)-benz-amide (3a)

Yellow crystals; m.p. 190-191OC, IR (KBr, cm-1):3308 (N-H), 1485 (C=N), 3055 C-H (CH2), 2966 (C-H, Ar) 1526 (C=C), 1634 (C=O), 789 (C-Cl), 675-870 (CH bend. Ar). 1H NMR (400 MHz, DMSO-d6, δ,ppm): 7.4-8.1 (m, 9H, ArH), 4.92 (s, 2H, NCH2N), 9.0(s, 1H, NH), 5.19 (s, 2H, CH2). Analysis: calcd. forC16H14ClN3O: C 64.11, H 4.71, N 14.02%; found: C65.06, H 4.41, N 13.9%. MS: m/z = 300.2 (M+1).

N-(2-phenyl-benzimidazol-1-yl methyl)-benzamide(3b)

Brown crystals; m.p. 95-197OC, IR (KBr, cm-1):3308 (N-H), 1488 (C=N), 3056 C-H (CH2), 2965

Table 2. Data of dock score and interactive amino acids.

No. R Dock score Number of hydrogen bonds Amino acid involved in bonding

1 H- -6.5 - -

2 CH3- -6.5 - -

3 ClCH2- -9.0 2 Arg255, Glu193

4 C2H5- -6.9 - -

5 C3H8- -6.5 1 Lys194

6 C4H10- -6.8 - -

7 NH2- -6.6 1 Gln192

8 -6.7 1 Arg255

9 SH- -6.2 - -

10 SHCH2- -6.5 - -

11 C6H5- -8.1 - -

12 (2-C5H4N)- -7.1 1 Ala98

13 (3-C5H4N)- -8.3 1 Asn292

14 (2-OH-C6H4)- -8.1 1 Gly99

15 (2-Cl-C6H4)- -8.9 1 Glu193

16 (3-Cl-C6H4)- -8.1 - -

17 (4-Cl-C6H4)- -8.6 1 Glu193

18 (2-Br-C6H4)- -8.7 1 Arg255

19 (3-Br-C6H4)- -8.3 1 Glu193

20 (4-Br-C6H4)- -8.4 1 Glu193

21 (4-NO2-C6H4)- -8.9 1 Asn292

22 (C6H5-CH2)- -6.7 - -

23 (4-F-C6H4)- -8.3 - -

24 (2-NH2-C6H4)- -8.3 1 Glu193

25 SC-558 -7.6 2 Gly29, Gly32


(C-H, Ar) 1526 (C=C), 1634 (C=O), 675-870 (CHbend. Ar). 1H NMR (400 MHz, DMSO-d6, δ, ppm):7.4-8.32 (m, 14H, ArH), 4.93 (s, 2H, NCH2N), 9.0(s, 1H, NH). Analysis: calcd. for C21 H17 N3 O: C77.04, H 5.23, N 12.84%; found: C 76.3, H 5.20, N12.72%. MS: m/z = 329.2 (M+1).

N-(2-pyridin-3-yl-benzimidazol-1-yl methyl)-benz-amide (3c)

Yellow crystals; m.p. 205-206OC, IR (KBr, cm-1):3308 (N-H), 1486 (C=N), 3053 C-H (CH2), 2966 (C-H, Ar), 1527 (C=C), 1635 (C=O), 675-870 (CHbend. Ar). 1H NMR (400 MHz, DMSO-d6, δ, ppm):7.2-8.96 (m, 13H, ArH), 4.9 (s, 2H, NCH2N), 9.5 (s,1H, NH). Analysis: calcd. for C20 H16 N4 O: C 73.15, H4.9, N 17.6%; found: C 72.06, H 5.6, N 16.92%. MS:m/z = 329.3 (M+1).

N-[2-(2-hydroxyphenyl)-benzimidazol-1-yl methyl)]-benzamide (3d)

White crystals; m.p. 180-182OC, IR (KBr, cm-1):3310 (N-H), 1491 (C=N), 3057 C-H (CH2), 2964(C-H, Ar) 1527 (C=C), 1633 (C=O), 675-870 (CHbend. Ar). 1H NMR (400 MHz, DMSO-d6, δ, ppm):7.04-8.18 (m, 13H, ArH), 4.91 (s, 2H, NCH2N), 9.0(s, 1H, NH), 13.6 (br,s, 1H, OH). Analysis: calcd. forC21 H17 N3 O2: C 73.45 , H 4.99 , N 12.24%; found: C72.06, H 4.63, N 11.92%. MS: m/z = 344.8 (M+1).

N-[2-(2-chlorophenyl)-benzimidazol-1-yl methyl]-benzamide (3e)

White crystals; m.p. 190-191OC, IR (KBr, cm-1):3307 (N-H), 1485 (C=N), 3048 C-H (CH2), 2963(C-H, Ar) 1526 (C=C), 1634 (C=O), 770 (C-Cl),675-870 (CH bend. Ar). 1H NMR (400 MHz,DMSO-d6, δ, ppm): 7.41-8.9 (m, 13H, ArH), 4.91 (s,2H, NCH2N), 9.01(s, 1H, NH). Analysis: calcd. forC21H16ClN3O: C 69.71, H 4.46, N 11.61%; found: C68.5, H 4.31, N 10.2%. MS: m/z = 362.2 (M+1),363(M+2).

N-[2-(3-chlorophenyl)-benzimidazol-1-yl methyl]-benzamide (3f)

White crystals; m.p. 200-202OC, IR (KBr, cm-1):3309 (N-H), 1485 (C=N), 3034 C-H (CH2), 2953 (C-H, Ar) 1527 (C=C), 1633 (C=O), 765 (C-Cl),675-870 (CH bend. Ar). 1H NMR (400 MHz,DMSO-d6, δ, ppm): 7.46-8.7 (m, 13H, ArH), 4.98 (s,2H, NCH2N), 8.9 (s, 1H, NH). Analysis: calcd. forC21H16ClN3O: C 69.71, H 4.46, N 11.6%1; found: C68.5, H 4.81, N 11.2%.

N-[2-(4-chlorophenyl)-benzimidazol-1-yl methyl]-benzamide (3g)

White crystals; m.p. 195-196OC, IR (KBr, cm-1):3307 (N-H), 1485 (C=N), 3045 C-H(CH2) 2952 (C-H, Ar.) 1525 (C=C), 1635(C=O), 764 (C-Cl)675-870 (CH bend.Ar). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.35-8.6 (m, 13H, ArH), 4.96 (s, 2H,NCH2N), 8.90 (s, 1H, NH). Analysis: calcd. forC21H16ClN3O: C 69.71, H 4.46, N 11.61%; found: C68.52, H 4.31, N 10.8%. MS: m/z = 363.9 (M+2).

N-[2-(2-bromophenyl)-benzimidazol-1-yl methyl]-benzamide (3h)

White crystals; m.p. 190-192OC, IR (KBr, cm-1):3309 (N-H), 1488 (C=N), 3056 C-H (CH2), 2967 (C-H, Ar) 1527 (C=C), 1635 (C=O), 869 (C-Br), 675-870 (CH bend. Ar). 1H NMR (400 MHz, DMSO-d6, δ,ppm): 7.4-8.1 (m, 13H, ArH), 4.91(s, 2H, NCH2N),8.9 (s, 1H, NH). Analysis: calcd. for C21H16BrN3O: C62.08, H 3.97, N 10.34%; found: C 63.3, H 2.98, N9.43%. MS: m/z = 406.2 (M+1), 407.3 (M+2).

N-[2-(3-bromophenyl)-benzimidazol-1-yl methyl]-benzamide (3i)

White crystals; m.p. 192-194OC, IR (KBr, cm-1):3307 (N-H), 1487 (C=N), 3034 C-H (CH2), 2966 (C-H, Ar) 1525 (C=C), 1634 (C=O), 865 (C-Br), 675-870 (CH bend. Ar). 1H NMR (400 MHz, DMSO-d6, δ,ppm): 7.1-8.7 (m, 13H, ArH), 4.98(s, 2H, NCH2N), 9.0(s, 1H, NH). Analysis: calcd. for C21H16BrN3O: C 62.08,H 3.97, N 10.34%; found: C 61.3, H 3.90, N 9.49%.

N-[2-(4-bromophenyl)-benzimidazol-1-yl methyl]-benzamide (3j)

Off white crystals; m.p. 195-196OC, IR (KBr,cm-1): 3308 (N-H), 1486 (C=N), 3056 C-H (CH2),2967 (C-H, Ar) 1527 (C=C), 1635 (C=O), 865(C-Br), 675-870 (CH bend. Ar). 1H NMR (400 MHz,DMSO-d6, δ, ppm): 7.2-8.6 (m, 13H, ArH), 4.97 (s,2H, NCH2N), 8.89 (s, 1H, NH). Analysis: calcd. forC21H16BrN3O: C 62.08, H 3.97, N 10.34%; found: C61.9, H 2.96, N 9.82%.

N-[2-(4-nitrophenyl)-benzimidazol-1-yl methyl]-benzamide (3k)

Yellow crystals; m.p. 180-183OC, IR (KBr, cm-1):3308 (N-H), 1463 (C=N), 3058 C-H (CH2), 2966 (C-H, Ar) 1524 (C=C), 1634 (C=O), 747 (C-NO2),675-870 (CH bend. Ar). 1H NMR (400 MHz,DMSO-d6, δ, ppm): 7.24-8.46 (m, 13H, ArH), 4.94(s, 2H, NCH2N), 8.90 (s, 1H, NH). Analysis: calcd.for C21H16N4O3: C 67.73, H 4.33, N 15.05%; found:C 66.3, H 4.28, N 14.96%. MS: m/z = 373.7 (M+1).

N-[2-(4-fluorophenyl)-benzimidazol-1-yl methyl]-benzamide (3l)


White crystals; m.p. 230-231OC, IR (KBr, cm-1):3310 (N-H), 1458 (C=N), 3055 C-H (CH2), 2927 (C-H, Ar) 1525 (C=C), 1634 (C=O), 848 (C-F), 675-870 (CH bend. Ar). 1H NMR (400 MHz, DMSO-d6,δ, ppm): 7.4-8.48 (m, 13H, ArH), 4.90 (s, 2H,NCH2N), 9.03 (s, 1H, NH). Analysis: calcd. forC21H16FN3O: C 73.03, H 4.67, N 12.17%; found: C72.7, H 3.98, N 11.82%. MS: m/z = 346.3 (M+1).

N-[2-(2-amino-phenyl)-benzimidazol-1-yl methyl]-benzamide (3m)

Yellow crystals; m.p. 206-208OC, IR (KBr, cm-1):3310 (N-H), 1486 (C=N), 3086 C-H (CH2), 2959 (C-H, Ar) 1525 (C=C), 1634 (C=O), 675-870 (CHbend. Ar). 1H NMR (400 MHz, DMSO-d6, δ, ppm):6.52-7.95 (m, 13H, ArH), 4.98 (s, 2H, NCH2N), 8.79(s, 1H, NH), 4.0 (s, 2H, NH2). Analysis: calcd. for

Table 3. Physical data of synthesized compounds.

Compound R Molecular Molecular Melting Yield Rf

formula weight point (OC) (%) value

3a ClCH2- C16H14ClN3O 299.75 190-191 76.5 0.59

3b C6H5- C21H17N3O 327.38 195-197 80.4 0.95

3c (3-C5H4N)- C20 H16 N4 O 328.37 205-206 79.7 0.85

3d (2-OH-C6H4)- C21 H17 N3 O2 343.38 180-182 75.3 0.84

3e (2-Cl-C6H4)- C21H16ClN3O 361.82 190-191 80.0 0.87

3f (3-Cl-C6H4)- C21H16ClN3O 361.82 200-202 71.3 0.86

3g (4-Cl-C6H4)- C21H16ClN3O 361.82 195-196 73.6 0.75

3h (2-Br-C6H4)- C21H16BrN3O 406.28 190-192 75.4 0.81

3i (3-Br-C6H4)- C21H16BrN3O 406.28 192-194 76.7 0.80

3j (4-Br-C6H4)- C21H16BrN3O 406.28 195-196 78.6 0.77

3k (4-NO2-C6H4)- C21H16N4O3 372.38 180-183 79.2 0.64

3l (4-F-C6H4)- C21H16FN3O 345.37 230-231 72.7 0.76

3m (2-NH2-C6H4)- C21H18N4O 342.39 206-208 78.5 0.88

Mobile phase -CHCl3 : CH3OH; 9.5 : 0.5.

Figure 1. Binding pose for compound 3a (dock score -9.0 kcal/M) within the domain of COX-2 receptor showing hydrogen bonding indashed green line


Figure 2. Binding pose for internal ligand SC-558 (dock score -7.6 kcal/M) within the domain of COX-2 receptor showing hydrogen bond-ing in dashed green line

Table 4. Acute toxicity data of synthesized compounds.

Compound Dose Number of animals per group Mortality

3a (1g/kg) 6 0

3b (1g/kg) 6 0

3c (1g/kg) 6 0

3d (1g/kg) 6 0

3e (1g/kg) 6 0

3f (1g/kg) 6 0

3g (1g/kg) 6 0

3h (1g/kg) 6 0

3i (1g/kg) 6 0

3j (1g/kg) 6 0

3k (1g/kg) 6 3

3l (1g/kg) 6 0

3m (1g/kg) 6 0


C21H18N4O: C 73.67, H 5.30, N 16.36%; found: C71.9, H 4.96, N 15.82%.

Pharmacological studies

AnimalsSwiss albino mice (20-25 g) and albino Wistar

rats (150-200 g) of either sex were used for currentanimal study. All the animals were housed in groupsof 6 per cage at a temperature of 25 ± 1OC and a rel-ative humidity of 45-55%. A 12 h dark and 12 hlight cycle was followed during the experiments.Animals were allowed free access to food and water

ad libitum. All the animal experiments were per-formed with the approval of the Institutional AnimalEthics Committee, Chitkara College of Pharmacy,Punjab, India (Regd. No. 1181/PO/Ebi/08/CPC-SEA).

Acute toxicity studiesThe acute toxicity studies were performed in

groups of six Swiss albino mice, weighing 25 ± 2 gwhich were fasted overnight and treated orally withtest compounds with a dose of 1000 mg/kg bodyweight in evaluating their toxic effect as per OECD

Table 5. Anti-inflammatory activity of tested compounds and diclofenac sodium.

No. Treatments Dose (mg/kg) Percentage (%) Percentage (%)

edema at 4 h reduction in edema

1. Vehicle control (DMSO) (1mg/mL) 100 ± 5.25 0.00

Diclofenac sodium 2.(positive control)

(50 mg/kg) 21.75 ± 1.25a 76.25 ± 3.75a

3. 3a (100 mg/kg) 29.50 ± 1.55a 70.50 ± 2.25a

4. 3b (100 mg/kg) 73.85 ± 3.55 26.15 ± 1.20

5. 3c (100 mg/kg) 68.35 ± 3.05 31.65 ± 1.29

6. 3d (100 mg/kg) 71.25 ± 3.25 28.75 ± 1.34

7. 3e (100 mg/kg) 30.34 ± 1.25a 69.66 ± 2.75a

8. 3f (100 mg/kg) 65.28 ± 3.75 34.72 ± 1.54

9. 3g (100 mg/kg) 34.18 ± 1.5a 65.82 ± 2.15a

10. 3h (100 mg/kg) 33.81 ± 1.2a 66.19 ± 2.25a

11. 3 (100 mg/kg) 60.23 ± 2.78 39.77 ± 1.46

12. 3j (100 mg/kg) 36.59 ± 1.45a 63.41 ± 2.10a

13. 3l (100 mg/kg) 50.25 ± 2.23 49.75 ± 1.95

14. 3m (100 mg/kg) 55.25 ± 2.25 44.75 ± 1.5

Values are the mean ± standard error of mean (SEM) (n = 6). aStatistically significant compared to control group (p ≤ 0.05). All the sta-tistical analyses were performed by one-way ANOVA followed by Tukeyís test as a post hoc analysis.

Figure 3. Secondary structure of PDB ID: 1CX2


(Organization for Economic Co-operation andDevelopment) 423 guidelines.

Anti-inflammatory activity by carrageenan-indu-ced rat paw edema method

For anti-inflammatory activity, Wistar rats ofeither sex were divided into vehicle control, stan-dard (positive control) and different test groups ofsix rats each. All test compounds, vehicle controland reference drug were given orally to the respec-tive groups as a suspension in dimethyl sulfoxide(10% DMSO in water, 10 mL/kg), one hour beforecarrageenan injection (31). The procedure followedwas, acute inflammation produced by injection ofcarrageenan (0.1 mL of 1% w/v suspension) (32), in

the right hind paw of the rats under the plantaraponeurosis. It was injected +1 h after the oraladministration of the drug. The paw edema volumewas calculated by plethysmograph using mercurydisplacement method at 0 h and 4 h (immediatelyafter injection and 4 h post injection of car-rageenan). The percent anti-inflammatory activitywas calculated according to the formula givenbelow:

Percentage inhibition of paw edema = (1ñVt/Vc) ◊ 100

where Vc represents an average increase in paw vol-ume (average inflammation) of the control group ofrats at a given time; and Vt was the average inflamma-tion of the test compound treated rats at the same time.

Figure 4. Analgesic activity of tested compounds and indomethacin; ap < 0.005 vs. Vehicle Control (10 % DMSO in water, 10 mL/kg ), (n

= 6/group), One-way ANOVA; SEM = Standard error of the mean. All statistical analyses were performed by one-way ANOVA followedby Tukeyís test as a post hoc analysis

Table 6. Percentage analgesic activity of tested compounds.

S. No. Treatment Dose (mg/kg) Percentage protection

1. Vehicle control (DMSO) 10 mL/kg 0

Indomethacin 2.(positive control)

30 mg/kg 66.06a

3. 3a 100 mg /kg 63.66a

4. 3e 100 mg/kg 61.03a

5. 3g 100 mg/kg 53.63a

6. 3h 100 mg/kg 56.74a

7. 3j 100 mg/kg 52.24a

Values are the mean ± SEM (n = 6). aStatistically significant compared to vehicle control group (10 % DMSOin water, 10 mL/kg ), (p ≤ 0.05). All statistical analyses were performed by one-way ANOVA followed byTukey's test as a post hoc analysis.


Acetic acid-induced writhing assayAcetic acid-induced writhing test was used to

evaluate the analgesic potential of most potent com-pounds showing anti-inflammatory activity (33).Swiss albino mice were divided into vehicle control,standard (positive control) and different test groupsof six mice each. The vehicle control group wasadministered p.o., with dimethyl sulfoxide (10%DMSO in water, 10 mL/kg), whereas positive con-trol [indomethacin (30 mg/kg)] and test compoundswere administered at a dose level of 100 mg/kg sus-pended in 10% DMSO p.o., 30 min before the i.p.injection of the acetic acid solution at a dose of 1mL/kg. The number of wriths per animal wasrecorded for 15 min. The analgesic activity was cal-culated using the following formula and expressedas percentage protection and the results are present-ed in Table 6 and Fig. 4.% Analgesic activity = No. of writhings of control ñNo. of writhings of test compound/No. of writhingsof control ◊ 100

Final effective dose of compounds was select-ed by performing pilot studies.

Ulcerogenic assayWistar rats of either sex weighing 150-200 g

were divided into respective groups like vehiclecontrol [2% sodium carboxymethyl cellulose(CMC)], reference control (indomethacin) and dif-ferent test compound groups (n = 6). The test com-pounds and reference drug were administered to ani-mals orally at a dose of 100 mg/kg and 30 mg/kg,respectively, by dissolving in 2% sodium car-boxymethyl cellulose (CMC) as a vehicle. After 6 h,the rats were sacrificed for ulcerogenic activity, andtheir stomach was removed. Formalin (10% v/v)was then injected into the suture ligated stomach forfurther examination. The stomach was opened,washed in normal saline, and examined under mag-

nifier for measuring the length of lesions. Theinduced ulcers appeared as small spots punctiformlesions and each was given a score between 1 and 4.Ulcers of 0.5 mm diameter were assigned a score of1 whereas ulcers of diameters 1 and 2 mm weregiven scores of 2 and 4, respectively. Stomach withno lesions was assigned a score of zero. Summed togive a total lesion score (in mm) for each animal, themean count for each group being calculated (34) andresults are depicted in Table 7.

Statistical analysisAll the results were expressed as the mean ±

standard error of mean (SEM). Data of the resultswere analyzed by using one-way variance(ANOVA) followed by Tukeyís test. A value of p <0.05 was considered as statistically significant. TheSIGMA Plot 13, Systat Software, Inc. 2107 NorthFirst Street, Suite 360, San Jose, CA 95131 USA,was used for statistical analysis.


Docking study

Before in vivo evaluation, it was thought wor-thy to study the interaction of compounds to be syn-thesized with COX-2 receptor using moleculardocking studies. The purpose of the study was toscreen the compounds in silico for further in vivoevaluation. Considering 1CX2 as target, the series ofcompounds were docked to get the best in silicoconfirmations in the domain of selective COX-2inhibitor.

In the present study, H-bonding and dockscore were kept in consideration to get the hitsamong the set of compounds for the analysis.From the in silico study, 13 hit compounds withdock score higher than that of internal ligand SC-558 (-7.6 kcal/M), were further synthesized and

Table 7. Ulcerogenic activity of tested compounds and indomethacin.

No Compound Lesion score (mm) (Mean ± SEM)

1. Vehicle control (2% CMC) 1.0 ± 0.25a

2. Indomethacin (reference control) 32.50 ± 2.50

3. 3a 13.90 ± 0.95a

4. 3e 15.85 ± 1.18a

5. 3g 18.90 ± 1.85

6. 3h 16.55 ± 1.25a

7. 3j 20.45 ± 2.05

Values are themean ± SEM (n = 6). aStatistically significant compared to indomethacin (p ≤ 0.05), All the sta-tistical analysis was performed by one-way ANOVA followed by Tukey's test as post hoc analysis.


evaluated for anti-inflammatory, analgesic andulcerogenic activity. The main amino acids whichplayed a vital role in interaction are Lys194,Asn292, Arg255, Gly99 and Glu193. Among theseries, compound 3a has displayed best dockscore of -9.0 kcal/mol with 2 H-bonds, length of2.216 Å and 2.036 Å, respectively, shown in Figu-re 1. Similarly, compound 3e showed dock scoreof -8.9 kcal/M with 1 hydrogen bond and mainamino acid which played interaction is Glu193.The parameters of grid box are given in Table 1.The data of dock score and interactive aminoacids of series are provided in Table 2. The inter-nal ligand (SC-558) displayed dock score of -7.6kcal/M with 2 hydrogen bonds, length of 2.226 Åand 2.136 Å, respectively and its interactions withthe target receptor is shown in Figure 2. The sec-ondary structure of the target receptor is shown inFigure 3. From the docking study, we predictedthat the compounds showing good dock score aswell as interaction with the receptor would pos-sess better activity.

Chemistry

In this study, on the basis of docking studiesout of 24 derivatives, 13 novel compounds incorpo-rating the scaffold of benzimidazole were synthe-sized and screened for anti-inflammatory, analgesicand ulcerogenic activity. Synthesis of the com-pounds was carried out as outlined in generalschemes. Benzamide (active hydrogen compound)was reacted with secondary amine (2-substitutedbenzimidazole) in the presence of formalin andconc. hydrochloric acid to furnish the Mannichbases. These were characterized on the basis of theirelemental and spectral analysis. From IR spectra, theappearance of peaks at 3308-3310 cm-1 and 1400-1500 cm-1 indicated the presence of NH of carbox-amide and C=N stretching of benzimidazole, respec-tively. From NMR spectra, a sharp singlet at 4.8-4.99 ppm ascertained the presence of CH2 attachedto NH in all the synthesized compounds. Theappearance of singlet at 8.0-8.9 ppm confirmed thepresence of NH (D2O exchangeable) of carboxamidein all the synthesized compounds. The appearanceof multiplet at 6.30-8.0 ppm indicated the presenceof aromatic and hetero-aromatic protons. In addi-tion, compound 3a showed singlet at 5.19 ppm for2H of CH2. The appearance of broad singlet at 13.6ppm confirmed the presence of one proton ofhydroxyl functionality in compound 3d. The calcu-lated molecular weight of the synthesized com-pounds was matched with an observed m/e value.Hence, data obtained were found to be in good con-

firmity with the calculated values of the proposedstructure. Physical data of synthesized compoundsare given in Table 3.

Animal studies

Acute toxicity of test compoundsOut of 13 compounds, only one compound 3k

showed toxic effect and mortality in mice at a doseof 1g/kg p.o., out of six animals in 3k treatmentgroup, 3 animals were dead after treatment. Rest allcompounds with a dose of 1g/kg, were nontoxic asthey have not shown mortality and adverse effects(lethargy and convulsions) in any group (12 groups).Acute toxicity data of synthesized compounds aregiven in Table 4.

Anti-inflammatory activity of test compoundsFrom Table 5, it was found that out of 12 com-

pounds, five compounds showed significant resultsin comparison with standard diclofenac sodium aspositive control. Amongst all the title compounds,3a, 3e, 3g, 3h and 3j showed significant (p < 0.05)potent anti-inflammatory activity as compared tovehicle control group (10% DMSO) and rest of thecompounds showed moderate or low activity.

Analgesic activity of test compoundsOn the basis of anti-inflammatory activity only

five compounds were selected for analgesic activity.Fig. 4 and Table 6 revealed that all the compounds3a, 3e, 3g, 3h and 3j showed significant (p < 0.05)potent analgesic and percentage protection analgesicactivity of test compounds, respectively, as com-pared to vehicle control group (10% DMSO).

Ulcerogenic activityOn the basis of efficacy and potency of anti-

inflammatory and analgesic activity, five com-pounds were selected for ulcerogenic studies. Thesecompounds also showed significant (p < 0.05)results for ulcerogenic activity as compared toindomethacin as reference drug. Table 7 revealedthat compound 3a, 3e and 3h were most potentamong five compounds used for respective study.

CONCLUSION

A series of N-(benzimidazol-1-yl methyl)-benza-mide derivatives were initially screened by docking sim-ulations using COX-2 receptor (PDB ID: 1CX2).Binding affinities of the synthesized compounds wereevaluated by using docking program Autodock Vina.The main amino acids that played a vital role in interac-tion with COX-2 receptor are Lys194, Asn292, Arg255,


Gly99 and Glu193. The designed derivatives havingdock score higher than internal ligand, were synthesizedwith remarkable yields and their structures were eluci-dated by spectral analysis. Selected compounds werescreened for their in vivo anti-inflammatory and anal-gesic activity and five out of twelve compounds showedsignificant results as compared to disease control groupsin animal studies. Based on anti-inflammatory and anal-gesic activity, compounds 3a (dock score -9.0 kcal/M),3e (dock score -8.9 kcal/M), 3g (dock score -8.6kcal/M), 3h (dock score -8.7 kcal/M) and 3j (dock score-8.4 kcal/M) were selected for ulcerogenic activity. Thecompounds 3a, 3e and 3h were found to be safe on gas-tric mucosa as indicated by their low lesion score 13.90± 0.95, 15.85 ± 1.18 and 16.55 ± 1.25, respectively, ascompared to indomethacin (32.50 ± 2.50). Structureactivity relationship revealed that compounds substitut-ed with electron withdrawing groups (Cl, Br) at orthoposition of phenyl ring and chloromethyl group at 2-position in benzimidazole nucleus were found to bemost active anti-inflammatory, analgesic and non-ulcerogenic agents. The pharmacological activitiesexhibited by newly synthesized compounds have con-firmed their novelty as safer therapeutic agents. Both thehypothesis (docking) and practice (anti-inflammatory,analgesic and ulcerogenic activity) reveal that N-(benz-imidazol-1-yl methyl)-benzamide derivatives act aspotent inhibitor of 1CX2 receptor. Hence it is concludedthat N-(benzimidazol-1-yl methyl)-benzamide deriva-tives with electron withdrawing substituents could beused for designing more potent anti-inflammatory andanalgesic agents with less gastric lesions. Further studiesof these derivatives are highly recommended to assessthe safety of novel compounds.

Acknowledgments

The authors are thankful to Management,Chitkara University, Punjab for providing necessaryfacilities to carry out this work. One of the authors isthankful for getting financial assistance fromDepartment of Science and Technology, New Delhi.

Conflict of interests

The authors declare that there is no conflict ofinterests.

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Received: 13. 08. 2016


Cancer still remains a mean threat to humanhealth; it is a major cause of death worldwide.Therefore, the development of highly efficient,selective and less noxious antitumor drug remainsan urgent need (1).

The design and synthesis of new pyrazolederivatives has been a unique position in the drugdiscovery due to their broad range of biologicalactivities, such as anti-inflammatory, antiangio-genic, antibacterial, antimicrobial, anticancer,antioxidant, anti-influenza and analgetic activities(2-9). Moreover, pyrazole unit are important corestructures in a number of natural products (10-13).

Pyrazole is versatile lead compound fordesigning potent anticancer compounds. Severalrecent studies suggest pyrazole derivatives as prom-ising anticancer agents, indicating their use in thedevelopment of new anticancer agents (14-18). Theimportant role of pyrazoles in antitumor agents dueto their good inhibitory activity againstBRAF(V600E), EGFR, telomerase, ROS ReceptorTyrosine Kinase and Aurora-A kinase (19). In addi-tion, N-substituted pyrazoles have been implement-ed as anti-leukemic, antitumor, anti-proliferative,anti-angiogenic, DNA interacting, proapoptotic,autophagy, and anti-tubulin agents. These com-

pounds exhibited remarkable anti-cancer effectsthrough inhibition of different types of enzymes,proteins and receptors which play critical role in celldivision (20).

On the other hand, we expected that compound4 and 5 are interesting pyrazole derivatives withpossible anticancer activity. In continuation to ourwork effort to develop new anticancer agents (21),we reported herein a series of novel functionalizedpyrazole derivatives starting from compounds (4) or(5) to evaluate their in vitro anti-tumor activitiesagainst HepG-2, PC-3 and HCT-116 human carci-noma cell lines using MTT assay.

EXPERIMENTAL

Chemistry

GeneralAll melting points were uncorrected and meas-

ured using an Electro-thermal IA 9100 apparatus(Shimadzu, Japan). Microanalytical data were per-formed by Vario El-Mentar apparatus (Shimadzu,Japan), National Research Centre, Cairo, Egypt. IRspectra were recorded (as potassium bromide pel-lets) using KBr disc technique on a Perkin-Elmer1650 Spectrophotometer, National Research Centre,

DESIGN, SYNTHESIS AND ANTICANCER ACTIVITY OF SOME NOVEL5-(4-METHOXYPHENYL)-2,4-DIHYDRO-3H-PYRAZOL-3-ONE

DERIVATIVES

USAMA FATHY1*, RASHA S. GOUHAR2 and HANEM M. AWAD3

1Appleid Organic Chemistry Department, 2Therapeutic Chemistry Department,3Tanning Materials and Leather Technology Department, National Research Centre,

33 El Bohouth St. (Former El Tahrir St.) - Dokki-Giza-Egypt-P.O.12622

Abstract: New pyrazole derivatives were designed and synthesized by the reaction of 5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (4) or 5-(4-methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (5) withseries of aldehydes, substituted amines, isatins and phenylisothiocynate to yield 6a-j, 7a-k, 9a-d and 10a-b,respectively. The synthesized compounds were examined in vitro for their anti-tumor activities against HepG-2, PC-3 and HCT-116 human carcinoma cell lines using MTT assay. Eight compounds showed good anticanceractivities against HCT-116 carcinoma cells and five compounds showed good anticancer activities against PC-3 cancer cells but all the compounds showed weak or no anticancer activities against HepG-2 liver cancer.

Keywords: pyrazoles, arylidines, hydrazones, isatin, anticancer

1427

* Corresponding author: e-mail: [email protected]; phone: +2 33371615; fax: +2 33370981

1428 USAMA FATHY et al.

Cairo, Egypt. NMR experiments were determinedon a JEOL-Ex-300 MHz in deuterated dimethyl sulf-oxide (DMSO-d6) and chemical shifts wereexpressed as parts per million; ppm (δ values)against TMS as an internal reference (Faculty ofScience, Cairo University, Cairo, Egypt). Massspectra were recorded on Shimadzu GCMS-QP-1000EX mass spectrometer at 70 eV (CairoUniversity, Cairo, Egypt).

Ethyl 3-(4-methoxyphenyl)-3-oxopropanoate (3)This compound was prepared according to the

reported method (22) by the reaction of 4-methoxyacetophenone (1) and diethyl carbonate (2) in thepresence of sodium hydride. Viscous oil, yield 93%;1H NMR (DMSO-d6, δ, ppm): 1.25 (t, 3H, J = 7 Hz,OCH2CH3), 3.79 (s, 3H, OCH3), 3.85 (s, 2H,COCH2CO), 4.15 (q, J = 7 Hz, 2H, OCH2CH3), 6.87(d, J = 7 Hz, 2H), 7.80 (d, J = 7 Hz, 2H, Ar-H).

5-(4-Methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (4)

This compound was prepared according to thereported method (23) by the reaction of ethyl 3-(4-methoxyphenyl)-3-oxopropanoate (3) and hydrazinehydrate refluxing in absolute ethanol for 10-15 min. Yield (94%); m.p. 220OC; IR (KBr, cm-1): 3150(NH), 1670 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.78 (s, 3H, -OCH3), 5.80 (s, 1H, -CH=), 6.96 (d, J= 9 Hz, 2H, ArH), 7.58 (d, J = 9 Hz, 2H, ArH),10.03(s, 1H, D2O exchangeable, NH); MS (C10H10N2O2),m/z = 191 (M+).

5-(4-Methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (5)

This compound was prepared according to thereported method (23, 24) by the reaction of ethyl 3-(4-methoxyphenyl)-3-oxopropanoate (3) and phenylhydrazine refluxing in acetic acid for 10 min.

Yield (96%); m.p. 135OC; IR (KBr, cm-1): 1690(C=O); 1H NMR (DMSO-d6, δ, ppm): 3.79 (s, 3H, -OCH3), 5.95 (s, 1H, -CH=), 6.97 (d, J = 9 Hz, 2H,ArH), 7.24 (t, J = 9 Hz, 1H, ArH), 7.45 (t, J = 9 Hz,2H, ArH), 7.77 (d, J = 9 Hz, 2H, ArH), 7.81 (d, J =9 Hz, 2H, ArH); MS (C16H14N2O2), m/z = 267 (M+).

Synthesis of 4-arylidene-5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-ones (6a-j)

To a mixture of 3-(4-methoxyphenyl)-1-pyra-zol-5(4H)-one (4) or 3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-5(4H)-one (5) (1 mM) and theappropriate aldehyde (1 mM) in absolute ethanol (25mL), few drops of piperidine were added. The reac-tion mixture was refluxed for 4 h, and then cooled to

room temperature. The precipitate was filtered,dried and washed with ethanol. The crude productwas recrystallized from EtOH/DMF to afford com-pounds 6a-j, respectively.

4-Benzylidene-5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (6a)

Yield (70%); m.p. 110OC; IR (KBr, cm-1): 3220(NH), 1720 (C=O; 1H NMR (DMSO-d6, δ, ppm):3.78 (s, 3H, -OCH3), 6.60 (d, J = 9 Hz, 2H, ArH ),6.94-7.89 (m, 6H, 4ArH + -CH=), 7.90 (d, J = 8 Hz,2H, ArH), 10.03 (s, 1H, D2O exchangeable, NH);MS (C17H14N2O2), m/z = 279 (M+).

4-(4-Bromobenzylidene)-5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (6b)

Yield (80%); m.p. 140OC; IR (KBr, cm-1): 3225(NH), 1715 (C=O; 1H NMR (DMSO-d6, δ, ppm):3.77 (s, 3H, -OCH3), 6.95 (d, J = 9 Hz, 2H, ArH of4-MeOC6H4), 7.39 (s, 1H, -CH=), 7.60 (d, J = 9 Hz,2H, ArH of 4-MeO-C6H4), 7.73 (d, J = 8 Hz, 2H,ArH of 4-BrC6H4), 7.88 (d, J = 8 Hz, 2H, ArH of 4-BrC6H4), 9.98 (s, 1H, D2O exchangeable, NH); MS(C17H13BrN2O2) m/z = 358 (M+).

4-(4-(Dimethylamino) benzylidene)-5-(4-meth-oxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (6c)

Yield (79%); m.p. 130OC; IR (KBr, cm-1): 3230(NH), 1670 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.04 (s, 6H, 2CH3), 3.78 (s, 3H, OCH3), 6.71 (d, J =8.5 Hz, 2H, ArH of N(CH3)2), 6.86 (d, J = 2.5 Hz,2H, ArH), 7.38 (s, 1H, -CH=), 7.60 (d, J = 9 Hz, 2H,ArH), 7.69 (d, J = 9 Hz, 2H, ArH), 9.67 (s, 1H, D2Oexchangeable, NH), MS (C19H19N3O2) m/z = 322(M+).

4-(4-Chlorobenzylidene)-5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (6d)

Yield (65%); m.p. 140OC; IR (KBr, cm-1): 3220(NH), 1680 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.77 (s, 3H, OCH3), 6.58-8.0 (m, 8H, 7ArH+-CH=),10.20 (s, 1H, D2O exchangeable, NH), MS(C17H12Cl2N2O2) m/z = 348 (M+).

4-(4-Hydroxy-3-methoxybenzylidene)-5-(4-meth-oxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (6e)

Yield (71%); m.p. 145OC; IR (KBr, cm-1):3411-3194 (NH+OH), 1670 (C=O); 1H NMR(DMSO-d6, δ, ppm): 3.78 (s, 3H, -OCH3), 3.81 (s,3H, -OCH3), 4.90 (s,1H, OH), 6.52-7.38 (m, 6H,5ArH+-CH=), 7.57 (d, J = 9 Hz, 2H, ArH ), 9.71 (s,1H, D2O exchangeable, NH), MS (C18H16N2O4) m/z= 325 (M+).

Design, synthesis and anticancer activity of some novel... 1429

4-(3,4-Dimethoxybenzylidene)-5-(4-methoxyphen-yl)-2,4-dihydro-3H-pyrazol-3-one (6f)

Yield (66%); m.p. 170OC; IR (KBr, cm-1): 3220(NH), 1665 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.77 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3), 3.87 (s,3H, -OCH3), 6.56-7.59 (m, 8H, 7ArH + -CH=),9.90(s, 1H, D2O exchangeable, NH), MS (C19H18N2O4)m/z = 339 (M+).

4-Benzylidene-5-(4-methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (6g)

Yield (75%); m.p. 225OC; IR (KBr, cm-1): 1660(C=O); 1H NMR (DMSO-d6, δ, ppm): 3.75 (s, 3H, -OCH3), 6.72 (d, J = 9 Hz, 2H, ArH of 4-MeOC6H4),7.07-7.20 (m, 7H of 2 phenyl groups), 7.22 (s, 1H, -CH=), 7.33 (t, J = 9 Hz, 1H, ArH ), 8.01 (d, J = 9Hz, 2H, ArH of 4-MeOC6H4), 8.21 (d, J = 9 Hz,ArH); MS (C23H18N2O2) m/z = 355 (M+).

4-(4-Bromobenzylidene)-5-(4-methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (6h)

Yield (70%); m.p. 115OC; IR (KBr, cm-1): 1655(C=O); 1H NMR (DMSO-d6, δ, ppm): 3.78 (s, 3H, -OCH3), 6.86 (d, J = 9 Hz, 2H, ArH of 4-MeOC6H4),7.20 (t, J = 9 Hz, 1H, ArH), 7.40 (t, J = 9 Hz, 2H,ArH), 7.50 (s, 1H, -CH=), 7.67 (d, J = 9 Hz, 2H,BrArH), 7.72 (d, J = 9 Hz, 2H, BrArH), 7.88 (d, J =9 Hz, 2H, MeOArH), 8.06 (d, J = 9 Hz, 2H, ArH),MS (C23H17BrN2O2) m/z = 434 (M+).

4-[4-(Dimethylamino)benzylidene]-5-(4-methoxy-phenyl)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one(6i)

Yield (90%); m.p. 145OC; IR (KBr, cm-1): 1665(C=O); 1H NMR (DMSO-d6, δ, ppm): 3.12 (s, 6H,2CH3), 3.85 (s, 3H, -OCH3), 6.80 (d, J = 9 Hz, 2H,ArH), 7.10 (d, J = 9 Hz, 2H, ArH), 7.22 (t, J = 9 Hz,1H, ArH), 7.40 (t, J = 9 Hz, 2H, ArH), 7.51 (s, 1H,-CH=), 7.61 (d, J = 9 Hz, 2H, ArH), 8.01 (d, J = 9Hz, 2H, ArH), 8.58 (d, J = 9 Hz, 2H, ArH),ArH);MS (C25H23N3O2) m/z = 398 (M+).

4-(4-Hydroxy-3-methoxybenzylidene)-5-(4-meth-oxyphenyl)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (6j)

Yield (60%); m.p. 180OC; IR (KBr, cm-1): 3150(OH), 1670 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.77 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3), 6.83 (d, J= 9 Hz, 1H, vaniline), 6.97 (d, J = 9 Hz, 2H,MeOArH), 7.04 (m, 2H, Vaniline), 7.20 (t, J = 9 Hz,1H, ArH), 7.40 (t, J = 9 Hz, 2H, ArH), 7.50 (s, 1H,-CH=), 7.78 (d, J = 9 Hz, 2H, MeOArH), 7.99 (d, J= 9 Hz, 2H, ArH), MS (C24H20N2O4) m/z = 401 (M+).

Synthesis of 5-(4-methoxyphenyl)-4-(2-arylhydraz-ineylidene)-2,4-dihydro-3H-pyrazol-3-one deriva-tives (7a-k)

The appropriate amine (1 mM) was dissolvedin concentrated HCl (15 mL) and water cooled in iceand then NaNO2 (1 mM) in water (10 mL) wasadded with stirring. A mixture of compound 4 or 5(0.1 mM), NaOAc (5 g), ethanol (50 mL) and water(20 mL) was prepared separately and cooled in ice.The diazonium salt solution was added slowly to thesecond solution, with ice cooling. The cooled mix-ture was stirred for 0.5 h and filtered to give coloredcrystals, which were crystallized from ethanol togive the corresponding hydrazones 7a-k, respective-ly (25).

5-(4-Methoxyphenyl)-4-[2-(p-tolyl)hydrazineyli-dene]-2,4-dihydro-3H-pyrazol-3-one (7a)

Yield (85%); m.p. 192OC; IR (KBr, cm-1): 3180(2NH), 1560 (C=O); 1H NMR (DMSO-d6, δ, ppm):2.32 (s, 3H, CH3), 3.82 (s, 3H, -OCH3), 7.05 (d, J = 9Hz, 2H, ArH), 7.26 (d, J = 9 Hz, 2H, ArH), 7.48 (d, J= 9 Hz, 2H, ArH), 7.99 (d, J = 9 Hz, 2H, ArH), 11.94(s, 1H, D2O exchangeable, NH), 13.80 (s, 1H, D2Oexchangeable, NH); MS (C16H14N4O2) m/z = 295 (M+).

5-(4-Methoxyphenyl)-4-[2-(4-methoxyphenyl)hydrazineylidene]-2,4-dihydro-3H-pyrazol-3-one(7b)

Yield (78%); m.p. 200OC; IR (KBr, cm-1):3300(2NH), 1670 (C=O); 1H NMR (DMSO-d6, δ,ppm): 3.77 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3),6.94 (d, J = 9 Hz, 2H, ArH), 7.0 (d, J = 9 Hz, 2H,ArH), 7.53 (d, J = 9 Hz, 2H, ArH), 8.01 (d, J = 9 Hz,2H, ArH), 11.72 (s, 1H, D2O exchangeable, NH),13.60 (s, 1H, D2O exchangeable, NH); MS(C17H16N4O3) m/z = 3244 (M+).

4-[2-(4-Fluorophenyl)hydrazineylidene]-5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one(7c)

Yield (78%); m.p. 175OC; IR (KBr, cm-1): 3450(2NH), 1660 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.81 (s, 3H, -OCH3), 6.97 (d, J = 9 Hz, 2H, ArH),7.04-7.33 (m, 2H, ArH), 7.57-7.64 (m, 2H, ArH),8.02 (d, J = 9 Hz, 2H, ArH), 11.81 (s, 1H, D2Oexchangeable, NH), 13.80 (s, 1H, D2O exchange-able, NH); MS (C16H13FN4O2) m/z = 313(M+).

4-[2-(4-Chlorophenyl)hydrazineylidene]-5-(4-meth-oxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (7d)

Yield (79%); m.p. 213OC; IR (KBr, cm-1): 3420(2NH), 1665 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.81 (s, 3H, -OCH3), 7.03 (d, J = 9 Hz, 2H, ArH),


7.46 (d, J = 9 Hz, 2H, ArH), 7.57 (d, J = 9 Hz, 2H,ArH), 8.01 (d, J = 9 Hz, 2H, ArH), 11.90 (s, 1H, D2Oexchangeable, NH), 13.81 (s, 1H, D2O exchangeable,NH); MS (C16H13ClN4O2) m/z = 329 (M+).

4-[2-(4-Bromophenyl)hydrazineylidene]-5-(4-methoxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (7e)

Yield (72%); m.p. 205OC; IR (KBr, cm-1): 3230(2NH), 1655 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.77 (s, 3H, -OCH3), 6.95 (d, J = 9 Hz, 2H, ArH),7.08 (d, J = 9 Hz, 2H, ArH), 7.59 (d, J = 9 Hz, 2H,ArH), 7.99 (d, J = 9 Hz, 2H, ArH), 11.99 (s, 1H,D2O exchangeable, NH), 13.60 (s, 1H, D2Oexchangeable, NH); MS (C16H13BrN4O2) m/z = 374(M+).

5-(4-Methoxyphenyl)-2-phenyl-4-2-phenylhydr-azineylidene-2,4-dihydro-3H-pyrazol-3-one (7f)

Yield (67%); m.p. 130OC; IR (KBr, cm-1): 3200(NH), 1660 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.78 (s, 3H, -OCH3), 6.95 (d, J = 9 Hz, 2H, ArH),7.12-68 (m, 8H, ArH), 7.72 (d, J = 9 Hz, 2H, ArH),7.80 (d, J = 9 Hz, 2H, ArH),12.01 (s, 1H, D2Oexchangeable, NH); MS (C22H18N4O2) m/z =371(M+).

5-(4-Methoxyphenyl)-2-phenyl-4-[2-(p-tolyl)hydr-azineylidene]-2,4-dihydro-3H-pyrazol-3-one (7g)

Yield (68%); m.p. 180OC; IR (KBr, cm-1): 3350(NH), 1655 (C=O); 1H NMR (DMSO-d6, δ, ppm):2.33 (s, 3H, CH3), 3.82 (s, 3H, -OCH3), 7.05 (d, J =9 Hz, 2H, MeOArH), 7.10-7.80 (m, 7H, ArH), 7.98(d, J = 9 Hz, 2H, MeOArH), 8.16 (d, J = 9 Hz, 2H,ArH), 13.76 (s, 1H, D2O exchangeable, NH); MS(C23H20N4O2) m/z = 385 (M+).

5-(4-Methoxyphenyl)-4-[2-(4-methoxyphenyl)hydrazineylidene]-2-phenyl-2,4-dihydro-3H-pyra-zol-3-one (7h)

Yield (71%); m.p. 160OC; IR (KBr, cm-1): 3360(NH), 1660 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.77 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3), 7.00 (d, J= 9 Hz, 2H, MeOArH), 7.05 (d, J = 9 Hz, 2H,MeOArH), 7.09-8.13 (m, 7ArH), 8.20 (d, J = 9 Hz,2H), 13.5 (s, 1H, D2O exchangeable, NH); MS(C23H20N4O3) m/z = 401 (M+).

4-[2-(4-Fluorophenyl)hydrazineylidene]-5-(4-methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyra-zol-3-one (7i)

Yield (73%); m.p. 165OC; IR (KBr, cm-1): 3270(NH), 1665 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.78 (s, 3H, -OCH3), 7.08 (d, J = 9 Hz, 2H,MeOArH), 7.19-7.75 (m, 7H, 2ArH), 8.04 (d, J = 9

Hz, 2H ArH), 8.12 (d, J = 9 Hz, 2H ArH), 13.2 (s,1H, D2O exchangeable, NH); MS (C22H17FN4O2) m/z= 389 (M+).

4-[2-(4-Chlorophenyl)hydrazineylidene]-5-(4-methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyra-zol-3-one (7j)

Yield (80%); m.p. 195OC; IR (KBr, cm-1): 3310(NH), 1660 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.78 (s, 3H, -OCH3), 7.01 (d, J = 9 Hz, 2H,MeOArH), 7.11 (t, J = 9 Hz, 1H, ArH), 7.36 (t, J =9 Hz, 2H, ArH), 7.40-8.00 (m, 4H, ArH), 8.03 (d, J= 12 Hz, 2H, ArH), 8.12 (d, J = 12 Hz, 2H, ArH),13.19 (s, 1H, D2O exchangeable, NH); MS(C22H17ClN4O2) m/z = 405 (M+).

4-[2-(4-Bromophenyl)hydrazineylidene]-5-(4-methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyra-zol-3-one (7k)

Yield (77%); m.p. 170OC; IR (KBr, cm-1): 3330(NH), 1665 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.82 (s, 3H, -OCH3), 7.06 (d, J = 9 Hz, 2H, MeOArH),7.24 (t, J = 9 Hz, 1H, ArH), 7.45 (t, J = 9 Hz, 2H,ArH), 7.64 (d, J = 9 Hz, 2H, ArH), 7.90 (d, J = 12 Hz,2H, ArH), 8.00 (d, J = 12 Hz, 2H, ArH), 8.13 (d, J =12 Hz, 2H, ArH), 13.60 (s, 1H, D2O exchangeable,NH); MS (C22H17ClN4O2) m/z = 500 (M+).

Synthesis of 3-[3-(4-methoxyphenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene] indolin-2-one deri-vatives (9a-d)

To a mixture of pyrazole 4 or 5 (1 mM) andisatin (1 mM) in glacial acetic acid (25 mL), 3 mMof anhydrous sodium acetate were added. The reac-tion mixture was refluxed for 4 h, and then cooled toroom temperature. The precipitate was filtered,dried and washed with ethanol. The crude productwas recrystallized from EtOH/DMF to afford com-pounds 9a-d, respectively.

3-[3-(4-Methoxyphenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]indolin-2-one (9a)

Yield (65%); m.p. >300OC; IR (KBr, cm-1):3250 (2NH), 1700-1660 (2C=O); 1H NMR (DMSO-d6, δ, ppm): 3.77 (s, 3H, -OCH3), 6.70-7.95 (m, 8H,2ArH), 10.45 (s, 1H, D2O exchangeable, NH isatin),11.56 (s, 1H, D2O exchangeable, NH); MS(C18H13N3O3) m/z = 319 (M+).

5-Chloro-3-[3-(4-methoxyphenyl)-5-oxo-1,5-dihy-dro-4H-pyrazol-4-ylidene] indolin-2-one (9b)

Yield (67%); m.p. 230OC; IR (KBr, cm-1): 3320(2NH), 1710-1665 (2C=O); 1H NMR (DMSO-d6, δ,ppm): 3.79 (s, 3H, -OCH3), 6.84 (d, J = 9 Hz, 2H,


ArH), 6.92-7.89 (m, 5H, 2ArH), 10.63 (s, 1H, D2Oexchangeable, NH isatin), 11.23 (s, 1H, D2O exchange-able, NH); MS (C18H12ClN3O3) m/z = 353 (M+).

3-[3-(4-Methoxyphenyl)-5-oxo-1-phenyl-1,5-dihy-dro-4H-pyrazol-4-ylidene] indolin-2-one (9c)

Yield (72%); m.p. >300OC; IR (KBr, cm-1):3200 (NH), 1700-1660 (2C=O); 1H NMR (DMSO-d6, δ, ppm): 3.79 (s, 3H, -OCH3), 6.70-7.98 (m,13H, 3ArH), 11.20 (s, 1H, D2O exchangeable, NH);MS (C24H17N3O3) m/z = 395 (M+).

5-Chloro-3-[3-(4-methoxyphenyl)-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-ylidene] indolin-2-one (9d)

Yield (69%); m.p. 240OC; IR (KBr, cm-1): 3150(NH), 1700-1655 (2C=O); 1H NMR (DMSO-d6, δ,ppm): 3.80 (s, 3H, -OCH3), 6.65-8.20 (m, 12H,3ArH), 10.85 (s, 1H, D2O exchangeable, NH); MS(C24H16ClN3O3) m/z = 429 (M+).

Synthesis of 4-[mercapto(phenylamino)methyl-ene]-5-(4-methoxyphenyl)-2,4-dihydro-3H-pyra-zol-3-one derivatives (10a-b)

To a mixture of compound (4) or (5) (1 mM)and KOH (2 mM) in DMF (25 mL) stirring for 30min, then 1.1 mM of phenylisothiocyanate were

added. The strring was continued overnight at roomtemp. Reaction mixture was neutralized with 1 MHCl in ice bath (pH = 7), The desired product wasisolated as precipitate after pouring reaction mixtureto an ice-cold water. The precipitate was filtered,washed with cold water, dried and recrystalizedusing absolute ethanol.

4-[Mercapto(phenylamino)methylene]-5-(4-meth-oxyphenyl)-2,4-dihydro-3H-pyrazol-3-one (10a)

Yield (83%); m.p. 125OC; IR (KBr, cm-1): 3320(2NH), 1680 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.82 (s, 3H, -OCH3), 7.06 (d, J = 9 Hz, 2H, MeOArH),7.24 (t, J = 9 Hz, 1H, ArH), 7.45 (t, J = 9 Hz, 2H,ArH), 7.64 (d, J = 9 Hz, 2H, ArH), 7.90 (d, J = 12 Hz,2H, ArH), 8.00 (d, J = 12 Hz, 2H, ArH), 8.13 (d, J =12 Hz, 2H, ArH), 13.60 (s, 1H, D2O exchangeable,NH); MS (C17H15N3O2S) m/z = 325 (M+).

4-[Mercapto(phenylamino)methylene]-5-(4-meth-oxyphenyl)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (10b)

Yield (77%); m.p. 160OC; IR (KBr, cm-1): 3340(NH), 1670 (C=O); 1H NMR (DMSO-d6, δ, ppm):3.82 (s, 3H, -OCH3), 7.06 (d, J = 9 Hz, 2H, MeOArH),7.24 (t, J = 9 Hz, 1H, ArH), 7.45 (t, J = 9 Hz, 2H,

Scheme 1. Synthesis of pyrazoles 4 and 5


ArH), 7.64 (d, J = 9 Hz, 2H, ArH), 7.90 (d, J = 12 Hz,2H, ArH), 8.00 (d, J = 12 Hz, 2H, ArH), 8.13 (d, J =12 Hz, 2H, ArH), 13.60 (s, 1H, D2O exchangeable,NH); MS (C23H19N3O2S) m/z = 401 (M+).

Biological evaluation

In vitro anticancer activity

Cell culture of HepG-2 (human liver carcino-ma), PC-3 (human prostate adenocarcinoma) and

HCT116 (human colorectal carcinoma) cell lineswere purchased from the American Type CultureCollection (Rockville, MD) and maintained inRPMI-1640 medium which was supplemented with10% heat-inactivated FBS (fetal bovine serum), 100U/mL penicillin and 100 U/mL streptomycin. Thecells were grown at 37OC in a humidified atmos-phere of 5% CO2.

Scheme 2. Synthesis of arylidines 6a-j and arylhydrazones 7a-k

Figure 1. Anticancer activity of 29 compounds against three cancer types, using MTT assay at 100 µg/mL


MTT cytotoxicity assay

The antitumor activity against HepG-2, PC-3and HCT-116 human cancer cell lines was estimat-ed using the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay,which is based on the cleavage of the tetrazoliumsalt by mitochondrial dehydrogenases in viable cells(25-27). Cells were dispensed in a 96 well sterilemicroplate (5 ◊ 104 cells/well), and incubated at37OC with series of different concentrations, inDMSO, of each tested compound or doxorubicin(positive control) for 48 h in a serum free mediumprior to the MTT assay. After incubation, media

were carefully removed, 40 µL of MTT (2.5 mg/mL)were added to each well and then incubated for anadditional 4 h. The purple formazan dye crystalswere solubilized by the addition of 200 µL ofDMSO. The absorbance was measured at 590 nmusing a SpectraMaxÆ ParadigmÆ Multi-Modemicroplate reader. The relative cell viability wasexpressed as the mean percentage of viable cellscompared to the untreated control cells.


All experiments were conducted in triplicateand repeated in three different days. All the values

Table 1. The anticancer IC50 values of 29 compounds using MTT assay against the three cancer types.

CompoundHCT-116 PC-3 HepG-2

IC50 (µg/mL)

4 70.06 ± 3.4 86.96 ± 5.7 165.62 ± 5.9

5 66.19 ± 2.9 70.94 ± 3.9 233.42 ± 8.8

6a 101.29 ± 5.1 172.97 ± 8.1 138.89 ± 6.8

6b 76.41 ± 4.1 871.68 ± 4.8 157.68 ± 7.1

6c 80.19 ± 4.2 128.86 ± 6.9 152.89 ± 6.3

6d 69.34 ± 3.8 111.25 ± 7.8 245.36 ± 8.9

6e 67.36 ± 2.8 105.31 ± 5.8 165.40 ± 7.3

6f 64.42 ± 5.1 96.54 ± 5.1 218.42 ± 6.9

6g 62.37 ± 4.3 80.55 ± 6.3 258.03 ± 8.1

6h 73.87 ± 3.6 74.06 ± 3,8 244.45 ± 7.3

6i 59.79 ± 3.9 67.76 ± 4.2 276.11 ± 5.9

6j 61.36 ± 4.2 85.84 ± 3.6 344.39 ± 8.7

7a 56.35 ± 5.7 63.23 ± 3.1 175.84 ± 5.3

7b 105.99 ± 6.9 > 1000 106.67 ± 5.4

7c 96.00 ± 7.1 > 1000 133.37 ± 5.2

7d 77.33 ± 4.6 167.21 ± 3.2 123.70 ± 6.1

7e 85.06 ± 6.1 194.48 ± 8.9 118.36 ± 4.8

7f 67.71 ± 4.5 94.94 ± 5.7 156.06 ± 3.9

7g 64.68 ± 5.1 91.60 ± 4.6 217.70 ± 7.1

7h 66.35 ± 4.3 101.86 ± 6.1 207.86 ± 4.9

7i 63.72 ± 2.1 81.08 ± 3.7 238.32 ± 7.4

7j 62.36 ± 2.5 76.18 ± 3.4 221.75 ± 7.2

7k 61.10 ± 5.0 72.71 ± 2.9 261.23 ± 8.1

9a 111.29 ± 3.8 > 1000 485.15 ± 9.2

9b 104.42 ± 7.1 > 1000 592.99 ± 9.7

9c 81.95 ± 4.6 132.79 ± 3.8 322.90 ± 8.9

9d 78.76 ± 4.3 143.88 ± 6.1 271.83 ± 7.9

10a 59.12 ± 4.0 68.20 ± 3.1 415.07 ± 9.5

10b 176.99 ± 8.9 > 1000 216.92 ± 7.1

Doxorubicin 73.50 ± 2.9 75.24 ± 4.1 67.9 ± 3.2


were represented as the mean ± SD. IC50s weredetermined by probit analysis using SPSS softwareprogram (SPSS Inc., Chicago, IL).


Chemistry

The synthetic approach was confined to threegeneral schemes to obtain the target compounds.The reaction of 4-methoxyacetophenone (1) anddiethyl carbonate (2) in the presence of sodiumhydride afforded the ethyl 3-(4-methoxyphenyl)-3-oxopropanoate (3). Compound 3 undergo smoothreaction with hydrazine hydrate or phenylhydrazinein heated ethanol or glacial acetic acid for 10 min toyield pyrazoles 4 and 5, respectively (Scheme 1).From the literature data, keto form of similar com-pounds exists in isolated solid compound or in lowpolarity solvents while hydroxyl form can be pre-ferred in polar solvent such as DMSO (24, 28). 1HNMR of pyrazoles 4 (R = H) and 5 (R = Ph) inDMSO, showed singlet signal corresponding to C = Hat δ 5.8-5.9 ppm which support the presence of com-pounds 4 and 5 in enol form (Scheme 1) where IR ofthese compounds were carried out in solid form KBradapting disc technique.

Furthermore, the active methylene group ofpyrazole ring of compounds 4 and 5 is a favorableunit to react with electrophiles usually resulting inthe formation of C=C in case of aldehydes or C=Nbond in case of dizonium chloride of anilines.Therefore, the reactivity of compounds 4 and 5

towards substituted aldehydes as carbon elec-trophiles and toward substituted dizonium chlorideof anilines were utilized. The treatment of com-pound 4 or 5 with a series of aldehydes in refluxedabsolute ethanol in the presence of piperidine asbasic catalyst afforded the corresponding 4-(2-ary-

lylidene)-5-(4-methoxyphenyl)-2-phenyl-2,4-dihy-dro-3H-pyrazol-3-one (6a-j) (Scheme 2).

The reaction of compound 4 or 5 with 2-ben-zylidenemalononitrile in refluxed absolute ethanolin the presence of piperdine, two expected routessuggested for the reaction product via cyclization orarylidine-exchange via losing of malononitrile,respectively, was leading to either 6-amino-3-(4-methoxyphenyl)-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile structure 8a-j (29, 30) or6a-j (31, 32), as outlined in Scheme 2. However inall cases, the reaction preceeded via loss of maloni-trile according to the proposed mechanism which isshown in Scheme 2, and the reaction product wasproved by (IR, MS, 1H NMR).

Furthermore, the treatment of compound 4 or 5with dizonium chloride salts of anilines in the pres-ence of anhydrous sodium acetate in ice bath yield-ed the corresponds 4-(2-(aryl)hydrazineylidene)-5-(4-methoxyphenyl)-2-phenyl-2,4-dihydro-3H-pyra-zol-3-ones (7a-j), respectively. The structures of thesynthesized compounds were confirmed on the basisof their IR, mass and 1H NMR spectroscopic analy-sis (Scheme 2). 1H NMR of compounds 7a-j showedthe appearance of D2O-exchangeable hydrazone NHas a singlet signal which demonstrates the presenceof these compounds in the form of hydrazone struc-ture (23, 25) and excludes the possible azo/hydrazotautomerization.

Moreover, compound 4 or 5 allowed to reactwith isatin derivatives in refluxing acetic acid in thepresence of anhydrous sodium acetate, furnished asingle product that was identified as 5-substituted-3-(3-(4-methoxyphenyl)-5-oxo-1,5-dihydro-4H-pyra-zol-4-ylidene)indolin-2-one 9a-d.

In addition, the reaction of compound 4 or 5

with phenylisothiocyanate in DMF, in the presenceof KOH, at room temperature afforded 4-mercap-

Scheme 3. Synthesis of compounds 9a-d and 10a-b


to(phenylamino)methylene-5-(4-methoxyphenyl)-2-2,4-dihydro-3H-pyrazol-3-ones 10a-b (Scheme 3).

Biological activity

Anti-tumor activity

The compounds were examined in vitro fortheir anti-tumor activities against HepG-2, PC-3 andHCT-116 human carcinoma cell lines using MTTassay. The percentage of the intact cells was meas-ured and compared to the control (Fig. 1). The activ-ities of these compounds against the three carcinomacells were compared with that of doxorubicin. Theobtained results showed that all compounds showeddose-dependent anticancer activities against thethree cancer cells.

From Figure 1 we can deduce that, at 100µg/mL, eight compounds (7a, 10b, 6i, 7k, 6j, 7j, 6g,7i) showed good anticancer activities against HCT-116 carcinoma cells. Fifteen compounds (6f, 7g, 5,7h, 6e, 7f, 6d, 4, 6h, 6b, 7d, 9d, 7c, 12b, 7e) showedmoderate activities and the rest of the compoundsshowed weak activities against HCT-116 cells. Inaddition, five compounds (7a, 6i, 10a, 5, 7k)showed good anticancer activities, six compounds(6h, 7j, 6g, 7i, 6j, 4) showed moderate antitumoractivity and the rest of the compounds showed weakor no antitumor activities against PC-3 cancer cells.Furthermore, all the compounds showed weak or noanticancer activities against HepG-2 liver cancer.The IC50 values are shown in Table 1.

Structureñactivity relationships in structures6aj, 7a-k, 9a-d and 10-b demonstrated that com-pounds with para electron-withdrawing substituent(-N(CH3)2, F, Cl, Br) showed higher anticanceractivities than those with electron-donating sub-stituent (-CH3, -OCH3). A comparison of the parasubstituent demonstrated that an electron-withdraw-ing group have improved anticancer activity and thepotency order is -N(CH3)2> F > Cl > Br, whereas thedonating group substituent had moderate effects inthe case of -N-Ph and had weak or no effects in thecase of -N-H. The derivatives with ñN-Ph showedstronger anticancer activities than ñN-H of the com-pounds.

CONCLUSION

In summary, we synthesized a series of pyra-zole derivatives and their structures confirmed byIR, mass and NMR spectroscopic analysis. Thesecompounds were examined for their anticanceractivities on three different human tumor cell lines.The results showed that the compounds 7a, 10b, 6i,7k, 6j, 7j, 6g, 7i showed good anticancer activities

against HCT-116 carcinoma cells. The compounds7a, 6i, 10a, 5, 7k showed good anticancer activitiesagainst PC-3 cancer cells and all the compoundsshowed weak or no anticancer activities againstHepG-2 liver cancer.

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13. Fustero S., Roman R., Sanz-Cervera J.F.,Simon-Fuentes A., Cunat A.C. et al.: J. Org.Chem. 73, 3523 (2008).

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Received: 16. 08. 2016


Quinazoline is considered one of the most impor-tant scaffolds in medicinal chemistry due to its diversebiological activities. Several quinazoline derivativespossess anticancer (1-9), antihypertensive (10), antivi-ral (11, 12), sedative (13), antibacterial (14-20), anti-inflammatory and analgesic activities (21-30).Hybridizaton of quinazolin-4-one with 2-imino pyri-dine, compound 1, (Fig. 1) shows potent anticanceractivity (31). While hybridization with pyran-3-car-bonitrile gives compound 2 with cytotoxic activityhigher than doxorubicin (31). 2,3 disubstituted quina-zoline moiety (compound 3) have promising anti-cancer activity (32) against MCF-7 cell line (Fig. 1).

Dibromoquinazoline derivatives bearing 4-chlorophenyl at position 2 and aniline derivatives atposition 3 (compound 4) exert powerful anti-breastcancer activity (33). In continuation of our researchprogram (28-36), our target in this work is tohybridize pyran or pyridine moiety with bromoquinazoline derivative bearing 4-chlorophenyl moi-ety at position 2 and aniline moiety at position 3hopping to obtain safe and potent anticancer deriva-tives against MCF-7 cell line.

EXPERIMENTAL

Chemistry

All melting points are measured using Electro-thermal IA 9100 apparatus (Shimadzu, Japan). IRspectra were recorded on Perkin Elmer 1650 spec-

trophotometer (USA), in Faculty of Science, CairoUniversity, Cairo, Egypt. 1H NMR spectra weredetermined on JOEL NMR FXQ-300 MHz usingTMS as an internal standard. 13C NMR (DMSO-d6)spectra were recorded at 100.62 MHz at the afore-mentioned research center in Cairo University. Massspectra were determined on at 70 eV EI Ms-QP1000 EX (Shimadzu, Japan). Microanalyses wereoperated using Vario EL elemental apparatus(Shimadzu, Japan).

6-{4-[6-Bromo-2-(4-chlorophenyl)-4-oxoquinaz-

olin-3(4H)-yl]phenyl}-2-oxo-4ó(substituted

aryl)-1,2-dihydropyridine-3-carbonitrile (IIIa-d)

General method

A mixture of ketone II (0.9 g; 0.002 M), ethylcyanoacetate (0.23 mL, 0.002 M), anhyd. ammoniumacetate (1.24 g; 0.016 M) and the appropriate aldehy-des namely, p-chlorobenzaldehyde, 3,4-dichloroben-zaldehyde, 3,4,5- trimethoxybenzaldehyde and/orfuran-2-carboxaldehyde in 10 mL n-butanol wasrefluxed for 6 h. The reaction mixture was concentrat-ed to half its volume under reduced pressure. Aftercooling, the formed precipitate was filtered off, airdried, and recrystallized from the proper solvent togive compounds IIIa-d, respectively.

6-{4-[6-Bromo-2-(4-chlorophenyl)-4-oxoquina-

zolin-3(4H)-yl]phenyl}-4-(4-chlorophenyl)-2-oxo-

1,2-dihydropyridine-3-carbonitrile (IIIa)

SYNTHESIS AND DESIGN OF NEW BROMOQUINAZOLINE DERIVATIVESWITH ANTI-BREAST CANCER ACTIVITY

MARWA F. AHMED1, 2* , DAREEN JAIASH2 and AMANY BELAL3

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo, Egypt2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Taif University, Taif 21974,

Kingdom of Saudi Arabia3Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt

Abstract: A new series of 6- bromoquinazolin-4(3H)-one derivatives were synthesized. Their chemical struc-tures were confirmed by spectral and elemental analysis. All synthesized compounds were tested in vitro againsthuman breast cancer cell line (MCF-7). Compounds IIIc, Vb and IIIa exerted powerful cytotoxic activity withIC50 (0.236, 0.316 and 0.359 µMl/mL), respectively.

Keywords: quinazolinone; anti-tumor, breast cancer

1437


1438 MARWA F. AHMED et al.

Crystallized from ethanol to give yellow crys-tals, m.p. 110OC in 70% yield. Analysis forC32H17BrCl2N4O2, calcd.: C, 60.02; H, 2.68; N,8.75%, Found: C, 60.17; H, 2.74; N, 8.69%. IRspectrum (KBr, cm-1) showed absorption bands at3380 (NH), 2225 (-CN), 1745 (C=O, pyridone),1685 (C=O, quinazolinone), and 1610 (C=C).1HNMR spectrum (DMSO-d6, δ, ppm) showed 6.2(1H, s, 1 H of pyridine), 7.3-8.2(m, 15H, Ar-H), andat 9.5 (1 H, s, NH proton, exchangeable with D2O).13C NMR (DMSO-d6, δ, ppm): 165, 161, 160.5, 159,157, 149, 136.4, 136, 133.9, 133.6, 132, 131.7, 130,129.1, 128.9, 128.7, 126.7, 126.6, 126.4, 124.5, 122,121.7, 116. MS: m/z = 639.


zolin-3(4H)-yl]phenyl}-4-(3,4-dichlorophenyl)-2-

oxo-1,2-dihydropyridine-3-carbonitrile (IIIb)

Crystallized from ethanol to give yellowish browncrystals, m.p. 115OC in 80% yield. Analysis forC32H16BrCl3N4O2, calcd.: C, 56.96; H, 2.39; N, 8.3%;found: C, 56.89; H, 2.42; N, 8.42%. IR spectrum (KBr,cm-1) showed absorption bands at 3385 (NH), 2230 (-CN), 1750 (C=O, pyridone), 1680 (C=O, quinazoli-none), and 1600 (C=C). 1HNMR spectrum (DMSO-d6,δ, ppm) showed 6.4 (1H, s, 1 H of pyridine), 7.4-8.6(m, 14H, Ar-H), and at 9.2 (1 H, s, NH, exchangeablewith D2O).13C NMR (DMSO-d6, δ, ppm): 168, 162,

161, 159, 157.5, 149, 136.5, 135.9, 133.9, 132.5, 132.2,132, 130.1, 129.2, 128.7, 128.1, 127.8, 126.7, 126.5,124.8, 124.5, 122, 121.7, 117, 105. MS: m/z = 672.


zolin-3(4H)-yl]phenyl}-2-oxo-4-(3,4,5-trimeth-

oxyphenyl)-1,2-dihydropyridine-3-carbonitrile

(IIIc)

Crystallized from methanol to give yellowcrystals, m.p.205OC in 75% yield. Analysis forC35H24BrClN4O5, calcd.: C, 60.40; H, 3.48 N, 8.05%;found: C, 60.50; H, 3.61; N, 8.12%. IR spectrum(KBr, cm-1) showed absorption bands at 3380 (NH),2235 (-CN), 1765 (C=O, pyridone), 1680 (C=O,quinazolinone), and 1600 (C=C). 1HNMR spectrum(DMSO-d6, δ, ppm) showed 3.9 (9H, s, OCH3), 6.4(1H, s, 1 H of pyridine), 7.2-8.7 (m, 13H, Ar-H) andat 11.5 (1 H, s, NH, exchangeable with D2O). 13CNMR (DMSO-d6, δ, ppm): 165, 162, 160.5, 159,157.4, 152, 149, 138.5, 136.3, 135, 132.3, 131.9,129, 128.7, 126.8, 126.7, 126.3, 126.2, 124.6, 124.3,121, 116.2, 116, 60, 56. MS: m/z = 696.


zolin-3(4H)-yl]phenyl}-4-(furan-2-yl)-2-oxo-1,2-

dihydropyridine-3-carbonitrile (IIId)

Crystallized from methanol to give browncrystals, m.p. 195OC in 80% yield. Analysis for

Figure 1. Anticancer quinazoline derivatives

Synthesis and design of new bromoquinazoline derivatives with... 1439

C30H16BrClN4O3, calcd.: C, 60.47; H, 2.71; N, 9.4%;found: C, 60.57; H, 2.83; N, 9.52%. IR spectrum(KBr, cm-1) showed absorption bands at 3370 (NH),222 (5-CN), 1755 (C=O, pyridone), 1670 (C=O,quinazolinone) and 1595 (C=C). 1HNMR spectrum(DMSO-d6, δ, ppm) showed 6.4 (1H, s, 1 H of pyri-dine), 7.1-8.4 (m, 14H, Ar-H) and at 10.0 (1 H, s,NH, exchangeable with D2O). 13C NMR (DMSO-d6,δ, ppm): 169.5, 164, 160.6, 160.1, 159.5, 149.9,147.7, 143.7, 136.3, 135.7, 132.2, 131, 129, 128.8,126.7, 126.6, 126.4, 124.6, 124.2, 122, 121.7, 121.3,117, 113, 110. MS: m/z = 596.

6-{4-[6-bromo-2-(4-chlorophenyl)-4-oxoquina-

zolin-3(4H)-yl]phenyl}-2-imino-4-(substituted

aryl)phenyl-1,2-dihydropyridine-3-carbonitrile

(IVa-d)

General method

A mixture of compound II (0.9 g, 0.002 M),malononitrile (0.12 mL, 0.002 M), anhyd. ammoni-um acetate ((1.24 g, 0.016 M) and the appropriatealdehydes namely, p-chlorobenzaldehyde, 3,4-dichlorobenzaldehyde, 3,4,5- trimethoxybenzalde-hyde and/or furan-2-carboxaldehyde in 10 mL n-butanol was refluxed for 6 h. After cooling, the reac-tion mixture was filtered and the precipitate wascrystallized from the proper solvent to give theiminopyridines IV a-d, respectively.

6-{4-[6-bromo-2-(4-chlorophenyl)-4-oxoquina-

zolin-3(4H)-yl]phenyl}-4-(4-chlorophenyl)-2-

imino-1,2-dihydropyridine-3-carbonitrile (IVa)

Crystallized from glacial acetic acid to give redcrystals, m.p. 145OC in 75% yield. Analysis forC32H18BrCl2N5O, calcd.: C, 60.12; H, 2.84; N,10.95%; found: C, 60.25; H, 2.92; N, 10.99%. IRspectrum (KBr, cm-1) absorption bands at 3335-3250(NH, =NH), 2215 (C=N), 1710 (C=O, quinazoli-none) and at 1600 (C=N). HNMR spectrum(DMSO-d6, δ, ppm) showed 7.4-8.3 (m, 16H, Ar-H )and 9.70, 11.60 (2H, 2s, 2NH, exchangeable withD2O). 13C NMR (DMSO-d6, δ, ppm): 170, 167, 163,162, 160, 148, 136.4, 135.7, 133.9, 133, 132, 130,129.1, 128.9, 128.7, 126.7, 126.5, 126.3, 124.6,124.3, 123, 121, 118, 115, 103. MS: m/z = 639.


zolin-3(4H)-yl]phenyl}-4-(3,4-dichlorophenyl)-2-

imino-1,2-dihydropyridine-3-carbonitrile (IVb)

Crystallized from methanol to give reddishbrown crystals, m.p. 132OC in 75% yield. Analysisfor C32H17BrCl3N5O, calcd.: C, 57.04; H, 2.54%; N,10.39; found: C, 57.15; H, 2.62; N, 10.41%. IR spec-trum (KBr, cm-1) absorption bands at 3335-3215

(NH, =NH), 2210 (C=N), 1720 (C=O, quinazoli-none), and at 1610 (C=N).1HNMR spectrum(DMSO-d6, δ, ppm) showed 7.6-8.5 (m, 15H, Ar-H)and 10.60, 12.30 (2H, 2s, 2NH, exchangeable withD2O). 13C NMR (DMSO-d6, δ, ppm): 173, 164, 162,161, 148, 136.2, 134, 133.3, 132.6, 132.3, 132,131.8, 130.1, 129.2, 128.7, 128, 127.7, 126.6, 126.5,124.6, 124.4, 124.3, 123, 121, 118.5, 116, 105. MS:m/z = 672.97.


zolin-3(4H)-yl]phenyl}-2-imino-4-(3,4,5-trimeth-

oxyphenyl)-1,2-dihydropyridine-3-carbonitrile

(IVc)

Crystallized from glacial acetic acid to givebrown crystals, m.p. 215OC in 70% yield. Analysisfor C35H25BrClN5O4, calcd.: C, 60.49; H, 3.63%; N,10.08; found: C, 60.52; H, 3.67; N, 10.10%. IR spec-trum (KBr, cm-1) absorption bands at 3335-3220(NH, =NH), 2200 (C=N), 1720 (C=O, quinazoli-none) and at 1610 (C=N).1HNMR spectrum(DMSO-d6, δ, ppm) showed 3.8 (9H, s, OCH3),6.55-8.4 (m, 14H,) and 9.70, 11.90 (2H, 2s, 2NH,exchangeable with D2O). 13C NMR (DMSO-d6, δ,ppm): 176, 166, 165, 163, 153, 150, 139, 137, 136,135, 132, 130, 129, 126.8, 126.7, 126.6, 126.4,124.5, 124.2, 123, 119, 117, 60, 56. MS: m/z = 695.


zolin-3(4H)-yl]phenyl}-4-(furan-2-yl)-2-imino-

1,2-dihydropyridine-3-carbonitrile (IVd)

Crystallized from ethanol to give brown crys-tals, m.p. 240OC in 75% yield. Analysis forC30H17BrClN5O2, calcd.: C, 69.84; H, 3.52%; N,11.77; found: C, 69.79; H, 3.57; N, 11.64%. IR spec-trum (KBr, cm-1) absorption bands at 3320-3210(NH, =NH), 2200 (C=N), 1710 (C=O, quinazoli-none) and at 1600 (C=N).1HNMR spectrum(DMSO-d6, δ, ppm) showed 6.9-8.4 (m, 15H, Ar-H)and 9.70, 11.20 (2H, 2s, 2NH, exchangeable withD2O). 13C NMR (DMSO-d6, δ, ppm): 169.5, 164,162, 150.1, 147.7, 144, 137, 135, 132.3, 131.7, 129,128.9, 126.9, 126.7, 126.4, 124.6, 124.3, 122, 121,115, 113, 109. MS: m/z = 595.

2-Amino-6-{4-[2-(furan-2-yl)-4-oxoquinazolin-

3(4H)-yl]phenyl}-4-(4-substituted phenyl)-4H-

pyran-3-carbonitrile (Va-d)

General method

A mixture of compound II (0.90 g, 0.002 M),malononitrile (0.12 mL, 0.002 M), and the appropri-ate aromatic aldehydes, namely, p-chlorobenzalde-hyde, 3,4-dichlorobenzaldehyde, 3,4,5- trimethoxy-benzaldehyde and/or furan-2-carboxaldehyde in few

1440 MARWA F. AHMED et al.T

able

1. E

ight

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e gr

owth

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the

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µg/m

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IC50

IC50

No.

Via

bilit

y %

(µg/

mL

)µM

/mL

03.

125

6.25

12.5

2550

100

200

400

IIIa

100

100

100

97.9

394

.69

87.0

664

.49

53.8

628

.45

230

0.35

9

IIIb

100

100

100

100

98.1

293

.76

84.2

365

.24

34.8

730

00.

444

IIIc

100

100

98.7

496

.02

89.4

976

.85

57.4

139

.13

22.3

414

10.

236

IIId

100

100

100

99.1

796

.04

89.7

681

.28

70.3

247

.68

380

0.63

7

IVa

100

100

100

100

98.2

590

.74

71.2

359

.79

30.5

726

70.

417

IVb

100

100

100

100

98.7

894

.52

87.1

670

.93

54.6

8>

400

IVc

100

100

100

100

100

100

98.7

991

.85

84.3

2>

400

IVd

100

100

100

100

98.3

991

.46

75.0

656

.42

31.5

725

20.

420

Va

100

100

100

100

96.3

887

.44

78.1

966

.41

35.1

830

50.

474

Vb

100

100

98.7

596

.63

89.0

478

.19

64.7

251

.69

28.1

621

40.

316

Vc

100

100

100

100

100

98.1

592

.72

78.5

756

.84

> 40

0

Vd

100

100

100

100

98.1

791

.43

79.4

564

.36

42.7

033

30.

557

drops of piperidine was refluxed for 8 h. Thereaction mixture was reduced to half its vol-ume under reduced pressure, and cooled. Thecrude precipitate was filtered off, washedwith cold water, and crystallized from theproper solvent to give aminopyrans Va-d,respectively.

2-Amino-6-{4-[6-bromo-2-(4-chlorophen-

yl)-4-oxoquinazolin-3(4H)-yl]phenyl}-4-(4-

chlorophenyl)-4H-pyran-3-carbonitrile (Va)

Crystallized from methanol to give yel-low crystals, m.p. 176OC in 75% yield.Analysis for C32H19BrCl2N4O2, calcd.: C,59.84; H, 2.98%; N, 8.72; found: C, 59.72; H,2.95; N, 8.80%. IR spectrum (KBr, cm-1)absorption bands at 3435-3360 (NH2), 2225 (-CN), 1685 (C=O quinazolinone), and at1600 (C=N). 1HNMR spectrum (DMSO-d6, δ,ppm) 4.1 (1H, d, 1 H of pyran), 5.1 (1H, d, 1H of pyran), 7.5-8.4 (m, 15H, Ar-H) and 9.8(2H, s, NH2, exchangeable with D2O). 13CNMR (DMSO-d6, δ, ppm): 168, 162, 160,149, 140.5, 140.2, 136.7,136.6, 135.8, 132.2,132,131.3, 130.4, 129, 128.9, 128.6, 128.4,126.7, 126, 125.9, 124.8, 124.5, 123, 121.7,120, 91.5, 59, 30 MS: m/z = 642.


yl)-4-oxoquinazolin-3(4H)-yl]phenyl}-4-

(3,4-dichlorophenyl)-4H-pyran-3-carboni-

trile (Vb)

Crystallized from ethanol to give browncrystals, m.p. 215OC in 70% yield. Analysis forC32H18BrCl3N4O2, calcd.: C, 56.79; H, 2.68; N,8.28%; found: %C, 56.79; H, 2.68; N, 8.39%.IR spectrum (KBr, cm-1) absorption bands at3440-3360 (NH2), 2220 (-CN), 1690 (C=Oquinazolinone), and at 1610 (C=N). 1HNMRspectrum (DMSO-d6, δ, ppm) showed 4.5 (1H,d, 1 H of pyran), 5.4 (1H, d, 1 H of pyran), 7.5-8.4 (m, 14H, Ar-H) and 10.1 (2H, s, NH2,exchangeable with D2O). 13C NMR (DMSO-d6,δ, ppm): 169, 163, 157, 148, 143, 141, 139,136, 132.8, 131.9, 130.5, 130.2, 130, 129,128.9, 128.5, 126.7, 126, 124.4, 124, 122, 119,92, 60, 32. MS: m/z = 677.


yl)-4-oxoquinazolin-3(4H)-yl]phenyl}-4-

(3,4,5-trimethoxyphenyl)-4H-pyran-3-car-

bonitrile (Vc)

Crystallized from glacial acetic acid togive yellow crystals, m.p. 240OC in 70%


Figure 2. Cell viability curves of all synthesized compounds


yield. Analysis for C35H26BrClN4O5, calcd.: C,60.23; H, 3.75; N, 8.03%; found: C, 60.35; H, 3.80;N, 8.12%.. IR spectrum (KBr, cm-1) absorptionbands at 3430-3350 (NH2), 2220 (-CN), 1685 (C=Oquinazolinone), and at 1610 (C=N).1HNMR spec-trum (DMSO-d6, δ, ppm) showed 3.8 (9H, s,OCH3), 4.2 (1H, d, 1 H of pyran), 5.1 (1H, d, 1 H ofpyran), 7.2-8.3 (m, 13H, Ar-H ) and 9.0 (2H, s, NH2,exchangeable with D2O). 13C NMR (DMSO-d6, δ,ppm): 165, 161, 159.9, 152.9, 148, 141, 136.5,136.3, 136, 135.5, 132.3, 131.9, 130, 129.1, 127,126.6, 125.9, 124.6, 124.3, 123, 122, 119, 106, 91,61, 56.2, 29.3. MS: m/z = 698.

2-Amino-6-{4-[6-bromo-2-(4-chlorophenyl)-4-

oxoquinazolin-3(4H)-yl]phenyl}-4-(furan-2-yl)-

4H-pyran-3-carbonitrile (Vd)

Crystallized from ethanol to give yellow crys-tals, m.p. 180OC in 70% yield. Analysis for

C30H18BrClN4O3, calcd.: C, 60.27; H, 3.03; N,9.37%; found: %C, 60.31; H, 3.12; N,9.30%. IRspectrum (KBr, cm-1) absorption bands at 3420-3350(NH2), 2200 (-CN), 1670 (C=O quinazolinone), andat 1600 (C=N).1HNMR spectrum (DMSO-d6, δ,ppm) showed 4.0 (1H, d, 1 H of pyran), 4.9 (1H, d,1 H of pyran), 7.0-8.2 (m, 14H, Ar-H) and 9.4 (2H,s, NH2, exchangeable with D2O). 13C NMR (DMSO-d6, δ, ppm): 163, 160.5, 158, 152.5, 148, 143, 140.5,136.4, 132.3, 131.9, 129, 128.5, 126.6, 126.4, 125.9,124.6, 124.3, 123, 119, 90, 31. MS: m/z = found:597.

Pharmacological screeningCell line propagation

The cells were propagated in Dulbeccoís mod-ified Eagleís medium (DMEM), supplemented with1% L-glutamine, 50 µg/mL gentamycin (37),HEPES buffer and 10% heat-inactivated fetal

Scheme 1. Synthesis of quinazoline derivatives IIIa-d


bovine serum. All cells were maintained in a humid-ified atmosphere at 37OC with 5% CO2 and weresubcultured two times a week. Cell toxicity wasmonitored by determining the effect of the test sam-ples on cell viability and cell morphology.

Cytotoxicity evaluation using viability assay

For cytotoxicity assay, the cells were seeded in96-well plate at a cell concentration of 1 ◊ 104 cellsper well in 100 µL of growth medium (37, 38). After24 h of seeding fresh medium containing differentconcentrations of the test sample was added. Serialtwo-fold dilutions of the tested chemical compoundwere added to confluent cell monolayers dispensedinto 96-well. The microtiter plates were incubated at37OC in a humidified incubator with 5% CO2 for aperiod of 48 h. Control cells were incubated with orwithout DMSO and without test sample. After incu-bation of the cells for at 37OC, various concentra-tions of sample were added, and the incubation wascontinued for 24 h and viable cells yield was deter-mined by a colorimetric method.

After the end of the incubation period, mediawere aspirated and the crystal violet solution (1%)was added for at least 30 min. The plates were rinsed

using tap water. Glacial acetic acid (30%) was thenadded and then the absorbance of the plates weremeasured after gently shaken on Microplate reader(TECAN, Inc.), using a test wavelength of 490 nm.All results were corrected for backgroundabsorbance detected in wells without added stain.Treated samples were compared with the cell controlin the absence of the tested compounds. All experi-ments were carried out in triplicate. The cell cyto-toxic effect of each tested compound was calculated.


Chemistry

Compounds I and II are synthesized accordingto reported method (18). One pot reaction of ketoneII with aromatic aldehydes, namely, p-chloroben-zaldehyde, 3,4-dichlorobenzaldehyde, 3,4,5-trimeth-oxybenzaldehyde and/or furan-2-carboxaldehydewith ethyl cyanoacetate afforded the correspondingpyridine-2(1H)-ones III a-d, respectively (Scheme1). The one pot reaction of II with the same aldehy-des and malononitrile in the presence of anhyd.ammonium acetate give the corresponding 2(1H)-iminopyridine derivatives IVa-d, respectively

Scheme 2. Synthesis of quinazolin-4-one derivatives IVa-d, Va-d


(Scheme 2), while the same one pot reaction of II

with malononitrile in piperidine, gave the corre-sponding 2-aminopyrans Va-d (Scheme 2).

Biological activity

In vitro cytotoxic activity

In this study a novel series of 6-bromo-2-(4-chlorophenyl)quinazolin-4(3H)-one derivatives weresynthesized (Schemes 1, 2). All synthesized com-pounds were screened for their in vitro cytotoxic activ-ities against MCF-7 cell line. Growth curves showedthat most our tested compounds exhibit significantefficacy against MCF-7 with IC50 values range (0.236ñ 0.637 µMol/mL). As shown in Table 1: compoundsIIIc, Vb and IIIa exerted the most potent cytotoxiceffect against MCF-7 with IC50 (0.236, 0.316 and0.359 µM/mL). Compounds IVa, IVd, IIIb, Vd andIIId exerted a moderate cytotoxic effect (IC50 0.417,0.420, 0.444, 0.474, 0.557 and 0.637 µMol/mL,respectively), while compounds IVb, IVc, and Vc

exerted a weak cytotoxic effect against MCF-7.

Structure - Activity Relationships

Concerning 6-{4-[6-bromo-2-(4-chlorophen-yl)-4-oxoquinazolin-3(4H)-yl]phenyl}-2-oxo-4-(substituted aryl)-1,2-dihydropyridine-3-carboni-trile (IIIa-d) derivatives, substitution with 4-chlorophenyl IIIa, or 3,4,5- trimethoxyphenyl IIIc

at position 4 gives the most potent cytotoxic activi-ty. While substitution at the same position with 3,4-dichlorophenyl IIIb, or with furyl moiety IIId givesmoderate cytotoxic effect.

On discussing the SAR of compounds 6-{4-[6-bromo-2-(4-chlorophenyl)-4-oxoquinazolin-3(4H)-yl]phenyl}-2-imino-4-(substituted aryl)phenyl-1,2-dihydropyridine-3-carbonitrile (IVa-d), substitutionwith 4-chlorophenyl IVa, or furyl moiety IVd givesmoderate cytotoxic effect. While substitution with3,4-dichlorophenyl IVb, or 3,4,5- trimethoxyphenylIVc decreases the cytotoxic activity.

On the other hand, substitution of 2-amino-6-{4-[2-(furan-2-yl)-4-oxoquinazolin-3(4H)-yl]phen-yl)-4-(4-substituted phenyl}-4H-pyran-3-carboni-trile with 3,4-dichlorophenyl at position 4 givescompound Vc with potent cytotoxic activity, whilesubstitution with 4-chlorophenyl Va, or furyl moietyVd gives moderate cytotoxic effect. Substitutionwith 3,4,5- trimethoxyphenyl IVc decreases thecytotoxic activity.

CONCLUSION

In summary, 6 bromo-2-(4-chlorophenyl)quin-azolin-4(3H)-one derivatives III-V were synthe-

sized. All synthesized compounds were tested invitro against human breast cancer cell line (MCF-7).Compounds IIIc, Vb and IIIa exerted powerfulcytotoxic activity with IC50 (0.236, 0.316 and 0.359µM/mL) respectively.

Acknowledgement

I deeply thank Ghadeer Manei Aljuaid (Labtechnician) and Sameerah Eid Alharthi (Lab techni-cian) faculty of pharmacy Taif University, for labfacilities and kind help during the research.

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9. Al-Obaid A.M., Abdel-Hamide S.G., El-KashefH.A., M.Abdel-Aziz A.A., El-Azab A.S. et al.:Eur. J. Med. Chem. 44, 2379 (2009).

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Received: 19. 09. 2016


Tuberculosis still remains a major publichealth threat despites years of research. The WorldHealth Organization (WHO) estimates, that TB isthe leading infections cause of death worldwide.Moreover, TB accounts the loss of two million livesannually as a result of e.g., presence of multidrug-resistant (MDR) and extensively drug resistant(XDR) strains of M. tuberculosis (1-3), co-infectionwith HIV, smoking, malnutrition, alcoholism andchronic lung disease.

Current recommended antitubercular strategyinvolve the use of first- and second-line multipleantibiotics such as: isoniazid (INH), rifampin (RIF),pyrazinamide, ethambutol and streptomycin, how-ever it is often insufficient (4-6). In the last decade,there has been growing interest in developing newsubstances that are efficient against M. tuberculosis.Among them, there are azole derivatives widelyknown as antifungal agents (7). It has been provedthat 14α-demethylase is an important Mtb proteinwhich takes part in sterol biosynthesis (8). Anenzyme with similar structure is present in fungi andis inhibited by different azole compounds. They actas antifungal agents. These observations were

proved on the example of econazole and clotrima-zole (9, 10). Results of anti-Mtb activity testsrevealed that both econazole and clotrimazole arepotent inhibitors of M. tuberculosis H37Rv with lowMIC values (9). Surprisingly, they were found to bemore active than rifampicin.

About twenty years ago, antitubercular activityof nitroimidazo[2,1-b]oxazoles derived from 2,4-dinitroimidazole, has been found (11). Unfortunately,the leading compound from this series ñ CGI-17341,which was active against MDR Mtb, also have hadstrong mutagenicity. Further, this undesired potencyhas been eliminated by modification of the sub-stituent on C-2 position in bicyclic system (12). Thesealterations allowed to found the most potent com-pound OPC-67683. Nowadays, this bicyclic nitroim-idazole undergoing clinical trials as promising tuber-culostatic agent. Another anti-Mtb nitroimidazolederivative with two conjugated rings is PA-824 ñ anitroimidazooxazine (13). It has activity against aero-bic and anaerobic populations of M. tuberculosis. Itacts as a pro-drug requiring bioreductive activation. Itis an inhibitor of mycolic acid and protein biosynthe-sis. PA-824 is currently in Phase III clinical trials.

STRUCTURE ñ TUBERCULOSTATIC ACTIVITY EVALUATION AMONG ISOMERIC BICYCLIC NITROIMIDAZOLES

JUSTYNA ØWAWIAK*, JACEK KUJAWSKI and LUCJUSZ ZAPRUTKO

Department of Organic Chemistry, Pharmaceutical Faculty, PoznaÒ University of Medical Sciences,Grunwaldzka 6, 60-780 PoznaÒ, Poland

Abstract: Despite many existing treatment methods, the threat of tuberculosis (TB) is still a reality for millionsof people worldwide. What is more, TB accounts the loss of two million lives annually as a result of e.g., pres-ence of multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis. In the lastdecade, there has been growing interest in developing new substances that are efficient against M. tuberculosis.Among them, there are azole derivatives widely known as antifungal agents. Nowadays, some bicyclic nitroim-idazoles (PA-824, CGI-17341, OPC-67683) undergoing clinical trials as promising tuberculostatic agents. Inour study, bicyclic 7-nitroimidazo[5,1-b]-2,3-dihydrooxazoles, isomeric with CGI-17341 and 3-hydroxy-8-nitroimidazo[5,1-b]-1,4,5,6-tetrahydropyrimidines, which are structural isomers of the structure of PA-824were synthesized and tested for their physicochemical properties. It has turned out that there are interesting dif-ferences between bicyclic isomers. In order to explain the reasons of the experimental disparities in biologicalactivity of tested compounds and referenced nitroimidazooxazine PA-824, it was decided to carry out a molec-ular docking simulations.

Keywords: tuberculosis, antitubercular activity, azoles, bicyclic nitroimidazoles, molecular docking simula-tions

1447

* Corresponding author: e-mail: [email protected]; phone: +48 61 8546 678; fax: +48 61 8546 680

1448 JUSTYNA ØWAWIAK et al.

Table 1. Physicochemical parameters for bicyclic nitroimidazole derivatives.

No. of H No. of H Compound MW < 500 Mi LogP bonds bonds TPSA Volume

< 5 acceptors donors

PA-824 357.288 3.977 7 0 82.118 282.457

OPC-67683 534.491 4.842 10 0 103.824 440.445

CGI-17341 183.167 1.181 6 0 72.884 153.724

1 169.140 0.482 6 0 72.884 136.922

2 183.167 0.570 6 0 72.884 153.483

3 183.167 0.985 6 0 72.884 153.724

4 197.194 1.072 6 0 72.884 170.285

5 231.211 1.701 6 0 72.884 191.769

6 245.238 1.788 6 0 72.884 208.330

7 248.036 0.843 6 0 72.884 155.048

8 262.063 0.930 6 0 72.884 171.609

9 227.220 0.829 7 0 82.118 196.097

10 241.247 0.917 7 0 82.118 212.658

11 260.253 1.217 7 1 87.116 220.388

12 274.280 1.305 7 1 87.116 236.949

13 294.698 1.895 7 1 87.116 233.924

14 308.725 1.983 7 1 87.116 250.485

15 339.149 2.026 7 1 87.116 238.274

16 353.176 2.114 7 1 87.116 254.835

17 386.149 2.300 7 1 87.116 244.378

18 400.176 2.388 7 1 87.116 260.939

19 240.263 0.957 7 1 87.116 215.946

20 254.29 1.045 7 1 87.116 232.507

21 240.263 0.642 7 1 87.116 215.731

22 254.29 0.729 7 1 87.116 232.292

1: R = -H, R1 = -CH3

2: R = -CH3, R1 = -CH3

3: R = -H, R1 = -CH2CH3

4: R = -CH3, R1 = -CH2CH3

5: R = -H, R1 = -Ph6: R = -CH3, R1 = -Ph7: R = -H, R1 = -CH2Br8: R = -CH3, R1 = -CH2Br9: R = -H, R1 = -CH2O-i-Pr10: R = -CH3, R1 = -CH2O-i-Pr

11: R = -H, R2 = -Ph12: R = -CH3, R2 = -Ph13: R = -H, R2 = -C6H4Cl14: R = -CH3, R2 = -C6H4Cl15: R = -H, R2 = -C6H4Br16: R = -CH3, R2 = -C6H4Br17: R = -H, R2 = -C6H4I18: R = -CH3, R2 = -C6H4I19: R = -H, R2 = -(CH2)3CH3

20: R = -CH3, R2 = -(CH2)3CH3

21: R = -H, R2 = -CH2CH(CH3)2

22: R = -CH3, R2 = -CH2CH(CH3)2

Structure - tuberculostatic activity evaluation among... 1449

These observations encouraged us to synthe-size a set of close structural analogs to CGI-17341and PA-824.

EXPERIMENTAL

Bicyclic 7-nitroimidazo[5,1-b]-2,3-dihydroox-azoles, isomeric with CGI-17341 and 3-hydroxy-8-nitroimidazo[5,1-b]-1,4,5,6-tetrahydropyrimidines,which are structural isomers of the structure of PA-824 were synthesized as it was described in (14) and(15).

Density functional calculations were executedand the geometries of referenced PA-824 and repre-sentative compound 14 were optimized at the DFTlevel of theory using the Gaussian 09 D.01 program(16), B3LYP functional, 6-31G(d,p) basis set, andConductor-like Polarizable Continuum Model(CPCM, water as a solvent) (17-19). The vibrationalfrequencies and thermodynamic properties were cal-culated by applying the ideal gas, rigid rotor, andharmonic oscillator approximations, energy mini-mum was confirmed by the frequency calculationfor all conformers, no negative frequencies weredetected in generated vibrational spectrum of ana-lyzed conformers. The binding affinity of the ana-logue 14 were obtained using AutoDock Vina tool(20). Water molecules were excluded from targetactive site.

RESULTS

As it is shown in Table 1, the obtained bicyclicnitroimidazoles, in most cases, are fully compatiblewith Lipinskiís rule of five (21). They have favor-able values of C log P (< 5), molecular weight lessthan 500 Daltons, no more than 10 hydrogen bondacceptors and 5 H-bond donors, low and moderateTPSA parameters.

The results listed in Table 1 show good molec-ular properties, similar to those that are characteris-tic for compounds PA-824, CGI-17341 and OPC-67683. Although lipophilicity values calculated forobtained compounds are rather favorable, both 7-nitroimidazo[5,1-b]-2,3-dihydrooxazoles and 3-hydroxy-8-nitroimidazo[5,1-b]-1,4,5,6-tetrahy-dropyrimidines do not exhibit in vitro activityagainst M. tuberculosis H37Rv in tested concentra-tions (14, 15). Results for selected products are col-lected in Table 2.

There have to be some other additional factorslike steric and electronic, which are responsible forappearance of tuberculostatic activity. In our previ-ous papers (14,15) we have taken the attempts of

Tab

le 2

. Ant

imyc

obac

teri

al a

ctiv

ity o

f in

vest

igat

ed c

ompo

unds

, as

MIC

val

ues

(µg/

mL

).

Myc

. tbc

. M

yc. t

bc.

Myc

. tbc

. 244

1M

yc. t

bc. 5

318

Myc

. tbc

.965

6 M

yc. t

bc.1

4023

Com

poun

dM

yc. t

bc.

456

(res

. 16

76 (

res.

(res

. SM

, IN

H,

(res

. SM

, IN

H,

(res

. SM

, IN

H,

(res

. SM

, IN

H,

Myc

.BC

GM

yc. a

vium

H37

Rv

INH

, RM

P)IN

H)

RM

P)R

MP,

EM

B)

RM

P)R

MP)

1>

10.0

> 10

.0>

10.0

xx

xx

> 10

.0>

10.0

2>

10.0

> 10

.0>

10.0

xx

xx

> 10

.0>

10.0

7>

10.0

> 10

.0>

10.0

xx

xx

> 10

.0>

10.0

8>

10.0

> 10

.0>

10.0

xx

xx

> 10

.0>

10.0

11>

25.0

xx

> 25

.0>

25.0

> 25

.0>

25.0

xx

12

> 25

.0x

x>

25.0

> 25

.0>

25.0

> 25

.0x

x

21

> 25

.0x

x>

25.0

> 25

.0>

25.0

> 25

.0x

x

22

> 25

.0x

x>

25.0

> 25

.0>

25.0

> 25

.0x

x

PA-8

24<

1.0

< 1.

0<

1.0

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d


explanation of this fact using e.g., atom ñ atom dis-tances and substrate ñ enzyme fit models.


It is known from the literature (22) that thereare some key structural features that are importantfor aerobic activity: the preference for the 4-nitroim-idazole, rigid bicyclic system, electron-donatingcomponent at the 2-position in imidazole ring andthe presence of hydrophobic substituent at six-mem-

bered saturated ring condensed with imidazole. Incase of 8-nitroimidazo[5,1-b]tetrahydropyrimidines,first and second condition was realized. Probably,the most essential requirement is the nature of thesubstituent at the C-2 position of the imidazole ring.The presence of alkyl group or hydrogen atom atthis position significantly reduces anti-Mtb activity(22). Considering the structural features of nitroimi-dazo[2,1-b]dihydrooxazoles having tuberculostaticpotency, it can be found some key factors: 4-nitro-derivatives are more active, the presence of small,

Figure 1. The structures of bicyclic nitroimidazole derivatives with antitubercular activity

Figure 2. Overlapping of PA-824 and compound 14

Structure - tuberculostatic activity evaluation among... 1451

lipophilic alkyl groups or alkyl halides as sub-stituents in saturated oxazole ring and oxygen atomat C-2 position of imidazole ring (23). In case of iso-meric nitroimidazo[5,1-b]dihydrooxazoles, elec-tron-donating atom is placed at C-5 position of imi-dazole ring. It seems that it is the essential hindrancein appearance of tuberculostatic activity among thebicyclic nitroimidazo[5,1-b]dihydrooxazole class.

Additionally, in order to explain the reasons ofthe experimental differences in biological activity oftested compounds and referenced nitroimidazoox-azine PA-824, it was decided to carry out a molecu-lar docking simulations. In computational medicinalchemistry, some of the most helpful information isobtained by visualising the 3D structures of pro-teins, chemical compounds, and protein ñ liganddocking poses (6). Public databases containing the3D conformations of proteins are widely used for insilico techniques. On the base of the data knownfrom the literature (24), the potential target binding

site has been identified ñ Deazaflavin-dependentnitroreductase (Ddn) from M. tuberculosis involvedin bioreductive activation of PA-824 (24). Also, it isknown, that the most potent aminoacids within ofDdn structure able to interact with PA-824 are: Tyr-130, Tyr-136, Ser-78, and Trp-20. In this analysis,nine conformers has been generated. It was foundthat only in four cases overlapping of PA-824 andcompound 14 is observed (Fig. 2).

Moreover, the energy value of the affinity(binding mode) of compound 14 is slightly highercomparing with PA-824: -5.2 and -5.6 kcal/M,respectively (Table 3).

Also, the range of energy between the mostpreferred to the less stable conformer is higher in thecase of 14 than in PA-824 (1.3 and 0.4 kcal/M),respectively. It indicates that compound 14 is lessstable than PA-824. The better efficacy of PA-824and Ddn bonding in comparison with analogue 14 isalso connected with higher lipophilicity (log P) of

Table 3. Energy values of tested compoundsCompound 14Detected 4 CPUsUsing random seed: 378963840

ModeAffinity Dist from best mode Dist from best mode(kcal/M) rmsd l. b. rmsd u. b.

1 -6.0 0.000 0.000

2 -5.8 3.422 5.220

3 -5.2 3.288 4.164

4 -5.2 3.572 5.451

5 -5.1 3.328 6.260

6 -4.8 3.058 6.131

7 -4.7 2.920 4.069

8 -4.7 12.783 13.881

PA-824Detected 4 CPUsUsing random seed: 824877764

ModeAffinity Dist from best mode Dist from best mode(kcal/M) rmsd l. b. rmsd u. b.

1 -5.9 0.000 0.000

2 -5.8 5.164 6.280

3 -5.7 1.940 2.637

4 -5.6 15.654 17.180

5 -5.6 13.635 15.709

6 -5.6 15.028 17.105

7 -5.5 14.791 16.898

8 -5.5 3.670 5.529

9 -5.5 13.407 15.612


native ligand (mi log P = 3.977 and 1.983, respec-tively). Similar trend is observed for log P that hasbeen determined with quantum methods in SMDmodel (25) (0.726 and 0.243, respectively). It isclear that PA-824 is better penetrating throughlipophilic cell membranes, that is why it showshigher biological activity, than tested ligand 14.

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25. Marenich A.V., Cramer C.J., Truhler D.G.: J.Phys. Chem. B 113, 6378 (2009).

Received: 29. 09. 2016


The number of life threatening infectious dis-eases and microbial multidrug-resistance are respon-sible for great number of deaths all over the world.Drug discovery programs have reported that manynatural and synthetic products are classified asantimicrobial agents. Quinoline derivatives whichpresent in several natural products (Cinchona alka-loids) produce diverse biological and pharmacolog-ical activities such as; antimalarial (1-3), anticancer(4-6), antituberculosis (7, 8), anti-inflammatory (9,10) analgesic (11), HIV-1 integrase inhibitors (12),antioxidant (13), anti-depressant (14), and anti-con-vulsant (15). Till date, this class plays an essentialrole in the field of antibiotic chemotherapy used forthe treatment of a large number of serious bacterialinfections (16-18). Many hypotheses have beenadvanced to account quinolineís mode of actions(19-21). These have included DNA binding by inter-calation quinoline drug with the microbial DNAthus, inhibits nucleic acid synthesis, replication andtranscription (22).

The most common powerful quinoline deriva-tives as antibiotics are fluoroquinolones (Fig. 1).

However, literature survey reported that most ofthem have adverse effects which may occur almostanywhere in the body. Most of fluoroquinolonedrugs have fluoride atom as a central part which hasthe unique ability to penetrate the blood-brain barri-er, entering the brain and causing undesirableeffects. One of the most common adverse effects ofthis type of antibiotic is disturbances of the CNS(23, 24). So, the main challenge that always facesthe researchers is discovering and developing newantimicrobial agents without or with the lowestadverse effects to treat serious microbial infections.Depending upon the above knowledge and in con-tinuation to our previous efforts dealing with syn-thesis of various 6-bromoquinaldine derivatives (25,26) of strong antimicrobial potency, the aim of thisstudy is to synthesize some new 6-bromoquinaldinederivatives incorporated to different biologicallyactive heterocycles that possess broad spectrumantimicrobial potency like, imidazole (27, 28), pyra-zole (29, 30), furane (31, 32) or to glycosidic moi-etes of reported antimicrobial activity through S- orN-linkages (33, 34) to evaluate their antimicrobial

SYNTHESIS AND ANTIMICROBIAL EVALUATION OF SOME NEW QUINALDINE DERIVATIVES

EBTEHAL S. AL-ABDULLAH1, NADIA G. HARESS1, MOGEDDA E. HAIBA1, 2*, NEAMA A. MOHAMED2 and AMANY Z. MAHMOUD3, 4

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia

2Department of Therapeutical Chemistry, Pharmaceutical and Drug Industries Division, National Research Center, Tahrir Street, Dokki, Cairo 12622, Egypt

3Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 11451, Riyadh 11451, Saudi Arabia

4Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71516, Egypt

Abstract: A new series of quinoline derivatives incorporated to glycosides and biologically active heterocyclicmoieties were synthesized starting with 6-bromo-4-hydroxyquinaldine (6-bromo-4-hydroxy-2-methylquino-line) compound 1. Some of the synthesized compounds were evaluated for their antibacterial and antifungalactivities. Most of the tested compounds exhibited potential antibacterial activity against Gram-positive bacte-ria, the detailed synthesis, spectroscopic data, antibacterial and antifungal evaluation of the synthesized com-pounds are reported.

Keywords: quinaldine derivatives, selenadiazole, imidazole, glycosides, antibacterial, antifungal

1453


1454 EBTEHAL S. AL-ABDULLAH et al.

activity against a number of Gram positive, Gramnegative bacteria as well as fungi. The obtained bio-logical results have been summarized in Table 1

Examples of different marketed fluoroquino-line drugs used as broad-spectrum antimicrobials areillustrated in Fig. 1


Chemistry

The synthetic routs of the desired compoundsillustrated in Schemes 1-3. Firstly, 6-bromo-4-hydroxyquinaldine, compound 1, was preparedaccording to the reported method (25), and wasreacted with phosphorus pentasulfide in refluxingdry xylene to afford the thiol derivative 2. The latter

compound 2 was coupled with a solution of the acti-vated cyclic bromo sugar 2, 3, 4, 6-tetra-O-acetyl-α-D-gluco or galacto/pyranosyl bromide at room tem-perature, in potassium hydroxide solution to affordthe corresponding thioglycoside derivatives 3a, b.Alternatively, refluxing of compound 1 with phos-phorus oxychloride afforded 6-bromo-4-chloro-quinaldine 4 (25), which in turn was converted to thecorresponding hydrazine derivative 5 (35) via thereaction of compound 4 with hydrazine hydrate(98%) in refluxing ethanol. Then, product 5 wasreacted with the activated cyclic bromo sugar 2, 3, 4,6-tetra-O-acetyl-α-D-gluco or galacto/pyranosylbromide in an acidic medium to afford the corre-sponding N-glycoside derivatives 6a, b. Addition-ally, condensation of compound 5 with furfural or

Table 1. Zone of inhibition of bacterial growth (mm) investigating antimicrobial activity of the tested compounds using agar well diffu-sion method.

Tested sampleGram positive Gram negative Fungus

B. subtilis S. aureus S. typhi E. coli K. pneumoniae P. aeruginosa C. albicans

2 14 14 - - - - -

3a 16 15 - - - - -

6a - - - - 10 - -

7a 12 15 - 13 - 15 15

15a 11 - - - - - -

16a 10 14 12 16 - - -

18 - 15 10 - - - -

Fluconazole - - - - - - 35

Ciprofloxacin 30 25 20 35 33 31 -

Table 2. Minimum inhibitory concentration [MIC](mg/mL) of the tested compounds against selected microorganisms strains using agardilution method.

Tested sampleGram positive Gram negative Fungus

B. subtilis S. aureus S. typhi E. coli K. pneumoniae P. aeruginosa C. albicans

2 4 4 - - - - -

15a 4 - - - - - -

3a 2 2 - - - - -

7a 0.5 0.25 - 2 - 4 4

6a - - - - 4 - -

16a 2 2 2 1 - - -

18 - 4 4 - - - -

Fluconazole µg/mL

- - - - - - 31

Ciprofloxacin µg/mL

12.5 15 20 15 12.5 12.5 -

Synthesis and antimicrobial evaluation of some new quinaldine derivatives 1455

benzaldehyde in glacial acetic acid yielded thehydrazone derivatives 7a, b, respectively. Further-more, the hydrazine derivative 5 was refluxed withmethyl or phenyl isothiocyanate in dry benzene togive the corresponding thiosemicarbazide deriva-tives 8a, b (Scheme 1).

Also, conversion of the chloroquinaldinederivative 4 to 4-(p-acetylanilino) derivative 9 wasproceeded according to the reported method (25).Compound 9 was condensed with thiosemicarbazideor phenyl thiosemicarbazide to afford the thiosemi-carbazone derivatives 10a, b (25) respectively. Eachof the derivatives 10a, b was reacted with phenacylbromide to afford the thiazole products 11a, b. Onthe other hand, compounds 10a, b were reacted withethyl bromoacetate in absolute ethanol containingfew drops of piperidine to give the thiazolidinonederivatives 12a, b. Moreover, compound 9 wasrefluxed with semicarbazide hydrochloride in thepresence of sodium acetate to give the semicar-bazide derivative 13. Oxidative cyclization of com-pound 13 with thionylchloride or selenium dioxidein glacial acetic acid afforded the correspondingthiadiazole or selenadiazole derivatives 14a, b,respectively (Scheme 2).

Also, the hydrazino derivative 5 was refluxedwith acetyl chloride or benzoyl chloride, to give thehydrazide derivatives 15a, b, respectively, whichunderwent cyclocondensation by refluxing in aceticacid to give the corresponding cyclized imidazo-quinoline derivatives 16a, b. Furthermore, com-pound 5 was reacted with 2-(ethoxymethylene) mal-ononitrile in absolute ethanol to give the pyrazolocarbonitrile derivative 17 from which, when treatedwith oxalyl chloride in dry benzene, at room tem-perature, the pyrazolo pyrimidine derivative 18 wasobtained (Scheme 3).

The structures of the new synthesized com-pounds were established and confirmed on the basesof their elemental analyses and spectral data (IR, 1H-NMR, 13C-NMR and MS.)

Biological evaluation

Some of the newly synthesized compoundswere screened for their antibacterial activity usingthe agar dilution method (36). Ciprofloxacin andfluconazole were used as reference drugs. Theresults were recorded for each tested compound asthe average diameter of inhibition zones of bacterialor fungal growth around the discs in mm. The mini-mum inhibitory concentration (MIC) measurementwas determined for compounds that showed signifi-cant growth inhibition zones using the agar dilutionmethod (36). The MIC (mg/mL) values of the activecompounds against the tested bacterial and fungalstrains are recorded in Table 2

Each of B. subtilis and S. aureus wereemployed as Gram positive microorganisms, while,E. coli, K. pneumonia and P. aeruginosa were usedas Gram negative bacteria. Also, C. albicans repre-sented the fungi in the present investigation. Themajority of the compounds showed an antibacterialeffect towards Gram positive bacteria that can bedescribed generally as moderate effect. Tables 1, 2depicts the antibacterial effect of the tested com-pounds measured as zones of inhibition as well asminimal inhibitory concentrations. The hydrazinederivative of the present nucleus (compound 7a) iseffective towards Gram positive bacteria (S. aureusand B. subtilis), some Gram negative (E. coli and P.aerugenosa) and C. albicans, a result that representsit as a promising broad spectrum antimicrobial.However, the hydrazide derivative (compound 15a)showed antibacterial effect only toward B. subtilis.The other compounds showed moderate effectsagainst the tested Gram positive bacteria as present-ed in (Table 1). Also, the tested compounds showedvariability in the MIC results. The results of themicrobial sensitivity of the selected Gram negativemicroorganisms revealed that, each of the imidazoderivative 16a and the pyrazolo pyrimidine deriva-tive 18 affected S. typhi with lower MIC in case ofthe derivative 16a (2 mg) about one half that of the

Figure 1. Some marketed quinolone drugs


derivative 18 (4 mg). Also, E.coli was sensitive tocompound 16 a with MIC of 1 mg. On the otherhand, K. pneumoniae and P. aeruginosa were mod-erately sensitive to compounds 6a and 7a, respec-tively, in almost equipotent effect. In an attempt toinvestigate the antifungal effect of the newly syn-thesized compounds, it was found that only deriva-tive 7a showed a moderate effect against C. albicans

(4 mg). Figure 2 summarizes the effects of the test-ed compounds against Gram positive micro-organ-isms B.subtilis and S. aureus. This figure shows thatthe compound 7a represented the lowest MIC (0.5,0.25 mg/mL). In general, the MIC results of the test-ed compounds with respect to B. subtilis rangedfrom 0.5 to 4 mg/mL, while for S. aureus liesbetween 0.25 and 4 mg/mL.

Scheme 1. Synthetic pathways of compounds 2-8a, b

dry xylene


(ppm) relative to tetramethylsilane (TMS) as inter-nal standard. Coupling constants are given in Hz.The mass spectra were recorded on GCMC-QP 1000EX Shimadzu Gas Chromatography MS spectrome-ter, Japan E.I.70 ev. Elemental analysis (C, H, N)were carried out at the Micro Analytical Center,Faculty of Science, Cairo University, Egypt, andwere in full agreement with the proposed structureswithin ± 0.2-0.3% of the theoretical values. Allreagents were of commercial quality and were used

EXPERIMENTAL

General

Melting points were obtained on a Barnstead9001 Electrothermal melting point apparatus and areuncorrected. IR spectra were recorded on a PerkinElmer FT-IR Spectrum BX Spectrometer at cm-1

scale using KBr discs. 1H-NMR and 13C-NMR wererecorded on a JEOL 300 MHz Spectrometer, Japanand chemical shift values were expressed in δ values


H2NCONHNH2HCl


without further purification. Reaction progress wasmonitored by analytical thin layer chromatography(TLC) on precoated (0.75 mm) silica gel GF254plates (E. Merck, Germany) and the products werevisualized by UV light.

Chemistry

6-Bromo-2-methylquinoline-4-thiol (2)To a mixture of the quinaldine derivative 1

(0.01 M) in dry xylene (10 mL), (0.02 M) of phos-phorus pentasulfide was added and heated underreflux for 6 h. After cooling, the solvent was evapo-rated under reduced pressure the solid formed waswashed well with water, filtered off, and recrystal-lized from DMF / water.

Yield 65%; m.p.: 235OC; IR (νmax/cm-1): 1610(C=C); 1H-NMR (DMSO-dd, δ, ppm): 2.2 (s, 1H,SH), 2.3 (s, 3H, CH3), 6.8 (s, 1H, H-3), 7.5-8.1 (m,3H, Ar); 13C-NMR (DMSO-dd, δ, ppm): 23.9 (CH3),119.8, 120.6, 128.5, 129.1, 132.02, 132.9, 136.4,146.8, 158.9 (Ar-C); MS: m/z (%): 253, 255 (M+,M+2, 8.98, 8.7) consistent with the molecular formu-la (C10H8BrNS).

6-Bromo-2-methyl-4-(2í,3í,4í,6í-tetra-O-acetyl-ββ-D-gluco and galactopyranosyl thio) quinoline (3a, b)

A mixture of compound 2 (0.01 M) in ethanol(10 mL) and a solution of aqueous potassiumhydroxide (0.01 M) dissolved in distilled water (5

mL) was added to a solution of 2, 3, 4, 6-tetra-O-acetyl-α-D-gluco-or galacto/pyranosylbromide(0.01 M) in acetone (7 mL). The reaction mixturewas stirred at room temperature for 5 h. After com-pletion of the reaction, the mixture was concentrat-ed under reduced pressure and poured onto ice-water. The solid formed was filtered off, washedwell with water and recrystallized from ethanol.

6-Bromo-2-methyl-4-(2í,3í,4í,6í-tetra-O-acetyl-ββ-D-galactopyranosyl thio) quinoline (3a)

Yield 70%; m.p.: 140OC; IR (νmax/cm-1): 1745(C=O); 1H-NMR (DMSO-dd, δ, ppm): 1.94-2.2 (m,12H, 4xOAc), 2.3 (s, 3H, CH3) 3.9 (m, 2H, 6í-H2),4.1-5.1 (m, 5H, H-5í, 4í, 3í, 2í, 1í), 6.8 (s, 1H, H-3),7.5-8.1 (m, 3H, Ar); 13C-NMR (DMSO-dd, δ, ppm):21.08 (4 x COCH3), 23.2 (CH3-quinaldine) 61.2(CH2, C-6í), 62.8, 63.9, 67.1, 70.8, 80.7 (C-5í, 4í, 3í,2í, 1í), 119.8, 120.6, 128.5, 129.1, 130.2, 132.9,143.4, 146.8, 158.9 (Ar-C and C=N); 168.6 (4 xC=O); MS: m/z (%): 583,585 (M+, M+2, 5.2, 5.0 ) con-sistent with the molecular formula (C24 H26 Br N O9 S)

6-Bromo-2-methyl-4-(2í,3í,4í,6í-tetra-O-acetyl-ββ-D-glucopyranosyl thio) quinoline (3b)

Yield 65%; m.p.: 127OC; IR (νmax/cm-1): 1744(C=O); 1H-NMR (DMSO-dd, δ, ppm): 1.92-2.1 (m,12H, 4 x OAc), 2.3 (s, 3H, CH3) 4.01 (m, 2H, 6í-H2),4.02-5.09 (m, 5H, H-5í, 4í, 3í, 2í, 1í), 6.85 (s, 1H,



H-3), 7.5-8.1 (m, 3H, Ar); 13C-NMR (DMSO-dd, δ,ppm): 21.08 (4 x COCH3), 23.2 (CH3-quinaldine),60.5 (CH2, C-6í), 61.3, 62.1, 65.1, 68.7, 80.3 (C-5í,4í, 3í, 2í, 1í), 119.8, 120.6, 128.5, 129.1, 130.2,132.9, 143.4, 146.8, 158.9 (Ar-C and C=N); 168.3(4 x C=O); MS: m/z (%): 583, 585 (M+, M+2, 3.6,2.9) consistent with the molecular formula (C24 H26

Br N O9 S).

1-(6-Bromo-2-methylquinolin-4-yl)-2-(2í,3í,4í,6í-tetra-O-acetyl-ββ-D-galacto and glucopyrano- yl)hydrazine (6a, b)

To a solution of compound 5 (0.01 M) in ace-tone (10 mL) 2, 3, 4, 6-tetra-O-acetyl-α-D-gluco- orgalacto/pyranosyl bromide (0.01 M) in acetone (15mL) was added followed by addition of few drops ofacetic acid, the reaction mixture was heated withstirring on a water bath at 55-65OC for 10 h. Afterthe reaction was completed, the solvent was concen-trated under reduced pressure. After cooling, theproduct was poured onto ice cold water. The solidformed was filtered off, washed with water, andrecrystallized from ethanol.

1-(6-Bromo-2-methylquinolin-4-yl)-2-(2í,3í,4í,6í-tetra-O-acetyl-ββ-D-galacto) hydrazine (6a)

Yield 72%; m.p.: 290OC; IR (νmax/cm-1): 3380(br., NH), 1742 (C=O); 1H-NMR (DMSO-dd, δ, ppm):1.97-2.02 (m, 12H,4xOAc), 2.31 (s, 3H, CH3), 3.91 (s,2H, 6í-H2) 4.02-5.36 (m, 5H, H-5í, 4í, 3í, 2í, 1í), 6.3(s, 1H, Ar, H-3), 7.6-8.1 (m, 3H, Ar), 9.8 (s, 1H, 1NH),10.5(s, 1H, NH);13C-NMR (DMSO-dd, δ, ppm): 21.85(4 x OAc), 22.91 (CH3-quinaldine), 60.91 (CH2, C-6í),61.93, 62.32, 64.34, 68.94, 71.63, (C-5í, 4í, 3í, 2í, 1í),110.71, 116.92, 117.6, 120.6, 129.2, 132.4, 146.6,147.49, 158.5 (Ar-C, C=N); 168.72 (4 x C=O); MS:m/z (%): 581, 583 (M+, M+2, 14.6, 14.0) consistent withthe molecular formula (C24 H28 Br N3 O9 ).

1-(6-Bromo-2-methylquinolin-4-yl)-2-(2í,3í,4í,6í-tetra-O-acetyl-ββ-D-gluco) hydrazine (6b)

Yield 70%; m.p.: 230OC; IR (νmax/cm-1): 3380(NH), 1747 (C=O), 1672 (C=N); 1H-NMR (DMSO-dd, δ, ppm): 1.97-2.01 (m, 12H, 4 x OAc), 2.3 (s, 3H,CH3), 3.91 (s, 2H, 6í-H2) 5.34-4.09 (m, 5H, H-5í, 4í,3í, 2í, 1í), 6.2 (s, 1H, Ar, H-3), 7.6-8.1 (m, 3H, Ar),9.1 (s, 1H, NH), 10.7(s, 1H, NH); 13C-NMR(DMSO-dd, δ, ppm): 21.85 (4 x OAc), 22.91 (CH3-quinaldine), 60.91 (CH2, C-6í), 61.93, 62.32, 64.34,68.94, 71.63, (C-5í, 4í, 3í, 2í, 1í), 110.71, 116.92,117.6, 120.6, 129.2, 132.4, 146.6, 147.49, 158.5(Ar-C, C=N); 168.12 (4 x C=O); MS: m/z (%): 581,583 (M+, M+2, 2.4, 2.1) consistent with the molecularformula (C24 H28 Br N3 O9 ).

1-(6-bromo-2-methylquinolin-4-yl)-2-((furan-2-yl)methylene) hydrazine (7a) and 1-(6-bromo-2-meth-ylquinolin-4-yl)-2- benzylidene hydrazine (7b)

To a solution of compound 5 (0.01 M) in gla-cial acetic acid (10 mL), the appropriate aldehydes(0.01 M), namely, furfural or benzaldehyde wasadded. The reaction mixture was refluxed for 1h.,after cooling the separated solid was collected by fil-tration, washed with water, dried and recrystallizedfrom ethanol

1-(6-Bromo-2-methylquinolin-4-yl)-2-((furan-2-yl)methylene) hydrazine (7a)

Yield: 80%, m.p.:> 300OC; IR (c νmax/cm-1):3260 (NH), 1637(C=N); 1H-NMR (CDCl3, δ, ppm):2.4 (s, 3H, CH3), 6.3 (s, 1H, Ar, H-3), 6.7-7.8 (m,6H, 5 Ar-H and N=CH), 8.1 (s, 1H, Ar, H-5) 9.3 (s,1H, NH); 13C-NMR (CDCl3, δ, ppm): 24.01 (CH3),107.2, 109.3, 109.8, 117.5, 118.4, 129.03, 132.8,133.6,133.9 142.8, 146.9, 149.4, 148.3, 158.8 (Ar-C, C=N), MS; m/z (%): 329, 331 (M+, M+2, 26.2,23.8) consistent with the molecular formula (C15 H12

Br N3 O).

1-(6-Bromo-2-methylquinolin-4-yl)-2-benzylidenehydrazine (7b)

Yield: 85%, m.p.:> 300OC; IR (νmax/cm-1): 3206(NH), 1637(C=N); 1H-NMR (CDCl3, δ, ppm): 2.4 (s,3H, CH3), 6.5 (s, 1H, Ar, H-3), 7.3-7.9 (m, 8H, 7 Ar-H and N=CH), 8.1 (s, 1H, Ar, H-5) 10.1 (s, 1H,NH); 13C-NMR (CDCl3, δ, ppm): 24.01 (CH3),107.2, 119.8, 120.7, 128.6, 128.9, 129.03, 129.1,131.5, 132.02, 132.9, 136.5, 142.9, 146.9, 158.8(Ar-C, C=N), MS; m/z (%): 339, 341 (M+, M+2, 38.3,35.0) consistent with the molecular formula (C17 H14

Br N3).

1-(6-Bromo-2-methylquinolin-4-yl)-4-methyl-thiosemicarbazide (8a), 1-(6-bromo-2-methylquino-lin-4-yl)-4-phenylthiosemicarbazide (8b)

A mixture of compound 5 (0.002 M) and thesuitable substituted isothiocyanate (0.002 M), name-ly methyl or phenyl isothiocyanate, in dry benzene(20 mL) was refluxed for 30 minutes. After cooling,the solid formed was washed with diluted ethanolfiltered off and recrystallized from dil. ethanol.

1-(6-Bromo-2-methylquinolin-4-yl)-4-methyl-thiosemicarbazide (8a)

Yield: 82%, m.p.: 261OC; IR (νmax/cm-1): 3313,3286 (3NH), 1620 (C=N); 1H-NMR (CDCl3, δ,ppm): 2.02 (s, 3H, CH3), 2.3(s, 3H, quinaldine-CH3),6.1 (s, 1H, H-3), 7.4-8.1 (m, 3H, Ar), 9.04 (s, 1H,NH), 9.5 (s, 1H, NH) 10.5 (s, 1H, NH); 13C-NMR


(CDCl3, δ, ppm): 24.2 (quinaldine-CH3), 28.2 (N-CH3), 110.71, 116.92, 117.6, 120.6, 129.2, 132.4,146.6, 147.49, 158.5 (Ar-C, C=N); 175.9 (C=S),MS; m/z (%): 324,326 (M+, M+2, 22.8, 21.5) consis-tent with the molecular formula (C12 H13 Br N4 S).

1-(6-Bromo-2-methylquinolin-4-yl)-4-phenyl-thiosemicarbazide (8b)

Yield: 85%, m.p.:> 300OC; IR (νmax/cm-1): 3290,3216 (3NH), 1623 (C=N); 1H-NMR (CDCl3, δ, ppm):2.3 (s, 3H, quinaldine-CH3), 6.5 (s, 1H, H-3), 6.7 (d, J= 8.7, 2H, Ar, H-2í,6í), 7.6-8.1 (m, 6H, Ar), 8.3 (s, 1H,NH), 9.8 (s, 1H, NH), 10.1 (s, 1H, NH); 13C-NMR(CDCl3, δ, ppm): 24.2 (quinaldine-CH3), 110.71,116.92, 117.6, 120.6, 123.6, 126.9, 128.7, 129.2,132.4, 136.8, 146.6, 147.49, 158.5 (Ar-C, C=N); 175.5(C=S), MS; m/z (%): 386, 388 (M+, M+2,18.3, 18.0)consistent with the molecular formula (C17 H15 Br N4 S).

1-[(6-Bromo-2-methylquinolin-4-yl)-4-ethylidene-benzenamine]-2-[2-(2,3-dihydro-2-phenylthiazol-4-ylidene)]hydrazine(11a), 1-[(6-bromo-2-methyl-quinolin-4-yl)-4-ethylidenebenzen-amine]-2-[2-(2,3-dihydro-2,3-diphenylthiazol-4-lidene)]hydr-azine (11b)

To a solution of the thiosemicarbazide deriva-tives 10a, b (0.01 M) in absolute ethanol (15 mL)phenacyl bromide (0.01 M) was added and the reac-tion mixture was heated under reflux for 5 h., theexcess solvent was evaporated under reduced pres-sure, after cooling, the solid formed was filtered off,dried and recrystallized from dil. ethanol.

1-[(6-Bromo-2-methylquinolin-4-yl)-4-ethyli-denebenzenamine]-2-[2-(2,3-dihydro-2-phenylthiazol-4-ylidene)]hydrazine (11a)

Yield: 79%, m.p.: 230OC; IR (νmax/cm-1): 3286,3168 (2NH), 1625 (C=N); 1H-NMR (CDCl3, δ,ppm): 2.03 (s, 3H, CH3), 2.3 (s, 3H, quinaldine-CH3),6.2 (s, 1H, H-3), 6.9-7.8 (m, 12H, 11 Ar-H and CHthiazolidine ), 8.1 (s, 1H, Ar, H-5), 9.04 (s, 1H, NH),9.8 (s, 1H, NH); 13C-NMR (CDCl3, δ, ppm): 19.3(CH3), 24.2 (quinaldine-CH3), 107.3, 109.5, 116.01,116.4, 118.1, 120.8, 123.6, 126.01, 127.5, 127.9,129.3, 129.9, 132.6, 143.02, 144.5, 146.7, 147.3,156.4, 157.6, 157.9, 159.02, (Ar-C, C=N); MS; m/z(%): 527, 529 (M+, M+2, 6.1, 5.7) consistent with themolecular formula (C27 H22 Br N5 S).

1-[(6-Bromo-2-methylquinolin-4-yl)-4-ethyli-denebenzenamine]-2-[2-(2,3-dihydro-2,3-diphenylthiazol-4-lidene)]hydrazine (11b)

Yield: 87%, m.p.: 200OC; IR (νmax/cm-1): 3313(NH), 1633 (C=N); 1H-NMR (CDCl3, δ, ppm): 2.1

(s, 3H, CH3), 2.3 (s, 3H, quinaldine-CH3), 6.2 (s, 1H,H-3), 6.5-7.8 (m, 17H, 16Ar-H and CH thiazoli-dine), 8.1 (s, 1H, Ar, H-5) 10.3 (s, H, NH); 13C-NMR(CDCl3, δ, ppm): 19.3 (CH3), 24.2 (quinaldine-CH3),106.4, 107.3, 115.6, 116.01, 116.4, 116.9, 118.1,120.8, 123.6, 126.01, 126.9, 127.5, 128.5, 129.3,129.9, 130.5, 132.6, 134.02, 140.3, 144.5, 146.7,147.3, 147.9, 159.02, 159.6 (Ar-C, C=N), MS; m/z(%): 603, 605 (M+, M+2, 12.1, 11.4) consistent withthe molecular formula (C33 H26 Br N5S).

2-[(6-Bromo-2-methylquinolin-4-yl)-4-ethyli-denebenzenamine-1-hydrazono)]thiazolidin-4-one(12a), 2-[(6-bromo-2-methylquinolin-4-yl)-4-ethylidenebenzenamine-1-hydrazono)]-3-phenyl-thiazolidin-4-one (12b)

To a solution of the thiosemicarbazide deriva-tives 10a, b (0.01 M) in absolute ethanol (15 mL),ethyl bromoacetate (0.01 M) was added and the mix-ture was heated under reflux for 3 h. After coolingthe solid formed was collected by filtration, andrecrystallized from dil. ethanol.

2-[(6-Bromo-2-methylquinolin-4-yl)-4-ethylidene-benzenamine-1-hydrazono)]thiazolidin-4-one(12a)

Yield: 77%, m.p.: 210OC; IR (νmax/cm-1): br.3321 (2NH), 1720 (C=O), 1635 (C=N); 1H-NMR(CDCl3, δ, ppm): 2.02 (s, 3H, CH3), 2.3 (s, 3H,quinaldine-CH3), 3.9 (s, 2H, thiazolidinone), 6.2 (s,1H, H-3), 7.0 (d, J = 7.8, 2H, Ar, H-2í, 6í), 7.6-7.9(m, 4H, Ar-H), 8.1 (s, 1H, Ar, H-5), 9.5 (s, 1H, NH),9.8 (s, 1H, NH); 13C-NMR (CDCl3, δ, ppm):19.3(CH3), 24.2 (quinaldine-CH3), 31.2 (CH2), 107.3,116.01, 116.4, 118.1, 120.8, 123.6, 129.3, 129.9,132.6, 144.5, 146.7, 147.3, 157.9, 159.2, 162.1 (Ar-C,C=N), 170.5 (C=O); MS; m/z (%): 467, 469 (M+,M+2,17.6, 16.8) consistent with the molecular formula(C21 H18 Br N5 O S).

2-[(6-Bromo-2-methylquinolin-4-yl)-4-ethylidene-benzenamine-1-hydrazono)]-3-phenylthiazolidin-4-one (12b)

Yield: 81%, m.p.: 180OC; IR (νmax/cm-1): 3260(NH), 1728 (C=O), 1635 (C=N); 1H-NMR (CDCl3,δ, ppm): 2.1 (s, 3H, CH3), 2.36 (s, 3H, quinaldine-CH3), 4.1 (s, 2H, thiazolidinone), 6.3 (s, 1H, H-3),6.9 (d, J = 7.8, 2H, Ar, H-2í, 6í, of benzeneamine),7.2-7.9 (m, 9H, Ar-H), 8.1 (s, 1H, Ar, H-5), 9.7 (s,1H, NH); 13C-NMR (CDCl3, δ, ppm): 20.1 (N-CH3),24.3 (quinaldine-CH3), 32.3 (CH2), 107.3, 116.01,116.4, 118.1, 120.3, 120.8, 123.6, 123.9, 128.5,129.3, 129.9, 132.6, 134.7, 144.5, 146.7, 147.3,157.9, 159.2, 162.1, (Ar-C, C=N), 170.1(C=O); MS;


m/z (%): 543, 545 (M+, M+2, 29.8, 28.1) (C27 H22 BrN5 O S).

1-[(6-Bromo-2-methylquinolin-4-ylamino)-4-phenylethylidine] semicarbazide (13)

A mixture of semicarbazide hydrochloride(0.01 M), and crystalline sodium acetate in water(10 mL) was added while stirring to a solution ofcompound 9 (0.001 M) in ethanol (20 mL). Stirringwas continued for 15 min, at 5OC, the solid productwas filtered off, washed with water, dried andrecrystallized from ethanol

Yield: 90%, m.p.: 275OC; IR (νmax/cm-1): 3413(NH2), 3276 (br, 2NH), 1680 (C=O); 1H-NMR(CDCl3, δ, ppm): 2.1 (s, 3H, CH3), 2.3 (s, 3H,quinaldine-CH3), 6.07 (s, 1H, NH2), 6.3 (s, 1H, H-3),6.6 (d, J = 8.7, 2H, H-2í, 6í), 6.9-8.1 (m, 5H, Ar-H),9.04 (s, 1H, NH), 10.2 (s, 1H,NH); 13C-NMR(CDCl3, δ, ppm): 22.5 (CH3), 24.2 (quinaldine-CH3),107.3, 116.01, 116.4, 118.1, 120.8, 123.6, 129.3,129.9, 132.6, 144.5, 147.3, 157.9, 160.2, 161.7,162.1 (Ar-C, C=N and C=O), MS; m/z (%): 411,413 (M+, M+2, 24.5, 23.0) consistent with the molec-ular formula (C19H18BrN5O).

4-[(6-Bromo-2-methylquinolin-4-yl)-(1, 2, 3-thia-diazol-4-yl)] benzenamine (14a)

To a stirred solution of the semicarbazide 13

(0.001 M) in acetic acid (2 mL), at zero OC, thionylchloride (0.025 M) was added dropwise and theresulting mixture was further stirred for 30 min atthis temperature, then, the reaction mixture was leftat room temperature overnight, and a saturated solu-tion of sodium bicarbonate was added. The productwas extracted with chloroform (40 mL), the organiclayer was washed well with water, dried with anhy-drous sodium sulfate. After evaporation of the sol-vent under reduced pressure, the solid formed wasfiltered off, dried and recrystallized from DMFaffording the title compound.

N-[4-(1, 2, 3-thiadiazol-4-yl) phenyl]-6-bromo-2-meth-ylquinolin-4-amine (14a)

Yield: 89%, m.p.:> 300OC; IR (νmax/cm-1):3313, (NH), 1615(C=N); 1H-NMR (CDCl3, δ, ppm):2.3 (s, 3H, quinaldine-CH3), 6.2 (s, 1H, H-3), 6.5 (d,J = 7.5, 2H, H-2í, 6í), 7.1-8.1 (m, 6H, 5H, Ar and1H, thiazolyl), 8.9 (s, 1H, NH); 13C-NMR (CDCl3, δ,ppm): 24.2 (CH3), 107.3, 115.9, 116.3, 118.1, 120.8,122.6, 127.5, 129.3, 129.9, 132.6, 144.5, 146.7,147.3, 159.2, 157.9 (Ar-C, C=N); MS; m/z (%):396,398 (M+, M+2, 32.3, 26.9) consistent with themolecular formula (C18 H13 Br N4 S)

4-[6-(Bromo-2-methylquinolin-4-yl)-(1, 2, 3-sele-nadiazol -4-yl)] benzenamine (14b)

To a mixture of compound 13 (0.001 M) inboiling acetic acid (10 mL), powdered seleniumdioxide (0.007 M) was added portionwise, aftercomplete addition, the reaction mixture was stirredfor 4 h., then, cooled and poured onto ice-water, thesolid formed was collected by filtration, washedwell with water, dried and recrystallized fromDMF/water

Yield: 85%, m.p.: 295OC; IR (νmax/cm-1): 3370,(NH), 1625 (C=N); 1H-NMR (CDCl3, δ, ppm): 2.3(s, 3H, quinaldine-CH3), 5.9 (s, 1H, selenadiazole-H), 6.2 (s, 1H, H-3), 6.9-8.1 (m, 7H, Ar-H), 10.5 (s,1H, NH); 13C-NMR (CDCl3, δ, ppm): 24.2 (CH3),107.3, 111.5, 116.01, 116.4, 118.1, 120.8, 123.6,126.9, 129.3, 132.6, 144.5, 146.7, 147.3, 159.2,157.9 (Ar-C,C=N); MS; m/z (%): 443, 445 (M+, M+2,50.6, 21.4) consistent with the molecular formula(C18 H13 Br N4 Se).

1-(6-Bromo-2-methylquinolin-4-yl)- acetohydrazide(15a), 1-(6-bromo-2-methylquinolin-4-yl)-benzohydr-azide (15b)

A mixture of compound 5 (0.01 M) and thesuitable acid chloride, namely acetyl chloride orbenzoyl chloride (5 mL) was refluxed for 3 h. Then,the excess acid chloride was evaporated underreduced pressure and the solid product was washedwith cold water, collected by filtration and recrys-tallized from ethanol.

1-(6-Bromo-2-methylquinolin-4-yl)-acetohydraz-ide (15a)

Yield: 73%, m.p.:> 300OC; IR (νmax/cm-1): br.3288 (2NH), 1663 (C=O), 1591 (C=N); 1H-NMR(CDCl3, δ, ppm): 2.1 (s, 3H, COCH3), 2.3 (s, 3H,quinaldine-CH3), 6.2 (s, 1H, H-3), 7.6-8.1 (m, 3H, Ar-H); 13C-NMR (CDCl3, δ, ppm): 24.2 (quinaldine-CH3),24.6 (CH3), 107.3, 116.4, 118.1, 120.8, 129.3, 132.6,146.7, 147.3, 158.02 (Ar-C, C=N), 165.1 (C=O), MS;m/z (%): 293, 295 (M+, M+2, 10.7, 7.8) consistent withthe molecular formula (C12 H12 Br N3 O)

1-(6-Bromo-2-methylquinolin-4-yl)-benzohy-drazide (15b)

Yield: 76%, m.p.: > 300OC; IR (νmax/cm-1): br.3310 (2NH), 1700 (C=O), 1630 (C=N); 1H-NMR(CDCl3, δ, ppm): 2.3 (s, 3H, quinaldine-CH3), 6.3 (s,1H, H-3), 7.5-8.2 (m, 8H, Ar-H) 13C-NMR (CDCl3,δ, ppm): 24.2 (CH3), 107.3, 116.4, 118.1, 120.8,126.5, 128.01, 129.3, 131.4, 132.6, 133.6, 146.7,147.3, 158.02 (Ar-C, C=N), 164.1 (C=O), MS; m/z(%): 355, 357 (M+, M+2, 16.4, 15.0) (C17 H14 Br N3 O).


8-Bromo-2,4-dimethyl-1H-imidazo[4,5-c]quino-line (16a), 8-bromo-4-methyl-2-phenyl-1H-imi-dazo[4,5-c]quinoline (16b)

A mixture of compounds 15a, b (0.01 M) andacetic acid (25 mL) was refluxed for 3 h. The reac-tion mixture was evaporated under reduced pres-sure, after cooling, the reaction mixture was pouredonto ice-water and the solid formed was washedwith water during filtration, dried and recrystallizedfrom ethanol.

8-Bromo-2,4-dimethyl-1H-imidazo[4,5-c]quino-line (16a)

Yield: 69%, m.p.: 195OC; IR (νmax/cm-1): 3231(NH), 1591 (C=N); 1H-NMR (CDCl3, δ, ppm): 2.2(s, 3H, CH3), 2.4 (s, 3H, quinaldine-CH3), 7.6-8.1(m, 3H, Ar), 9.2 (s, 1H, NH) 13C-NMR (CDCl3, δ,ppm): 19.1(CH3), 21.2 (quinaldine-CH3), 119.6,120.9, 121.9, 121.7, 127.6, 129.01, 132.8, 142.6,145.8, 158.9, (Ar-C, C=N); MS; m/z (%): 275, 277(M+, M+2, 50.7, 21.9) consistent with the molecularformula (C12 H10 Br N3).

8-Bromo-4-methyl-2-phenyl-1H-imidazo[4,5-c]quinolone (16b)

Yield: 70%, m.p.: 154OC; IR (νmax/cm-1): 3316(NH), 1627 (C=N); 1H-NMR (CDCl3, δ, ppm): 2.4(s, 3H, quinaldine-CH3), 7.1-7.9 (m, 7H, Ar), 8.1 (s,1H, Ar-H9), 9.3 (s, 1H, NH); 13C-NMR (CDCl3, δ,ppm): 21.2 (CH3), 119.6, 120.9, 121.9, 121.7, 126.1,127.6, 127.9, 128.04, 129.01, 129.9, 132.8, 142.6,145.8, 158.9 (Ar-C, C=N); MS; m/z (%): 337, 339(M+, M+2, 5.5, 5.0) consistent with the molecular for-mula (C17 H12 Br N3).

5-Amino-1-(6-bromo-2-methylquinolin-4-yl)-1H-pyrazole-4-carbonitrile (17)

To a solution of compound 5 (0.01 M) inabsolute ethanol (15 mL), 2-(ethoxymethylene) mal-ononitrile (0.02 M) was added, the reaction mixturewas heated under reflux for 3 h., the excess solventwas evaporated under reduced pressure, after cool-ing, the solid formed was filtered off, dried andrecrystallized from ethanol.

Yield: 90 %, m.p.:> 300oC; IR (νmax /cm-1):3412 (NH2), 2219 (CN); 1H-NMR (CDCl3, δ, ppm):2.3 (s, 3H, CH3), 4.01 (s, 1H, NH2), 6.9 (s, 1H, H-3),7.3 (s, 1H, pyrazole), 7.5-8.1 (m, 3H, Ar ) 13C-NMR(CDCl3, δ, ppm): 24.1 (CH3), 91.5, 111.7, 115.9,119.6, 125.8, 127.1, 127.9, 130.4, 140.5, 142.7,143.1, 147.9, 157.1(Ar-C, C=N), MS; m/z (%): 327,329 (M+, M+2, 31.8, 28.4) consistent with the molec-ular formula (C14 H10 Br N5).

1-(6-Bromo-2-methylquinolin-4-yl)-4,5-dihydro-4-oxo-1H-pyrazolo[3,4-d]pyrimidine-6-carbon-ylchloride (18)

To a mixture of compound 17 (0.01 M) in drybenzene (15 mL) oxalyl chloride (0.015 M) wasadded. The mixture was stirred at room temperaturefor 24 h., the solid product was collected by filtra-tion, washed well with water, dried and recrystal-lized from ethanol.

Yield: 88%, m.p.: 288OC; IR (νmax/cm-1): 3215(NH), 1723, 1675 (2C=O); 1H-NMR (CDCl3, δ,ppm): 2.3 (s, 3H, Ar-CH3), 6.9 (s, 1H, H-3), 7.5-8.1(m, 4H, 3Ar and 1H, pyrazole). 8.9 (s, 1H, NH), 13C-NMR (CDCl3, δ, ppm): 24.1(CH3), 106.3, 111.7,112.9, 119.6, 127.1, 127.9, 130.4, 132.5, 140.9,146.7, 147.9, 158.1, 159.9 (Ar-C, C=N), 161.4,165.8 (2C=O), MS; m/z (%):417, 419 (M+, M+2, 4.4,6.1) consistent with the molecular formula (C16 H9

Br Cl N5O2).

Antimicrobial evaluation

Some of the synthesized new compounds wereinvestigated for their antimicrobial effect towardeach of Gram positive as well as Gram negative bac-teria and fungi. Strains used to detect the antimicro-bial activity of the prepared compounds included,Staphylococcus aureus ATCC 29213 and Bacillussubtilis ATCC 3366 as Gram positive, Pseudomo-nas aeruginosa ATCC 27853, Escherichia coliATCC 25922, Salmonella typhi ATCC 25566 andKlepsiella pneumoniae ATCC 13883 as Gram nega-tive, as well as Candida albicans NRRL Y-477 topresent fungus.

The agar dilution method was used to study themicrobiological inhibition of the prepared com-pounds. The MuellerñHinton agar plates were pre-pared and adjusted to contain serial twofold dilu-tions of the tested compounds in-house. Each platewas inoculated with a specific microorganism at 0.5McFarland and incubated at 37OC 24 h for.

The MIC was considered the lowest concentra-tion of an antimicrobial agent that completely inhib-ited growth on the agar as detected visually.Ciprofloxacin and fluconazole were used as refer-ence drugs.

CONCLUSION

The most common powerful quinoline deriva-tives as antibiotics are fluoroquinolones. However,literature survey reported that most of them haveadverse effects which may occur almost anywherein the body. Depending upon the above knowledgeand in continuation to our previous efforts dealing


with the synthesis of various 6-bromoquinaldinederivatives of strong antimicrobial potency, a novelseries of bromoquinaldine derivatives incorporatedinto or fused to various biologically active hetero-cyclic ring systems or glycosides were synthesizedand evaluating some of them as antibacterial andantifungal. All the tested compounds exhibitedpotential antibacterial activity against Gram-posi-tive bacteria except compound 6a which internsappeared to have activity against the Gram negativeK. pneumoniae. From that we can conclude that sub-stituted quinaldine compounds may provide moreactivity by further study trying to modify their sub-stitutions to give new derivatives with expectedimproved antimicrobial activity and that may be thesubject of our future work.

Acknowledgment

This research project was supported by a grantfrom the ìResearch Center of the Female Scientificand Medical Collegesî, Deanship of ScientificResearch, King Saud University.

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Received: 15. 02. 2016


A cream is a heterogeneous formulation that isprepared by mixing two liquids that cannot be mixedwithout the help of a third ingredient known asemulsifying agent. One out of these two liquidsserves as the dispersion medium while the other isdistributed throughout the dispersion medium in theform of droplets. The range of these droplets in thecreams used for pharmaceutical purpose is about 0.1to 50 µm (1). An oil-in-water (o/w) cream is one inwhich oil is present as droplets in the dispersionmedium that is water while the reverse is true forwater-in-oil (w/o) emulsion. When a small amountof oil is required like in shaving or conditioningcreams, o/w-emulsions are used while the w/o emul-sions are desired where more oily preparation isrequired like emollient creams and sunscreens.There is a number of emulsifiers that are used to for-mulate the emulsions but these may cause irritancyand sensitivity to the user. So it is necessary todevelop the formulation using emulsifying agentsthat do not cause such adverse reactions. Nonionic

surfactants as emulsifiers are considered safe withregard to such adverse reactions (2).

Various processes can account for the instabil-ity of the emulsions such as creaming, Ostwaldripening, phase separation, rupture and coalescence(3). Physical and chemical stability of the formula-tions can be assessed promptly by keeping them atdifferent temperatures for some time. By rheologicalmeasurement one can estimate not only the physicalstability but also the quality, effectiveness and pur-pose of system (4).

Bombax ceiba belongs to the familyBombacaceae and commonly known as silk cottontree. It is a remarkable medicinal plant of Indiantropical and subtropical regions including Pakistan.In old-style medicine systems that are still in prac-tice in India like Ayurveda, Siddha and Unani, it isused to cure many diseases like sexual incapacity,vaginal infections and to stop bleeding from wounds(5). Many diseases can be cured by using differentparts of B. ceiba. Stem bark acts as acrid, demulcent,

NATURAL DRUGS

FORMULATION OF A CREAM CONTAINING EXTRACT OF BOMBAX CEIBAAND ITS EX VIVO CHARACTERIZATION UNDER VARIOUS

STORAGE CONDITIONS

MASOOD-UR-REHMAN1* and NAVEED AKHTAR1

1Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, the Islamia University ofBahawalpur 63100, Bahawalpur, Pakistan

Abstract: This study was aimed to formulate a cream (W/O emulsion) containing Bombax ceiba (stem bark)extract and evaluate its stability by exposing it to different storage conditions. The formulation was preparedusing ABIL EM90 as an emulsifying agent, liquid paraffin oil and water by heating the aqueous phase at 70-75OC and the oil phase at the same temperature and then adding aqueous phase to oil phase with continuous stir-ring. This newly prepared formulation was stored for a period of 12 weeks at 8OC, 25OC, 40OC and 40OC/75%RH (relative humidity) and assessed for any change in physical characteristics (color, liquefaction, phase sep-aration) from time to time at certain intervals. The formulation presented better physical stability throughoutthe study period. Only slight organoleptic changes were observed in the samples stored at higher temperatures.Rheological observations indicated the shear thinning effect (pseudo plastic behavior) of the formulation hav-ing the value of flow index less than 1. Microscopy showed that the droplet size of internal phase was increasedmore in the formulations kept at the higher temperature (40OC). It can be said that newly developed cream con-taining B. ceiba extract exhibited good physical characteristics indicating its stability. The cream might be usedas a skin care formulation for different cosmetic purposes.

Keywords: Bombax ceiba, cream, stability, droplet size, rheology, phase separation

1465

* Corresponding author: e-mail: [email protected]; tel: +92 323 6535564; fax: +92 62 9255243

1466 MASOOD-UR-REHMAN and NAVEED AKHTAR

diuretic, anti-inflammatory, slightly astringent andtonic. It can be used for facial illnesses such asmarks, acne vulgaris, pigmentation disorder, inflam-mation, blister and burning sensation. Stem barkcontains lupeol, β-sitosterol, shamimicin and api-genin (6).

The aim of the present study was to prepareand investigate physical stability of the cream con-taining B. ceiba extract that could serve as a cos-metic product and contribute to the skin care. Thestability of the formulation was evaluated using dif-ferent parameters by placing at different storageconditions over time.

EXPERIMENTAL

Plant material identification

Bombax ceiba bark was collected locally fromBahawalpur, Pakistan and validated by theCholistan Institute of Desert Studies (CIDS), TheIslamia University of Bahawalpur, Pakistan. Avoucher specimen (No. 3524/CIDS/IUB) was issuedafter depositing the sample in the herbarium ofCIDS, The Islamia University of Bahawalpur,Pakistan. ABIL EM 90 or cetyl PEG/PPG-10/1dimethicone, was obtained from the Franken(Germany) and paraffin oil from Merck (Germany).

Extraction and cream preparation

Bombax ceiba stem bark was collected locallyand extracted with 80% aqueous-methanol.Previously weighed contents of oily phase including

paraffin oil (14%) and emulsifier (ABIL-EM 90)(4.5%) were heated up to 75 ± 1OC. Aqueous phasecomprising of water (77.5%) and B. ceiba extract(4%) was also heated to the same temperature (75 ±1OC). Subsequently, aqueous phase was added to theoil phase with constant stirring with the help of astirrer (Euro-Star, IKA D 230, Germany) at a speedof 2000 rpm for 10 min till the complete addition ofaqueous phase, 2 to 3 drops of rose oil as fragrantwere added during stirring. Mixer speed was adjust-ed to 1000 rpm after the aqueous phase was addedand continued for almost 10 min to homogenize theingredients. This process of emulsification was fur-ther carried on with reduction in speed (500 rpm) forcomplete homogenization until the emulsion wascooled down to room temperature.

Characterization of the cream

Examination was done by keeping the samplesof the cream at four different conditions of storagei.e., 8OC, 25OC, 40OC and 40OC + 75% RH (relativehumidity) for 12 weeks to assess the stability underthese conditions. The cream was evaluated physical-ly (color, liquefaction and phase separation) underthese conditions. Centrifugation (CentrifugeMachine, Hettich EBA 20 Germany) was carried outat a speed of 5000 rpm and at 25OC for 30 min byemploying the sample (few grams) in a disposablecentrifugal tube. Rheological parameters for the for-mulations were determined at 25OC after preparationand at different time intervals for 12 weeks, taking0.5 g of the sample. Rheology was determined using

Table 1. Variation in physical characteristics of the formulation at various time intervals stored at 8OC, 25OC, 40OC and 40OC/75% RH.

Physical Storage Days

characteristic temperature 0 Week 4 Week 8 Week 12 Week

Liquefaction 8OC - - - -

25OC - - - -

40OC - - - +

40OC/75% RH - - - -

Color 8OC LP LP LP LP

25OC LP LP LP LP

40OC LP LP LP LP

40OC/75% RH LP LP LP LP

Phase separation 8OC - - - -

25OC - - - -

40OC - - + +

40OC/75% RH - - - -

- = No change; + = slight change; LP = light pink.

Formulation of a cream containing extract of Bombax ceiba... 1467

a CP 41 spindle of a cone-plate rheometer(Brookfield DV-III Ultra). Values for consistencyindex (related to the system viscosity) and flowindex (related to the system pseudo plasticity) wereobtained by the power law. Increased shear stresseswere applied on the samples and change in viscosi-ty was observed.

Power law

The power law equation is as shown in Eq. 1.τ = kDn (1)

where τ = shear stress, D = yield stress (stress at zeroshear rate), k = plastic viscosity, and n = shear rateThe calculated parameters for this model are flowindex (no units), consistency index (cP) and confi-dence of fit (%).

Microscopic examination

Stability of the creams can be established by anessential characteristic that is the droplet size ofinternal phase. With the help of an optical micro-scope (Eclipse E200, Nikon, Japan), to which aCCD camera was attached the droplet size wasdetermined. The images were processed using minisee software (V. 1.1). Firstly, a minute quantity ofsample stored at different temperatures was taken onthe slide then it was diluted with the continuousphase and covered with the cover slip. After that itwas observed under the microscope by the 100×lens. The droplet diameter was determined by stagemicrometer and graticule. The micrographs obtainedare shown (Fig. 2).

RESULTS

The creams were examined on a physical(color, phase separation and liquefaction) basis andresults are shown in Table 1. It was noted that nochange in the color of the formulation occurred.There was no phase separation and liquefactionfound except to slight phase separation and lique-faction at 40OC on the 90th day of study was found.

In the present study, centrifugation test of freshlyprepared sample and samples kept at different stor-age conditions was done. These evaluations werecarried out for a period of 12 weeks at different stor-age conditions (8OC, 25OC, 40OC, and 40OC + 75%RH) and at specific time intervals.

We also evaluated the influence of storage con-ditions on the viscosity of the preparation by sub-jecting the formulations to the rheometer. Figure 1presents the rheograms of the formulations stored atdifferent temperatures. Measurements were record-ed for the flow index and consistency index for theformulations and are shown in Table 2.

The droplet size is also an important character-istic of the emulsion so it was measured according tothe procedure stated earlier. Figure 2 reveals themicroscopic images of the cream containing B.ceiba extract. It was found that the droplet size wasnot only increased with time but this increase wasalso more prominent at higher temperature.

DISCUSSION

Storage at various temperatures is a well-knownmethod to explore the stability of an emulsion. Thismethod is easy to carry out because breaking of theemulsions is accelerated by thermal stress (7). Thefreshly prepared formulation was light pink in colorand sustained the color throughout the study period (12weeks). This resistance in change of color revealed thestability of the formulation at different storage condi-tions i.e., 8OC, 25OC, 40OC, and 40OC + 75% RHthroughout the period of investigation. Phytochemicalstudies on various parts of B. ceiba exposed that it isrich in phenolic compounds (8). These phenolic com-pounds help retain the original color of the formula-tions by inhibiting the growth of microbes (9).

Flow characteristic of the cream is indicated byits viscosity and stability of emulsion can be assessedby determining the time and temperature effect uponviscosity (10). All samples kept at 8OC, 25OC and40OC + 75% RH were stable and no liquefaction was

Table 2. Flow index and consistency index values determined at various time intervals after storage at 8OC, 25OC, 40OC and 40OC/75% RH.

Time 8OC 25OC 40OC 40OC/75% RH

FI CI CF FI CI CF FI CI CF FI CI CF

0 Week 0.58 1485 99.6 0.58 1485 99.6 0.58 1485 99.6 0.58 1485 99.6

4 Week 0.57 1125 99.8 0.51 1036 99.8 0.66 1005 97.5 0.70 1121 99.6

8 Week 0.55 1045 99.7 0.61 850 99.4 0.68 801 96.8 0.67 905 99.7

12 Week 0.50 985 99.3 0.56 710 98.1 0.70 589 94.6 0.64 786 99.0

FI = Flow index; CI = Consistency index; CF = Confidence of fit (%)


observed. Very little liquefaction was seen in thesample kept at 40OC on 90th day. With the passage oftime it is likely that some time and temperature driv-en processes reduce the viscosity of preparation lead-ing to liquefaction (11). The droplets of internalphase move upward or downward causing creamingor sedimentation due to the density dissimilarity ofthe two phases (12). Slight phase separation wasdetected in the sample placed at 40OC on 90th day ofobservation. During creaming/sedimentation thedroplets tend to grow leading to decrease in the totalinterfacial energy. This shift to a larger mean dropletsize due to the coalescence of smaller droplets caus-es separation of the phases in some cases (12) whilethe emulsion remains stable at low temperaturesbecause viscosity did not change (13).

Type of the emulsion can be determined byusing different methods. We used electrical conduc-tivity test for this purpose. Emulsion with water asan external phase can conduct the electricity because

electricity can pass through the water while oil is apoor conductor of the electric current so when oil isthe dispersion medium there will be no electricalconductivity (14). No electrical conductivity wasfound in the formulation under test so we can saythat it was water in oil emulsion.

Physical stability of the creams can be evaluat-ed by using the techniques such as centrifugationthat can enhance the instability in the formulations(7). It was noted that after centrifugation there wasno phase separation found in the samples kept at8OC, 25OC and 40OC + 75% RH till the end of study.The appropriate speed of the mixer during theprocess of emulsification can prevent the formula-tion from breakage under accelerated studies (10).However, the sample exposed to 40OC exhibitedphase separation after 60 days in the centrifugationtest. By applying the centrifugal force creaming/sed-imentation can be augmented due to change in thedroplet size, structure and distribution (12).

Figure 1. Rheograms of formulation at various time intervals A) Formulation stored at 8OC, B) Formulation stored at 25OC, C) Formulationstored at 40OC, D) Formulation stored at 40OC/75% RH

Formulation of a cream containing extract of Bombax ceiba... 1469

Right consistency (rheology) for a good sensa-tion of the skin and better spreading is desired stan-dard for the emulsions used as personal care prod-ucts. Rheology can be used to display the informa-tion about the stability of the emulsion (15). In thecurrent study it was observed that the formulationsdepicted shear thinning effect because viscosity ofthe formulations decreased with the increase inshear stress as can be incurred from the values ofthe flow index that are less than 1. It is the proper-ty that is very much required for good applicationand good feel of the formulation. It is necessary toexpose the formulation to different temperaturesand obtain the rheological data to get the valuableinformation about the product stability and consis-tency (15). The slight decrease in the consistencyindex was found in the formulations. Consistencyindex represents the viscosity of the formulationsand it is known that consistency index normallydecreases upon storage (4).

Physicochemical characteristics including thedroplet size and rheological characteristics can beused to assess the emulsion stability. The dropletsize of the disperse systems like emulsions can beachieved by using numerous methods (16). In thecurrent investigation micrographs indicated thatspherical globules of 2-4 µm were present in thefreshly prepared formulations. These values arewithin the given range as mentioned in the literature

for the pharmaceutical emulsions (1). After keepingthe formulations on different storage conditions itwas seen that the droplet size increased differently atvarious temperatures. The droplet size was incre-ased up to 10.4 µm and 12.2 µm, respectively, at 8OCand 25OC after 12 weeks of storage. While thedroplet size of the formulations kept at 40OC and40OC/75% RH were found to be 17 µm and 25 µmafter 12 weeks. It can be seen that at higher temper-ature the droplet size of the formulation wasincreased more as compared to the formulationskept at lower temperature. There is some evidencethat the stability of the emulsion at lower tempera-tures may be because of the stable film of emulsifi-er around the droplets that allows less contact ofglobule during storage. Slight decrease in the vis-cosity with time at higher temperatures, 40OC and40OC/75% RH allows more probability of thedroplet size increase by coalescence (17).

CONCLUSION

From the current study, we can conclude thatthe cream containing Bombax ceiba stem barkextract in concentration of 4% exhibited promisingstability and physicochemical characteristics at dif-ferent storage conditions. This formulation showedno change in color or any liquefaction however aslight phase separation was seen at 40OC. The stabil-

Figure 2. Micrographs of formulation A) Freshly prepared B) Formulation after 12 weeks storage at 8OC, C) Formulation after 12 weeksstorage at 25OC, D) Formulation after 12 weeks storage at 40OC/75% RH E) Formulation after 12 weeks storage at 40OC


ity of the cream can be increased by storing at lowtemperatures while higher temperature can affect itnegatively. The formulation can offer a good deliv-ery system for skin rejuvenation agents. Further invivo studies have to be done to discover the benefitsof the cream as a phytocosmetic agent.

Acknowledgment

The authors thank to the Chairman and Dean ofthe Faculty of Pharmacy & Alternative Medicine,The Islamia University of Bahawalpur, Pakistan forproviding the lab facilities to conduct the study andthe moral support given to them.

Conflict of interest

There is no conflict of interest associated withthis work.

REFERENCE

1. James S., James C.B., Encyclopedia of Pharma-ceutical Technology. p. 1066, Second Edition,Marcel Dekker, Inc. (USA) 2002.

2. Chain I.P., Wan-GU C., Seong J.L.: Aust.Rheol J. 15, 125 (2003).

3. Roland I., Piel G., Delattre L., Evrard B.: Int. J.Pharm. 263, 85 (2003).

4. Guaratini T., Gianeti M.D., CamposP.M.B.G.M.: Int. J. Pharm. 327, 12 (2006).

5. Anandarajagopal K., Sunilson J.A.J., Ajayku-mar T.V., Ananth R., Kamal S.: Eur. J. Med.Plants 3, 99 (2013).

6. Verma V., Jalalpure S.S., Sahu A., BhardwajL.K., Prakesh Y.: Int. Pharm. Sci. 1, 62 (2011).

7. Masmoudia H., Dr¥eaua Y.L., Piccerelleb P.,Kister J.: Int. J. Pharm. 289, 117 (2005).

8. Dar A., Faizi S., Naqvi S., Roome T., RehmanS.Z.U. et al.: Biol. Pharm. Bull. 28, 596 (2005).

9. Ali A., Akhtar N.: Trop. J. Pharm. Res. 13, 677(2014).

10. Waqas M.K., Akhtar N., Khan H.M.S., MustafaR., Murtaza G.: Lat. Am. J. Pharm. 33, 731(2014).

11. Herbert A.L., Martin M.R., Gilbert S.B.:Pharmaceutical dosage, Disperse Systems. p.199 and 285, Vol. 1, Marcel Dekker, New York1989.

12. Badolato G.G., Aguilar F., Schuchmann H.P.,Sobisch T., Lerche D.: Prog. Colloid Polym.Sci. 134, 66 (2008).

13. Nour A.H., Yunus R.M.: J. Appl. Sci. 6, 2895(2006).

14. Khan B.A., Akhtar N., Khan H.M.S., WaseemK., Mahmood T. et al.: Afr. J. Pharm.Pharmacol. 5, 2715 (2011).

15. Tadros T.: Adv. Colloid Interface Sci. 108, 227(2004).

16. Baby A.R., Santoro D.M., Velasco M.V.R.,Serra C.H.D.R.: Int. J. Pharm. 361, 99 (2008).

17. Tcholakova S., Denkov N., Ivanov I.B.,Marinov R.: Bulg. J. Phys. 31, 96 (2004).

Received: 2. 08. 2016


Nardostachys jatamansi (D. Don) DC; belongsto Valerianaceae family. It has an important place intraditional medicine in the Indian subcontinent andthe Middle East, being used mainly as a tranquilizerand CNS sedative. Moreover, it is also used for gas-trointestinal hyperactivity (1). The roots ofNardostachys jatamansi are used traditionally in thetreatment of convulsive ailments, epilepsy, hysteria,heart palpitations, boils, diseases of the eyes, itch,etc. (2). In the Unani system of medicine, Sunbul-ul-tib (Nardostachys jatamansi) has been mentioned asa heptatonic, cardiotonic, diuretic and analgesic (3).

For the medicinal purpose, rhizomes of N. jata-mansi are mostly used. Macroscopically, the rhi-zome of the plant is cylindrical and elongated in

shape, and fine fibers in a network cover it.Phytochemical analysis showed the presence ofalkaloids, amino acids, sugars and tannins in hot andcold methanolic extracts (4). Valeranone sesquiter-penes are the principal active constituents in N. jata-mansi oil and it causes sleep induction (1). It con-tains many other sesquiterpenes including jata-mansinone, jatamansinol, oroseolol, oroselone,seselin, jatamol A and B, valeranal, nardostachyin,nardosinone, spirojatamol, jatamansic acid, nar-dostachone, calarenol, coumarin, jatamansin, xan-thogalin, seychelane and seychellene (5).

Six Gram negative (Shigella dysenteriae,Corynebacterium striatum, Proteus vulgarisEscherichia coli, Pseudomonas aeruginosa, Kleb-

EVALUATION OF ANTIBACTERIAL AND CARBONIC ANHYDRASEINHIBITORY POTENTIAL OF METHANOLIC EXTRACT OF

NARDOSTACHYS JATAMANSI (D. DON) DC RHIZOMES

TAYYEBA REHMAN1, SAEED AHMAD2, WAHEED MUMTAZ ABBASI1, ASHFAQ AHMAD3,MUHAMMAD BILAL1, MUHAMMAD MOHSIN ZAMAN1, AYMEN OWAIS GHAURI1,

ADEEL ARSHAD2 and KHALID AKHTAR1

1University College of Conventional Medicine, 2Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine,

The Islamia University of Bahawalpur, Bahawalpur, Pakistan3Middle East Technical University, Ankara, Turkey

Abstract: Many antimicrobial drugs are going to become resistant to different pathogens, so the discovery ofnew antimicrobial products is an important public health concern. Nardostachys jatamansi (D. Don) DC(Valerianaceae) is an important traditional herbal medicine used as tranquillizer, CNS sedative, antiepilepsy,cardiotonic, diuretic, heptatonic, analgesic and in boils, itch and eye diseases. Carbonic anhydrase inhibitors arethe potential source of treatment in case of glaucoma, hypertension and epilepsy and also used as diuretics. Theaim of present study was to evaluate the antibacterial activity of methanolic extract of N. jatamansi against dif-ferent bacterial strains and to estimate its carbonic anhydrase inhibitory potential. Antibacterial activity wasevaluated by agar well diffusion assay and broth microdilution assay. The study revealed that N. jatamansiextract is sensitive to all tested bacterial strains. The zones of inhibition and MIC ranged from 8-22 at a con-centration of 1-5 mg/mL and 0.3-0.6 mg/mL, respectively. Methanolic extract of N. jatamansi showed markedinhibition of carbonic anhydrase (IC 50 712.41 ± 0.001 µg/mL) when compared with standard acetazolamide.The results of the study suggest that N. jatamansi may be a valuable plant source of medicinally useful activecompounds that can be helpful in bacterial infections and showed some relation with the traditional use of thisplant in various diseases.

Keywords: Nardostachys jatamansi, antibacterial, carbonic anhydrase inhibitor

1471

* Corresponding author: [email protected]; phone: +923347822181

1472 TAYYEBA REHMAN et al.

siella pneumoniae) and two Gram positive bacteria(Bacillus subtilis, Staphylococcus aureus) wereincluded in the study. These bacterial strains wereselected as these are the commonly infection causingbacteria in humans as Bacillus subtilis involve in thevarious allergic conditions of respiratory track, foodpoisoning and eye infections (6). Staphylococcusaureus is responsible for a variety of diseases includ-ing skin (boils, itch), soft tissue, bone, joint, foodpoisoning, cardiovascular, respiratory and woundinfections. Pseudomonas aeruginosa can causeinfections such as urinary tract infections, pneumo-nia, ear and eye infections and traumatic woundinfections. Klebsiella pneumoniae causes urinarytract infections, wound infections, cholecystitis,meningitis and endocarditis. Escherichia coli is anopportunistic organism causing pneumonia and sep-sis in immunocompromised host and meningitis (7).

Carbonic anhydrase inhibitors can be used forthe treatment of many diseases. Carbonic anhydraseinhibitors are the first line treatments for glaucoma,cancer, osteoporosis, obesity, neurological disor-ders, as well as for gastric and duodenal ulcers. Itmay also act as diuretics and antiepileptic (8).

Many studies have been conducted to find theeffects of N. jatamansi in CNS diseases (9). A liter-ature search found that carbonic anhydrase inhibi-tion and antibacterial activity of Nardostachys jata-mansi methanolic extract against selected bacterialstrains have not been reported so far.

In this study, antibacterial activity against someselected bacterial strains and carbonic anhydrase inhi-bition of N. jatamansi methanolic extract aredescribed for the first time providing the basis forresearch to find new entities against different dis-eases.

EXPERIMENTAL

Reagents

Tris (Invitrogen: cat# 15504-020), HEPES(bioworld: cat#40820000-1), carbonic anhydrase(Sigma-Aldrich, C2624, P-Code: 1001584424), 4-nitrophenyl acetate (Sigma-Aldrich, N8130,lot#BCBK4587V), acetazolamide (Sigma-Aldrich,Lot BCBK5191V, P-Code 101400375, ≥ 99% pow-der), dimethyl sulfoxide (Merck, Germany), nutrientagar (Merck, Germany), nutrient broth (Merck,Germany). Ciprofloxacin (Novidat), Registration #012066, Sami Pharmaceuticals (Pvt.) Ltd.

Apparatus

Micro plate reader (Synergy HT BioTekÆUSA), Digital rotary evaporator apparatus,

(Heidolph Laboratory, Germany), pH meter (WTWseries Inolab), digital weighing balance (Uni Bloc,Shimadzu, AUW220D).

Collection and identification of plant sample

Dried plant material was purchased from thelocal market and identified by the botanist, Dr.Sarwar, Lecturer, The Islamia University, Bahawal-pur (Voucher No. 2205/L.S). The voucher specimenwas deposited in Botany Department, The IslamiaUniversity, Bahawalpur, Pakistan.

Preparation of extract

The plant extract was prepared by macera-tion method. Plant material was powdered in theelectric grinder; 100 g of dried powdered plantmaterial was taken in the amber colored glass bot-tle and 400 mL of methanol was added to it. Thematerial was soaked for 15 days with occasionalshaking. After 15 days, the soaked material wasfiltered through muslin cloth and then by Whatman# 1 filter paper by using Buchner funnel. Theprocess was repeated three times with 200 mLmethanol to extract maximum contents and mate-rial (10). The solvent was evaporated by usingrotary evaporator. The remainder was collected inlittle glass bottles and stored at 4OC until next use.It was the crude methnolic extract of N. jataman-si rhizomes.

A stock solution of freeze dried extract 5 mgplant extract/mL of DMSO was prepared and serialdilutions in the range of 0.5-5 mg/mL prepared fromthe stock solution.

Preliminary phytochemical screening

N. jatamansi rhizome extract was subjected topreliminary qualitative phytochemical screening foralkaloids (Dragendorffís and Mayerís test),flavonoids (sodium hydroxide), tannins (ferric chlo-ride test) and phenols (ferric chloride test) (11).

Bacterial strains and growth media

Staphylococcus aureus (S.A) ATCC-6538,Pseudomonas aeruginosa (P.A) ATCC-9027, pur-chased from Microbiologics Inc. Escherichia coli(E.C), Klebsiella pneumonia (K.P), Shigella dysen-teriae (S.D), Corynebacterium striatum (C.S),Bacillus subtilus (B.S), Proteus vulgaris (P.V) pur-chased from first fungal culture Bank of Pakistan(FCBP), Institute Of Agricultural Sciences,University of The Punjab, Lahore, Pakistan; acces-sion numbers were 12, 14, 72, 147, 174, 368, respec-tively. All the bacterial strains were grown on nutri-ent agar at 37OC for 24 h.

Evaluation of antibacterial and carbonic anhydrase inhibitory potential of... 1473

Synthetic antibacterial and carbonic anhydrase

inhibitor

Ciprofloxacin (Novidat 200/100 mg/mL) wasused as standard antibacterial drug and acetazol-amide was used as a standard carbonic anhydraseinhibitor.

Preparation of inoculum

Nutrient agar media were prepared by dis-solving 28 g of nutrient agar powder in 1000 mL ofdistilled water, heated until bubbles appeared andthen sterilized at 121OC and 15 psi in autoclave for15 min. Bacterial inoculums were prepared from24 h old pure culture on nutrient agar. Bacterialcolonies were grown in nutrient broth for 24 h. In1 liter of distilled water, 8 g of broth was dissolvedand kept in an autoclave (for sterilization) for 20min at 121OC at 15 psi. In Erlenmeyer flasks, 50mL of broth and 50 µL of culture stock solutionwas added and mounted on the horizontal shaker.After 24 h, cell turbidity was checked spectroscop-ically in comparison to that of 0.5 McFarland stan-dard. Then, the inoculum was used for the antibac-terial activity.

In vitro susceptibility tests

Agar well diffusion method

The agar well diffusion method was performedfor the determination of antibacterial activity of N.jatamansi methanolic extract. Prior to being testedfor the antibacterial activity, crude plant extract wasdissolved in DMSO and stock solution of 5 mg/mLwas prepared. The experiment was performed by themethod of (12) with slight modifications. 20 mL ofMueller Hinton agar was placed in Petri dishes andallowed to solidify. A suspension of the microor-ganism of 60 µL was evenly spread on the surface ofMueller Hinton agar with sterile cotton-tipped swab.Wells of 6 mm in diameter were made on solid agarsurface with the help of cork borer in each Petri dish.20 µL of extract solution was added to each well.Petri dishes were placed in the incubator at 37OC for24 h. After 24 h, the zones of inhibitions were meas-ured to estimate the antibacterial activity. Theexperiment was done in triplicate and results weretaken as an average of the three tests. The test wasalso performed with standard ciprofloxacin andmethanol.

Broth micro dilution method

Antibacterial analysis and determination ofminimum inhibitory concentration (MIC) of theextract and ciprofloxacin against different bacterialstrains were performed by broth micro dilution

method. This assay was carried out by the method of(13) with slight modifications. The test was per-formed in sterile 96-well micro plates. Total mixturevolume in a well was 200 µL, contained 20 µL ofmethanolic extract solution and 180 µL suspensionof bacterial culture. At 540 nm absorbance wasmeasured and this was taken as pre read. Then for16-24 hours, the plates were incubated at 37OC.After read was measured at 540 nm and the differ-ence between pre read and after read was taken as anindex of bacterial growth. All readings were taken astriplicate. Results are mean of triplicate (n = 3, ±S.E.M). Standard drug was ciprofloxacin andinstead of test sample methanol was added in theassay as the negative control.

The % inhibition was calculated by the follow-ing formula

Inhibition (%) = 100 × (X ñ Y) / Xwhere X = absorbance in negative control with bacterialcultureY = absorbance in test sample with bacteria

Serial dilutions of the test samples were madeto calculate the minimum inhibitory concentration.EZ-Fit5 Perrella Scientific Inc. Amherst USA soft-ware was used for MIC calculation.

Carbonic anhydrase inhibition assay

Carbonic anhydrase inhibition assay was per-formed according to (8) with slight modification. Inthis test, the formation of 4-nitrophenol was meas-ured, that is a yellow color compound. 4-Nitrophenolwas formed by the hydrolysis of 4-nitrophenylacetate. The experiment was performed in 20 mMbuffer of 7.4 pH containing Tris and HEPES. Foreach sample reaction well included following items:140 µL of buffer, 20 µL of the freshly prepared solu-tion of enzyme (0.1 mg/mL of deionized water) ofpurified bovine erythrocyte CA-II and 20 µL of thetest compound. The test compound was incubated for15 min at 25OC and pre-read was taken at 400 nm byusing Synergy HT BioTekÆ USA micro plate reader.

The reaction started by the addition of 4-nitro-phenyl acetate. 4-Nitrophenyl acetate was added in20 µL at the concentration of 0.7 mM, diluted inethanol and incubated at the same conditions for 30min and after read was taken at 400 nm. The reac-tion performed in triplicate with different concentra-tions. Assay was also performed with methanol forpossible inhibition of enzyme as the extraction ofplant was done with methanol. Percent inhibitionwas measured by formula given below;

% inhibition = 100 ñ (absorbance of test compound/absorbance of control) × 100



All the measures were done in triplicate andresults were expressed as the mean ± S.E.M. One-way ANOVA followed by Tukey post hoc test wasused for statistical analysis. A p-value ≤ 0.05 wasconsidered significant.

RESULTS

Preliminary phytochemical analysis of extractshowed the presence of alkaloids, flavonoids, tan-nins and phenols.

The growth inhibition value of methanolicextracts of N. jatamansi on different bacterial strains

the Table 1. Antibacterial activities of methanolic extracts of N. jatamansi against bacterial strains, mean ± SEM of measured zones ofinhibitions (mm) in experimental groups (n = 3).

Bacterial strains Zone of inhibition (mm) mean ± SEM*

1 mg/mL 2 mg/mL 4 mg/mL 5 mg/mL Ciprofloxacin

Staphylococcus aureus 12.6 ± 0.5 14.1 ± 0.28 16 ± 1 21 ± 1 26 ± 1a

Bacillus subtilis 14 ± 1 16.6 ± 0.5 18.8 ± 1.2 22 ± 1 30 ± 0.5b

Escherichia coli 11 ± 0.5 14.6 ± 0.5 17.3 ± 0.5 19 ± 0.5 32 ± 0.5c

Klebsiella pneumoniae 10.3 ± 0.5 12 ± 0.5 17 ± 0.2 21 ± 0.2 34 ± 0.5d

Shigella dysenteriae 8 ± 1 10 ± 0.5 14 ± 1 17 ± 0.5 35 ± 0.5e

Corynebacterium striatum 11 ± 0.5 13 ± 0.5 15 ± 0.5 18 ± 0.1 28 ± 1f

Proteus vulgaris 11 ± 0.2 14 ± 0.1 17 ± 0.5 19 ± 0.5 29 ± 0.5g

Pseudomonas aeruginosa 8 ± 0.5 13 ± 0.2 16.5 ± 0.5 18 ± 1 33 ± 0.5h

*SEM, standard error of mean; a Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM)for the control was 32 ± 0.5; b Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM) forthe control was 35 ± 1; c Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM) for thecontrol was 35 ± 1; d Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM) for the con-trol was 30 ± 1; e Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM) for the controlwas 30 ± 1; f Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM) for the control was26 ± 1; g Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM) for the control was 30 ±1; h Mean ± SEM in each column differs significantly (< 0.05). Diameter of inhibition zones (Mean ± SEM) for the control was 32 ± 1.

Table 2. MIC of methanolic extract of N. jatamansi and ciprofloxacin against bacterial strains.

Bacterial strains N. jatamansi MIC (mg/mL) Ciprofloxacin MIC (µg/mL)

Staphylococcus aureus 0.4 1

Bacillus subtilis 0.3 1

Escherichia coli 0.5 0.015

Klebsiella pneumoniae 0.4 1

Shigella dysenteriae 0.7 2

Corynebacterium striatum 0.6 8

Proteus vulgaris 0.5 5

Pseudomonas aeruginosa 0.6 5

Table 3. In vitro carbonic anhydrase inhibitory activity of N. jatamansi methanolic extract.

Test substance IC50 (Mean ± S.E.M)*

N. jatamansi 712.41 ± 0.001 µg/mL

Acetazolamide (Standard) 0.03 ± 0.00 µg/mL or 0.14 µM

*IC50 readings are mean ± S.E.M of 3 readings

Evaluation of antibacterial and carbonic anhydrase inhibitory potential of... 1475

is shown in Table 1. All bacterial strains showedmarked antibacterial activity. The diameters ofgrowth of inhibition zone of extract at various con-centrations were between 8-22 mm. The diameters ofthe zone of inhibitions were decreased by reducingthe concentration of extract. Ciprofloxacin showedzone of inhibitions in a range of 26-35 mm. Themethanolic extract showed the highest inhibitionagainst Bacillus subtilis and least against Shigelladysenteriae. However, methanol showed no inhibi-tion zone against any bacterial strain. There was asignificant difference between the zone of inhibitionvalues of ciprofloxacin and extract (p < 0.05).

Table 2 summarizes the results of antibacterialactivity as MIC through broth microdilutionmethod. The MIC values of extract ranged from 0.3-0.7 mg/mL. Results showed that the highestinhibitory activity of the plant extract was foundagainst Bacillus subtilis (MIC 0.3 mg/mL) and thelowest against Shigella dysenteriae (MIC 0.7mg/mL). The results obtained from ciprofloxacinshowed resistance to all selected bacterial strainsrepresenting MIC ranged from 0.015-8 µg/mL.There was a significant difference between MIC val-ues of ciprofloxacin and extract (p < 0.05). It is evi-dent from the results of the study that the highest thezone of inhibition, the lowest the minimum inhibito-ry concentration. In present study, the N. jatamansiextract showed highest zone of inhibition (22 mm)with the lowest MIC (0.3 mg/mL) against Bacillussubtilis. The lowest zone of inhibition was againstShigella dysenteriae (17 mm) with MIC (0.7mg/mL).

In this study, N. jatamansi methanolic extractshowed a marked inhibition of carbonic anhydrase(IC50 712.41 µg/mL) as represented in Table 3.Maximum inhibition of carbonic anhydrase (87%)was with the stock solution of the methanolic extract(1 mg/mL in DMSO). Methanol has no effect oncarbonic anhydrase inhibition.

DISCUSSION

Drugs from natural sources are preferredbecause they perceived drug likeness and biologicalfriendliness than synthetic compounds (14). Drugresistance due to an inappropriate and wide use ofsynthetic medicines is a major issue. Moreover, thereare various side effects of these commercially avail-able drugs (15). The study was conducted to investi-gate antibacterial activity of methanolic extract of N.jatamansi against S. aureus, P. aeruginosa, E. coli, K.pneumoniae, S. dysenteriae, C. striatum, B. subtilis,P. vulgaris, common clinical bacteria that can cause

infection in humans. Antibacterial activity of differ-ent types of extract of N. jatamansi was reportedagainst some bacterial strains (16), the screening ofmethanolic extract on all these bacterial strains andtheir minimum inhibitory concentrations (MICs)were not revealed before.

Based on agar well diffusion assay results of B.subtilis, S. aureus, K. pneumoniae showed the high-est antibacterial activity while others showed mod-erate antibacterial activity. The similar findingswere reported by (16) that the mixture of dichlo-romethane and methanol extract of N. jatamansishowed marked inhibition of B. subtilis, S. aureus,K. pneumoniae. However, Kumar (16) reported thelack of activity of essential oil and mixture of di-chloromethane and methanol extract of N. jataman-si against P. aeruginosa and E. coli.

It has been frequently reported that antibacter-ial activity of medicinal plants is mostly due to thepresence of alkaloids, flavonoids, tannins and triter-penoid in the plant extracts (17). The phytochemicalevaluation of this extract has proved the existence ofalkaloids, flavonoids and tannins in the methanolicextract, which might be the possible reason lyingbehind antimicrobial potential of this extract. Thepresence of alkaloids and tannins in methanolicextract of N. jatamansi was also reported in anotherwork (4).

Antibacterial activity of extract against differ-ent bacterial strains was dose dependent. Activitywas decreased by decreasing the concentration ofextract. N. jatamansi extract has antibacterial effectsmay be by direct action of the extract on structureand metabolism of bacteria. The study was the firstone that determined MIC of N. jatamansi extract.The role of MIC determination lies in the fact thatlower the MIC, more chances of it being useful formedicinal purposes. The lower the doses required toachieve therapeutic effect have chances of lowertoxicity and side effects (18).

Staphylococcus aureus skin and soft tissueinfections cause minor boils or abscesses and canlead to severe infections as endocarditis (19). Theroots of N. jatamansi are used traditionally in thetreatment of boils and itch, etc. (2). Results of thisstudy showed that N. jatamansi extract has markedinhibition against S. aureus (MIC 0.4 mg/mL) thusproviding the basis for its traditional use in boilsthrough antibacterial effects.

The results of present study presented themarked inhibition of carbonic anhydrase, so it canbe speculated that traditional effects of N. jataman-si as diuretic, antiepileptic effect and in eye diseasesmay be due to carbonic anhydrase inhibition. N.


jatamansi extract can be used for clinical treatmentof various diseases as a syrup. Formulation contain-ing N. jatamansi as an important ingredient hasreduced febrile convulsions in children during anexperimental clinical trial (20). So, traditionalantiepileptic effect of N. jatamansi that is proved incurrent study through in vitro carbonic anhydrasepotential was also confirmed by a clinical trial.Moreover, anticancer effects of N. jatamansi thatcan be speculated from the current study due to car-bonic anhydrase inhibitory potential is supported bycytotoxic activity of N. jatamansi extract againstlung and prostate cancer cell lines (21).

CONCLUSION

N. jatamansi inhibited carbonic anhydrase suf-ficiently and showed significant antibacterial activi-ty against different bacterial strains. Thus, the tradi-tional use of it in various diseases is related to itsreported activities. Moreover, the results of thestudy suggest that N. jatamansi may be a valuableplant source of medicinally active constituents thatcan be useful for the treatment of diseases.

REFERENCES

1. Houghton P.J.: J. Pharm. Pharmacol. 51, 505(1999).

2. Dweck A., MRSC FLS.: Medicinal and aromat-ic plants industrial profile. Peter Black Cosme-tics & Toiletries 1996.

3. Ali S., Ansari K.A., Jafry M., Kabeer H.,Diwakar G.: J. Ethnopharmacol. 71, 359 (2000).

4. Jha S.V., Bhagwat A.M., Pandita N S.: Phcog.J. 4, 16 (2012).

5. Singh A., Kumar A., Duggal S.: AJPRHC. 1,276 (2009).

6. Ihde D.C., Armstrong D.: Am. J. Med. 55, 839(1973).

7. Engelkirk P.G., Duben-Engelkirk Janet L.:Laboratory diagnosis of infectious diseases:Essentials of diagnostic microbiology. Lippin-cott Williams & Wilkins, Baltimore 2008.

8. Ashiq U., Jamal R.A., Saleem M., Mahroof-Tahir M.: Arab. J. Chem. 24 (2015).

9. Mathew M., Subramanian S.: PLoS One 9,e86804 (2014).

10. Ashraf M., Ahmad K., Ahmad I., Ahmad S.,Arshad S. et al.: J. Med. Plant Res. 5, 2086 (2011).

11. Padmavathy S., Mekala V.: Int. J. Res. Pharm.Biomed. Sci. 4, 1 (2013).

12. Dogruoz N., Zeybek Z., Karagoz A.: IUFS J.Biol. 67, 17 (2008).

13. Kaspady M., Narayanaswamy V.K., Raju M.:Lett. Drug Des. Discov. 6, 21 (2009).

14. Koehn F.E., Carter G.T.: Nat. Rev. DrugDiscov. 4, 206 (2005).

15. MÈgraud F.: Therap. Adv. Gastroenterol. 5, 103(2012).

16. Kumar V.P., Chauhan N.S., Padh H., Rajani M.:J. Ethnopharmacol. 107, 182 (2006).

17. Cowan M.M.: Clin. Microbiol. Rev. 12, 564(1999).

18. Srivastava G., Jain R., Vyas N., Mehta A.,Kachhwaha S., Kothari S.: Int. J. Pharm. Biol.Sci. 4, 825 (2013).

19. McCaig L.F., McDonald LC., Mandal S.,Jernigan D.B.: Emerg. Infect. Dis. 12, 1715(2006).

20. Majumdar R., Bagade A., Phadke M.: IndianPract. 54, 729 (2001).

21. Saetung A., Itharat A., Dechsukum C.,Wattanapiromsakul C., Keawpradub N.,Ratanasuwan P.: Songklanakarin J. Sci.Technol. 27, 469 (2005).

Received: 29. 08. 2016


Liver, the second largest organ of body respon-sible for a number of important functions includingproduction of bile, urea and plasma proteins, excre-tion of bilirubin, cholesterol, hormone and drugs andalso metabolism of protein, vitamin, minerals, car-bohydrates and fats (1), is most susceptible to toxic-ity (2). Hepatotoxicity is caused by chemicals thatdamage hepatic vasculature, hepatocytes and biliaryepithelial cells either directly or indirectly by pro-ducing their reactive metabolic species, hence illic-iting an immune reaction and resulting in hepaticinjury (3). Most of the toxins have minimum liverdamaging potential which can be reversed by termi-nating the use of offending agent. In overdose,causative mediator results in hepatic necrosis and ifnot treated timely and effectively, may lead to death(3). Moreover, it is estimated that over 900 drugshave potential to cause hepatic damage that is whymost marketed drugs with hepatotoxic potentialhave been withdrawn (4). Drug induced liver injury

can be either intrinsic or idiosyncratic (4) and resultsfrom inhalation, ingestion and parenteral adminis-tration of drugs. Certain drugs cause hepatic injuryeven at therapeutic doses (5), for instance paraceta-mol, diclofenac, isoniazid, halothane, erythromycin,penicillamine and phenytoin. Moreover, reactivemetabolites of various drugs interact with essentialmacromolecules such as proteins, lipids or nucleicacids, leading to protein dysfunction, lipid peroxida-tion, DNA damage and oxidative stress and finallyresulting in hepatic damage (6). Around the world,every year about 20,000 deaths occur followed byhepatic diseases (5).

Current therapy of liver disorders include inter-ferons, lamivudine, adefovir, ribavirin and cortico-steroids. But these medications owe many limita-tions such as low efficacy, high risk of adverseeffects and high price. Therefore, use of herbalremedies in treating liver maladies has been increas-ing nowadays. Various herbs are known for pos-

EVALUATION OF HEPATOPROTECTIVE POTENTIAL OF EUPHORBIAPROSTRATA AIT EXTRACT AGAINST CHEMICALLY INDUCED

HEPATOTOXICITY IN ALBINO MICE

ALAMGEER1*, KHADIJA1, ZAIN NASIR1, MUHAMMAD NAEEM QAISAR1, AMBREEN MALIK UTTRA1, KIFAYAT ULLAH KHAN2, IKRAM ULLAH KHAN3*,

MUHAMAMD SALEEM3, HASEEB AHSAN1, HIRA ASIF1, AMBER SHARIF1, WAQAS YOUNIS1

and HUMA NAZ3

1Department of Pharmacology, Faculty of Pharmacy University of Sargodha, Pakistan2Drug Control Office Gilgit-Baltistan, Pakistan

3Faculty of Pharmacy, GC University, Faisalabad, Pakistan

Abstract: Hepatic diseases are serious health issues as various pharmaceuticals, xenobiotics,environmental/chemical pollutants, some natural products and dietary supplements when used in overdose havetendency to cause hepatotoxicity. Present study was aimed at investigating hepatoprotective activity of aqueousmethanolic extract of Euphorbia prostrata against paracetamol and carbon tetrachloride induced hepatotoxicityin mice, using silymarin as a standard drug. Hepatoprotective effects were accessed by investigating serummarker enzymes (aspartate transaminase, alanine transaminase, alkaline phosphatase), total bilirubin, albuminand total protein as well as histopathological analysis. Euphorbia prostrata exhibited significant reduction inserum marker enzymes at 250 mg/kg and 500 mg/kg doses. Moreover, histopathological studies also support-ed biochemical estimation. It could be concluded that aqueous methanolic extract of Euphorbia prostrata pos-sesses hepatoprotective potential against carbon tetrachloride and paracetamol induced hepatic injury whichmight to due to anti-oxidant activity displayed by flavonoids and polyphenolics.

Keywords: Euphorbia prostrata, hepatotoxicity, marker enzyme, paracetamol, carbon tetrachloride

1477

* Corresponding author: e-mail: [email protected]; [email protected]

1478 ALAMGEER et al.

sessing hepatoprotective properties, derived fromdifferent cultural sources (7). Euphorbia prostrataAit is an annual herb, mainly found in tropic andsubtropic areas naturally present in Asia, Africa andmany other parts of world. It is used traditionally asanti-oxidant (8) and to inhibit HIV-1 and hepatitis Cvirus proteases (9, 10). It has been validated forpharmacological effects like anti-inflammatoryactivity (11), hemorrhoid (12), diarrhea (13), anti-bacterial, anti-fungal activity (14) and diabetes (15).The purpose of present study was to pharmacologi-cally investigate the hepatoprotective effect ofEuphorbia prostrata using animal models of liverinjury thus supporting its customary use in liver ail-ments.


Chemicals

The chemicals used were methanol, chloro-form, ether, normal saline, paracetamol, carbontetrachloride, silymarin (all from Sigma Aldrich).All the other chemicals used were of analyticalgrade.

Plant material

The aerial parts of Euphorbia prostrata Ait (2kg) were collected from Dhillam Ballaggan, Sialkot,Punjab, Pakistan from April to May 2015. Plant wasidentified and authenticated by Professor Dr. Ashiq,Department of Botany, University of Agriculture,Faisalabad. The washed and shade dried plant mate-rial was ground into powder with a Chinese herbalgrinder.

Preparation of plant extract

Aqueous methanolic (30 : 70) extract of aerialparts of Euphorbia prostrata was prepared usingcold maceration technique. Coarse powder of plantwas soaked in 3 L of 70% methanol and kept atroom temperature for 3 days (72 h) with occasionalstirring daily, followed by filtration after 3 days.This process was repeated thrice. Afterwards, all thefiltrates were combined and again filtered throughmuslin cloth and Whatman filter paper I. The filtratewas dried and concentrated under reduced pressurein rotary evaporator at 50OC. The solid extract thusformed was stored in a capped container in refriger-ator. The color of crude extract was dark brown(16).

Animals used

Young and healthy albino mice (20-40 g) ofboth sexes (4-5 months) were used. The mice were

housed under standard conditions of temperature (23to 25OC), relative humidity (55%) with 12 h lightand 12 h dark cycle at animal house of University ofSargodha, Sargodha. They were fed with standardpellet diet and tap water ad libitum. All the experi-ments performed complied with the rules ofNational Research Council (17).

Paracetamol (PCM) induced hepatotoxicity

In the dose response experiment, albino micewere randomly divided into 5 groups (n = 4). GroupI (Normal control group) animals received normalsaline 1 mL/kg, p.o. for 7 days. Group II (Diseasedcontrol) mice were given normal saline, 1 mL/kgp.o. for 7 days. Group III (Standard group) micewere administered silymarin, 100 mg/kg p.o. for 7days. Group IV and V (Experimental groups) ani-mals were given Euphorbia prostrata extract, 250mg/kg/d and 500 mg/kg/d p.o. for 7 days, respec-tively.

On 7th day, 1 h after administering normalsaline (disease control group), silymarin, 250 mg/kgand 500 mg/kg of drug extract to Group II, III, IVand V, respectively, paracetamol (250 mg/kg) wasgiven orally. After 24 h of administration, mice weresacrificed under mild ether anesthesia and hepato-protective activity was assessed (18).

Carbon tetrachloride (CCl4) induced hepatotoxic-

ity

Albino mice were randomly divided into 5groups (n = 4). Group I (Normal control group) wasgiven distilled water 1 mL/kg p.o. for 7 days fol-lowed by administration of olive oil (1 mL/kg, s.c.)on 7th day, 1 h after feeding distilled water. Group II(Diseased control) mice were administered distilledwater (1 mL/kg, p.o.) for 7 days. Group III(Standard group) animals received silymarin, 100mg/kg p.o. for 7 days. Group IV and V (Exper-imental groups) mice were given Euphorbia prostra-ta extract, 250 mg/kg/d and 500 mg/kg/d p.o. for 7days, respectively.

On 7th day, 1 h after administration of distilledwater, sylimarin, 250 mg and 500 mg of Euphorbiaprostrata extract to Group II, III, IV and V, respec-tively, carbon tetrachloride (20% v/v in olive oil) 1mL/kg was given subcutaneously. After 24 h ofadministration, mice were sacrificed under mildether anesthesia and hepatoprotective activities wereevaluated (19).

Assessment of hepatoprotective activity

The hepatoprotective activity was appraisedbiochemically as well as histopathologically. After

Evaluation of hepatoprotective potential of Euphorbia prostrata... 1479

24 h of drug treatment, animals were anesthetizedusing ether. Blood was withdrawn from each mousethrough carotid artery and collected in centrifuga-tion tubes and was allowed to clot for 30 min atroom temperature. Serum was separated by centrifu-gation at 3000 rpm for 15 min and biochemicalparameters like alanine aminotransferase (ALT),aspartate aminotransferase (AST), alkaline phos-phatase (ALP), albumin, bilirubin and total proteinwere evaluated. The hepatic tissues were alsoexcised quickly, washed with saline and stored in10% formalin. Four representative sections weretaken and submitted in one block. The histopatho-logical examination of these sections revealed archi-tecture of liver (5).


The results were expressed as the means ±standard error of mean (S.E.M). One-way ANOVA

followed by Dunnet test was applied usingGraphpad prism software. Significance was set at95% (p < 0.05).

RESULTS

The present study revealed that in paracetamolinduced intoxication, Euphorbia prostrata extractexhibited highly significant reduction in alanineaminotransferase (ALT) and aspartate aminotrans-ferase (AST) (p < 0.01) and alkaline phosphatase(ALP) (p < 0.001) at 250 mg/kg dose. Moreover,Euphorbia prostrata extract caused highly signifi-cant (p < 0.001) reduction in activities of alanineaminotransferase (ALT) and aspartate aminotrans-ferase (AST) at 500 mg/kg as compared to diseasecontrol group. However, Euphorbia prostrata extractproduced non-significant changes in levels of alka-line phosphatase (ALP) at 500 mg/kg. Also, non-

Table 1. Effect of methanolic extract of Euphorbia prostrata on biochemical parameters in paracetamol induced hepatotoxicity.

GroupsTotal bilirubin ALT AST ALP Albumin Total protein

(mg/dL) (U/L) (U/L) (U/L) (g/dL) (g/dL)

PCM (250 mg/kg)

0.83 ± 0.01 82.66 ± 15.45 85.33 ± 18.98 209.33 ± 5.20 4.30 ± 0.88 7.53 ± 0.62

Normal control(N.S 1 mL/kg)

0.53 ± 0.06ns 79.33 ± 4.05ns 72.33 ± 2.33** 135.66 ± 5.36** 3.00 ± 0.05ns 5.86 ± 0.46ns

Silymarin + PCM(100 mg/kg/10mL)

0.53 ± 0.03ns 19.00 ± 1.52*** 14.66 ± 1.85*** 193.33 ± 4.41*** 3.90 ± 0.57ns 6.53 ± 0.31ns

EP + PCM(250 mg/kg)

0.46 ± 0.03ns 50.00 ± 4.58*** 40.66 ± 2.33*** 234.66 ± 6.38*** 3.93 ± 0.58ns 7.60 ± 0.51ns

EP + PCM(500 mg/kg)

0.60 ± 0.00ns 21.00 ± 0.57*** 32.00 ± 1.73*** 206.00 ± 12.74ns 2.83 ± 0.06ns 6.13 ± 0.13ns

Results are expressed as the mean ± SEM (n = 4), where, ns = (p > 0.05), * = (p < 0.05), ** = (p < 0.01), *** = (p < 0.001) vs. PCM (250mg/kg). PCM=Paracetamol, EP=Euphorbia prostrata

Table 2. Effect of methanolic extract of Euphorbia prostrata on biochemical parameters in carbon tetrachloride induced hepatotoxicity.

GroupsTotal bilirubin ALT AST ALP Albumin Total protein

(mg/dL) (U/L) (U/L) (U/L) (g/dL) (g/dL)

CCl4

(1 mL/kg) 1.60 ± 0.15 175.00 ± 15.37 72.00 ± 16.16 441.00 ± 28.93 6.70 ± 1.35 9.33 ± 0.17

Normal control(N.S 1mL/kg)

0.53 ± 0.06ns 79.33 ± 4.05*** 72.33 ± 2.33ns 135.66 ± 5.36*** 3.00 ± 0.05ns 5.86 ± 0.46ns

Silymarin + CCl4

(100 mg/kg/10mL) 0.63 ± 0.03ns 47.66 ± 4.63*** 42.33 ± 9.06*** 162.00 ± 7.57*** 3.10 ± 0.10ns 5.83 ± 0.44ns

EP + CCl4

(250 mg/kg)0.50 ± 0.05ns 51.66 ± 5.36*** 42.66 ± 1.76*** 186.66 ± 4.41*** 3.46 ± 0.27ns 6.46 ± 0.12ns

EP + CCl4

(500 mg/kg)0.63 ± 0.03ns 34.66 ± 2.40*** 29.00 ± 1.73*** 158.66 ± 4.66*** 3.20 ± 0.15ns 6.30 ± 0.25ns

Results are expressed as the means ± SEM (n = 4), where, ns = (p > 0.05), * = (p < 0.05), ** = (p < 0.01), *** = (p < 0.001) vs. CCl4

(1mL/kg). CCl4=carbon tetrachloride, EP=Euphorbia prostrata


significant changes in levels of total bilirubin, albu-min and total protein at both concentrations wereobserved as illustrated in Table 1.

Likewise, in carbon tetrachloride inducedhepatotoxicity model, methanolic extract signifi-cantly (p < 0.001) decreased the levels of alanineaminotransferase (ALT), aspartate aminotransferase(AST) and alkaline phosphatase (ALP) at both dosesbut there were non-significant changes in the levelsof bilirubin, albumin and total protein at both dosesas presented in Table 2.

Additionally, histopathologic studies demon-strated that, in normal control group, normal archi-tecture of hepatocytes was observed with intact cellnuclei and regular portal vein. While, in paracetamolintoxicated group, severe histopathological changeswere observed such as infiltration of inflammatorycells, fatty changes, necrosis and ballooning degen-eration of hepatocytes, granuloma and malignancy.Silymarin (standard) treated group unveiled thathepatocytes were arranged in single plate with cen-tral veins and porta hepatis and no inflammatory ornecrotic changes were noticed in standard group.Moreover, no granuloma or malignancy was evi-dent. While, among extract treated groups, 250mg/kg exhibited partial protection of hepatocytesand prevented histopathological changes associated

with hepatotoxicity induced by acetaminophen, buthepatocytes were mildly infiltrated by inflammatorycells. However, no necrosis, granuloma or malig-nancy was seen. While, at 500 mg/kg, hepatocyteswere arranged in single plate, central vein and portahepatis were also clearly discernible. There were nosigns of inflammatory and necrotic changes, granu-loma or malignancy (Fig. 1).

The liver sections of carbon tetrachloride treat-ed mice divulged characteristic centrilobular patternof degeneration, liver fibrosis, hyperemia aroundcentral vein, wide vacuolar degeneration of hepato-cytes, lymphocyte infiltration, derangement of hepa-tocyte cord and necrosis at periphery of central vein.Whereas, in normal control group, central vein, por-tal space and hepatocytes were normal. However,silymarin distinctly protected liver from carbontetrachloride induced damage as revealed byhistopathological analysis. Similarly, Euphorbiaprostrata extract provided hepatic protection compa-rable to standard group particularly at 500 mg/kgdose (Fig. 2).

DISCUSSION

Liver is the central organ having astoundingrole in metabolism, excretion and detoxification of

Figure 1. Histopathological assessment of liver after administration of Euphorbia prostrata in paracetamol treated mice: A) Normal con-trol group B) PCM intoxicated group C) Silymarin 100 mg/kg D) EP (250 mg/kg) E) EP (500 mg/kg)

A

D E

B C


chemotherapeutic agents, xenobiotics and environ-mental pollutants. It is involved in maintenance,regulation of body homeostasis and various bio-chemical pathways to growth, nutrient provisionand fight against disease, energy generation andreproduction. Various liver maladies like hepatitis,cirrhosis and fatty liver disease originate because ofexposure to environmental toxins, generation offree radicals, deprived food habits, alcohol abuseand numerous prescribed and over-the-counterdrugs (20). The available synthetic drugs are lesseffective in treating hepatic diseases owing to theirnumerous adverse effects and low therapeuticpotential. Therefore, use of herbal medicines hasbeen increasing gradually in both developed as wellas developing countries as they are economical withsubstantial biological effects and free from toxicprofile. Furthermore, plant based preparations playa noteworthy role in regeneration of liver cells andacceleration of healing process and hence manage-ment of numerous liver ailments (21). Currently,most effective therapy for hepatic ailments are con-stituents derived from herbal drugs such as gly-cyrrhizin (Glycyrrhiza glabra) and silymarin(Silybum marianum) in Japan, catechin(Anacardium occidentalis) in Europe, and chizan-drins (Schizandra chinensis) in China (22).Similarly, in present study, hepatoprotective activi-ty of aqueous methanolic extract of Euphorbia pros-trata was evaluated using paracetamol (PCM) and

carbon tetrachloride (CCl4) as hepatotoxins to sci-entifically support its folkloric use.

These hepatotoxic agents cause liver damageresulting in elevation of marker enzymes includingalanine aminotransferase (ALT) and aspartateaminotransferase (AST) and alkaline phosphatase(ALP). Carbon tetrachloride is a hepatotoxic agentthat undergoes biotransformation in endoplasmicreticulum by CYP 450 and produces trichloromethylfree radical i.e. CCl3 that interact with cellular pro-teins and lipids. The trichloromethylperoxyl radicalmore potentially attacks lipids of endoplasmic retic-ulum causing lipid peroxidation and alter calciumhomeostasis resulting in cell death and release ofenzymes in circulation. In current study, methanolicextract of Euphorbia prostrata markedly decreasedlevels of liver marker such as enzymes alanineaminotransferase (ALT), aspartate aminotransferase(AST) and alkaline phosphatase (ALP), total biliru-bin and albumin comparable to that of standard drugsilymarin. However, in carbon tetrachloride intoxi-cation, Euphorbia prostrata extract reduced the lev-els of total bilirubin in dose independent manner,which might be due to either enzyme/receptor satu-ration or genetic variations. Likewise, paracetamolis a well-known and most commonly used analgesicand anti-pyretic drug. Hepatotoxic doses of parac-etamol deplete normal stores of hepatic glutathione.CYP 450 enzymes such as CYP2E1 and CYP1A2metabolize paracetamol and forms NAPQI (N-

Figure 2. Histopathological examination of liver after administration of Euphorbia prostrata in CCl4 treated mice: A) The Normal control,B) CCl4 intoxicated group, C) silymarin 100 mg/kg, D) EP (250 mg/kg), E) EP (500 mg/kg)

A

D E

B C


acetyl-p-benzo-quinoneimine) that is an alkylatingmetabolite. The P450 gene is highly polymorphic innature as CYP2D6, third isoenzyme, is associatedwith individual variation towards paracetamol toxic-ity. Paracetamol is metabolized into NAPQI to alesser extent by CYP2D6 but in ultra-rapid metabo-lizers, this enzyme causes hepatotoxicity.Glutathione is a natural anti-oxidant, but in paraceta-mol toxicity NAPQI becomes irreversibly conjugat-ed with sulfhydryl group of glutathione, thus causingits depletion. NAPQI causes hepatic damage byreleasing inflammatory mediators such as TNF-α,responsible for tissue necrosis (23). In paracetamolintoxicated mice, plant extract showed a dosedependent decrease in the levels of alanine amino-transferase (ALT), aspartate aminotransferase (AST)and alkaline phosphatase (ALP), which might be dueto stabilization of plasma membrane, as well asrepair of hepatic tissue damage caused by carbontetrachloride and paracetamol (24). Moreover, plantextract significantly decreased albumin level, butsurprisingly it exhibited concentration independentbehavior in case of total bilirubin which might be dueto hereditary disparity or receptor saturation produc-ing less than expected response at higher dose as incarbon tetrachloride model. However, in previousstudies it has been revealed increase in serum totalbilirubin level to be owing to defective bile excretionby liver, associated with loss of integrity of liver andeventually tissue necrosis. Hence, this results inincrease in binding, conjugating and excretorycapacity of liver cells, relative to erythrocyte degen-eration rate. Moreover, depletion in serum bilirubinlevel by plant extract might be due to its ability toalleviate biliary dysfunction during paracetamolinduced hepatotoxicity in prophylactic studies (24).Besides, histopathological examination also support-ed hepatoprotective ability of Euphorbia prostrata.

Since, it has been found that flavonoidsthrough their free radical scavenging activity areaccountable for hepatoprotection. Thus, liver dam-age induced by carbon tetrachloride and paraceta-mol is oxidative in nature, usually reversed by anti-oxidants by stabilizing cell membrane and repairingliver tissue damage. Moreover, various studiesrevealed hepatoprotective action of alkaloids due totheir anti-oxidant property (25). Also, hepatoprotec-tive agents act by inhibiting aromatase activity ofCYP 450 which favors liver regeneration (26).Hence, current study has revealed hepatoprotectiveaptitude of Euphorbia prostrata against carbon tetra-chloride (CCl4) and paracetamol (PCM) inducedliver damage, which could be due to its anti-oxidantactivity, as it contains glycosides, flavonoids, phe-

nols, polysaccharides, anthraquinones, phlobotan-nins, saponins and various other alkaloids (27, 28).Furthermore, in another study it has been ascer-tained that aqueous methanolic extract of E. prostra-ta possesses noticeable scavenging properties andscavenge DPPH, which might be owing to the pres-ence of phenolic and polyphenolic compounds thatsignificantly inhibit oxidative stress caused by freeradicals (29). Moreover, it has been avowed thatEuphorbia thymifolia possesses hepatoprotectivepotential due to marked anti-oxidant and inhibitorylipid peroxidation (LPO) activity (30, 31). So,Euphorbia prostrata being the member of same fam-ily and genus might have exerted hepatoprotectiveeffect due to its strong anti-oxidant and lipid perox-idation inhibition activity and owing to the presenceof flavonoids and phenolics. Further studies andinvestigations are required to prove hepatoprotectivepotential of active constituents of plant extract.

CONCLUSION

Euphorbia prostrata is a medicinally valuableplant and its hepatoprotective activity might be dueto flavonoid and phenolic constituents which exhib-it anti-oxidant and anti-lipid peroxidation properties.However, actual mechanism is not known and itneeds to be investigated.

Acknowledgment

The authors are thankful to University ofSargodha for providing all chemicals and materialrequired for research work and also grateful to staffof Animal House of University Sargodha for theirhelp and support.

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24. Sabbani P.K., Thatipelli R.C., Surampalli G.,Duvvala P.: J. Drug Metab. Toxicol. 7, 1(2016).

25. Sarkar C., Bose S., Banerjee S.: Indian J. Exp.Biol. 54, 705 (2014).

26. Seakins A., Robinson D.: Biochem. J. 86, 401(1963).

27. Singla A.K., Pathak K.: J. Ethnopharmacol. 29,291 (1990).

28. Bakhshi G.D., Langade D.G., Desai V.S.:Bombay Hosp. J. 50, 577 (2008).

29. Ahmad M., Shah A.S., Khan R.A., Khan F.U.,Khan N.A. et al: Afr. J. Pharm. Pharmacol. 5,1175 (2011).

30. Lin C.C., Cheng H.Y., Yang C.M., Lin T.C.: J.Biomed. Sci. 9, 656 (2002).

31. Singh S.K., Prabha T.: Indian J. Pharm. Sci. 67,736 (2005).

Received: 30. 08. 2016


Lovastatin (C24H36O5) is a potent drug that isused to lower blood cholesterol level of humans. Itactually belongs to a group of fungal secondarymetabolites known as statins which also include var-ious other cholesterol-lowering drugs such aspravastatin, simvastatin, mevastatin, etc. Lovastatinand pravastatin are natural statins; simvastatin issemi-synthetic while atorvastatin and fluvastatin aresynthetic statins. Natural statins can be producedthrough microbial fermentation (1). Lovastatin is areversible competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase which catalyzes a rate-limiting stepin the biosynthesis of cholesterol (Fig. 1A & B) (2).They are the most efficient agents for reducing plas-ma cholesterol, being also appreciated for their goodtolerance. The beneficial effects of the HMG CoAreductase inhibitors are usually attributed to theircapacity to reduce the endogenous cholesterol syn-thesis (3). Since mevalonate, the product of HMGCoA reductase reaction is the precursor not only forcholesterol, but also for many other nonsteroidalisoprenoidic compounds, inhibition of this keyenzyme may result in pleiotropic effects (4).

The first discovered statin, mevastatin, wasisolated from Penicillium citrinum and Penicilliumbrevicompactum (5). Among the genus Aspergillus,the most important species for statin production areA. terreus, A. flavipes, A. flavus, A. umbrosus, and A.parasiticus (4, 6). However, some hyper-producingstrains of A. terreus produce high amount of lovas-tatin under submerged fermentation, but it is notgenerally considered as safe drug and not suitablefor direct consumption by human beings. Liquidstate fermentation is not preferably chosen due tolow yield, extensive down streaming processing andthe consequent high capital and high operatingexpenses therefore, now it is replaced by solid statefermentation (6). The UV radiation is the most con-venient of all mutagens to use, and it is also veryeasy to take effective safety precautions against it. Itgives a high proposition of pyrimidine dimmers andincludes all types of base pair substitutions (7). Cellimmobilization is considered a promising approachfor enhancing the fermentation processes. It facili-tate continuous operation over a prolonged period,offers possible recycling of immobilized beads andsimple way for harvesting of products, reactor pro-

STRAIN IMPROVEMENT OF ASPERGILLUS TERREUS FOR HYPER-PRODUCTION OF LOVASTATIN AND IMMOBILIZATION OF

MUTANT ATU-06 STRAIN FOR REPEATED BATCH CULTURE PROCESS

SADIA JAVED1*, AMAIR RAZA1, SAQIB MAHMOOD2, MUNAZZAH MERAJ3, FARAH NAZ4

and SAMEERA HASSAN4

1Department of Applied Chemistry & Biochemistry, Government College University, Faisalabad, Pakistan2Department of Botany, Government College University, Faisalabad, Pakistan

3Department of Biochemistry, Peoples University of Medical and Health Sciences, Nawabshah, Pakistan4Centre of Excellence in Molecular Biology (CEMB), Lahore, Pakistan

Abstract: The aim of this work was to hyper-produce lovastatin by strain improvement of Aspergillus terreusS-24 through UV mutagenesis using corn cobs as carrier substrate in solid state fermentation. Ten positivemutants were selected on agar plates for still plate experiments. ATU-06 mutant strain showed 45.63 ± 1.74mg/100 mL lovastatin concentration higher than parent strain (18.74 ± 1.68 mg/100 mL) in pre-optimized fer-mentation medium. Fermentation parameters pH (6), temperature (30OC), inoculum size (2 mL) moisture con-tents (60%) and incubation time (120 h) were optimized for both mutant and parent strains. Kinetic growthparameters also showed that mutant strain ATU-06 showed greater stability and improved results as comparedto parent strain. Finally, the mutant Aspergillus terreus ATU-06 strain was immobilized by entrapping it in cal-cium alginate beads and agar for repeated use.

Keywords: mutation, lovastatin, Aspergillus terreus, immobilization, kinetic parameters

1485

* Corresponding author: e-mail: [email protected], [email protected]; phone: +92-333-6510755, +92-41-8505391

1486 SADIA JAVED et al.

ductivity and higher efficiency of catalysis anddevelopment of economical methods for loweringoperational cost of industrial processes (8).

Therefore, the present study was conducted forhyper production of cholesterol lowering druglovastatin produced by improving Aspergillus ter-reus strain on exposure to UV irradiation. Strainimprovement for the production of alkaline lovas-tatin has been reported by few scientists but there isan information gap regarding use of these inexpen-sive materials for considerably high production ofvalue added products by strain improvement and theimmobilization of mutant cells for repeated batchculture or multiple uses of these immobilized cells.

EXPERIMENTAL

Microorganism procurement and culture mainte-

nance

The pure local culture strain of A. terreus S-24was obtained from Institute of Microbiology,

University of Agriculture, Faisalabad, Pakistan. Theculture of A. terreus S-24 (stock culture) was main-tained in a refrigerator (4OC) on nutrient agar medi-um (Oxoid, Hampshire, UK).

Mutagenesis by UV lamp

For hyper-production of lovastatin, the sporesof A. terreus S-24 (1 ◊ 107 spores/mL) were mutatedby using UV lamp (UV-A, 20W, Phillips, λ360, dis-tance of the lamp from the Petri plates was 20 cm).These spores (10 mL) were exposed to ultravioletlight. The sample (1 mL) was withdrawn after 30min duration till 180 min. The optimum dose wasselected by preparing the survival data (9).

Selection of mutant

In the dark room, the 0.1 mL of these sporedilutions were streaked on nutrient agar plates(Oxoid, Hampshire, UK), that contain 0.1% triton X-100 to inhibit the growth of other fungal colonies. Allthese plates were kept at 37OC for 48 hours in an

Figure 1. (A) Structure of lovastatin and (B) mechanism of action of lovastatin

Strain improvement of Aspergillus terreus for hyper-production of 1487

incubator and counted the fungal colonies in eachplate (10). The mutants were randomly selected fromthe plates having at least 80-85% death rate or 15-20% survivors, especially those which showed mor-phological changes. Mutants were transferred to freshslants and incubated at 30OC for 6 days. All the select-ed mutants were tested for lovastatin production.

Analysis of lovastatin

The identifications and quantifications oflovastatin were carried out on the culture filtrate byHPLC method (11). Fungus secrets the lovastatin inits β-hydroxyacid form which are quite stable form

in solution and eluted first from column than lactoneform of the drug. The filtered broth was diluted 10-fold with acetonitrile-water (1 : 1 by volume). Thesamples were analyzed by using an HPLC (Hitachi)with UV detector (Hitachi L-2400) at 238 nm andHitachi L-2130 (C-18) column. The solvent was amixture of acetonitrile and 0.1% phosphoric acid(60 : 40 by volume) with flow rate of 1.5 mL/min.The sample injection volume was 20 µL.

Optimization of fermentation parameters

Different parameters were optimized for pro-duction of lovastatin by Aspergillus terreus S-24

Table 1. Effect of UV radiation dose to formulate the kill curve.

UV treatment time (min) CFU/mL Ln CFU/mL %age of survival

0 15◊1010 25.73 100

20 7◊109 22.66 88.06

40 13◊108 20.98 81.53

60 14◊107 18.75 72.87

80 18◊106 16.70 64.9

100 8◊105 13.59 52.8

120 7x104 11.15 43.33

140 9x103 9.1 35.3

160 2x102 5.29 20.5

180 5x10 3.91 15.15

210 2 0.69 2.68

Values (Mean ± SD) are average of three samples; CFU: Colony forming units/mL; %age of survival = (Colony countafter exposure time t) ◊100/Total colonies; % age of killing = 100 - %age of survival

Table 2. Estimation of lovastatin production by parent and mutant Aspergillus terreus strain.

Aspergillus terreus Dry cell mass Lovastatin Strain (mg/mL) (mg/100 mL)

Parent 5.37 ± 1.25 16.74 ± 1.09

ATU-01 4.22 ± 0.61 27.23 ± 1.81

ATU-02 4.15 ± 0.14 31.87 ± 0.76

ATU-03 5.16 ± 0.13 37.54 ± 1.01

ATU-04 5.29 ± 1.12 39.77 ± 0.61

ATU-05 4.28 ± 1.31 29.65 ± 1.11

ATU-06 5.37 ± 0.29 51.66 ± 0.88

ATU- 07 4.17 ± 1.31 25.62 ± 1.31

ATU-08 3.27 ± 1.29 43.58 ± 0.83

ATU- 09 4.14 ± 1.41 34.35 ± 0.51

ATU-10 6.64 ± 0.61 44.64 ±1.19

Values (Mean ± SD) are average of three samples, Analyzed individually in triplicate (n = 1 ◊ 3 ◊ 3) p ≤ 0.05;Vogel medium (KH2PO4, 0.5; peptone, 0.1; yeast extract, 0.2, NH4NO3, 0.2; (NH4)2SO4, 0.4; MgSO4, 0.02; glu-cose, 50%; trisodium citrate, 0.5g/100 mL); ATU, UV treated Aspergillus terreus; Temperature, 30OC,Incubation time 48 h


parent and Aspergillus terreus ATU-06 mutantstrain. The Vogel medium was fermented in tripli-cate in flasks with Aspergillus flavus containingselected substrate wheat bran at varying pH, tem-perature, inoculum size, moisture contents, fermen-tation time and nitrogen sources.

Immobilization of mutant ATU-06

All the immobilization processes were per-formed under aseptic conditions. The cell pellet ofA. terreus ATU-06 was obtained in logarithmicphase of growth was collected by centrifugation at5000 rpm in centrifuge (Mikro 20 Hettich,Germany). 0.38 g fungal cells were obtained fromculture mixed with 10 mL sodium alginate solutionat a concentration of 3% (w/v) through a sterilepipette. Beads of 2 mm diameter were obtained bydropping mixtures into sterile CaCl2 (0.1 M). Thebeads from 10 mL gel were used for inoculation of25 mL of the production medium (12). For entrap-ment in agar (0.38 g) and agarose (0.6 g) wet cellswere mixed with 10 mL of 3% agar solution at 45OC.The mixture was allowed to quickly cool at 4OC, cutinto 2 ◊ 2 ◊ 2 mm fragments and transferred to 25mL of the production medium.

Repeated batch experiment

The experimental set up was similar to theprocess described above. Every 96 h, immobilizedcells were removed, washed with saline and reculti-vated into fresh medium. The process was repeatedfor several batches till the beads started disintegrat-ed.


Statistical significance of the differencesbetween mean values was assessed two way analy-sis of ANOVA under CRD and DMR test usingMinitab 2000 version 13.2 statistical software(Minitab Inc., Pennsylvania, USA). A probability

value of p ≤ 0.05 was considered to denote a statis-tically significant difference (13).

RESULTS

Induction of mutation

In present investigation, efforts have beenmade to improve the native Aspergillus terreus strainby UV irradiation for enhanced production of lovas-tatin. In the case of UV irradiation treatment above15.5% survivor rate was obtained after 180 min,which decreased gradually with the increase ofexposure time (Table 1). These mutants were furtherevaluated through secondary metabolite productionby solid state fermentation. The selected 10 mutantsfrom UV mutants of Aspergillus terreus were usedfor the production of lovastatin (selected on thebasis of their ability to produce maximum lovas-tatin), using bagasse for the selection of best mutant.Among theses, the maximum lovastatin production(51.66 ± 0.88 mg/100 mL) with dry cell mass (6.64± 0.61 g/L) was observed for the mutant ATU-06using bagasse (Table 2). The results indicated ATU-06 was the most hyper-active mutant, givingapproximately 5.67 fold more lovastatin over theparent strain (16.74 ± 1.09 mg/ 100 mL) in similarculture conditions.

Kinetic parameters

Comparison of lovastatin production by parentAspergillus terreus S-24 strain and mutant ATU-06strain is presented in Table 3. The parent and mutantstrain of Aspergillus terreus showed maximumlovastatin production 12.74 ±1.09 mg/100 mL and28.31±0.96 mg/100 mL before optimization of envi-ronmental factors and it was further improved to27.66 ± 0.88 mg/100 mL and 87.33 ± 1.94 mg/100mL, respectively, on post optimization. The growthparameters for pre and post optimization of growthyield coefficients, volumetric rates and specific sub-

Table 3. Comparison of lovastatin production parameters pre and post optimization of the fermentation process among parent and select-ed Aspergillus terreus mutant ATU-06 in solid state fermentation.

Parameters Aspergillus Cell biomass Sugar consumed Lovastatin terreus (g/L) (g/L) (mg/100 mL)

Pre Parent 5.71 ± 1.21 30.1 ± 1.86 12.74 ± 1.09

Optimization Mutant 4.22 ± 1.01 15.21 ± 0.85 28.31 ± 0.96

Post Parent 5.42 ± 0.52 25.7 ± 1.84 27.66 ± 0.88

Optimization Mutant 3.67 ± 0.41 7.28 ± 1.92 87.33 ± 1.94

Optimum pH (6), temperature 30OC, inoculum size 2 mL, moisture contents 60% and incubation time 120 h for both par-ent and mutant strain; Values (Mean ± SD) are average of three samples, Analyzed individually in triplicate (n = 1 ◊ 3 ◊3) p ≤ 0.05; Lovastatin was produced in basal medium containing initial sugar concentration 50 g/L (w/v).


strate rates were also studied. The mutant strain ofAspergillus terreus ATU-06 significantly improvedthe values of Yx/s, (drug produced/g glucose con-sumed), Yp/x, (drug produced/g cell formed) andYp/s (Cell formed/g sugar consumed) (1.86 mg/g,5.42 mg/g and 3.66 mg/g/h) over the parental strainAspergillus terreus (0.42 mg/g, 2.23 mg/g and 5.27mg/g/h) before optimization, and on optimization itwas observed that these growth parameters were fur-ther improved by parent (1.07 mg/g, 9.53 mg/g and5.09 mg/g/h) as well as mutant strain (11.99 mg/g,23.79 mg/g and 1.98 mg/g/h), respectively (Table 4).On monitoring the culture for these parameters Qs (gsugar consumed/L/h), qs (g sugar consumed/L/h) and

Yx/s (drug produced/g glucose consumed) it wasobtained a significant increase in these variablesindicating that mutant derivative was a faster grow-ing organism. It is also inferred from the results thatmaximum drug was produced (QP) 1.69 g/L/h bymutant strain of Aspergillus terreus ATU-06 and itwas increased to 5.23 g/L/h on optimization of fer-mentation parameters.

Immobilization of Aspergillus terreus ATU-06

mutant

The best mutant Aspergillus terreus ATU-06was immobilized on calcium alginate and agar byentrapment method. It was obtained that effective-

Figure 2. Lovastatin production by free and immobilized cells by repeated batch fermentation of Aspergillus terreus ATU-06

Table 4. Comparison of kinetic parameters for lovastatin productivity among parent and selected Aspergillus terreus mutant ATU-06 insolid state fermentation.

Kinetic Aspergillus Pre optimization Post optimizationparameters terreus Parent ATU-06 Parent ATU-06

Aµ (h-1) 0.105 0.156 0.141 0.187

Substrate BYx/s 0.42 1.86 1.07 11.99

consumption CYp/x, 2.23 5.42 9.53 23.79

parameters Dqs (g/g/h) 5.27 3.60 5.09 1.98EQs (g/L/h) 1.806 0.9126 1.542 0.437FQx (g/L/h) 0.342 0.25 0.32 0.22

Product GYp/s 0.189 0.277 0.21 0.50formation Hqp (Product/h/g) 1.21 1.99 2.49 5.34parameters IQp (Product/L/h) 0.7644 1.69 3.09 5.23

Product was produced in basal medium, initial sugar concentration was 50 g/L (w/v); Aµ (h-1), Specific growth rate; BYx/s, drug produced/gglucose consumed; CYp/x, drug produced/g cell formed; Dqs (g/g/h), g sugar consumed/ g cells/h; EQs (g/L/h), g sugar consumed/L/h; FQx(g/L/h), g cells formed/L/h; GYp/s, Cell formed/g sugar consumed; Hqp (Product/g/h), drug produced/h/g cells; IQp (Product/L/h), drug pro-duced/L/h


ness of the immobilization was less than one (0.683,0.689 and 0.72 for agar, agarose and calcium algi-nate immobilized cells, respectively) and the activi-ty of immobilized cells was lower than free cells(Table 5). On comparison of both carriers for immo-bilization it was concluded that calcium alginatebeads exhibited 0.72 effectiveness factor higher thanagar and agarose (i.e. 0.683 and 0.689).

The possibility of repeated use of immobilizedcells of Aspergillus terreus was studied. It was foundthat multiple uses of immobilized cells were possi-ble as compared to free cells. Results have shown inFigure 2 indicated that free cells could not retainedtheir ability to produce lovastatin consistently after2 cycles with residual activity of 47.90% whileimmobilized cells showed the ability to producelovastatin after five cycles with 62.9% residualactivity. This loss in activity was due to destructionof cells as well as autolysis during the centrifugationand washing process.

DISCUSSION

Industrial strain improvement plays a key rolein the development of microbial fermentationprocess commercially. The improvement of lovas-tatin producing strain has been carried out by muta-genesis and selection. The most employed techniquehas been the induction of mutation in parental strainby mutagens (9). The mutants produced as a resultof induced mutation are used for commercial sec-ondary metabolite production (10). Among allmuatagens, UV radiation, chemical mutagens andgamma radiations are often used. In present study,strain of Aspergillus terreus S-24 was improved byUV irradiation for hyper production of lovastatin(Table 1 and 2). Similar trend of decrease in surviv-ability with increase in exposure time has also beenreported by some other investigators (14). Geneticchanges in organisms by UV irradiation increase theyields of certain chemicals by the organisms.

Samiee et al. (15) found Aspergillus terreus as a bestlovastatin (55 mg/L) producing strain out of 110fungal strains including some selected strains.Mukhtar et al. (14) reported the mutant strain UV-4gave the maximum lovastatin production (1396.09 ±0.08 µg/mL) with a dry cell mass of 19.47 ± 0.11mg/mL. All these findings indicated that the surviv-ability of parent strain depended on the nature of themicroorganism, treatment period and the type ofmutagens.

Optimization of environmental factors formaximum production of secondary metabolites isvery important. The findings of Mukhtar et al., (14)were in accordance to our results. They also report-ed the increase in lovastatin production on optimiza-tion of fermentation conditions by Aspergillus ter-reus NRRL-265 mutant strain. Potential kineticparameters for production of lovastatin byAspergillus terreus S-24 and mutant Aspergillus ter-reus ATU-06 during growth in optimized media insolid state fermentation are in Table 3 and 4. Iftikharet al. (16) observed Yp/x and Yp/s 1040 IU/g and2080 IU/g in shake flask while 1650 IU/g and 2750IU/g in fermentor during study of production oflipase by hyper producing mutant of Rhizopusoligosporus DGM: 31. Nadeem et al. (17) reportedthat maximum Yp/x and Yp/s were 264.28 PU/g ofcell biomass and 99.3 PU/g of glucose, respectively,after incubation and maximum cell growth rate0.308 by B. licheniformis N-2 strain after 8 h ofinoculation. He et al. (18) found maximum growthrate of 0.17 by Bacillus sp. after 6 h of inoculation.A maximum growth rate of 0.7, biomass (0.023g ofcells/g of glucose) and product yield (0.0211 U/g)for Bacillus L21 was reported by Genckal et al. (19).

On immobilization of Aspergillus terreus ATU-06 mutant strain it was evident that reuse of straincould be possible (Table 5 and Fig. 2) Our resultswere in close agreement to the Ahmad and Abdel-Fattah (20) findings who also reported lower activi-ty of the immobilized cells when they immobilized

Table 5. Production of lovastatin by free and immobilized Aspergillus terreus ATU-06 cells.

Carrier Lovastatin Effectiveness factor

(mg/100 mL) of immobilizationA

Free cells 87.33 ± 1.94 1.00

Agar 5 9.73 ± 1.78 0.683

Agarose 60.22 ± 1.33 0.689

Calcium alginate 63.13 ± 1.99 0.72

Values (Mean ± SD) are average of three lovastatin production samples, analyzed individually in triplicate (n = 1 ◊ 3 ◊ 3) p ≤ 0.05;AActivity of immobilized cells/activity of the free cells of the same amount


bacterial cells on different carriers by differentmethod. Less accessibility of nutrients to immobi-lized cells and low availability of oxygen can causedecreased activity of immobilized cells.Immobilization also changes the metabolic and mor-phological changes in the cells. Farag et al. (21)reported that Aspergillus terreus cells are reused foreight successive cycles but the activity increasedslightly up to four cycles then the reuse cultureexhibited a gradual decrease up to eight run. Woolimmobilized Bacillus licheniformis ATTC 21415was useful for 5 batches reported by Ahmed andAbdel-Fattah (20).

CONCLUSION

It was concluded from above study that Asper-gillus terreus ATU-06 showed better results for theproduction of lovastatin and immobilized cells weremore efficient for lovastatin production than freecells for repeated use. The present data would cer-tainly help to ascertain the ability of ATU-06 mutantas potential source of hypocholesterolemic moleculelovastatin to be used in clinical treatments.


The authors declare no potential conflicts ofinterest with respect to the research, authorship,and/or publication of this article.

Acknowledgment

The data of the paper are the part of the M.Phil. thesis of second author (Amair Raza).

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Received: 4. 10. 2016


Glucotoxicity, lipotoxicity, and glucolipotoxi-city are secondary phenomena that are proposed toplay a role in all forms of type 2 diabetes. Theunderlying concept is that once the primary patho-genesis of diabetes is established, involving proba-bly both genetic and environmental forces, hyper-glycemia and very commonly hyperlipidemia ensueand thereafter exert additional damaging or toxiceffects on the β cells. Elevations of plasma fatty acidlevels that often accompanying insulin resistance (1,2) may also play a pathogenic role in the early stagesof the disease as glucose levels begin to rise outsideof the normal range, as well as their contribution onthe deterioration of β cell function after the onset ofthe disease (3).

The abnormal metabolic medium created byhyperglycemia or the diabetic state initiates series of

dysfunctional responses culminating in the develop-ment of premature cardiovascular disease (4, 5). Theoxidative stress pathway serves, as a common ele-ment, to link all the major pathways implicated indiabetic vasculopathy (6). Hyperglycemia inducesoxidative stress in vascular cells by enhancing theproduction of reactive oxygen species (ROS). Theseresults in damage to cellular proteins, membranelipids, DNA and activation of nuclear factor-κB(NF-κB) (5).

Fruits and vegetables are primarly food sourcesproviding essential nutrients for sustaining life. Theyalso contain a variety of phytochemicals such as phe-nolics and flavonoids providing important health ben-efits. Thus, regular consumption of fruits and vegeta-bles is associated to reduced risks of chronic diseases,such as cancers and cardiovascular disease (7).

THERAPEUTIC EFFECT OF BRASSICA RAPA VAR RAPIFERA IN TYPE 2 DIABETES INDUCED BY A HIGH FAT-SUCROSE DIET OF

PSAMMOMYS OBESUS

LEILA SMAIL1*, SIHEM BERDJA1, BOUALEM SAKA2, SAMIA NEGGAZI, SALIHA BOUMAZA1,YASMINA BENAZZOUG3, GHOUTI KACIMI4, LYNDA BOUDARENE2

and SOUHILA AOUICHAT BOUGUERRA1

1Laboratory of Physiology of Organisms, Team of Cellular and Molecular Physiopathology, Faculty of Biological Sciences, University of Sciences and Technology, Houari Boumedienne,

(USTHB) BP 32 El Alia, 16011 Algiers, Algeria2Laboratory of Organic and Functionally Analysis, Faculty of, Chemistry,

USTHB, BP 32 El Alia, 16011Algiers, Algeria3Team of Biochemistry and Extracellular Matrix Remodeling, Faculty of Biological Sciences,

USTHB, BP 32, El Alia, 16011 Algiers, Algeria4Laboratory of Biochemistry of Central Hospital of Army, Ain Naadja, 16005 Algiers, Algeria

Abstract: This study was made to investigate the effect of a diet rich in fat and sucrose and therapeutic involve-ment of aqueous extract of Brassica rapa (Br). Diabetic rats were submitted to a natural diet supplemented inhigh fat (10%) high sucrose (20%)/per day for 9 months. After this period, we have administered the treatmentwith aqueous extract of Br via oral gavage at 200 mg/kg during 20 days. The biochemical parameters weremeasured in the plasma and the inflammatory-oxidative stress markers were measured in the supernatant ofaorta and heart tissues. The results showed that Br extract reduced the metabolic disorders, the inflammatoryand oxidative markers in plasma and homogenate tissue of rats. These results suggested that the aqueous extractof Br enhanced the anti-diabetic, anti-inflammatory and antioxidant defense against reactive oxygen speciesproduced under type 2 diabetes and hyper-insulinemic conditions.

Keywords: type 2 diabetes, insulin, Brassica rapa, inflammation, oxidative stress

1493

* Corresponding author: e-mail:[email protected]; phone: 0213 792533364

1494 LEILA SMAIL et al.

Brassica Rapa (Br) has active biological com-pounds such as flavonoids (isorhamnetin, kaempfer-ol and quercetin glycosides), phenylpropanoidsderivatives (8), indole alkaloids and sterol glucosides(9). Indole fraction from Brassica rapa roots isknown to possess anti-inflammatory potential viainhibition of pro inflammatory mediators such TNFαand interleukin-6 (10), antidiabetic effect in diabetesmellitus type 2 (11, 12). We found out that it wasinteresting to induce a glucolipotoxicity in vivo by ahigh fat high sucrose diet, in Psammomys obesus andanalyze the Brassica rapa therapeutic effect.

EXPERIMENTAL

Biological material

The present study was approved by theInstitutional Animal Care and Use Committee of theUniversity of Bab Ezzouar (Algiers, Algeria; Permitnumber for the present research project:(F00220110048) and has been achieved accordingto the Executive Decree no. 10ñ90 completing theExecutive Decree no. 04ñ82 of the AlgerianGovernment, establishing the terms and modalitiesof animal welfare in animal facilities.

Psammomys obesus gerbils also known as theìfat sand ratî were captured in the Algerian Sahara.During two week acclimation period, the animalswere fed with the halophilic plants, rich in water andmineral salts (13), that they normally eat in thedesert. They were put in individual cages under con-trolled temperature and lighting conditions, withfree access to food and water. They were dividedinto four groups. The first group (n = 8 normal con-trol), the second group (n = 8 normal treated with Br200 mg/kg during 20 days by oral gavage) were fed,during 9 months, by natural plants from the samehalophilic family during 9 months, but growingalong the edge of sea (salicornia; composition per100 g: water 80.8 g; mineral salts 6.9 g; lipids 0.4 g;proteins 3 g; carbohydrates 8.4 g; and 45-50kcal/100 g). The third group (n = 8 diabetic control)and the fourth group (n = 8 diabetic treated with Br200 mg/kg during 20 days by oral gavage) received

during 6 months a high-fat (10%) and high sucrose(20%) diet, comprising the salicorne plants plus thedaily addition of half of cooked egg yolk and 20 g ofsaccharose per day (composition of egg per 100 g:water 40-60 g; proteins 13.5 ñ 17.5 g; carbohydrates0.2 g; lipids 30 ñ 31 g; cholesterol 1.2 ñ 1.3 g; and370 ñ 400 kcal/100g).

Preparation of aqueous extract of Brassica rapavar rapiferra

Fresh roots of B. rapa var. rapiferra were col-lected from Algeriaí markets in February 2012.Voucher specimen (INA/P/No 6) has been kept inthe herbarium of the Department of Botany,National Institute of Agronomy (INA), Algiers,Algeria. The roots were separated from turnip tops,each cut into small slices and dried in shade till com-plete drying. The plant material was pulverized intopowdered form. The aqueous extract was preparedby decocting powdered roots (100 g) three times tillcomplete exhaustion, then the collected aqueousextract was lyophilized (Cryodos 80, -75OC, 5 m3/h)to obtain extract in yield of 0.15%. The extract wasstored in sealed glass vials at ± 4OC prior to be test-ed and analyzed.

Chemical study

Total phenolic content

Total phenolic contents of the extract weredetermined using FolinñCiocalteu reagent accordingto the method of (14), using gallic acid as a standard,and as modified by (15). An aliquot (0.2 mL) ofextract solution containing 1000 µg of extract wasmixed with 46 mL of distilled water and 1 mL ofFolin-Ciocalteu reagent in a volumetric flask. Afterspending 3 min in the dark, 3 mL of sodium carbon-ate solution (7.5%) was added. Absorbance at 740nm was measured in a spectrophotometer(Shimadzu 1800, Mulgrave, Victoria, Australia)after shaking and spending an additional 2 h in thedark. The total phenolic content was evaluated froma standard calibration curve of gallic acid, andresults were expressed as microgram of gallic acidequivalents per milligram of extract (µgGAE/mg).

Table 1. Total phenolic and flavonoid contents and antioxidant activity of Brassica rapa var. rapiferra (Br).

Extract / Total phenolic Total flavonoids DPPH Standards content (µg GAE/mg) (µg QE/mg) (IC50)

Aqueous extract 9.41 ± 0.18 1.01 ± 0.09 2100 ± 13

BHA n.a n.a 21.28 ± 0.12

BHT n.a n.a 12.76 ± 0.08

Each value is expressed as the means ± standard deviations for triplicate experiments. n.a: not applied.

Therapeutic effect of Brassica rapa var rapifera in type... 1495

Determination of total flavonoids

The total flavonoids were determined accord-ing to the Dowd method described by (15). 4 mL ofdiluted solution of extract was mixed with 4 mL ofaluminium trichloride solution (2% in methanol).After 15 min, the absorbance was measured at 415nm. Quercetin was used as reference compound toproduce the standard curve. The results areexpressed as µgQE/mg.

Antioxidant activity: Scavenging effect on DPPH

radical

The DPPH free radical scavenging assay wascarried out as described by (16). The aqueous extractwas dissolved in methanol. Sample of 25 µL of eachconcentration (100, 200, 400, 600, 800 and 1000µg/mL) were added to the DPPH methanol solution(60 µM, 975 µL). After 30 min of incubation at25OC, the absorbance at 517 nm was measured byusing UV spectrophotometer (Jasco, V-530).Ascorbic acid and α-tocopherol were used as refer-ence compounds. The radical scavenging activitywas then calculated from the equation: % of radicalscavenging activity = [(Abscontrol ñ Abso)/Abscontrol] ◊100, where Abscontrol is the absorption of the blanksample and Abssample is the absorbance of the testedextract.

Biological study

Analytical methods

The animals were bled from the retro-orbitalvenous plexus; this technique eliminates the use ofanesthetic agents affecting measurements of bio-chemical parameters. Blood, collected in dries tubes,was centrifuged at 3000 rpm for 10 min and stored at-30OC. Blood glucose, triglyceride, cholesterol andprotein were measured by enzymatic colorimetricmethod using a test kit of Biosystem. Blood insulinwas determined, by radioimmunoassay, using CIStest kit (ORIS INDUS) CPK. In addition, uric acidwas determined by the automate CKL, 0-323.

Isolation of tissue

At the end of the experiment, animals werekilled after anesthesia with urethane (25%). Theaorta and heart tissues were immediately excised,frozen in liquid azote and stored at -80OC. The tissuewas extracted in ice-cold solubilization buffer byusing a motor-driven Potter homogenizer (20 mM/LHEPES, 8 mM/L EDTA, 0.2 mM/L Na3VO4, 10mM/L Na4P207, 2.5 mM/L phenylmethylsulfonylfluoride, 1 mg/mL aprotinin, 2.5 mg/mL benzami-dine, 2.5 mg/mL pepstatin, 2.5 mg/mL leupeptin,160 mM/L NaF, 2 mM/L dichloroacetic acid, 1%

Triton X-100, pH 7.4. After 20 min at 4OC, the sam-ples were centrifuged at 20000 × g, the supernatantswere stored at -80OC.

Determination of malondialdehyde (MDA)

The MDA was evaluated after reaction withthiobarbituric acid (TBA) (Sigma) (17). The sam-ples were centrifuged at 10000 × g for 20 min at 4OCin buffered (Na2HPO4/NaH2PO4) 0.2 M, pH 6.5. TheMDA contained in the supernatant in the presence of10% TCA reacts with TBA and causes the formationof a complex read at 532 nm.

Catalase activity determination (CAT)

CAT activity was determined by monitoringthe disappearance of H2O2 at 240 nm. The reactionmixture contained 50 mM K-phosphate buffer (pH7) 0,33 mM H2O2 and enzyme extract (18).

Measurement of inflammation insulin pathway

markers

The assessment was determined by immunoen-zymatic assay. Invitrogen ELISA Kits were used formeasuring the levels of different markers in the tis-sue. IRS 1 [S312], AKT [S p 473], and NF-κB p65.The estimation is made by Elisa reader at 450 nm(BioTek Instruments).


Data were analyzed with ANOVA using STA-TISTICA version 6 and completed with HSD Tukeytest. The results were expressed as the mean ± stan-dard deviation.

The differences at level p < 0.05 were consid-ered to be statistically significant.


Chemical study

Total phenolics and flavonoids contents

The aqueous extract showed a weak amount ofphenolic and flavonoid compounds with values of9.41 ± 0,18 µg GAE/mg and 1,01 ± 0,09 µg QE/mg,respectively (Table 1).

Antioxidant activity

The principle antioxidant activity was based onthe availability of electrons to neutralize any freeradicals. In the present study, the antioxidant activi-ty was evaluated using scavenging DPPH free radi-cals assay. The results of the aqueous extract andthose controls (BHA and BHT).

The investigated aqueous extract of Brassicarapa showed a weak antiradical activity with IC50


value of 2100 ± 13 µg/mL, which was extremelylower than the high antioxidant effect of BHA andBHT (IC50 = 21.28 ± 0,12 µg/mL and 12.76 ± 0.08µg/mL, respectively) (Table 1).

Effect of Br on biochemical parameters of PoAs concerns animals submitted to natural diet

supplemented with high fat-sucrose diet (P diabetic)during 9 months of experiment, we noted a highincrease of serum glucose, proteins and dyslipi-demia which was marked by TC, TG, VLDL, LDLand decreased HDL in P diabetic. Therefore, a high-ly significant increase, of cardiac marker CPK andLDH and kidney marker creatinine were observed inP diabetic rats compared to P control. The treatmentwith aqueous extract of Brassica rapa (Br) amelio-rated the deregulation observed under a high fat highsucrose diet (Table 2a).

Effect of Br redox statues of PoThe elevation of plasma and erythrocyte MDA

level in the group of P diabetic was highly signifi-

cant but reversed with the Cat in this condition. Thetreatment with of Br prevented this oxidative stress(Table 2b).

The evaluation of insulinemia showed anincrease after 3 months and a decrease after 9 monthof experiment (Fig. 1).

Effect of Br on inflammatory parameters and

redox statues of PoIn both aorta and heart tissue, the AKT level

was decreased while the NFκB level was increasedin P diabetic. The treatment with Br modulated theseparameters. The evaluation of redox status parame-ters showed an increase of MDA and a decrease ofCat levels in aorta and heart of P diabetic comparedto P control, and reversed with treatment of Br in Pdiabetic +Br (Table 3 and 4).

This study emphasized on the protective effectin vivo of aqueous extract of Brassica rapa (Br) in ahigh fat-sucrose diet treated diabetic rats.

Total phenolics and flavonoids contents weredetermined according to their importance as an

Table 2a. Effect of aqueous extract of Br in glucose, lipid profile, CPK and protein level after the administration of high fat-sucrose dietin Psammomys obesus (Po).

Parameters P control P control + Br P diabetic P diabetic + Br

Glucose (g/L) 0.65 ± 0.05 0.62± 0.06 1.81± 0.12 **** 1.15± 0.13++

Triglyceride (TG) (g/L) 0.38 ± 0.08 0.37 ± 0.09 3.46 ±167**** 1.68 ± 0.4++

Cholesterol total (TC)(g/L) 0.9 ± 0.06 0.81± 0.02 4.88 ± 0.22**** 2.35 ± 0.53++

LDL-chol. (mM/L) 0.54 ± 0.26 0.49 ± 0.01 27.55 ± 3.18**** 0.64 ± 0.14++++

HDL- chol. (mM/L) 1.89 ± 0.21 1.71 ± 0.1 1.05 ± 0.08** 5.37 ± 0.67++++

Creatinine (U/L) 28 ± 1.41 25 ± 0.01 114 ± 17.61**** 44.75 ± 14.08++++

CPK (U/L) 4 ± 0.1 4 ± 0.5 642.5± 114.49**** 90.75 ± 7.41++++

LDH (U/L) 4 ± 2.82 3 ± 1.13 92 ± 10.10**** 13.75 ± 4.27++++

Protein (mg/dL) 0.67±0.03 0.66 ±0.06 0.87 ± 0.00** 0.66 ± 0.03++

Data are expressed as the mean ± S.E.M; (n = 8). **p < 0.001, ****p < 0.0001 (P diabetic vs P control), ++p < 0.001, ++++p < 0.0001 (Pdiabetic + Br vs. P diabetic); P control: Psammomys obesus control; P control + Br: Psammomys obesus control treated with extract aque-ous of Brassica rapa; P diabetic: Psammomys obesus received a high-fat (10%) and high sucrose (20%) diet; P diabetic + Br: Psammomysobesus received a high fat-sucrose diet and treated with extract aqueous of Brassica rapa.

Table 2b. Evaluation of the oxidative stress parameters in the blood of Po submitted at high fat-sucrose diet and treated with aqueousextract of Br.


Catalase (U/L) 0.17 ± 0.01 0.35 ± 0.03 0.07 ± 0.00**** 0.61 ± 0.01++

Catalase eryth. (U/L) 1.43 ± 0.26 2.19 ± 0.08 1.20 ± 0.08 2.36 ± 0.64++++

MDA (µM/L) 72.8 ± 5.31 71.2 ± 3.65 149.8 ± 2.69**** 71.4 ± 0.89++++

MDA eryth (µM/L) 87 ± 1.41 80 ± 0.56 108 ± 1.58**** 92.2 ± 3.96++++

Data are expressed as the mean ± S.E.M; (n = 8). **p < 0.001, ****p < 0.0001 (P diabetic vs P control), ++p < 0.001, ++++p < 0.0001 (Pdiabetic + Br vs P diabetic); Eryth.: erythrocyte.


antioxidant compounds. In fact, there is a relation-ship between the antioxidant ability and the totalphenol contents. Phenolic antioxidants are productsof a secondary metabolism in plants, and the antiox-idant activity is mainly induced in their redox prop-erties and chemical structure, which can play animportant role in chelating transitional metals,

inhibiting lipoxygenase and scavenging free radicals(19). The principle antioxidant activity is based onthe availability of electrons to neutralize any freeradicals. In the present study, the antioxidant activi-ty was evaluated by determining IC50, using scav-enging DPPH free radicals. The value of IC50 wasused in the in vitro study. This activity could be

Figure 1. Effect of aqueous extract of Br in insulinemia after the administration of high fat-sucrose diet in po. Data are expressed as the

mean ± S.E.M; (n = 8). ****p < 0.0001, **p < 0.01 (P diabetic vs. P control), NS, ++++p < 0.0001 (P diabetic + Br vs. P diabetic).

Table 3. Effect of aqueous extract of Br in the inflammation-oxidative stress parameters of the aorta after the administration of high fat-sucrose diet in Po.


MDA (pM/100 g) 2.68 ± 2.27 2.65 ± 2.00 179.75± 44.8**** 3.03 ± 1.04++++

Catalase(UI/min/g protein/100 g)

35.93 ± 5.74 33.8 ± 2.6 6.74 ±139**** 24.38 ± 8.10++++

AKT (pg/mL) 1.15 ± 0.53 1.04 ± 0.43 0.41 ±0.29**** 1.01 ± 0.04++++

NFκB (ng/mL) 9.80 ± 0.63 9.76 ± 0.54 92.70 ± 0.63**** 16.3 ± 0.59++++

Data are expressed as the mean ± S.E.M; (n = 8). ****p < 0.0001 (P diabetic vs.. P control), ++++p < 0.0001 (P diabetic + Br vs P dia-betic)

Table 4. Effect of aqueous extract of Br in the inflammation-oxidative stress parameters of the heart after the administration of high fat-sucrose diet in Po.


MDA (pM/100g) 1.21 ± 0.27 1.18 ± 0.10 1.98 ± 0.08** 0.89 ± 0.20++

Catalase(UI/min/g protein/100 g)

17.63 ± 11.52 16.33 ± 0.69 10.72 ± 3.53** 30.80 ± 24.46

AKT (pg/mL) 0.15 ± 0.03 0.14 ± 0.02 0.01 ± 0.00**** 0.16 ± 0.01++

NFκB (ng/mL) 4.22 ± 1.32 4.20 ± 8.84 5.92 ± 0.08 4.06 ± 1.78

Data are expressed as the mean ± S.E.M; (n = 8). **p < 0.001, ****p < 0.0001 (P diabetic vs. P control), ++p < 0.001, (P diabetic + Brvs. P diabetic)

insulin(IU/L)


explained by the presence of the phenolics com-pounds which are related to different mechanisms,such as free radical-scavenging, hydrogen-donation,singlet oxygen quenching, metal ion-chelation, andacting as substrates for radicals such as superoxideand hydroxyl (20).

In our study, we found a hyperglycemia asso-ciated to hypertriglyceridemia and hypercholes-terolemia characteristic of type 2 diabetes (21-23).After 20 days of treatment with aqueous extract ofBrassica rapa (200 mg/kg) ameliorated this bio-chemical parameters in diabetic rats. Our resultswere according to (24) in alloxan diabetic rats andtreated with Brassica rapa (200 mg/kg), in strepto-zotocin diabetic rats and treated with different doseof Brassica nigra (25) and in diabetic rats treatedwith Brassica oleracea (26).

The hypoglycemic effect of the seed extract ofBrassica juncea was attributed to the stimulation ofglycogen synthesis leading the increase of hepaticglycogen content and the suppression of glycogenphosphorylase and other glyconeogenic enzymesactivity (27). Of the same, oral administration ofBNO (Brassica Nigra Oil) 1000 mg/kg body weightsignificantly increased the hepatic glycogen levelsin STZ diabetic rats, possibly because of reactivat-ing the glycogen synthase system as a result ofincreased insulin secretion (28). Our results showedan increase of cardiac markers (CPK and LDH) andmarkers of renal function (creatinine). The elevationof CPK is an indicator of cardiac complicationscharacterized by muscle cells lysis (29). The Brextract treated in diabetic rats showed a reduction ofCPK and creatinine levels and our results are similarto (30) in high fat diet and STZ diabetic rats andtreated with Tannin fraction (100 and 200 mg/kg).

Therefore, our results showed that in diabeticanimals the level of MDA was high but the level ofCAT was low in the plasma, erythrocyte and wasrestored to normal values after the treatment with Brextract. Our results were according to (24) in allox-an diabetic rats treated with Brassica extract and inSTZ diabetic rats and treated with flavonoid (31,32). Franciscoa et al., 2009 showed that ethanolicextract of Br is full of phenolic antioxidants particu-larly flavonols and hydroxycinnamic acid (33).These compounds have strong and direct antioxidanteffects and induce expression of different genesinvolved in metabolic enzymes encoding which areeffective in reducing the incidence of diseases risks(34). Karpen et al. reported an elevated levels oflipid peroxides in the plasma of diabetic rats (35).Lipid peroxide-mediated tissue damage is resultedby in the development of both type I and II diabetes.

Low levels of lipid peroxides stimulate the secretionof insulin but when the concentration of endogenousperoxides increases, it may initiate uncontrolledlipid peroxidation, thus leading to cellular infiltra-tion and islet cell damage in type I diabetes (36).The increased lipid peroxidation in the tissues ofdiabetic animals may be induced by the observedincrease in the concentration of TBARS in the liverand kidney of diabetic rats (37).

Catalase is a hem-protein which is responsiblefor the detoxification of significant amounts of H2O2

(38). Reduced activities of catalase in the liver andpancreas during diabetes were reported, resulting ina number of deleterious effects due to the accumula-tion of superoxide radicals and hydrogen peroxide(39). CAT protects tissues from extreme toxichydroxyl through decomposing of hydrogen perox-ide (40). A dual action of polyphenols as bothantioxidant and prooxidant, has been demonstratedin cell culture systems with the common plantpolyphenols, quercetin and epigallocatechin-3-gal-late. These polyphenols have been shown todecrease production of ROS in the cells but alsotrigger the production of peroxides in the medium inthe presence of H2O2 (41, 42). There is a clear linkbetween hyperglycemia and active oxygen/nitrogenspecies in experimental and clinical types of dia-betes (43). Accumulation of reactive oxygen species(ROS) due to oxidative stress is also instrumental inthe expression of cell death as ROS can easily reactwith and oxidize vital cellular components such aslipids, proteins and DNA (44).

In our study, we showed a compensatoryhyperinslinemia associated with hyperlipidemiaafter 3 months of a high fat-sucrose diet indicator ofinsulin resistance. (45) suggested that impairedblood lipids were the characteristic of subjects withinsulin resistance, especially circulating FFAs (46).Fatty acid mobilization from adipose tissue is sensi-tive to insulin. Insulin is most potent acting in thesuppression of adipose tissue lipolysis. A rise ofplasma insulin concentration of only 5 IU/mLinhibits lipolysis by 50%, whereas a reduction inbasal insulin levels results in a marked accelerationof lipolysis (47). Our treatment ameliorates thesedisorders. In (28) it was demonstrated that BNOincreased plasma insulin concentrations in diabeticrats. Insulin levels higher than those of the controlgroup may result an inhibition of lipolysis anddecreased plasma triglyceride and cholesterol levels.These observations were supported by a recent evi-dence that FFAs activate directly macrophage tosecrete pro-inflammatory cytokines that render mus-cle cells insulin resistant (48). Meanwhile, the role


of pro-inflammatory cytokines in regulating insulinsensitivity has been suggested by several lines ofevidence. To this effect, subjects with T2DM exhib-ited higher serum levels of pro-inflammatorycytokines such as TNFα, IL-1β, and IL-6 (49).Moreover, FFAs contribute on the increased pro-duction of reactive oxygen species and lead to theactivation of stress-sensitive signaling pathwaysunder hyperglycemic status (50). The polyphenolsof quercetin had anti-oxidative and anti-inflammato-ry activities (12).

CONCLUSIONS

In conclusion, the present study has demon-strated the potency of aqueous extract of Brassicarapa (Br) to ameliorate hyperglycemia, hyperlipi-demia and insulin resistance in diabetic rats.

Brassica extract suppressed oxidative andinflammation stress in vivo in plasma and aorta andheart tissue in rats fed a high fat-sucrose diet. Theseresults suggest that aqueous extract of Brassica rapa(Br) may have an important implications for the pre-vention and early treatment of T2DM.

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Received: 18. 10. 2016


Compatibility of drotaverine hydrochlorideanion with human gastric juice makes the solubilityof hydrotropic adduct in vivo a condition whichshould translate into optimal biopharmaceuticalparameters of the type tmax, cmax and the AVC area (1-3).

The ability to generate amorphous structures ñin the process of formulation of a pharmaceuticalagent solid form ñ which significantly alter the rateof therapeutic agent dissolution and balance theadsorption isotherm described by the Freundlichequation (x/m = K ∑ c) is the morphological featureof the crystallographic system of therapeutic agenthydrochlorides (hydrotropic adduct) (4-7).

The amorphous form of the therapeutic agent ñin the solid state, has a high value of the internalenthalpy, thus devoid of its own crystal system, andthermodynamically, it will tend to crystallize in theprocess of compression (8-14).

Applicatively amorphous systems are charac-terized by enhanced solubility, which favors signifi-cant improvement of in vitro and in vivo bioavail-ability.

Maintaining thermodynamic stability of theamorphous system in the process of production ispossible by experimental selection of excipients inappropriate proportion or their individual share inexplicit granulometric form (15-21).

PHARMACEUTICAL TECHNOLOGY

MECHANISTIC MODELS IN THE ASSESSMENT OF DROTAVERINEHYDROCHLORIDE SOLUBILITY AFTER TABLET DISINTEGRATION

IN THE PRESENCE OF SELECTED EXCIPIENTS IN PHARMACOPOEIALACCEPTOR FLUID

GRZEGORZ DO£ GA1*, ZBIGNIEW MARCZY—SKI2 and MARIAN M. ZGODA3

1Sanofi-Aventis Ltd., Lubelska 52, 35-233 RzeszÛw, Poland2Department of Pharmacy, Chair of Applied Pharmacy, Medical University,

MuszyÒskiego 1, 90-151 £Ûdü, Poland3Extramural Doctoral Studies, Chair of Applied Pharmacy, Medical University,

MuszyÒskiego 1, 90-151 £Ûdü, Poland

Abstract: Research studies have been carried out on the possibility of producing a solid dosage form with dro-taverine hydrochloride by direct compression technique with the use of individual excipients such as: Vivapur,Prosolv 90 HD, Emdex, Emcompress, lactose and Pearlitol 200 SD. Granulometric parameters of tablet mass,model dosage forms as well as a reference form were analyzed. The determined pharmaceutical availability inthe form of a Q% coefficient was in the time function (t, min) an inspiration for estimating the rate of dissolu-tion of the therapeutic agent in the presence of an excipient and for assessing the impact of the grain structureand surface in the environment of 0.1 M of HCl on the process of its sorption. Kinetic equations of ì0î and ìIîorder and Higuchi, Korsmeyer-Peppas and Hixson-Crowell models, based on the thesis that the process of dis-solution (release) is consistent with Fickís law, were used to describe the process of dissolution of drotaverinehydrochloride in 0.1 M HCl. It results from the analysis of approximation equations, both in the kinetic andmechanistic model (Higuchi, Korsmeyer-Peppas, Hixson-Crowell models), that symmetrically to the dissolu-tion process there comes to adsorption of the therapeutic agent by granulometric grains which points to itsdeficit in the acceptor fluid (Freundlich adsorption isotherm).Formulation with Prosolv 90HD is characterizedby a profile of the rate of dissolution of drotaverine hydrochloride that is compatible with a reference form.Granulometric combination of lactose with Vivapur 101 is also worthy of note from the application point ofview.

Keywords: drotaverine hydrochloride, excipients, tablet, pharmaceutical availability, mathematical models

1501


1502 GRZEGORZ DO£ GA et al.

The aim of the research studies ofapplication nature was to produce amodel dosage form with drotaverinehydrochloride by direct compressiontechnique in different compression con-ditions with individual excipient and toevaluate their impact on the rate of dis-solution process and the rate of masstransfer through the phase boundary.

The model tablet masses (granu-lates) were subjected to morphologicalevaluation and there were made tabletsof the hardness in the range of 35-45 N(22, 23).

The determination of drotaverinehydrochloride pharmaceutical avail-ability in 0.1 M HCl was performed andthe obtained results were the basis forthe use of model systems of mathemat-ical equations for reliable assessment ofthe complex process of the effect of theexcipients on the dissolution rate of thetherapeutic agent (24-28).

The obtained results enable opti-mal design and production of therapeu-tically effective solid oral dosage formwith therapeutic agent hydrochloridei.e., hydrotropic structure of a therapeu-tic agent of high solubility in modelpharmacopoeial acceptor fluids (29,30).

EXPERIMENTAL

Reagents

Therapeutic agent: drotaverinehydrochloride: 6,7-diethoxy-1-[(3,4-diethoxyphenyl)methylene]-1,2,3,4-tetrahydroisoquinoline hydrochloride;C24H32ClNO4

Excipients: Microcrystalline cel-lulose, Ph. Eur., NP., JP: Vivapur 101(φ = 50-65 µm, d = 0.26-0.31 g/cm3),Vivapur 102 (φ = 90-100 µm, d = 0.28-0.33 g/cm3), Vivapur 200 (φ=190-250µm, d = 0.31-0.33 g/cm3), Vivapur 12(φ = 160-180 µm, d = 0.30-0.36 g/cm3);C6nH10n + 205n + 1, J. Rettenmaier andSon (Germany) (31). Prosolv HD 90,(silicified microcrystalline cellulosewith 2% silicon oxide, φ = 90 µm, d =0.42 g/cm3), J. Rettenmaier and Son(31). Lactose, C12H22O11, BorculaDomo-Holland, FP VI, Eur. Pharm. VI,T

able

1. P

resc

ript

ion

com

posi

tion

of m

odel

tabl

ets

with

dro

tave

rine

hyd

roch

lori

de p

rodu

ced

by d

irec

t com

pres

sion

met

hod.

Exc

ipie

nts

Ref

eren

ce

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

form

ulat

ion

No.

1N

o. 2

No.

3N

o. 4

No.

5N

o. 6

No.

7N

o. 8

No.

9

Dro

tave

rine

hydr

ochl

orid

e40

mg

40 m

g40

mg

40 m

g40

mg

40 m

g40

mg

40 m

g40

mg

40 m

g

Lac

tose

++

●●

●●

●●

●●

Viv

apur

101

●●

+●

●●

●●

●●

Viv

apur

102

●+

●+

●●

●●

●●

Viv

apur

200

●●

●●

+●

●●

●●

Viv

apur

12

●●

●●

●+

●●

●●

Pros

olv

90 H

D●

●●

●●

●+

●●

●

EM

DE

X●

●●

●●

●●

+●

●

Em

com

pres

s●

●●

●●

●●

●+

●

Pear

litol

200

SD

●●

●●

●●

●●

●+

Cor

n st

arch

+●

●●

●●

●●

●●

Povi

done

K-2

5+

●●

●●

●●

●●

●

Tal

c+

++

++

++

++

+

Mag

nesi

um s

tear

ate

++

++

++

++

++

Mechanistic models in the assessment of drotaverine hydrochloride... 1503T

able

2. G

ranu

lom

etri

c pa

ram

eter

s of

tabl

et m

ass

prep

ared

for

dir

ect c

ompr

essi

on.

Gra

nulo

met

ric

Ref

eren

ce

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

para

met

ers

form

ulat

ion

No.

1N

o. 2

No.

3N

o. 4

No.

5N

o. 6

No.

7N

o. 8

No.

9

Ang

le o

f re

pose

[O]

42.1

32.8

38.1

30.7

36.2

44.0

40.4

38.6

39.8

39.4

Flow

abili

tys/

100g

56.5

96.4

83.2

39.5

48.9

21.8

22.3

25.2

25.8

26.6

Bul

k w

eigh

t

[g/m

L]

0.51

60.

472

0.37

40.

385

0.41

60.

428

0.51

00.

632

0.83

60.

492

Wei

ght a

fter

ta

ppin

g [g

/mL

]0.

694

0.65

90.

502

0.55

20.

537

0.56

70.

595

0.76

60.

926

0.59

8

Car

r in

dex

25.6

528

.38

25.5

026

.25

27.4

024

.51

14.2

917

.49

9.72

17.7

3

Mas

s m

oist

ure

[%]

5.18

3.29

4.34

4.23

4.50

4.76

4.62

7.50

5.67

2.04

d =

2r [

mm

]7.

07.

07.

07.

07.

07.

07.

07.

07.

07.

0m

t [m

g]14

014

014

014

014

014

014

014

014

014

0

Tab

le 3

. Mor

phol

ogic

al p

aram

eter

s of

tabl

ets

with

dro

tave

rine

hyd

roch

lori

de p

rodu

ced

by d

irec

t com

pres

sion

met

hod.

Tab

let m

orph

olog

ical

Ref

eren

ce

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

Form

ulat

ion

para

met

ers

form

ulat

ion

No.

1N

o. 2

No.

3N

o. 4

No.

5N

o. 6

No.

7N

o. 8

No.

9

App

lied

com

pres

sion

35.7

41.2

43.7

44.8

43.5

42.6

42.7

44.7

43.4

42.3

- lim

it 35

-45N

[+2]

[-4]

[-12

][-

9][-

7][-

7][-

8][-

3][+

8][-

3]

Com

pres

sion

an

d ha

rdne

ss [

N]

34.8

60.7

106.

387

.682

.883

.283

.659

.7o

59.9

Fria

bilit

y(%

)0.

350.

070.

070.

000.

070.

130.

000.

210.

280.

07

Dis

inte

grat

ion

time

[min

]11

.33

25.1

912

.02

21.3

510

.31

6.56

12.1

018

.01

>120

23.1

5


1.2.4.EMDEX (dextrates, USP/NF ñ spray crys-talline dextrin with small amount of oligosaccha-rides, Eur. Pharm. IV-VI), J. Rettenmaier and Son(Germany) (31). EMCOMPRESS, calcium hydro-gen phosphate ñ CaHPO4 ∑ 2H2O, J. Rettenmaier andSon, Eur. Pharm. IV-VI (Germany). Mannitol-PEARLITOL 200 SD, D-mannitol, C6H14O6,(Roquette-France), FP VI, Eur. Pharm. IV. Cornstarch (Roquette-France), FP VI, Eur. Pharm. IV.Magnesium stearate ñ mixture of magnesiumstearate (C18H35O2)2Mg, and magnesium palmitate(C16H31O2)2Mg ñ Peter Graven, Daichi, FP.VI.Povidone K-25 (Kollidon 25), (C6H9NO)n-poly(1-(-2-oxo-1-pyrrolidinyl) ethylene, ñ BASF A.G., FP.VI, Eur. Pharm. IV. Talc ñ natural magnesium sili-cate, Mg3H2Si4O12 ñ Luzenac Naintsch, FP. VI., Eur.Pharm. V.

Manufacturing technology and physicochemical

tests of tablets

Tablet base was prepared by blending the ther-apeutic agent with excipients in a high-speed mixer(Fukae Powtec). Then, lubricants were added and allcomponents were mixed again in the final phase ofthe process.

The obtained tablet formulation was passedthrough a sieve of mesh ϕ = 1.00 mm and subjectedto morphological evaluation by determining:

ñ bulk and tapped density (Erweka SVM 202)ñ the tablet mass angle of repose (Erweka

GTE)ñ tablet mass moisture content (dryer Mettler

LP 16).After measurements, model tablet formulations

were compressed using Manesty BB3 Rotary TabletPress with tablet output/h up to 20-25.000. In orderto obtain comparable results of the compressionprocess there were determined:

ñ initially the force of compression for whichthe reference tablet hardness was within the range35-45 N; the value of this compression wasdescribed as 1,

ñ compression of the model batches of tabletswas also performed in the range of 35-45 N byincreasing the compression which was described as(+) or by decreasing it (-).

The obtained model tablets were subjected tothe following morphological tests:

ñ analysis of uniformity of mass,ñ determination of hardness, thickness and

diameter (ñ apparatus for tablet hardness testingPTB 311 E PHARMA TEST),

ñ determination of abrasiveness (ñ apparatusfor testing tablet friability PHARM TEST PTF ER),

Tab

le 4

. Pha

rmac

eutic

al a

vaila

bilit

y -

rele

ase

coef

fici

ent Q

of

drot

aver

ine

hydr

ochl

orid

e fr

om m

odel

dos

age

form

.

Rel

ease

coe

ffic

ient

- Q

%*

t, m

in √

tR

efer

ence

Fo

rmul

atio

n Fo

rmul

atio

n Fo

rmul

atio

n Fo

rmul

atio

nFo

rmul

atio

n Fo

rmul

atio

n Fo

rmul

atio

n Fo

rmul

atio

nFo

rmul

atio

n fo

rmul

atio

nN

o. 1

No.

2N

o. 3

No.

4N

o. 5

No.

6N

o. 7

No.

8N

o. 9

5/2.

236

35.6

535

.06

28.9

425

.68

21.4

322

.65

37.9

223

.30

15.4

624

.72

10/3

.162

64.4

661

.35

49.5

545

.45

36.4

138

.43

67.1

240

.87

29.3

447

.24

15/3

.872

85.5

480

.00

64.5

761

.22

47.9

849

.78

85.9

355

.36

41.4

662

.89

20/4

.472

94.3

193

.62

77.6

573

.29

55.9

159

.15

96.7

767

.52

53.4

974

.08

25/5

.000

99.1

210

1.94

87.8

784

.87

63.4

065

.94

101.

9277

.91

64.2

584

.47

30/5

.477

103.

6610

6.57

93.9

992

.58

70.3

272

.12

103.

1185

.36

84.4

788

.38

* nu

mer

ical

val

ues

of th

e re

leas

e co

effi

cien

t Q a

re th

e m

ean

of 3

-5 m

easu

rem

ents

.

Mechanistic models in the assessment of drotaverine hydrochloride... 1505

ñ determination of disintegration time (ñ disintegra-tion test apparatus Erweka).

Prescription composition of model formula-tions and the determined and calculated morpholog-ical parameters are demonstrated in Tables 1-3.

Profiles of drotaverine hydrochloride pharmaceu-

tical availability from model tablets in the envi-

ronment of pharmacopoeial acceptor fluids

The determination of the rate of dissolution ofdrotaverine hydrochloride from model tablets in the

Figure 1. Physicochemical processes observed during tablet disintegration in the presence of granulometrically defined excipient grain

1. Phase of tablet disintegration

2. Mixed phase after the process of tablet disintegration


Table 5. Correlation equations describing the kinetics of drotaverine hydrochloride solubility in 0.1 M HCl in the presence of an excipi-ent, p = 0.05

Medium Correlation Equation slopeformulation

Function Equation coefficient Ñr2î Ñaî Ñbî

log y = a + b∑1/x 0.9983 2.1098 -2.8145Q = F(t)y = a + b∑log x 0.9921 -24.6941 89.4753

Reference y = a + b∑1/x 0.9964 151.7962 -262.9316formulation Q = f(√t) log y = a + b∑1/x 0.9941 2.3644 -1.78570.1 M HCl y = a + b∑log x 0.9921 -24.6825 178.9410

3√100 ñ Q = f(t) y = a + b∑log x 0.9591 6.8157 -.8121

y = a + b∑log 0.9921 -24.6948 89.4753Q = F(t)y = a + b∑1/x 0.9848 113.7634 -407.8371

Formulation log y = a + b∑1/x 0.9992 2.3787 -1.8610No. 1 Q = f(√t) y = a + b∑log x 0.9984 -31.7444 189.7685

0.1 M HCl x log y = a + b∑log x 0.9881 1.1347 1.2578

y = a + b∑log x 0.9451 6.5658 -3.44733√100 ñ Q = f(t)y = a + b∑y 0.9163 4.3141 -0.1028

y = a + b∑log 0.9960 -33.1367 85.2896Q = F(t) log y = a + b∑log x 0.9961 1.0440 0.6646

log y = a + b∑log x 0.9862 2.0423 -3.0217

Formulation y=a+b∑log x 0.9967 -34.6695 172.9230No. 2 Q = f(√t) log y = a + b∑log x 0.9953 1.0037 0.1799

0.1 M HCl log y=a + b∑1/x 0.9991 2.3342 -1.9920

y = a + b∑log x 0.9451 6.5658 -3.44733√100 ñ Q = f(t)y = a + b∑x 0.9163 4.3141 -0.1028

log y = a + b∑log x 0.9938 0.9233 0.7196Q = F(t)y = a + b∑x 0.9885 17.3653 2.6561

Formulation y = a + b∑x 0.9994 -20.4866 20.8926No. 3 Q = f(√t) log y = a + b∑l/x 0.9983 2.3409 -2.1079

0.1 M HCl log y = a + b∑log x 0.9971 0.9234 1.4392

y = a + b∑x 0.9992 4.6262 -0.09313√100 ñ Q = f(t)log y = a + b∑x 0.9885 0.7092 0.0141

y = a + b∑log x 0.9967 -23.3011 63.4020Q = F(t) log y = a + b∑log x 0.9959 0.9234 0.6438

log y = a + b∑1/x 0.9872 1.9194 -2.9304

Formulation y = a + b∑x 0.9992 -11.3131 15.0018No. 4 Q = f(√t) log y = a + b∑log x 0.9985 2.1861 -1.9328

0.1 M HCl log y = a + b∑1/x 0.9969 0.8865 1.3191

y = a + b∑x 0.9981 4.6942 -0.08923√100 ñ Q = f(t)log y = a + b∑x 0.9849 0.7096 0.0132

y = a + b∑log x 0.9952 -24.1156 62.4215Q = F(t) log y=a + b∑log x 0.9969 0.8865 0.6595

y=a + b∑x 0.9871 15.9066 1.9048

Formulation log y = a + b∑1/x 0.9991 2.1957 -1.8973No. 5 Q = f(√t) y = a + b∑x 0.9984 -10.4342 15.3051

0.1 M HCl y = a + b∑log y 0.9967 -232.6678 127.6607

log y = a + b∑x 0.9993 0.6576 0.00553√100 ñ Q = f(t) y = a + b∑x 0.9976 4.4741 -0.0465

1/y = a + b∑x 0.9986 0.2151 0.0035

log y = a + b∑1/x 0.9984 2.1097 -2.6761Q = F(t) y = a + b∑log x 0.9901 -22.8102 87.5929

y = a + b∑x 0.9263 38.0092 2.5210

Formulation y = a + b∑log 1/x 0.9962 152.0911 -257.8573No. 6 Q = f(√t) log y = a + b∑1/x 0.9936 2.3516 -1.6971

0.1 M HCl y = a + b∑log x 0.9901 -20.7998 -1.9920

y = a + b∑x 0.9966 4.4535 -0.04853√100 ñ Q = f(t) log y = a + b∑x 0.9994 0.6562 -0.0058

1/y = a + b 0.9996 0.2152 0.0037


Table 5. Cont.

Medium Correlation Equation slopeformulation

Function Equation coefficient Ñr2î Ñaî Ñbî

log y = a + b∑log x 0.9979 0.8698 0.7311Q = F(t) y = a + b∑log x 0.9922 -36.4508 80.6995

y = a + b∑x 0.9901 15.0286 2.4775

Formulation y = a + b∑x 0.9995 -20.1652 19.4604No. 7 Q = f(√t) log y = a + b∑log x 0.9979 0.8698 1.4622

0.1 M HCl log y = a + b∑1/x 0.9974 2.3091 -2.1381

y = a + b∑log x 0.9711 6.6663 -3.72173√100 ñ Q = f(t)log y = a + b∑log x 0.9452 1.1163 -0.6784

y = a + b x 0.9982 5.4006 2.3386Q = F(t)log y = a + b∑log x 0.9996 0.5843 0.8751

Formulation log y = a + b∑x 0.9932 0.7406 0.2185No. 8 Q = f(√t) log y = a + b∑log x 0.9982 0.5446 1.8350

0.1 M HCl log y = a + b∑1/x 0.9868 2.3430 -2.6542

y = a+b∑x 0.9998 4.6199 -0.07223√100 ñ Q = f(t) log y = a + b∑x 0.9956 0.6853 -0.0095

1/y = a + b∑x 0.9851 0.1890 0.0068

y = a + b∑log x 0.9983 -32.2177 84.1120Q = F(t) log y = a + b∑1/x 0.9942 2.0384 -3.3097

log y = a + b∑log x 0.9893 0.9252 0.7172

Formulation log y = a + b∑1/x 0.9996 2.3434 -2.1204No. 9 Q = f(√t) log y = a + b∑log x 0.9983 -35.2103 168.2207

0.1 M HCl y = a + b∑x 0.9934 -17.2464 20.0362

y = a + b∑x 0.9682 4.8585 -0.07013√100 ñ Q = f(t) log y = a + b∑x 0.9430 0.7085 -0.0088

1/x = a + b∑x 0.9118 0.1779 0.0061

presence of excipients was performed according tothe standard pharmacopeia by a basket method (thevolume of acceptor fluid 0.1 M HCL ñ 1000 cm3, 50rot/min) using a system for testing the release offline with the collector of Varian fraction connectedwith the apparatus of high performance chromatog-raphy Agilent 1100 series.

The numerical value of the absorbance of thestandard solution and the tested solution was read ata wavelength λ = 353 nm.

The amount of drotaverine hydrochloride (x, Q%)released from the tablet and dissolved in the modelacceptor fluid was calculated from the dependence:

As ∑ mw ∑ Pw ∑ 2.5x(Q%) = ñññññññññññññññ

Aw ∑ 40where: As ñ test sample absorbance, mw ñ weight ofthe standard (mg), Aw ñ standard sampleabsorbance, Pw ñ content of drotaverine hydrochlo-ride in the reference form 40 ñ declared content ofthe therapeutic agent in a single tablet, 2.5 ñ dilutioncoefficient.

The calculated release coefficient Q(%) wasthe base for the application of mathematical models,which in a reliable way enable to estimate the

dynamics of the process of dissolution of drotaver-ine hydrochloride in the presence of excipients inthe pharmacopoeial acceptor fluid.

The obtained results are demonstrated in Table 4.

Kinetic models of drotaverine hydrochloride phar-

maceutical availability

The process of pharmaceutical availability(Q%) of model formulations with drotaverinehydrochloride (hydrotropic adduct of the therapeuticagent) should take place in acceptor fluid symmetri-cally to a progressing disintegration and developedin the time (t, min) surface of the tablet mass.

Thus, mechanistic models based on Fickísequation as well as kinetic models based on physicallaws were applied for the complex process of disso-lution of drotaverine hydrochloride in the presenceof excipients of different granulometric parameters.

Taking the above into consideration, the fol-lowing model equations were applied to describe therate of the therapeutic agent dissolution:

Q (%) = f (t,min); Q (%) = a + k ∑ t(1) log Q (%) = f (t,min); log Q (%) = a + k ∑ t0 and I-order kinetics equations


(3) Q (%) = f (√t); Higuchi model Q (%) = k ∑ t1/2

which is the base for Fickís diffusion model,(4) log Q (%) = f (log t) ñ Korsmeyer-Peppas

modelQ (%) = k∑tn, which in the logarithmic system

has the form log Q (%) = log K + n log t (applica-tively the used form is Q (%) = a = n log t; where a= log K),

(5) Qo1/3(%) ñ Qt (%) = KH∑t ñ Hixson-Crowell,

which in application has the form ñ Qt (%) = f(t,min).

It is assumed in kinetic models of 0 and I-orderthat the rate of therapeutic agent release is independ-ent of the function of time (t, min). However, in theKorsmeyer-Peppas model and in Higuchi model,which are based on the thesis that the release processis compatible with Fickís law, its rate varies with time.

Figure 2. The course of correlation between pharmaceutical availability ñ Q% and disintegration time of a model tablet in time function ñt, min.: Q % = ¶ (t, min.)

Figure 3. The course of correlation between pharmaceutical availability ñ Q%, and √t in Higuichi model: Q% = f (√t, min.)



It results from prescription compositions ofthe model dosage form, demonstrated in Table 1,that using the optimal content of talc and magne-sium stearate as a lubricant in the process of effec-tive compression, there was used for their produc-tion only one excipient with defined granulometricparameters (formulation No. 2-9). The referenceand No. 1 (32) formulations were manufacturedwith increased share of excipients (Table 1) whichwas reflected in the parameter assessing the tabletmass flow (flowability) and tapped bulk density(Table 2).

An angle of repose of tablet mass for modelformulations remains an interesting applicationparameter of applicative significance in the processof tablet stamping (formulation No. 2-9) in relationto the reference formulation.

It represents, with symmetrical filling of theniche and lack of the tendency to tablet mass bridg-ing, the condition for statistical distribution of thetherapeutic agent in the produced tablet. This is con-firmed by the determined morphological parametersof the produced tablets which at comparable com-pression are characterized by their proper hardnessand ñ with maintained grain granulometric size andthe ability to water sorption ñ the effective disinte-gration time is its emanation (Table 3).

Comparative studies were conducted in theenvironment of model pharmacopoeial acceptorfluid (0.1 M HCl) compatible with the hydrotropicform of drotaverine hydrochloride on the dissolutionrate of the therapeutic agent in the presence ofexcipients. The calculated pharmaceutical availabil-ity coefficients ñ Q(%) in the function of exposuretime are shown in Table 4. The time range of thecarried out research resulted from the electrical dis-integration time of this form of tablets (~ 20 min)provided by pharmacopoeial standard (33).

Demonstrated in Table 4 results of pharmaceu-tical availability ñ Q(%) allowed to trace the processof drotaverine hydrochloride dissolution in the pres-ence of excipients in mathematical models (kineticsolubility of 0 and I-order) and in Higuichi andKorsmeyer-Peppas models associated with Fickíslaw (34) as well as in Hixson-Crowell model, whichtakes into account the topological dimension of thetherapeutic agent hydrotropic molecule.

Analyzing the processes related to the disinte-gration of the tablet in the model acceptor fluid (0.1M HCl, 200 m Osm/dm3), symmetrically ongoingprocesses associated with drotaverine hydrochloridemolecule dissolution (hydration-solvation) should

be taken into account, as well as their physicaladsorption on the surface of homogeneous adsorbent(excipient) combined with filling of the granulomet-ric grain space with a solution the structure of whichis not biodegradable during GI tract transit (35, 36).With the significant concentration gradient betweenthe solution in the structure of the granulometricgrain and the external solution (acceptor fluid), theequilibrium in the concentrations will be reachedduring the mixing process. In this situation, theequation in the mathematical model of Korsmeyer-Peppas ~Q (%) = k ∑ tn represents a significantlycompatible reference to the empirical Freundlichequation ñ y (x/m) = k ∑ c1/n, which describes theprocess of physical adsorption of the therapeuticagent on the surface of the excipient (solid adsor-bent), Figure 1.

The demonstrated in Table 4 calculated coeffi-cients of release Q (%) were the basis for tracing thedependence Q (%) = f (t, min) (Fig. 2) and fordescribing their course at a coefficient of probabili-ty p = 0.05 with correlation equations which are pre-sented in Table 5.

It results from the analysis of disintegration ofmodel tablets and from the dissolution rate of dro-taverine hydrochloride in 0.1 M HCl (Table 4, Fig.2) that in the kinetic model ñ Q(%) = f (t, min) theircourses formed three independent sets in relation tothe reference formulation (RF).

Formulation No.1 (lactose + Vivapur 10) andNo.6 (Prosolv 90HD) were found in the first set,symmetrical in relation to the reference formulation. Surprisingly, formulations: No. 2 (Vivapur 101),No. 3 (Vivapur 102), No. 7 (EMDEX) and No. 9(Perlitor 200 SD) were in the second set, whereas inthe third set formulations: No. 4 (Vivapur 200), No.5 (Vivapur 12) and No. 8 (Emcompress).

It results from Table 5 approximation equationsof high correlation coefficient r2 ≥ 0.990 that in thekinetic model the equations: y = a + b ∑ log x and logy = a + b ∑ 1/x describe the model formulations com-patible with the reference formulation (RF).

However, for other sets, the profile of depend-ence of which ñ Q (%) = f (t, min) is similar toFreundlich adsorption isotherm (y = x/m = k ∑ c 1/η;log y = log k + 1/η log c) the dominating correlationequation is ñ log y = a + b ∑ log x supplemented indi-vidually for the formulations with the equations ofthe type: y = a + b ∑ x, y = a + b ∑ log x and log y =a + b 1/x∑

In Higuichi model also three sets were notedwith the same formulation composition of excipientsfor the course of functional correlation ñ Q(%) = f (√t),Fig. 3.


The course of the correlation Q(%) in the func-tion of √t for the reference (RF) and model formula-tions No.1 and No. 6 is described in the order ofnumerical value of the correlation coefficient by theequations of the type:

y = a + b ∑ 1/x, log y = a + b ∑ 1/x, and y = a + b ∑ log x

However, for the second and third set consist-ing, according to the course of the function in theorder of model formulations: No. 2, No. 3, No. 9,No. 7, No. 5, No. 4 and No. 8, maintaining theabove-mentioned notification, i.e., according to thenumerical value of ìr2î coefficient, their course isdescribed by equations of the type: log y = a + b ∑ logx and log y = a + b ∑ 1/x with supplementation ofequations of the type: y = a + b ∑ log x and y = a + b∑ x(!) for individual formulations.

Axiologically reassessing the observed phe-nomena and processes that accompany the mecha-nism of drotaverine hydrochloride dissolution in 0.1M HCl in the presence of excipients, it should beemphasized that on its own only Prosolv 90 HD ofparticle size 90-110 µm and density 0.35-0.50 g/cm3

(17) makes it possible to produce a model tabletmorphologically and kinetically compatible with thereference formulation (RF). Formulation No. 1 isalso worth noting with lactose and Vivapur 101 ofparticle size 50 (65) µm and density 0.26-0.31 g/cm3

in its prescription composition (31).Thus, the individual use of Vivapur: ñ 101, ñ

102, ñ 12 and 200 in the formulation of a model

tablet results in pharmacopoeial conditions (0.1 MHCl) in temporary loss of drotaverine hydrochloridecontent in the acceptor fluid due to the binding oftherapeutic agent solution by the granulometricgrain structure as well as in physical adsorption ofthe therapeutic agent. Microcrystalline cellulose(Vivapur 101-200) is not biodegradable in the gas-trointestinal tract but still in time function osmolarequilibrium is determined between the solution inthe grain structure and molecular solution in the gas-trointestinal tract (small intestine) in the process ofmass exchange at the phase boundary.

In the Hixson-Crowell model, which takes intoaccount the topological dimension of therapeuticagent hydrotropic particle (Ro= 3√[η] ∑ Mmicel/0.005,Robs= 3√0.09549 ∑ [η] ∑ Mmiceli) (34-36), the course ofthe correlation 3√100 ñ Q(%) = f (t, min) is of regres-sive character (Fig. 4) with maintained division intothree sets.

Particular attention should be paid to thecourse of the correlation ñ 3√100 ñ Q (%) = f (t, min)(Fig. 4) in the class of microcrystalline celluloseVivapur 101, ñ 102, ñ 200, ñ 12 and Provasol 90 HDin the presence of which the complex process of dro-taverine hydrochloride dissolution at substantiallyhigh values of the correlation coefficient r2 ≥ 0,990is described by equations of the type: y = a ñ b ∑ xand y = a ñ b ∑ log x supplemented by correlations1/y = a ñ b ∑ x and log y = a ñ b ∑ log x.

For excipients soluble in 0.1 M HCl such asEMDEX and Pearlitol 200 SD and insoluble ñ

Figure 4. The course of correlation3√100ñ Q in time function t, min. for the process of pharmaceutical availability of drotaverine hydrochlo-

ride in the model


Emcompress, the course of the correlation inHixson-Crowell model 3√100 ñ Q (%) = f (t, min) atvariable numerical value of the correlation coeffi-cient r2 in the range 0.968 - 0.999 is generallydescribed by equations of the type y = a ñ b ∑ x andlog y = a ñ b ∑ x.

The analysis demonstrated that with all thecomplexity of the assessment of the process of phar-maceutical availability in Hixson-Crowell model,the approximation equations of the type: y = a ñ b ∑x, y = a - b ∑ log x or log y = a ñ b ∑ x, which basi-cally describe the course of the correlation 3√100 ñQ (%) = f (t, min), do not reflect in full the process-es associated with the physicochemical and granulo-metric properties of the excipients.

CONCLUSIONS

1. The obtained results of the research havedemonstrated that only SiO2 silicified microcrys-talline cellulose Prosolv 90H allows independentproduction of model dosage form with drotaverinehydrochloride of morphological and kinetic parame-ters compatible with the reference formulation bydirect compression technique.

Applicative combination of lactose withVivapur 10 is worth noting, which also allows toproduce a model dosage form of expected morpho-logical and pharmacokinetic parameters.

2. Analyzing the course of the correlation inthe kinetic model ñ Q (%) = f (t, min), Higuichimodel ñ Q (%) = f () and in Korsmeyer-Peppasmodel ñ log Q% = f (log t) Table 4 ñ with the wholecomplexity of the process of drotaverine hydrochlo-ride dissolution in 0.1 M HCl in the presence ofexcipients such as Vivapur, EMDEX, Emcompressand Pearlitol 200 SD, symmetrical in time processof adsorption of the therapeutic agent solution bygranulometric grain (Freundlich adsorptionisotherm) should be noted, which is manifested byits deficit in the acceptor fluid. This is fully reflect-ed in approximation equations for the kinetic model,Higuichi model and above all for Korsmeyer-Peppasmodel.

3. A significant and independent of the testedkinetic and diffusion models is the fact that in theclass of Vivapur excipients (microcrystalline cellu-lose) there is observed, due to the sorption of thetherapeutic agent solution in 0.1 M HCl by the struc-ture and surface of the excipient, a temporarydecrease of its content in the acceptor fluid with theincrease of the grain granulometric size (Vivapur101 ñ 102 ñ 12 ñ 200 ˆ in µm) ñ a decrease of thenumerical value of coefficient Q% in relation to the

reference formulation. Its diffusion from the struc-ture of the grain to the external solution depends onthe gradient of osmolarity of molecular solution inthe process of mass exchange at the boundary phase(small intestine).

4. Physicochemical and granulometric proper-ties and, above all, the character of the impact on thesurface and in the structure of the grain of excipientsfind full confirmation in the Hixson-Crowell mathe-matical model ñ ñ Q (%) = f (t), which takes intoaccount hydrodynamic diameter of the therapeuticagent particle. The above is reflected in correlationequations demonstrated in Table 4. It results fromthe carried out studies that excipients such asVivapur, EMDEX, Emcompress and Pearlitol200SD require in application formulations the use ofother excipients (e.g., sugar alcohols) whichdecrease their ability to sorption.

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Received: 8. 08. 2016


Limited gastric residence time has complicatedthe oral sustained drug delivery system. As themajority of the drugs are better absorbed from thestomach or the upper part of small intestine, bothprevention in complete drug release in the absorp-tion zone and reduction in the efficacy of adminis-tered dose can occur due to the rapid gastrointestinaltransit (1, 2). For a number of drugs showing limit-ed bioavailability as a result of having narrowabsorption window in the upper segment of GIT,hydrodynamically balanced system (HBS); one ofthe advancements of floating drug delivery approachpromises various applications. It helps in prolongingthe gastric residence time (GRT) and increasing thequantity of drug that reaches at its site of absorptionto a maximum level, thereby improving bioavail-ability (3).

A unique antidepressant, venlafaxine (1-[2-(dimethylamino)-1-(4-methoxyphenyl)ethyl cyclo-hexanol hydrochloride) is referred to as a serotonin,norepinephrine-dopamine reuptake inhibitor (4).Venlafaxine acts as an antidepressant by inhibitingthe neuronal uptake of serotonin, norepinephrine and

to a lesser degree dopamine (5, 6). Venlafaxine has avery short steady state half-life of approximately 5 ±2 h. So in order to maintain the adequate plasma lev-els of the drug, two or three times daily administra-tion is required (7). Due to its short half-life, patientsare also directed to strictly comply with the medica-tion regimen and not to miss even a single dose, asmissing a dose can produce withdrawal symptoms(8). In this situation, such a dosage form is necessarywhich is capable of releasing the drug in sustainedmanner that will also simplify the therapeutic regi-men by avoiding the requirement to administer theformulation frequently. Fewer incidences of nauseaand dizziness have also been reported with the use ofsuch types of formulations (9). The bioavailability ofvenlafaxine is low ranging from 10-45%. Accordingto BCS, it falls under the class I since it is havinghigh solubility and high permeability. It is also freelysoluble in water (572 mg/mL) (10). These propertiesof venlafaxine together with the requirement ofreduction in its frequency of administration make itan ideal candidate for the development of sustainedrelease gastroretentive dosage form.

FORMULATION, DEVELOPMENT AND IN VITRO EVALUATION OF A NOVEL SUSTAINED RELEASE HYDRODYNAMICALLY BALANCED

GASTRORETENTIVE DOSAGE FORM FOR VENLAFAXINE HCl

AQEEL ASLAM1*, SAJID BASHIR1 and BABAR MURTAZA1

1Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan

Abstract: The present study was aimed to formulate, develop and evaluate hydrodynamically balanced systemof an antidepressant, venlafaxine hydrochloride as a single unit floating capsule by the application of Box-Behnken statistical design with 3 factors, 3 levels and 15 runs. Formulations were prepared by physical blend-ing of the drug and polymers in varying ratios. The contents of hydroxyl propylmethylcellulose K15M (A), eth-ylcellulose (B) and cellulose acetate phthalate (C) were used as independent variables whereas the dependentvariables considered were the cumulative percentage drug release at 2 h (Y1), 8 h (Y2), 12 h (Y3) and total float-ing time (Y4). Mathematical model was used to evaluate the main effects and interaction terms quantitatively.Formulation optimization was done by setting the target values for the response variables. The optimized for-mulation was observed to fulfill the requirements of an optimum controlled release floating dosage form sinceit better regulated the drug release (Y1; 4.28%, Y2; 70.83% and Y3; 80.92%) and also floated well for more than16 h. It can be concluded that the hydrodynamically balanced system of venlafaxine hydrochloride can be suc-cessfully formulated as an approach to increase the gastric residence time and hence improving its bioavail-ability.

Keywords: hydrodynamically balanced system, venlafaxine HCl, Box-Behnken statistical design, formulationoptimization

1513


1514 AQEEL ASLAM et al.

Response surface methodology is extensivelyused to optimize formulations by the selection of asuitable experimental design because by using it, adeeper understanding of a process or product isobtained as well as robustness of that product can beestablished (11). Box-Behnken statistical design isan advancement from factorial designs that havebeen used in response surface modeling and opti-mization so widely (12, 13).

This study was conducted with an aim todevelop floating gastroretentive capsule formulationincorporating 150 mg venlafaxine HCl along withswellable hydrophilic polymer HPMC K15M andrelease modifiers such as EC and CAP which wouldrelease the drug in stomach and upper part of GIT ina controlled manner. Since venlafaxine has betterabsorption from these regions, gastroretention of thedosage form will improve its oral bioavailability.


Materials

Venlafaxine HCl was received as a gift samplefrom M/s Global Pharmaceuticals (Pvt.) Ltd., plot22-23, Industrial triangle Kahuta road, Islamabad,Pakistan. HPMC K4M, HPMCK15M (hydroxylpropylmethylcellulose), EC (ethylcellulose), CAP(cellulose acetate phthalate), acetone extra pure(99%) and HCl (37% pure) were obtained fromSigma-Aldrich Laborchemikalien GmbH. D-30926Seeize. Empty hard gelatin capsule shells (size # 00)were obtained from Qaiser Scientific store, Lahore,Pakistan.

Drug-excipients interaction study and identifica-

tion

Fourier transform infrared spectroscopy (FTIR)This study was done by adopting the method

described previously (14). An infrared spectrumof pure drug, each excipient and physical mixtureof optimized formulation was recorded usingFTIR spectrophotometer (IR Prestige 21, Shima-dzu). The scanning range was 4000-500 cm-1 andthe IR spectra of samples were obtained usingKBr disc method. To detect any chemical interac-tion, any alteration in the spectrum pattern of drugdue to presence of polymers was investigated.

UV spectroscopy (determination of λλmax)As described by Pawar HA and Dhavale R

(14), the UV spectrum of venlafaxine HCl solution(concentration; 3 µg/mL) in 0.1 M HCl (pH 1.2) wasrecorded in the range of 200-400 nm on doublebeam UV-visible spectrophotometer (UV 1700Shimadzu). The spectrum and wavelength of maxi-mum absorption were recorded.

Preparation of standard curve

For this purpose, procedure used by (14) wasused with some modifications. The absorbance ofvenlafaxine HCl solutions (concentration; 3, 6, 9,12, 15, 18, 21, 24, 27, 30 and 33 µg/mL) was meas-ured at 225 nm against blank i.e., 0.1 M HCl on dou-ble beam UV-visible spectrophotometer (UV 1700Shimadzu). The coefficient of correlation and equa-tion for the straight line were also determined.

Table 1. Independent variables and their levels in Box-Behnken statistical design.

Level (mg per capsule)

Parameter Low Middle High

Amount of HPMC K15M (A) -1 (200) 0 (250) +1 (300)

Amount of EC (B) -1 (50) 0 (100) +1 (150)

Amount of CAP (C) -1 (20) 0 (40) +1 (60)

Table 2. Composition of different formulations as per design.

Ingredients (mg)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15

Venlafaxine hydrochloride

150 150 150 150 150 150 150 150 150 150 150 150 150 150 150

HPMC K15M 300 200 300 200 250 250 250 300 200 200 250 250 250 250 300

EC 100 50 100 150 150 150 100 50 100 100 50 100 100 50 150CAP 60 40 20 40 60 20 40 40 60 20 20 40 40 60 40

Formulation, development and in vitro evaluation of a novel... 1515

Box-Behnken statistical design

One of the widely used response surfacedesigns, a Box-Behnken statistical design with 3factors, 3 levels, and 15 runs with triplicate centerpointswas employed for the formulation of hydrody-namically balanced system of venlafaxine HCl.Formulation design, optimization and other investi-gations were done using Statgraphics CenturionXVI software from Stat-Point Technologies, USA,version 15.2.0 (64 bit). The independent variables orthe factors were the amount of HPMC K15M (A),EC (B) and CAP (C). Levels of these factors wereset in the formulation design on the basis of theresults of preliminary study and were coded as -1, 0,and +1 as shown in Table 1. The responses selectedwere the cumulative percentagedrug release at 2 h(Y1), 8 h (Y2) and 12 h (Y3) and the total floatingtime (Y4). Formulation optimization was done bysetting targets for these response variables.

Preparation of capsules

Preparation of EC granulesFirstly, EC granules were prepared by

weighing 50 mg of EC and then dissolving it in 1mL of acetone. Then, this solution was granulatedwith already weighed drug content. After this, thegranules were air dried, passed through ASTM #25 mesh and finally the granules equivalent to150 mg of venlafaxine HCl were weighed sepa-rately. Similar procedure was adopted for prepar-ing EC granules containing 100 mg and 150 mg ofEC.

Filling of capsulesBy the use of low density floating polymer

such as HPMC K15M and release modifying agentssuch as EC and CAP, single unit floating capsules ofvenlafaxine HCl were formulated that wouldachieve a density lower than the gastric fluids afteradministration and hence would float. The composi-tion of designed 15 formulations has been listed inTable 2. EC granules equivalent to one hundred andfifty milligrams of venlafaxine HCl and differentquantities of polymers were weighed accurately andblended together for 10 min manually. Finally, thegranules and polymer blend was filled into theempty hard gelatin capsule shells of size # 00 man-ually.

In vitro analysis

Evaluation of in vitro drug releaseFor this purpose, method described by (14) was

used with some modification. USP type II (paddle)apparatus (Pharma test) was used to perform drug

release studies in vitro. The operating speed for thepaddle was 50 rpm and the apparatus was main-tained at temperature 37 ± 5OC. A single HBS cap-sule was transferred into dissolution vessel contain-ing 900 mL of 0.1 M HCl (pH 1.2) as the dissolutionmedium. Then from the dissolution vessel, 5 mL ofaliquots was removed at specified time intervalssuch as after 1, 2 (Y1), 3, 4, 5, 6, 7, 8 (Y2), 9, 10, 11and 12 h (Y3). The removed portion was replacedwith equivalent volume of fresh dissolution mediummaintained at the same temperature. The collecteddissolution samples were filtered using Whatmanísfilter paper and then after making suitable dilution(i.e., 1 mL of filtrate was taken and diluted with 0.1M HCl, pH 1.2 to make the final volume 5 mL),these were used to determine released venlafaxineHCl concentrations at 225 nm by using a doublebeam UV-visible spectrophotometer (UV 1700Shimadzu).

Evaluation of in vitro floating propertiesThe in vitro floating properties of HBS capsules

were determined visually. The capsules wereimmersed in 900 mL of 0.1 M HCl (pH 1.2) in USPtype II apparatus (Pharma test). The operating speedfor the paddle was 50 rpm and the apparatus wasmaintained at temperature 37 ± 5OC. The time requiredfor the capsules to rise to the surface was taken asfloating lag time and the time for which the capsulesconstantly remained buoyant on the surface of themedium was taken as the total floating time (Y4).

Drug release kinetics

The kinetic analysis of in vitro release data ofvenlafaxine HCl from different HBS capsules wasdone by fitting it into various mathematical modelssuch as zero order, first order, Higuchi, Hixson andCrowell powder dissolution, and KorsmeyerñPep-pas model. By calculating the r2 values, the fitness ofthe data was determined (15). This was done byusing excel solver add-in (16).

Zero-order model

It describes the systems where the drugrelease rate is independent of concentration of thedissolved substance (17).

F = Ko t (1)where, the fraction of drug that has been released intime t is represented by F, and Ko is representing thezero-order release constant and t is time in h.

First-order model

It suggests that the drug release rate dependson its concentration (18).


lnF = K1stt (2)where, the fraction of drug that has been released intime t is represented by F, and K1st is representingthe first-order release constant and t is time in h.

Higuchi model

It is a square root model used for describing therelease of drugs incorporated into semisolid andsolid matrices developed by Higuchi et al. (19). Ithas been shown in the equation given below,

F = KH√t (3)where, F represents the fraction of drug released intime t, and KH is the Higuchi dissolution constantand t is time in h.

Hixson and Crowell powder dissolution model

This model developed by Hixson and Crowellis represented by the following equation (20),

F = 100 (1 ñ (1 ñ KHCt)3) (4)where, F represents the fraction of drug released intime t, and KHC is the dissolution constant and t istime in h.

KorsmeyerñPeppas model

They developed a simple mathematical model,relating exponentially the drug release from a poly-meric system to the elapsed time (t) (21).

F = KKPtn (5)where, F represents the fraction of drug released intime t, KKP is the rate (kinetic) constant incorporat-

Figure 1. FTIR of venlafaxine

Figure 2. FTIR of the physical mixture of optimized formulation


Figure 3. UV spectrum of venlafaxine hydrochloride in 0.1 M HCl

Figure 4. Calibration curve of venlafaxine hydrochloride in 0.1 M HCl

ing the properties of macromolecular polymeric sys-tem and drug, n is the diffusional (release) exponent,this indicates the drug release mechanism and t istime in h. The value of n is determined by linearregression of log F vs. log t. For a capsule, a cylindri-cal geometry is considered for the determination ofvalues of n for all these HBS venlafaxine capsules. Ifit is 0.45, in indicates Fickian diffusion. If it is greaterthan 0.45 but less than 0.89, it indicates anomalous ornon-Fickian diffusion and if it is equal to or greaterthan 0.89, it shows case-II transport (22).


Drug-excipients interaction study and identifica-

tion

Fourier transform infrared spectroscopy (FTIR)

A slight variation was observed in the charac-teristic bands of FTIR spectrum of venlafaxine HClafter doing pre-formulation studies revealing no

chemical interaction. These spectra have been givenin Figures 1 and 2.

UV spectroscopy (determination of λλmax)

The UV spectrum of venlafaxine HCl showedthat the wavelength of maximum absorbance wasobtained at 225 nm. The UV spectrum has beenshown in Figure 3.

Calibration curve of venlafaxine

The calibration curve was found to be linear inthe range of 3-33 µg/mL and straight line equationwas obtained having the correlation coefficientvalue of 0.9935. The calibration data of Venlafaxinehas been shown in Table 3 and the calibration curvehas been shown in Figure 4.

Formulation development

Initially, HPMC K4M was evaluated for itsfloating characteristics but satisfactory results were


Figure 5. (A-D). Main effects plot for Y1-Y4, respectively. Where HPMC: hydroxypropylmethyl cellulose, EC: ethyl cellulose and CAP:cellulose acetate phthalate

A

B

C

D


not obtained. Then, another grade of HPMC i.e.,HPMC K15M was examined and as a result ofobtaining desired results, was chosen as a low den-sity floating polymer. After the application of Box-Behnken statistical design, fifteen formulationswere prepared and then evaluated for four respons-es such as Y1, Y2, Y3 and Y4. In vitro drug releaseand buoyancy study revealed that values ofresponses for these fifteen formulations variedmarkedly indicating strong relationship betweenresponses and the factors as shown in Table 4. The

range of response Y1 was from 32.45% in F5 to43.32% in F11. Response Y2 ranged from 70.71%in F15 to a maximum of 90.29% in F2. Similarly,another response Y3 was in the range of 81.18% inF15 to 99.25% in F2 and Y4 was ranging from 10 hin F2 to 16 h in F1.

In vitro drug release

It has been observed that all designed formula-tions showed high values of Y1. Such a high releasecould be due to the probable reason that the gel layerformed by HPMC K15M that is effective in control-ling drug release is not generated immediately. Aslong as the gel barrier is being formed, there is higherosion rate and subsequent high drug release ini-tially (23-25). Then, as the hydration of polymeroccurs, the drug release slows down. So, in case ofY1, amount of HPMC K15M proved to be insignifi-cant (p > 0.05). When ANOVA was performed at95% confidence interval to estimate the significanceof the model, the factors that were significant in con-trolling drug release at the end of 2 h were theamount of EC (p < 0.05) and the amount of CAP (p< 0.05). It was also observed that Y1 and both theamount of EC and CAP have an inverse relationshipdepicting that as the amount of EC and CAP areincreased, drug release is decreased and vice versa.For instance, F5 showed the least value of Y1

(32.45%) as it contained the maximum amount of

Table 4. Observed responses in designed fifteen HBS formulations.

ResponsesFormulations

(Y1) (Y2) (Y3) (Y4)% % % h

F1 32.53 76.35 89.29 16

F2 36.43 90.29 99.25 10

F3 39.17 75.27 88.55 14

F4 34.27 74.94 87.97 10

F5 32.45 73.53 84.23 13

F6 37.26 72.03 83.73 13

F7 35.35 78.76 89.29 14

F8 36.85 81.41 93.94 16

F9 32.95 81.08 89.88 10

F10 41.91 80.49 92.37 12

F11 43.32 83.98 98.17 13

F12 35.44 80.41 94.19 13

F13 36.18 80 91.12 14

F14 33.78 81.58 96.76 14

F15 35.19 70.71 81.18 16

Table 3. Calibration data of venlafaxine (n = 4).

Concentration (µg/mL) Absorbance

3 0.1081 ± 0.00011

6 0.2043 ± 0.0003

9 0.3062 ± 0.00023

12 0.4571 ± 0.0001

15 0.5350 ± 0.00011

18 0.6333 ± 0.00021

21 0.7362 ± 0.00026

24 0.8025 ± 0.00019

27 0.9221 ± 0.00011

30 1.1124 ± 0.00013

33 1.2050 ± 0.00018


Figure 6. (A-D). Contour plot for Y1-Y4, respectively. Where HPMC: hydroxypropylmethyl cellulose, EC: ethyl cellulose, CAP: celluloseacetate phthalate and TFT: total floating time. The amounts of HPMC, EC and CAP are in mg. The drug release at 2h, 8 h and 12 h is in %

A

B

C

D


EC 150 mg and CAP 60 mg. F7 showed 35.35% andF12 showed 35.44% of Y1 since both were formu-lated with EC 100 mg and CAP 40 mg. On the otherhand, F11 showed the highest value of Y1 (43.32%)as the amount of EC was 50 mg and CAP 20 mgonly.

As the time passed on, the gel layer formed byHPMC K15M became effective and played animportant role in controlling drug release at the endof 8 h along with the hydrophobic nature of EC coat-ing as a result of lacking any of the hydrophilicgroups (hydroxyl or carboxylic). In case of Y2, theamount of CAP proved to be insignificant (p >0.05). While the amount of HPMC K15M and theamount of EC was statistically significant (p < 0.05)as determined after performing ANOVA at 95%confidence interval. It was also observed that Y2 andboth the amount of HPMC K15M and EC have aninverse relationship depicting that as the amount ofHPMC K15M and EC are increased, drug release isdecreased and vice versa. For instance, F15 showedthe least value of Y2 (70.71%) as it contained themaximum amount of HPMC K15M 300 mg and EC150 mg. F7, F12 and F13 showed cumulative per-centage drug release of 78.76%, 80% and 80.41%,respectively, as these were formulated with HPMCK15M 250 mg and EC 100 mg. On the other hand,F2 showed the highest value of Y2 (90.29%) as theamount of EC was 50 mg and HPMC K15M 200 mgonly.

A majority of the HBS capsules extended therelease of venlafaxine HCl for more than 12 h. Thecomplex interaction between diffusion, swelling anderosion mechanisms is known to be involved in thedrug release from these hydrophilic matrices. Thehydration of HPMC controls all of these mecha-nisms. It also forms the gel barrier through whichthe drug diffuses (23). Higher concentration of thepolymer causes greater amount of gel to be formedand subsequent increase in the diffusion path lengthfor the drug due to which drug release from the for-mulation is retarded. Increase in the polymer pro-portion also cause decrease in the growth of erosion,diffusion and swelling fronts due to the formation ofa stronger gel layer and subsequent difficult entry ofmedium into the matrix (24, 26-28).

After performing ANOVA at 95% confidenceinterval to estimate the significance of the model,the amount of both HPMC K15M and EC wasproved to be significant in controlling the cumula-tive percentage drug release at 12 h (p < 0.05) andthe amount of CAP was insignificant (p > 0.05) Itwas also observed that Y3 and both the amount ofHPMC K15M and EC have an inverse relationshipdepicting that as the amount of HPMC K15M andEC are decreased, drug release is increased and viceversa. For instance, F2 showed the highest value ofY3 (99.25%) as the amount of HPMC K15M and ECwere at low level i.e., 200 mg and 50 mg, respec-tively. F7 showed 89.29%, F13; 91.12% and F12;

Table 5. Drug release kinetics of all the fifteen formulations.

Formulation Zero-order First-order Higuchi Hixson-Crowell Korsmeyer- Peppas

code K0 r2 K1st r2 KH r2 KHC r2 KKP r2 n

F1 9.9375 0.7738 0.1850 0.9807 26.5876 0.9785 0.0510 0.9631 22.3117 0.9959 0.5939

F2 11.2722 0.8558 0.2312 0.9567 30.0153 0.9600 0.0625 0.9810 22.7053 0.9970 0.6491

F3 10.0768 0.6270 0.1946 0.9645 27.1163 0.9929 0.0531 0.9202 25.6318 0.9952 0.5303

F4 9.9651 0.6008 0.1896 0.9377 26.8184 0.9882 0.0519 0.8932 25.7072 0.9895 0.5228

F5 9.6991 0.6390 0.1799 0.9602 26.0840 0.9917 0.0496 0.9158 24.4214 0.9947 0.5354

F6 9.7210 0.4258 0.1852 0.9209 26.2980 0.9929 0.0506 0.8347 27.5323 0.9948 0.4753

F7 10.2451 0.6738 0.1991 0.9664 27.5185 0.9922 0.0543 0.9374 25.1998 0.9974 0.5473

F8 10.6100 0.7183 0.2123 0.9737 28.4545 0.9885 0.0575 0.9584 25.1693 0.9979 0.5658

F9 10.2487 0.8053 0.1952 0.9856 27.3787 0.9742 0.0536 0.9799 22.1991 0.9977 0.6122

F10 10.6930 0.5823 0.2215 0.9675 28.8144 0.9959 0.0595 0.9266 27.9898 0.9965 0.5156

F11 10.8932 0.7489 0.2220 0.9503 29.1639 0.9725 0.0598 0.9447 24.9747 0.9864 0.5831

F12 10.6158 0.7364 0.2122 0.9795 28.4528 0.9864 0.0576 0.9660 24.7768 0.9981 0.5742

F13 10.2631 0.8205 0.1946 0.9774 27.3903 0.9701 0.0534 0.9754 21.8113 0.9970 0.6218

F14 10.4168 0.8661 0.1966 0.9502 27.7045 0.9509 0.0540 0.9620 20.5308 0.9916 0.6599

F15 9.1629 0.4910 0.1640 0.9196 24.7488 0.9989 0.0455 0.8407 25.1894 0.9991 0.4905


94.19% of Y3 as these were formulated with 250 mgof HPMC K15M and 100 mg of EC. While the leastcumulative percentage drug release was shown byF15 (81.18%) since the amount of HPMC K15Mand EC was 300 mg and 150 mg, respectively, inthat formulation.

In vitro buoyancy

A majority of the formulations floated well formore than 12 h. The increased gel strength of thepolymeric combination matrices might be the prob-able reason. The mechanism responsible to makethese capsules buoyant could be the rapid hydrationand swelling of polymeric matrices resulting in theformation of a floating mass (29). The factors thatwere found to play significant role in governing Y4

after performing ANOVA at 95% confidence inter-val, were A (p < 0.05) and AC (p < 0.05). It was alsoobserved that the matrix integrity of these HBS cap-sules was satisfactory. Main effects plots and con-

tour plots for different formulation factors have beenshown in the Figure 5 (A-D) and Figure 6 (A-D),respectively.

Drug release kinetics

Table 5 lists various kinetic models computedfor all the HBS gastroretentive formulations.Venlafaxine release data were evaluated by zero-order, first-order, and Higuchi and Hixson-Crowellmodels. Some of the formulations followed first-order release patterns because the plots of the per-cent cumulative drug release versus the square rootof time were found to be linear. Hence, the mecha-nism of drug release from these formulations wasfound to be diffusion-controlled such as F1, F9 andF13. The rest of the formulations followed Higuchimodel since the regression coefficient (r2) values areranging from 0.9509 to 0.9989, suggesting that therelease of a solid drug from a hydrophilic matrixinvolved the simultaneous penetration of the sur-

Figure 7. Kinetic evaluation of the optimized formulation: Korsmeyer-Peppas plot

Table 6. Multiple response optimization.

Response (Range observed in F1 - F15) Target value

% Cumulativerelease at the end of, Low High

(i) 2 h 32.45 43.32 minimize

(ii) 8 h 70.71 90.29 minimize

(iii) 12 h 81.18 99.25 minimize

Total floating duration (h)

10 16 maximize


rounding liquid, dissolution of the drug, and leach-ing out of the drug through interstitial channels orpores. This was further augmented when the releasedata were subjected to Korsmeyer-Peppas equationwhich is used to describe the anomalous releasebehavior from the matrix. In the present study, thevalues of n, calculated per the algorithm proposedby Peppas and Sahlin (31), ranged between 0.4753-0.6599. The values of the kinetic constant (KKP),which is a direct function of matrix solubility, werefound to increase with increases in the amount ofeither polymer (32, 21). It should be noted that K1

has much lower values when compared to K0, clear-ly indicating that the release of venlafaxine was pri-marily controlled by non-Fickian diffusion depict-ing that swelling and erosion of the HBS gastrore-tentive capsules go on to increase in association withan increase in the content of any of the polymers.

Data analysis

Mathematical relationships for the measuredresponses and the independent variables or factorswere generated with the help of softwareStatgraphics Centurion XVI and are shown inEquations 6-9. These equations represent the quan-titative effect of variables (A, B, C) and their inter-actions on the response. Coefficient with more thanone factor term and those with higher order termsrepresent interaction terms and quadratic relation-ship, respectively. A positive sign represents syner-gistic effect, while a negative sign indicates antago-nistic effect or an inverse relationship between thefactors and response (30). Correlation coefficient(r2) for the equations indicates the percentage vari-ability in model fitting for that particular variable.The adjusted r2 value is more suitable for comparingmodels with different number of independent vari-ables which can be obtained by including only sta-tistically significant (p < 0.05) coefficients in theequation. The adjusted r2 value for responses Y1, Y2,Y3 and Y4 were found to be 90.78%, 93.64%,85.02% and 86.02%, respectively, which indicategood fit.

Y1 = 62.6204 ñ 0.0293A ñ 0.0915B ñ 0.6505C + 0.0006AC + 0.0012BC+ 0.0025C2 (6)r2 = 96.7056%, r2 (adjusted) = 90.78%

Y2 = 116.078 ñ 0.1222A ñ 0.2342B + 0.1750C + 0.0005AB + 0.0001AC ñ 0.0002B2 +0.001BC-

0.0037C2 (7)r2 = 97.7292%, r2 (adjusted) = 93.64%

Y3 = 97.7071 ñ 0.1061A ñ 0.0997B + 0.1289C ñ 0.0003A2 ñ 0.0001AB + 0.0008AC + 0.0005BC ñ

0.0017C2 (8)r2 = 94.6498%, r2 (adjusted) = 85.02%

Y4 = -2.95833 + 0.1017A ñ 0.0242B ñ 0.1771C ñ 0.0002A2 + 0.001AC ñ 0.0001B2 ñ

0.0003BC ñ 0.0005C2 (9)r2 = 95.0057%, r2 (adjusted) = 86.02%

Formulation optimization and evaluation

For formulation optimization, target values forthe responses (Y1-Y4) were set on the basis of invitro drug release and buoyancy study of multipletrial formulations run in the laboratory (Table 6).Composition of the optimized formulation given bythe software using this technique known as ìmulti-ple response optimizationî is shown in Table 7.

Optimized formulation was prepared by themethod as described before. Then, it was evaluatedfor Y1, Y2, Y3 and Y4 by the method as describedbefore. Furthermore, the drug release data wereanalyzed kinetically by fitting into various mathe-matical models as described above. The results ofall these parameters have been shown in Tables 8,9 and Figure 7. The desirability was maximized bythe use of optimized formulation over the indicatedregion to 0.916206 as shown in Figure 8.

It was also observed that the optimized formu-lation fulfilled the requirements of an optimum con-trolled release dosage form since it better regulatedthe drug release at the end of 2 h (34.28%), 8 h(70.83%) and 12 h (80.92%). The validity of thedeveloped model is indicated by the point that therelease profile predicted by the software was foundto be very close to that observed experimentally(Table 8). The release profile of the optimized for-

Table 7. Composition of the optimized formulation.

Composition Amount per capsule (mg)

Venlafaxine HCl 150

Amount of HPMC K15M (A) 299.999

Amount of EC (B) 149.999

Amount of CAP (C) 59.999

Table 8. Response variables of the optimized formulation(observed and predicted).

Response variables Predicted Observed

Y1 (%) 33.2004 34.28

Y2 (%) 71.5634 70.83

Y3 (%) 81.5411 80.92

Y4 (h) 16.0417 >16


mulation could be best expressed by Korsmeyer-Peppas equation as plots showed highest linearitywith r2 of 0.9939 (Fig. 5) and the value of n was0.483 (Table 9) concluding that it followed anom-alous transport. Optimized formulation remainedfloated for more than 16 h in vitro. Therefore; inaddition to the drug release control for an extendedduration, the optimized formulation also showed theexcellent floating potential that is the prerequisitefor prolonged gastric residence of the formulation.Furthermore, in vivo and pharmacokinetic studiesare required to be carried out.

CONCLUSION

This study examines the preparation of a hydro-dynamically balanced system containing venlafaxineHCl using the synthetic polymers HPMC K15M aslow density hydrophilic polymer, EC and CAP asrelease modifiers. A systematic study using a Box-

Behnken statistical design revealed the most suitableamount of HPMC K15M, EC and CAP in the hydro-dynamically balanced system. The optimized formu-lation fulfilled all the requirements of the target setand showed suitable values of cumulative percentagedrug release at 2 h, 8 h, 12 h and total floating time.The drug release pattern followed Korsmeyer-Peppas model with anomalous transport mechanism.The applicability of the statistical optimization tech-niques in predicting the composition of an optimizedformulation is clearly indicated by the presentresearch. It can also be concluded that the hydrody-namically balanced system of venlafaxine HCl canbe successfully formulated as an approach toincrease the gastric residence time and henceimproving its bioavailability. Indeed, this approachmight be a better alternative to the conventionaldosage form. However, clinical studies should beconducted with optimized formulation in order to co-relate the in vitro and in vivo performance.

Figure 8. Contour plot showing effect of HPMC K15M, EC and CAP on desirability factor. Where HPMC: hydroxypropylmethyl cellu-lose, EC: ethyl cellulose and CAP: cellulose acetate phthalate. The amounts of HPMC, EC and CAP are in mg

Table 9. Drug release kinetics of the optimized formulation.

No. Kinetic model Value of constant Value of r2 Value of ëní

1 Zero-order K0 = 8.181 0.4723 -

2 First-order K1st = 0.156 0.9392 -

3 Higuchi KH = 24.142 0.9930 -

4 Hixson-Crowell KHC = 0.043 0.8638 -

5 Korsmeyer-Peppas KKP = 25.007 0.9939 0.483


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Received: 19. 08. 2016


The use of Laplace transforms of a function,LF(t), and the inverse operation, L-1 , may be appliedto obtain solutions to the systems of linear differen-tial equations of the first-order. However, sufficientmatrix algebra is needed to use the above transforms(1). That operation transforms differentiation intomultiplication as well as integration into division(1). The Laplace transform enables complex rateexpressions to be manipulated easily by convention-al algebraic techniques (2). However, it is true withregards to a one-compartment body model. The anti-Laplace of the resulting complicated transforms maybe found only in an extensive table of Laplace trans-forms if two-compartment body model is concerned.Therefore, according to Gibaldi and Perrier (2), andBenet (3, 4), it is easier to use a general partial frac-tion theorem for obtaining inverse Laplace trans-forms (4). To illustrate the application of thisapproach for solving linear differential equations ofthe first-order a two-compartment pharmacokineticmodel with the first- or zero-, or bolus (instanta-neous) input will be employed. It should be men-tioned that the application of the Laplace transformsto a one-compartment body model is much easierand has been explained quite simply (2, 5, 6).

A two-compartment model with intravenous

bolus injection

The experimental Laplace transform for thedisposition function of the central compartment, ds,C

in a linear two-compartment mammillary modelwhere elimination of drug from central compartmentoccurs has been provided (2-4):

ΠN

i=2(s + Ei)

ds,C = ññññññññññññ (1)Π

N

i=1(s + λi)

where: E1 = k10+k12 and E2 = k21, and λi is a dispositionrate constant, which may be expressed in terms ofthe above individual intercompartmental transferrate constants and elimination rate constants, Π ñcontinued product, s ñ the Laplace operator whichreplaces the time domain of a rate equation.The model concerned is presented below:

k12

Xc ↔ XT

k21

↓k10

where XC and XT are the amounts of a drug as a func-tion of time in central and tissue compartments, andk12, k21, and k10 are the first-order rate constants of

APPLICATION OF LAPLACE TRANSFORMS TO A PHARMACOKINETICOPEN TWO-COMPARTMENT BODY MODEL

TADEUSZ W£ADYS£AW HERMANN

Department of Physical Pharmacy and Pharmacokinetics, PoznaÒ University of Medical Sciences, 6 åwiÍcickiego St., 60-781 PoznaÒ, Poland

Abstract: The anti-Laplace of complicated transforms for two-compartment differential equations may befound only in an extensive table of Laplace transforms which usually are not available. Therefore, a general par-tial fraction theorem was used for obtaining their inverse Laplace transforms. First, the disposition function ofthe central compartment in a linear two-compartment mammillary model, ds,C must be written down. Second,except for that disposition function appropriate intake functions were considered with intravenous instanta-neous bolus injection, constant zero-order rate infusion, and intramuscular first-order single dose injection ofindobufen (Ibustrin). The product of the disposition function and the appropriate input function yields theexperimental Laplace transforms for the amount (concentrations) of drug in the central compartment, as,C (XC).The input functions for the above mentioned routes of drug administration are equal: X0 (the dose), k0 ∑ (1 ñeñT∑s)/S, and ka ∑ F ∑ X0/ (s + ka), respectively. The equations derived are also illustrated in four figures.

Keywords: two-compartment body model, Laplace transforms, the inverse Laplace, partial fraction theorem,disposition function, different intake function: bolus injection, infusion, intramuscular injection

1527

* Corresponding author: e-mail: [email protected]; phone: +48 61 693 101 555

1528 TADEUSZ W£ADYS£AW HERMANN

transfer a drug from the central compartment to thetissue compartment and vice versa as well as elimi-nation rate constant from the central compartment,respectively. The product of the input, ins, and dis-position functions yields the Laplace transform forthe amount of drug in the central compartment, as.c

as,C = ins ∑ ds,C (2)where ins = X0 (a single dose injected) for the abovemodel.Therefore

ΠN

i=2(s + Ei)

as,C = X0 ∑ ññññññññññññ (3)Π

N

i=1(s + λi)

The above equation may be rewritten for thecentral compartment amounts of drug followingintravenous bolus injection

X0 ∑ (s + E2) P(s)as,C = ññññññññññññññññ = ñññññññññ (4)

(s + λ1) ∑ (s + λ2) Q(s)The anti-Laplace of the resulting transform, L-1 maybe obtained by use of a general partial fraction theo-rem. If the quotient of two polynomials P(s)/Q(s) isgiven as above, then

P(s) P(λi)L-1 ññññññ = ΣN

i=1ñññññññññ ∑ eλi ∑ t (5)

Q(s) Qi(λi)The roots of the polynomial, Q(s) are λ1 = ñ α and λ2

= ñ β. The term Qi(λi) may be defined as follows.When

i = 1 Qi(λi) = (λi + β) = (β ñ α)i = 2 Qi(λi) = (λ2 + α) = (α ñ β) (6)

Hence the solution for the amount of drug in thecentral compartment, XC, applying the general par-tial fraction equation, is obtained

ΠN

i=2(Ei ñ λl)

XC = X0 ∑ ΣN

l=1ññññññññññ ∑ eλi ∑ t =

ΠN

i=1(λi ñ λl)

X0(K21 ñ α) X0(K21 ñ β)= ññññññññññ ∑ eñα ∑ t + ññññññññññ ∑ eñβ∑ t

(β ñ α) (α ñ β)X0XC = ññññññ [(α ñ k21) ∑ eñα ∑ t + (k21 ñ β) ∑ eñβ∑ t

α ñ βX0(α ñ k21) X0(k21 ñ β)

when ññññññññññññ = A i ñññññññññññññ = B (7)α ñ β α ñ βXC = A ∑ eñα ∑ t + B ∑ eñβ∑ t (8)

A plot of the logarithms of drug plasma con-centrations (C = XC/Vd) versus time according to the

Figure 1. Semilog plot of plasma levels vs. time after intravenous bolus administration of the iodine salt to a healthy volunteer describedby biexponential equation and the method of residuals (2, 6)

Application of Laplace transforms to a pharmacokinetic... 1529

above equation will yield a biexponential curve(Fig. 1).

The disposition constants α and β may beobtained applying the method of residuals (Fig. 1).

A two-compartment model with a first-order

input process

For a drug that enters the body by an apparentfirst-order absorption process (generally via the oralor intramuscular routes) and distributes in the bodyaccording to a two-compartment model:

k12

Xa →ka XC ↔ XT

k21

↓k10

where: Xa ñ amount of drug at an extravascular siteof absorption as a function of time , ka ñ the first-order rate constant of absorption.

The disposition function for the central com-partment is identical to the disposition function foran intravenous bolus injection

ΠN

i=2(s + Ei)

ds,C = ññññññññññññ (9)Π

N

i=1(s + λi)

However, the input function is different to describefirst-order absorption (2, 5, 6)

ka ∑ F ∑ X0ins = ñññññññññññññññ (10)s + ka

where F determines the fraction of drug absorbed.The Laplace transform for the amount of drug

in the central compartment equals the product of thedisposition and input functions

ka ∑ F ∑ X0 ∑ Π2

i=2(s + Ei)

as,C = ñññññññññññññññññññ (11)(s + ka)∑ Π

2

i=1(s + λi)

Solving for the amount of drug in the centralcompartment by taking anti-Laplace yields

Π2

i=2(Ei ñ ka)

XC ∑ ka ∑ F ∑ X0 ∑ ñññññññññññññññññññ ∑ eñka∑ t +Π

2

i=1(λi ñ ka)

Π2

i=2(Ei ñ λl)

+ ka ∑ F ∑ X0 ∑ Σ2

i=1ññññññññññññññññññ ∑ eñλl∑ t (12)

(ka ñ λl) ∑ Π2

i=1(λi ñ λl)

k21 ñ kaXC = ka ∑ F ∑ X0 ∑ ñññññññññññññññññññ ∑ eñka∑ t +(λ1 ñ ka) ∑ (λ2 ñ ka)

k21 ñ λ1+ ka ∑ F ∑ X0 ∑ ñññññññññññññññññññ ∑ eñλ1∑ t +(ka ñ λ1) ∑ (λ1 ñ λ2)

k21 ñ λ2+ ka ∑ F ∑ X0 ∑ ñññññññññññññññññññ ∑ eñλ2∑ t

(ka ñ λ2) ∑ (λ2 ñ λ1)

When the hybrid rate constants α and β (1) aswell as the other constants B, A, and C0 ñ the corre-sponding zero-time intercepts obtained by themethod of residuals, are substituted, respectively,the equation may be transformed to a simpler form:

k21 ñ kaXC ∑ ka ∑ F ∑ X0 ∑[ ñññññññññññññññññññ ∑ eñka∑ t +(α ñ ka) ∑ (β ñ ka)

k21 ñ α k21 ñ β+ ññññññññññññññ ∑ eñα t + ñññññññññññññññ ∑ eñβ∑ t

(ka ñ α) ∑ (β ñ α) (ka ñ β) ∑ (α ñ β)XC = B ∑ eñβ∑ t + A ∑ eñα∑ t ñ C0∑ eñka∑ t (13)

The plots of the above equation presented onboth a graph and on a semilog papers are very char-

Figure 2. Serum concentrations of (-)-R-indobufen enantiomer vs. time after single 200 mg intramuscular dose administration of racemicindobufen (Ibustrin) to a healthy volunteer described by TopFit 2.0 computer program for an open two-compartment body model (6, 7)

1530 TADEUSZ W£ADYS£AW HERMANN

Figure 3. Semilog plot of serum (-)-R-indobufen enantiomer vs. time after single 200 mg intramuscular dose administration of racemicindobufen (Ibustrin) to a healthy volunteer described by TopFit 2.0 computer program for an open two-compartment body model (6, 7)

Figure 4. Semilog plot of plasma levels vs. time of a drug that confers two-compartment model characteristics, following constant rateintravenous infusion to steady-state ( ___ ) and following the rapid intravenous injection of a dose that gives an initial drug concentra-tion equal to the steady-state concentration (_ _ _) (2)

Application of Laplace transforms to a pharmacokinetic... 1531

acteristic for a two-compartment model (Fig. 2 and3).

The experimental data presented in Fig. 2 and3 have been excerpted from a published article (7).

A two-compartment model for an intravenous

infusion

k12

→k0 XC ↔ XT

k21

↓k10

where: k0 ñ the zero-order rate of drug infusion (con-stant).

The disposition function for the central com-partment is identical to the disposition function foran intravenous bolus injection.

s + E2ds,C = ññññññññññññññññññ (14)(s + λ1) ∑ (s + λ2)

Multiplication of this disposition function bythe input function for an intravenous infusion begin-ning at time zero (i.e., ins = k0 ∑ (1 ñ e-T∑s)/s (2, 5, 6)yields the following Laplace transform for theamount of drug in the central compartment

k0 ∑ (s + E2) ∑ (1 ñ e-T∑s)as,C = ññññññññññññññññññ (15)

s ∑ (s + λ1) ∑ (s + λ2)where T is the duration of an infusion.

The above two polynomials fulfill the require-ments for the use of a partial fraction theorem forobtaining inverse Laplace transforms. Hence thesolution for the amount of drug in the central com-partment XC as a function of time may be written

k0 ∑ (E2 ñ λ1) ∑ (1 ñ e-λl∑T)Xc = ññññññññññññññññññññññ ∑ eñλl∑ t +

ñ λ1 ∑ (λ2 ñ λ1)

k0 ∑ (E2 ñ λ2) ∑ (1 ñ e-λ2∑T)+ ññññññññññññññññññññññ ∑ eñλ2∑ t (16)

ñ λ2 ∑ (λ1 ñ λ2)The roots of the polynomial, Q(s) were speci-

fied previously, and the equation may be rewrittenk0 ∑ (α ñ k21) ∑ (1 ñ e-λl∑T)

Xc = ññññññññññññññññññññññ ∑ eñα∑t + α ∑ (α ñ β)

k0 ∑ (β ñ k21) ∑ (1 ñ e-β∑T)+ ññññññññññññññññññññññ ∑ eñβ∑t(17)β ∑ (β ñ α)

where: k0 is the zero-order rate of the infusion.

It should be noted that the above single equa-tion describes the amount of drug in the central com-partment as a function of time while the infusion isbeing carried out and after infusion stops. While theinfusion is continuing, T = t and varies with time

k0 ∑ (α ñ k21) ∑ (e-ñα∑t ñ e-ñ2α∑t)Xc = ññññññññññññññññññññññ + α ∑ (α ñ β)

k0 ∑ (β ñ k21) ∑ (e-ñβ∑t ñ e-ñ2β∑t)+ ññññññññññññññññññññññ (18)β ∑ (β ñ α)

However, when infusion ceases, time tbecomes a constant corresponding to T ñ the dura-tion of the infusion. It should be apparent that uponstopping the infusion, drug concentrations in theplasma decline in a biexponential manner (Fig. 4).

It can be seen from Fig. 4 that the biexponen-tial characteristics of the drug is more evident fol-lowing the bolus injection than after terminating theinfusion.

Acknowledgment

This publication has been written to givethanks to the Almighty God for my 80th birthdayanniversary which hopefully I am supposed to sur-vive on June 13, 2017.

REFERENCES

1. Wagner J.G., Pernarowski M.: Biopharma-ceutics and relevant pharmacokinetics, DrugIntelligence Publications, Hamilton, Illinois1971.

2. Gibaldi M., Perrier D.: Pharmacokinetics, 2nd

edn., Marcel Dekker, New York and Basel1982.

3. Benet L.Z.: J. Pharm. Sci. 60, 1593 (1971).4. Benet L.Z.: J. Pharm. Sci. 61, 536 (1972).5. Hermann T.W.: Farm. Pol. 71, 289 (2015).6. Hermann T.W.: Pharmacokinetics. Theory and

practice. PZWL, Warszawa 2002.7. G≥Ûwka F.K.: Chirality 12, 38 (2000).

Received: 9. 09. 2016


Oral route is the most popular and preferredmethod of drug application (1-3). Orodispersiblefilms and oral lyophilizates are relatively novel soliddosage form which placed in the mouth quickly dis-solve or disintegrate in the saliva without necessityof drinking water. Their advantages include no riskof choking, easier acceptance and dosage in com-parison with conventional solid forms of the drug(4-7). Different manufacturing technologies oforodispersible forms have been developed.Lyophilizates can be prepared by freeze-drying,while films are obtained by solvent casting, hot-meltextrusion, sublimation and compression methods (8-13). Freeze-drying involves freezing of solution,suspension or emulsion containing drug with excip-ients followed by sublimation of ice. Rapid coolingresults in freezing the system with simultaneouscrystallization of ice in the form of fine crystals,which sublimate at reduced pressure, formingporous tablet matrix. Due to high porosity, orallyophilizates disperse in the oral cavity within a fewseconds (14-16). In the solvent casting method, the

active substance, polymer and excipients are dis-solved or suspended in water or in a mixture ofwater/organic solvents. The prepared mass is thenpoured into glass forms followed by solvent evapo-ration. The obtained films are further divided intopieces with the size corresponding to a properdosage of drug (17, 18).

The main component in oral lyophilizates isgelatin, while in orodispersible films ñ polymer or amixture of polymers which provide optimal elastic-ity. Gelatin is a mixture of purified protein fractionsobtained by acid hydrolysis of animal origin colla-gen. It provides a uniform dispersion of the drugsubstances and does not increase the disintegrationtime of obtained lyophilizates (17). Polymers oftenused in films production include pullulan, povidone,chitosan or hypromellose (PharmacoatÆ 606,MethocelÆ, MetoloseÆ) (18). PharmacoatÆ 606 ishydrophilic polymer forming transparent, odorlessand tasteless films. It is a non-ionic cellulose etherwith an average molecular weight from 10 000 to150 000 Da (19).

FORMULATION AND CHARACTERIZATION OF LORATADINE CONTAINING ORODISPERSIBLE LYOPHILIZATES AND FILMS

ALEKSANDRA AMELIAN1, 2, EMILIA SZYMA—SKA1, and KATARZYNA WINNICKA1*

1 Department of Pharmaceutical Technology, Medical University of Bia≥ystok, Mickiewicza 2c, 15-222 Bia≥ystok, Poland

2Department of Clinical Pharmacy, Medical University of Bia≥ystok, Mickiewicza 2a, 15-222 Bia≥ystok, Poland

Abstract: Orodispersible formulations are relatively novel solid dosage forms that involve rapid disintegrationor dissolution in the mouth. They enable an easy application and originally were developed to address swal-lowing difficulties of conventional tablets and capsules. The aim of this study was to develop and quality eval-uate oral lyophilizates and orodispersible films with loratadine. Based on the preliminary studies, optimal com-position of lyophilizates and films were chosen for further studies. Oral lyophilizates were obtained by freeze-drying process using different concentration of gelatin, mannitol and sodium bicarbonate. Films were preparedby solvent-casting method with different concentrations of hypromellose and glycerol used as plasticizer.Obtained formulations were characterized for drug content, disintegration time, organoleptic and mechanicalproperties. In order to identify surface morphology of obtained solid dosage forms, scanning electronmicroscopy was used. The results showed that designed dosage forms with loratadine were characterized byappropriate mechanical properties, the uniform content of the drug substance and low moisture content.Disintegration time measured in vivo was = 36 s for films and = 9 s for lyophilizates.

Keywords: oral lyophilizates, orodispersible films, loratadine, solvent-casting method, freeze-drying method

1533

* Corresponding author: e-mail: [email protected]; phone: 48 85 7485615, fax: 48 85 7485616

1534 ALEKSANDRA AMELIAN et al.

Loratadine is a second-generation histamine H1

receptor antagonist commonly used in the treatmentof allergic rhinitis and urticaria. It is a white, crys-talline powder not soluble in water. Loratadine is agood candidate for fast disintegrating oral prepara-tions, because it is characterized by an acceptabletaste and it does not require taste masking (20, 21).

The aim of this work was to design and char-acterize oral lyophilizates and orodispersible filmswith loratadine. The films were obtained by casting-solvent method using PharmacoatÆ 606 and glycerol,while lyophilizates were formulated by freeze-dry-ing process using different concentration of gelatin,mannitol, and sodium bicarbonate. The obtaineddosage forms were characterized for uniformity ofweight and thickness, moisture content and drugloading. Disintegration time of the films andlyophilizates were measured in vivo and in vitro bytwo independent methods. The morphology of for-mulations was determined by scanning electronmicroscopy. The sensory evaluation in vivo was alsoperformed. Mechanical properties of the films weredetermined by using TA.XT. Plus Texture Analyzerand expressed by three different parameters: percentof elongation, tensile strength and Youngís modu-lus. Additionally, film folding endurance was exam-ined.

EXPERIMENTAL

Materials

Hypromellose (PharmacoatÆ 606) was obtainedfrom ShinEtsu Chemical, Tokyo, Japan. Polyethyl-ene glycol was purchased from Donauchem,Warsaw, Poland. Gelatin (type B), sodium bicar-bonate, mannitol and glycerol were purchased from

Sigma Aldrich, Steinheim, Germany. Water wasdistilled and passed through a reverse osmosis sys-tem Milli-Q Reagent Water System (Billerica, MA,USA). PCV blisters (diameter of 13 mm and depthof 5 mm) were obtained from Fagron, KrakÛw,Poland.

Methods

Preparation of oral lyophilizates

Oral lyophilizates were obtained by the freeze-drying method. Gelatin was dissolved in distilledwater at about 40OC to obtain concentrations of 0.5and 1%. Different amounts of mannitol (2.5, 5.0,10.0 and 20.0%) and sodium bicarbonate (0.25 and0.5%) were added to gelatin solution (Table 1).Then, loratadine was suspended in a solution so thatthe amount of 0.5 g of final suspension contained 10mg of drug. Obtained suspensions were dosed intoPCV blisters (0.5 g into each blister slot), frozen at -20OC for 10 min and freeze-dried (lyophilizerFreeZone System, Labconco, Kansas, MO, USA).In order to set the optimal parameters of the freeze-drying process to obtain product of the desired prop-erties, a number of preliminary tests were conductedand the experimental parameters were chosen.Experiments were carried out under the followingconditions: primary drying for 4 h at -48OC, pressureof 0.08 mbar with gradually increasing the tempera-ture to 20OC. In the second drying step, the temper-ature was set to 20OC for 1 h and vacuum pressure of0.08 mbar. After preliminary studies, formulations(L2, L3, L7 and L8) with short disintegration timeand optimal mechanical strength (these formulationsdid not crumble and were easily removed from theblister) were prepared with loratadine (L2L, L3L,L7L and L8L).

Table 1. Composition of oral lyophilizates placebo.

Formulation Gelatin (g) Mannitol (g) Sodium bicarbonate (g) Water (g)

L1 2.5 0.25

L2 5.0 0.25

L3 1.0 10.0 0.25

L4 5.0 0.50

L5 20.0 0.25

L6 2.5 0.25up to 100.0

L7 5.0 0.25

L8 0.5 10.0 0.25

L9 5.0 0.50

L10 20.0 0.25

Formulation and characterization of loratadine containing... 1535

Preparation of orodispersible films

Films were prepared by the solvent castingmethod. Film forming agent (PharmacoatÆ 606) wasused in three concentrations: 2.5%, 5% and 10%.Effect of plasticizer type (glycerol, polyethyleneglycol and mixture of both) with different concen-tration was additionally evaluated. An overview onfilm composition is given in Table 2. After prelimi-nary studies, formulations (F1, F2, F3), whichformed smooth, non-sticky, fast disintegrating andeasily peelable films were selected for loratadineloaded films preparation (F1L, F2L and F3L).PharmacoatÆ 606 was dissolved in distilled water attemperature of 50OC. Glycerol was dissolved in thepolymer solution and homogenized 1 h using a

magnetic stirrer. After that, 2 g of loratadine wasadded. Obtained suspensions were casted onto aPetri dish and dried at temperature room for 24 h.After drying, films were cut into rectangular piecesof 2 cm × 3 cm, which contained 10 mg of loratadineeach.

Evaluation of oral lyophilizates and orodis-

persible films

Uniformity of weight and thicknessWeight and thickness of obtained formulations

were evaluated according to the specifications ofEuropean Pharmacopoeia (22). Film thickness wasmeasured by a micrometer (precision ± 0.001 mm,Helios-preisser, Gammertingen, Germany) at three

Table 2. Composition of orodispersible films placebo.

Plasticizer type (g) Formulation

Glycerol Polyethylene glycolPharmacoatÆ 606 (g) Water (g)

F1 1.0

F2 2.0

F3 3.0

F4 1.0

F5 2.0 up to 100.0

F6 3.0

F7 1.0 1.0

F8 2.0 2.0

F9 3.0 3.0

F10 1.0

F11 2.0

F12 3.0

F13 1.0

F14 2.0 5.0 up to 100.0

F15 3.0

F16 1.0 1.0

F17 2.0 2.0

F18 3.0 3.0

F19 1.0

F20 2.0

F21 3.0

F22 1.0

F23 2.0 10.0

F24 3.0

F25 1.0 1.0

F26 2.0 2.0

F27 3.0 3.0


different positions on the film. Lyophilizates thick-ness was measured using calibrated digital caliper(Beta 1651 DGT, Milan, Italy). Films and lyophi-lizates were weighed individually using an elec-tronic balance (XA 60/220, Radwag, Radom,Poland).

Drug contentLoratadine content uniformity in films and

lyophilizates was tested by dissolving the dosageform in 10 mL of 0.1 M HCl. Then, 1 mL of thesolution was withdrawn and diluted by a mobilephase. After filtration through 0.45 µm celluloseacetate filters (Millipore, Billerica, MA, USA), con-centration of loratadine was determined by theHPLC system Agilent Technologies 1200 equippedwith a G1312A binary pump, a G1316A thermostat,a G1379B degasser and a G1315B diode arraydetector (Agilent, Waldbronn, Germany). Data col-lection and analysis were performed usingChemstation 6.0 software. Isocratic separation wasachieved on a Zorbax Eclipse XDBxC18, 4.6 × 150mm, 5 µm column (Agilent, Waldbronn, Germany).Mobile phase was 0.025 M sodium phosphate bufferpH 3.7 and acetonitrile (1 : 4; v/v), the flow rate was1.0 mL/min and UV detection was performed at awavelength of 247 nm (23-26). For injection into theHPLC system, 20 µL of sample was used. The reten-tion time of loratadine was 3.5 min. Standard cali-bration curve was linear over the range of 1 ñ 100µg/mL with the correlation coefficient (R2) 0.999.

Sensory evaluation Sensory evaluation of roughness and taste of

dosage forms was carried out by six healthy volun-teers (Research Ethics Committee at MedicalUniversity of Bia≥ystok approval number R-J-002/262/2014). Before the assay, potential probantswere tested by the sensitivity of taste. For this rea-son, basic taste solutions with different concentra-tions of model substances: sour (tartaric acid), sweet(sucrose), salty (sodium chloride) and bitter (quininehydrochloride) in distilled water were prepared. Forfurther studies volunteers with the highest taste sen-sitivity were selected (27). A numerical scale wasused with the following values: 0 ñ pleasure/notrough; 1 ñ slightly pleasure/slightly rough; and 2 ñunpleasure/rough.

Morphology analysis Morphology analysis was performed using

scanning electron microscope (Hitachi S4200,Tokyo, Japan). Samples were sputter-coated withgold before imaging.

Evaluation of disintegration time

In vivoDisintegration time of the lyophilizates and

films in the oral cavity was evaluated by six healthyvolunteers. The study protocol was approved by theResearch Ethics Committee at the MedicalUniversity of Bia≥ystok (number R-J-002/262/2014)and complied with the principles of the Declarationof Helsinki. After the mouth was rinsed with puri-fied water, lyophilizate or film was hold in themouth without chewing until the dosage form disin-tegrated. The time required for the complete disinte-gration in the oral cavity was noted.

In conventional disintegration apparatusDisintegration time of formulations was meas-

ured using conventional apparatus (Erweka ED-2L,Heusenstamm, Germany) according to EuropeanPharmacopoeia (22). Distilled water was used asdisintegration medium.

On Petri dishPetri dish with a diameter of 7 cm was filled

with 7 mL of water and the lyophilizate or film (n =6) was carefully put in the center. Time needed tocompletely disintegrate into fine particles was meas-ured (28).

Moisture contentContent of moisture in films and lyophilizates

was assessed using moisture analyzer balance(Radwag WSP 50SX, Radom, Poland).

Evaluation of mechanical film propertiesMechanical properties were examined using

Texture Analyzer TA.XT. Plus (Stable Micro-systems, Godalming, UK) and expressed by threedifferent parameters ñ percent of elongation (E%),tensile strength (TS) and Youngís modulus (E).Experimental parameters of the process were chosenduring preliminary tests and set as follows: pre-testspeed 1.00 mm/s, test speed 1.00 mm/s, post-testspeed 1.00 mm/s, distance 3.0 mm, strain 10.0%,trigger force 0.001 N, break sensitivity 0.107 N. TSwas measured by applied stress and calculated bydividing the force per area. The E% and E were cal-culated by following equations:

L ñ L0E% = ññññññ × 100L0

δE = ññññε

where: L0 ñ length of the sample prior to the meas-urement, L ñ length of the sample after the measure-ment, δ ñ tension, ε ñ linear deformation (29).


Additionally, film folding endurance wasexamined and the number of folds necessary tobreak the film was defined (30).


The choice of the excipients in the design ofdosage forms is critical because it influences theirproperties and quality. The short disintegration timeand appropriate mechanical strength are crucial fac-tors in orodispersible delivery systems. The proper-ties of orodispersible films and oral lyophilizatesmainly depend on: amount and type of polymer,matrix forming agent, amount of solvents, and typeof manufacturing process. In the case of films, typeand amount of plasticizer is also important (2-5). Inorder to determine the optimal composition, variousexcipients in different amounts have been examined.For this purpose, 10 different formulations of orallyophilizates (Table 1) and 27 of orodispersible filmformulations (Table 2) were designed and exam-ined.

In order to develop a composition of orallyophilizates, the effect of gelatin, mannitol andsodium bicarbonate was tested. Based on the place-bo lyophilizates evaluation, it was shown that lowerconcentration of mannitol and higher concentrationof sodium bicarbonate resulted in too crumbly, notdurable texture. Interestingly, there were no signifi-cant differences in disintegration time between for-mulations prepared with various gelatin concentra-tions (data not shown). All placebo lyophilizates hada very low moisture content (from 0.2% in L2 to 1%in L3 ). Lyophilizates containing mannitol as a fillerat the amount of 5 and 10% and with lower (0.25%)amount of sodium bicarbonate (L2, L3, L7, L8)were characterized by sufficient mechanical proper-ties, which are very important in further processessuch as packaging, opening and removing the drugfrom the packaging, thus they were selected for fur-ther studies with loratadine. Lyophilizates with theaddition of 0.5% of sodium bicarbonate as disinte-grating agent were crumbly and it was impossible toremove them from blisters.

Table 3. Physicochemical characteristics of orodispersible films and oral lyophilizates with loratadine.

Disintegrating time (s)Formu- Weight Thickness Drug content Moisture contentlation (mg) (µm/mm) (mg) (%) In vivo Conventional On Petri

apparatus dish

Films

F1L 242.0 ± 2.0 42.0 ± 2 µm 10.0 ± 1.0 0.35 ± 0.2 34.0 ± 1.1 30.0 ± 1.2 38.0 ± 1.1

F2L 269.0 ± 1.8 44.0 ± 1 µm 9.8 ± 0.8 0.72 ± 0.1 34.0 ± 0.9 30.0 ± 1.0 37.0 ± 1.2

F3L 312.0 ± 1.2 48.0 ± 3 µm 10.3 ± 0.5 1.0 ± 0.2 36.0 ± 1.2 31.0 ± 0.9 40.0 ± 1.5

Lyophilizates

L2L 63.7 ± 0.9 2.0 mm 9.93 ± 0.3 0.5 ± 0.1 9.0 ± 0.5 6.0 ± 0.5 9.0 ± 0.6

L3L 63.2 ± 1.1 2.0 mm 8.75 ± 0.6 0.75 ± 0.2 9.0 ± 0.6 6.0 ± 0.6 7.0 ± 0.5

L7L 64.3 ± 1.2 2.0 mm 10.73 ± 0.2 0.9 ± 0.1 8.0 ± 0.5 6.0 ± 0.6 6.0 ± 0.5

L8L 64.4 ± 0.8 2.0 mm 9.3 ± 0.5 0.9 ± 0.1 8.0 ± 0.5 6.0 ± 0.5 6.0 ± 0.5

Table 4. Sensory evaluation of designed orodispersible films and oral lyophilizates with loratadine.

VolunteerTaste/Roughness a)

F1L F2L F3L L2L L3L L7L L8L

A 0 0 0 0 0 0 0

B 0 0 0 0 0 0 0

C 1 0 1 0 0 0 0

D 0 0 0 0 0 0 0

E 0 0 0 0 0 0 0

F 0 0 1 0 0 0 0

a)Scored as follows: 0 = pleasure/not rough; 1 = slightly pleasure/slightly rough; 2 = unpleasure/rough


Figure 1. Representative SEM images of oral lyophilizate placebo formulation L2 (A), lyophilizate with loratadine L2L (B), placebo filmF2 (C), and film with loratadine F2L (D) 10 000 ×

To choose optimal excipients and their concen-trations for placebo orodispersible films, the effectof polyethylene glycol, glycerol and PharmacoatÆ

606 on their physico-chemical properties have beenexamined. Disintegration time of films was mainlyinfluenced by the polymer concentration. With theincreased amount of polymer, increased disintegra-tion time was observed. The shortest disintegrationtime was noted in formulations containing 2.5%Pharmacoat 606Æ, therefore this concentration ofpolymer was used to preparing films with lorata-dine. Films containing glycerol as plasticizer pos-sessed better mechanical strength compared to for-mulations with polyethylene glycol. Films withpolyethylene glycol addition were characterized bygreater moisture content and tended to stick togeth-er. Moreover, volunteers claimed that films with theaddition of polyethylene glycol or polyethylene gly-col/glycerol mixture were characterized by unpleas-ant taste. Interestingly, mixture of both plasticizers

did not improve the mechanical properties of films(data not shown). Films placebo F1, F2, and F3 werechosen for preparing formulations with loratadine.

Characteristics of films and lyophilizates withloratadine is shown in Table 3. Thickness uniformi-ty is extremely important as it directly influencesdosage of drug. Thickness of lyophilizates was 2mm and for orodispersible films it ranged from 42.0to 48.0 µm. Loratadine loading was uniform (22) inall formulations (for orodispersible films, it was inthe range between 9.8 and 10.3 mg and forlyophilizates ñ from 8.75 to 10.73 mg). All preparedfilms possessed low moisture content (0.35 ñ 1%),did no stick and showed no blooming.

Disintegration time is crucial feature of orodis-persible dosage forms and depends on the volume ofmedium used (31, 32). As volume of saliva in theoral cavity is less than 7 mL, therefore it is reallyimportant to measure disintegration time not only inthe conventional pharmacopoeial apparatus. Petri

A

C

B

D


Figure 2. Mechanical properties of orodispersible films with loratadine expressed as percent at elongation (A), tensile strength (B), Youngísmodulus (C) and folding endurance (D)

F1L F2L F3L

F1L F2L F3L

F1L F2L F3L

F1L F2L F3L

A

B

C

D

Per

cent

at c

long

atio

n (E

%)

Tens

ile s

tren

ght (

TS

- N

/m2 )

Youn

gís

mod

ulus

(E

- N

/cm

3 )

Fol

ding

end

uran

ce (

amou

nt)


dish with 7 mL of water was used as an alternativemethod to evaluate the in vitro disintegration rate.The values of disintegration time measured by con-ventional apparatus for films and lyophilizates were= 31 s and 6 s, respectively. Disintegration timeevaluated in vivo for films was = 36 s and forlyophilizates = 9 s.

Obtained lyophilizates were characterized byporous structure (Fig. 1 A, B), which allows pene-tration of saliva into the matrix, resulting in rapiddisintegration. In the surface analysis of films, cohe-sive and homogeneus matrix was observed. Absenceof pores and surface uniformity depicts good quali-ty of films (Fig. 1 C, D). Moreover, the structure offilms and lyophilizates showed regular arrangementof loratadine crystals (Fig. 1 B, D).

Organoleptic properties like taste, mouth-feel-ing and roughness are of considerable importancein orally disintegrating dosage forms design, there-fore the sensory evaluation was also performed.Prepared films and lyophilizates were characterizedby very pleasant taste and no roughness was report-ed (Table 4).

Mechanical properties of films containingloratadine expressed by three different parameters ñpercent at elongation (E%), tensile strength (TS) andYoungís modulus (E) are presented in Figure 2 A-C.Tensile strength (TS) is defined as maximum stressapplied at which the film breaks and it assesses themechanical strength of films. Youngís modulus isthe measure of film stiffness (32-34). Elastic andflexible film is characterized by low value of TS andhigh value of percent of elongation. These parame-ters are very important during removal, cutting,packaging, transport and storage of films (35).Mechanical properties such as tensile strength andpercent of elongation are influenced by plasticizeradded to the formulation (32, 36). It was shown thatglycerol as plasticizer in combination withPharmacoatÆ 606 as membrane-forming substanceensured appropriate mechanical properties ñobtained films were transparent, elastic and flexible.Folding endurance is another parameter determiningthe mechanical properties of a film. It is measuredby repeatedly folding a film at the same point untilit breaks. Folding endurance value is a numer oftimes the film is folded without breaking. Higherfolding endurance value depicts more mechanicalstrength of a film. As mechanical strength is gov-erned by plasticizer concentrations, it is clearly evi-dent that plasticizer concentration also indirectlyaffects folding endurance value (34, 36). For formu-lation F1L folding endurance was 61, for F2L it was90, and for formulation containing the highest

amount of plasticizer ñ F3L, folding endurance was100 (Fig. 2 D).

CONCLUSIONS

In this work, formulation of loratadine inorodispersible films and oral lyophilizates wasachieved. Obtained dosage forms possessed veryshort disintegration time evaluated in vivo (= 36 sfor films and = 9 s for lyophilizates). All formula-tions were characterized by uniformity of the drugcontent, weight and thickness. Prepared films andlyophilizates with loratadine showed pleasant tasteand mouth feeling which is very important for oral-ly administrated products. Glycerol as plasticizer incombination with PharmacoatÆ 606 ensured appro-priate mechanical properties ñ films were transpar-ent, elastic and flexible. Designed formulations aresupposed to be promising alternative for conven-tional solid dosage forms with loratadine.

REFERENCES

1. Sznitowska M., P≥aczek M., Klunder M.: ActaPol. Pharm. 62, 29 (2005).

2. El Meshad A.N., El Hagrasy A.S.: Pharm. Sci.Tech. 12, 1384 (2011).

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Received: 13. 09. 2016


Hypertension is the prominent cause for stroke,cardiovascular disorder and renal disease which fur-ther lead to death of an individual. It has been fore-cast that there will be a relative increase of about24% in the occurrence of hypertension in the devel-oped countries from 2000 to 2025 (1). Karen et al.,in their study surveyed that the number of adultswith hypertension has doubled from 1995 to 2005(2). So the treatment of hypertension has become theforemost requirement to have a control on increas-ing death rate. Lowering blood pressure is the lead-ing treatment to ensure a downfall in the risk of car-diovascular morbidity and mortality. The renin-angiotensin system plays a commanding role inblood pressure regulation and is the major target formany antihypertensive drugs like olmesartanmedoxomil (OLM) which is a selective AT1 subtypeangiotensin II receptor antagonists and is approvedfor effective treatment of hypertension, kidney dis-ease and heart failure (3, 4). Clinical trials in hyper-tensive patients with OLM revealed tremendous

pharmacological actions and a good tolerance with-out any serious side effect (5). Moreover, the effi-ciency of OLM has been revealed to be much high-er or at least equivalent to many other commonlyprescribed antihypertensive agents (6). It is also apromising drug for diminishing the vascular risk inhigh-risk elderly patients with new-onset diabetes. Itis commercially available as conventional tablets inthe strength of 10 and 20 mg. The maximum dose inthe treatment of hypertension is 40 mg (7).However, these marketed formulations of OLM areavailable in crystalline form which is having lowsolubility and dissolution rate. One of its commer-cial tablet formulations under the brand nameìBenicarî possesses oral bioavailability of only 26%in healthy humans (8). Even the remaining unab-sorbed drug leads to gastrointestinal side effectssuch as dyspepsia, abdominal pain, gastroenteritisand nausea. This reduced oral bioavailabilty ofOLM is due to its low aqueous solubility (< 7.75µg/mL) (9).

EFFECT OF POLYMER AND METHOD ON PARTICLE SIZE AND CRYSTALLINITY OF OLMESARTAN MEDOXOMIL DURING

THE FORMULATION OF SOLID DISPERSIONS

GURJEET SINGH1, GHANSHYAM DAS GUPTA2* and SHAILESH SHARMA2

1Department of Pharmaceutical Sciences, IKG Punjab Technical University, Kapurthala 144603, Punjab, India

2Department of Pharmaceutics, ASBASJSM College of Pharmacy, Bela, Ropar 140111, Punjab, India

Abstract: This study explores the use of modified locust bean gum and poloxamer 188 as carriers to improvethe dissolution rate of olmesartan medoxomil; whilst also investigating the effect of technique (lyophilizationand modified solvent evaporation) used for the preparation of solid dispersions. Both carriers along with meth-ods showed significant variability in particle size and, in turn, the solubility profile of olmesartan medoxomilindicating the great influence of carrier/method used. The solid dispersion prepared with lyophilization methodusing modified locust bean gum as a carrier showed reduced particle size of 58.8 ± 1.8 nm as indicated by par-ticle size analysis resulting in higher solubility (1097 ± 15.92 µg/mL) and dissolution efficiency (84.39% after120 min) as compared to other carrier and method. The resulting solid dispersions were characterized using X-ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) andtransmission electron microscopy (TEM), which further supported the findings. As indicated by TEM, opti-mized formulation showed spherical and reduced particles of around 34-36 nm which may have contributed tothe improved dissolution rate of drug. The optimized solid dispersion was then formulated into fast dissolvingtablet to achieve instantaneous and enhanced release for the better and immediate treatment of hypertension.

Keywords: solubility, freeze drying, particle size, fast dissolving tablet, modified locust bean gum

1543

* Corresponding author: e-mail: [email protected]; phone: +91 9417862160

1544 GURJEET SINGH et al.

Improvement in oral bioavailability of OLMcan augment its clinical efficacy and can reduce theoral dose required to achieve equivalent effect and,in turn, diminish the side effects. This oral bioavail-ability of poorly water soluble drug can be increasedby increasing its solubility. Various techniques suchas complex formation (10), solid dispersion (11-13),liquisolid compact (14), nanosuspension (8),nanoemulsion (15) and self-nanoemulsifying drugdelivery systems (16) have been reported forimproving the solubility of OLM. Many authorshave classed the solid dispersion technique as one ofthe most effective and economical method forimproving dissolution rate (17, 18) and has beenwidely explored for enhancing the solubility and, inturn, efficacy of many pharmaceutical agents viz.ezetimibe (19), tacrolimus (20), meloxicam (21),tovaptan (22), etc. The fact behind the enhancementin solubility attained by the formation of solid dis-persions using hydrophilic carrier is the reduction inparticle size, amorphization of a crystalline drug,reduced agglomeration, increased wet ability andmolecular dispersibility. The prominent effect ofparticle size reduction on the drug dissolution ratehas not been studied so far for any drug formulatedas solid dispersion. According to FeundlichñOst-wald equation, the reduction of particle size intonanometric range enhances dissolution rate of drugnot only due to increased surface area but also dueto increased saturation solubility (23).

The aim of the present study is to enhance thesolubility and dissolution rate of OLM and comparethe potential of a natural (modified locust bean gum)and synthethic (poloxamer 188) carrier along withthe method (lyophilization and modified solventevaporation) for the preparation of solid dispersions

and also to determine the effect of individual carri-er/ method on particle size of solid dispersions. Thismight assist the future research in selecting the mostappropriate carrier and method for the developmentof solid dispersion formulations which is a criticalparameter for producing formulations with desiredsolubility and dissolution profile. The study alsorelates to the fabrication of optimized formulationinto fast dissolving tablet to achieve instantaneousrelease for the better and immediate treatment ofhypertension.


Olmesartan medoxomil was obtained as a giftsample from Cipla Ltd., Mumbai. Locust bean gumwas obtained from Wilson Laboratories, Mumbai.Poloxamer 188 was purchased from Sigma Aldrich,Mumbai. Methanol and ethanol were purchasedfrom S.D. Fine Chemicals, Mumbai. Camphor waspurchased from Loba Chem Pvt. Ltd., Mumbai.Crospovidone was purchased from SignetChemicals Pvt. Ltd., Mumbai. All other chemicalsused were of analytical grade.

Preparation of modified locust bean gum

Locust bean gum was modified according tothe method described by Murali Mohan Babu et al.(24). For the preparation of modified locust beangum, powdered locust bean gum was heated in hotair oven (Decibel, Chandigarh, India) at 120OC for 2h. The prepared modified locust bean gum was thensieved (100 mesh) and stored in an air tight contain-er at 25OC. Both locust bean gum and modifiedlocust bean gum were then characterized for viscos-ity using Rheometer (Rheolab QC, Anton Paar,

Table 1. Characterization of locust bean gum and modified locust bean gum.

Parameter LBG MLBG

10 1903 ± 26 192.1 ± 1.6

Viscosity Shear 20 1629 ± 19 169.1 ± 1.9

(cps) Rate (s-1) 30 1414 ± 17 150.9 ± 1.2

40 1251 ± 13 137.2 ± 1.5

Swelling index (%) 1494 ± 12 1319 ± 15

Hydration Capacity 2.9 ± 0.03 2.93 ± 0.02

Moisture Sorption Capacity (%) 7.4 ± 0.18 12.96 ± 0.21

Angle of Repose 36.67 ± 0.9 36.29 ± 0.8

Carr's Index (%) 21.50 ± 0.7 20.82 ± 0.5

Data are expressed as the mean ± S.D. (n = 3). * LBG = Locust bean gum, MLBG = Modified Locust bean gum

Effect of polymer and method on particle size and crystallinity of... 1545

Tab

le 2

. Com

posi

tion

and

char

acte

riza

tion

of p

repa

red

solid

dis

pers

ions

of

olm

esar

tan

med

oxom

il. Cha

ract

eriz

atio

n

Poly

mer

Form

ulat

ion

Dru

g:

Met

hod

% Y

ield

% D

rug

Part

icle

Sa

tura

tion

solu

bilit

y co

deca

rrie

r ra

tious

edco

nten

tsi

ze(µ

g/m

L)

Pure

Dru

g-

--

--

9 ±

0.2

PMO

1-

--

--

66 ±

3.7

6

OL

M1

1 : 1

Mod

ifie

d so

lven

t eva

pora

tion

94.5

± 1

.298

.4 ±

1.3

974.

1 ±

3.3

192

± 2.

45

OL

M2

1 : 2

Mod

ifie

d so

lven

t eva

pora

tion

91.7

± 1

.097

.6 ±

1.0

956.

2 ±

3.0

237

± 5.

98

OL

M3

1 : 3

Mod

ifie

d so

lven

t eva

pora

tion

82.1

± 1

.594

.8 ±

1.4

916.

3 ±

2.5

389

± 5.

19

OL

M4

1 : 4

Mod

ifie

d so

lven

t eva

pora

tion

73.5

± 0

.995

.8 ±

1.1

877.

3 ±

4.1

557

± 11

.95

PLX

M 1

88O

LM

51

: 5M

odif

ied

solv

ent e

vapo

ratio

n68

.8 ±

0.8

98.6

± 1

.087

1.8

± 3.

259

3 ±

10.4

8

OL

M6

1 : 1

Lyo

phili

satio

n92

.7 ±

1.3

96.5

± 0

.959

7.6

± 1.

735

8 ±

7.45

OL

M7

1 : 2

Lyo

phili

satio

n83

.1 ±

1.1

99.5

± 0

.856

1.9

± 3.

253

7 ±

6.85

OL

M8

1 : 3

Lyo

phili

satio

n74

.1 ±

0.9

96.8

± 1

.153

3.9

± 2.

978

9 ±

7.58

OL

M9

1 : 4

Lyo

phili

satio

n72

.5 ±

0.7

97.9

± 1

.251

4.3

± 2.

595

7 ±

13.8

5

OL

M10

1 : 5

Lyo

phili

satio

n70

.1 ±

0.9

98.1

± 0

.851

5.5

± 4.

596

3 ±

12.5

7

PMO

2-

--

--

79 ±

2.3

OL

M11

1 : 1

Mod

ifie

d so

lven

t eva

pora

tion

94.8

± 1

.199

.1 ±

1.2

893.

9 ±

1.5

278

± 6.

81

OL

M12

1 : 2

Mod

ifie

d so

lven

t eva

pora

tion

92.9

± 1

.098

.0 ±

1.1

881.

7 ±

2.7

399

± 7.

92

OL

M13

1 : 3

Mod

ifie

d so

lven

t eva

pora

tion

94.1

± 0

.696

.1 ±

1.0

806.

6 ±

2.9

581

± 9.

62

OL

M14

1 : 4

Mod

ifie

d so

lven

t eva

pora

tion

96.3

± 0

.998

.4 ±

1.3

238.

6 ±

3.1

649

± 5.

99

ML

BG

OL

M15

1 : 5

Mod

ifie

d so

lven

t eva

pora

tion

93.9

± 1

.297

.2 ±

1.1

769.

4 ±

2.1

746

± 13

.46

OL

M16

1 : 1

Lyo

phili

satio

n96

.9 ±

0.3

99.2

± 1

.242

8.9

± 2.

648

9 ±

12.8

3

OL

M17

1 : 2

Lyo

phili

satio

n94

.9 ±

0.9

98.2

± 1

.529

7.3

± 2.

169

3 ±

17.6

4

OL

M18

1 : 3

Lyo

phili

satio

n96

.7 ±

1.4

98.2

± 1

.017

5.9

± 1.

694

1 ±

11.8

9

OL

M19

1 : 4

Lyo

phili

satio

n93

.8 ±

1.9

98.2

± 1

.458

.8 ±

1.8

1097

± 1

5.92

OL

M20

1 : 5

Lyo

phili

satio

n95

.8 ±

1.0

97.3

± 1

.159

.5 ±

1.1

1102

± 1

9.51

Dat

a ar

e ex

pres

sed

as m

ean

± S.

D. (

n =

3)


Ostfildern, Germany) at different shear rate. Theywere also evaluated for their swelling index, hydra-tion capacity, moisture sorption capacity, angle ofrepose and Carrís index (24, 25).

Drug excipients compatibility studies

While designing the solid dispersions, it isimperative to give consideration to the compatibili-ty of drug and carrier used within the systems. It istherefore necessary to confirm that the drug is notshowing any incompatibility with the carrier underexperimental conditions (40 ± 5OC and 75 ± 5% RH)for at least 2 weeks. Desired quantity of drug withspecified excipients (poloxamer 188 and modifiedlocust bean gum) in the ratio 1 : 5 was taken andmixed thoroughly, sieved and filled in dried vials.The vials were examined daily at regular intervalsfor discoloration, clump formation and liquefaction.Also the FTIR spectra of pure drug, carrier and soliddispersion were obtained to determine the compati-bility. For FTIR, each sample of approximately 4mg was kept in FTIR spectrometer (ALPHA FTIR,Bruker, Germany) and spectra were recorded.

Formulation preparation

Modified solvent evaporation methodFor the preparation of solid dispersions, the

drug was dissolved in suitable quantity of methanolwith continuous stirring. Both the carriers (polox-amer 188 and modified locust bean gum) were dis-solved separately in appropriate quantity of water(up to the wet mass of carrier). The drug to carrierratios were varied from 1 : 1 to 1 : 5 to determine theeffect of increasing concentration of carrier. Thedrug solution was then added to the carrier solutionto form a clear solution. The entire solvent wasevaporated under reduced pressure at 60-70OC usingrota evaporator (RV-10, IKA, Bangalore, India).The obtained solid dispersions were dried, sievedand stored in a desiccator (25).

Lyophilization (Freeze drying method)Solid dispersions containing different drug to

carrier ratios from 1 : 1 to 1 : 5 were also preparedby freeze drying method. OLM was dissolved insufficient quantity of methanol to form phase I.Similarly, carriers were dissolved separately in

Figure 1. Viscosity profile of locust bean gum A and modified locust bean gum B


water to form phase II and both the phases weremixed together. Methanol was evaporated andresulted solution was freezed in a quick/deep freez-er (RQFV-265, REMI Elektrotechnik, Mumbai,India) at -20OC and was then lyophilized in a freezedryer (LyoQuest-55, Azbil Telstar Technologies,Terrassa, Spain) at a temperature of -45OC and avacuum of 0.200 Mbar. The freeze dried mass wasthen sieved through sieve no. 60 and stored in desic-cator (26, 27).

Characterization of solid dispersion formulation

Drug content of the prepared solid dispersionswas determined by dissolving solid dispersionsequivalent to 10 mg of OLM in methanol. The dilut-ed solution was then filtered through membrane fil-ters (Millipore Corp, Billerica, US) of size 0.45 µmand analyzed using high performance liquid chro-matography (HPLC) assay (1260 series, AgilentTechnology, Santa Clara, CA, USA) with mobilephase consisting of acetonitrile and 0.05 M sodiumdihydrogen phosphate in a ratio of 60: 40 (28). Themobile phase at flow rate of 1 mL/min was eluted in

BDS Hypersil C-18, 150 ◊ 4.6 mm, 5 µ column(Thermo Fisher Scientific Inc., USA) at room tem-perature and samples were analyzed at 257 nm usingUV diode array detector. The retention time of drugwas observed at 3.54 ± 0.05 min.

The percentage yield of each formulation wasdetermined according to the recoverable finalweight of solid dispersions using following eq.

Solubility of prepared solid dispersions

The solubility of solid dispersions was deter-mined by adding solid dispersions equivalent to 20mg of drug to 10 mL of triple distilled water inscrew capped vials. The vials were sealed and kepton isothermal water bath shaker (NSW-125, NarangScientific Works, New Delhi, India) at 3 ± 0.5OC for48 h. Then, the resulted samples were centrifugedand supernatant was filtered through membrane fil-ters (Millipore Corp., Billerica, US) of size 0.45 µm,which were diluted with triple distilled water andanalyzed by HPLC assay.

Figure 2. Particle size of solid dispersion prepared with modified locust bean gum using lyophilization method. A) Particle size B)Histogram; and prepared with poloxamer 188 using lyophilization method C) Particle size D) Histogram

Practical weight of solid dispersionPercentage yield = ñññññññññññññññññññññññññññññññññ (1)

Theoretical weight of solid dispersion


In vitro drug release study

In vitro drug release studies of prepared solid dis-persions and pure drug were performed in triplicateusing dissolution apparatus (DS 8000, LABINDIA,Navi Mumbai, India) in distilled water at 37 ± 0.5OCusing USP type II apparatus at 100 rpm. Powderedsolid dispersions equivalent to 40 mg of OLM wereadded to the dissolution medium. At appropriate timeintervals, 10 mL sample was withdrawn and replacedwith fresh dissolution medium to maintain the sinkconditions. The withdrawn samples were then filteredusing membrane filter (Millipore Corp., Billerica,MA) and analyzed for drug content using HPLC. Thedissolution efficiency (DE%) after 30 and 120 minwas determined using trapezoidal method and was cal-culated as the percentage area of the rectangle dividedby area of 100% dissolution at particular time.

Nanocharacterization using different techniques

Particle size analysisParticle size of the solid dispersion was deter-

mined using particle size analyzer (Nano ZS90,

Malvern Zetasizer, Malvern, UK). The sample wasdissolved in triple distilled water and subjected toparticle size analysis. This method also depicted theintensity distribution of the particles diameter andpolydispersity index which is a measure of unifor-mity in size distribution.

Differential scanning calorimetry (DSC)DSC analysis was performed using automatic

differential scanning calorimeter (Shimadzu DSC-60A). Each sample (3 mg) was weighed and ana-lyzed in pierced aluminium pans and analysis wascarried out under nitrogen purge at a heating rateof 10OC/min and temperature range of 50 to300OC.

X-ray diffractionX-ray diffraction patterns can be employed for

confirming the crystalline nature of the drug.Powder X-ray diffractometer (D8-Focus, Bruker,USA) was employed to carry out XRD of drug,polymers and solid dispersions using CuKα radiation

Table 3. Dissolution efficiency of olmesartan medoxomil solid dispersions.

Polymer Formulation code Method used DE30 DE120

Pure Drug - 6.65 12.31

PMO1 - 18.22 31.88

OLM1 Modified solvent evaporation 19.87 35.68




PLXM 188 OLM5 Modified solvent evaporation 25.44 54.41

OLM6 Lyophilization 23.84 52.36





PMO2 - 20.91 34.60





MLBG OLM15 Modified solvent evaporation 27.99 58.34







Tab

le 4

. Com

posi

tion

and

char

acte

riza

tion

of d

rug

free

tabl

et.

Ingr

edie

nts

(mg)

F1F2

F3F4

F5F6

F7F8

F9F1

0F1

1F1

2F1

3F1

4F1

5F1

6

Ac-

Di-

Sol

36

912

--

--

--

--

--

--

Cro

spov

idon

e-

--

-3

69

12-

--

--

--

-

Cam

phor

--

--

--

--

612

1824

--

--

Thy

mol

--

--

--

--

--

--

612

1824

Avi

cel p

H10

212

412

412

412

412

412

412

412

412

412

412

412

412

412

412

412

4

Lac

topr

ess

9191

9191

9191

9191

9191

9191

9191

9191

Man

nito

l67

6461

5867

6461

5864

5852

4664

5852

46

Tal

c6

66

66

66

66

66

66

66

6

Mg

stea

rate

99

99

99

99

99

99

99

99

Wei

ght (

mg)

298.

334

30

2.51

2 30

1.55

429

7.21

6 30

3.11

1 29

8.09

829

7.59

229

9.93

730

2.03

829

6.30

230

3.23

329

5.84

329

8.72

929

9.49

230

2.01

730

5.40

2±

2.91

8±

3.16

6±

4.18

2±

2.48

7±

2.91

2±

3.56

1±

4.82

3±

2.90

9±

3.96

7±

2.90

9±

3.98

7±

3.10

3±

2.63

9±

3.85

3±

2.67

8±

4.01

2

Har

dnes

s 3.

22.

03.

43.

23.

33.

23.

33.

63.

23.

33.

43.

12.

82.

92.

82.

8(k

g/cm

2 )±

0.09

6±

0.07

0±

0.10

3±

0.10

2±

0.11

2±

0.11

7±

0.12

9±

0.10

2±

0.11

5±

0.12

1±

0.10

2±

0.12

4±

0.05

6±

0.08

7±

0.14

0±

0.08

4

Fria

bilit

y (%

)0.

614

0.72

50.

663

0.68

70.

619

0.60

70.

570

0.67

30.

943

1.27

91.

602

1.91

60.

842

1.25

71.

678

1.87

4±

0.01

2±

0.03

8±

0.03

3±

0.02

7±

0.03

0±

0.04

3±

0.05

9±

0.02

7±

0.03

2±

0.05

1±

0.02

4±

0.05

3±

0.04

1±

0.04

0±

0.03

5±

0.04

1

Dis

intig

ratio

n 9

8360

3972

6841

3276

5237

2311

172

5841

time

(sec

)±

1.48

± 1.

69±

1.15

± 0.

81±

1.91

± 1.

63±

1.48

± 1.

12±

1.86

± 1.

02±

1.08

± 0.

43±

2.14

± 1.

38±

1.73

± 0.

78

Wet

ting

time

8467

5132

6153

3421

6741

2814

9461

4532

(sec

)±

1.69

± 1.

37±

2.48

± 1.

63±

3.26

± 1.

83±

2.41

± 2.

06±

1.48

± 3.

48±

2.30

± 1.

69±

2.08

± 2.

16±

3.27

± 1.

30

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a ar

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pres

sed

as th

e m

ean

± S.

D. (

n =

3).


in the angle 20 range from 5 to 80 with step size of0.02, step time of 18.7 min and scanning speed of4/min.

Scanning electron microscopy (SEM)The morphology of pure drug, carrier and pre-

pared solid dispersions was determined using scan-ning electron microscope (ZEISS SUPRA-55,Germany) operated at an accelerating voltage of 10kV and aperture was 20 µm. Samples were preparedby mounting powder on a brass stub using graphiteglue and coated with gold and then viewed underSEM.

Transmission electron microscopy (TEM)It was carried out in order to determine the

morphology (particle-shape and particle-size) of thesolid dispersions. When dispersed in water, soliddispersion either forms molecular dispersion or nano-dispersion with reduced particle size. The solid dis-persion was dissolved in triple distilled water and adrop was coated on a carbon coated copper grid of200 mesh size and dried prior to the observationunder transmission electron microscope, (HRTEM,

JEOL, JEM 2100, Japan) operated at an acceleratingvoltage of 200 kV with beam current of 100 µA(29).

Preparation of blank tablets for optimization of

different components

The drug free tablets were prepared in order tooptimize the type and concentration of sublimatingagent and superdisintegrant. All the raw materialswere passed through a screen (44 mesh). Camphor,crospovidone, microcrystalline cellulose, mannitoland lactopress were mixed using a glass mortar andpestle. The blends were lubricated with 1% w/w oftalc and 2% w/w of magnesium stearate, which werethen compressed in a eight station tablet punchingmachine (Lab-8, Saimach, Ahmedabad, India)equipped with flat face 8 mm punches. The tabletweight was adjusted to 300 mg. The tablets weredried for 6 h under vacuum at 50OC to render thetablets porous by sublimation of sublimating agent.

Experimental design of fast dissolving tablets

A 32 randomized full factorial design (DesignExpert version 10, State Ease Inc., USA) was used

Figure 3. Dissolution profile of solid dispersions prepared with poloxamer 188 using A) modified solvent evaporation method, B)lyophilization; and solid dispersions prepared with modified locust bean gum using C) modified solvent evaporation method, D) lyophiliza-tion


in order to investigate the dual influence of two for-mulation variables. In this design, 2 factors wereevaluated, each at 3 levels, and experimental trialswere performed at all 9 possible combinations. Theamounts of subliming agent, camphor (X1), and thesuperdisintegrant, crospovidone (X2), were selectedas independent variables. The disintegration timeand percentage friability were selected as dependentvariables. Checkpoint batch was also prepared toprove the validity of the evolved mathematicalmodel. In addition, contour plots were used tographically represent the effect of the independentvariables.

Evaluation of tablet properties

The strength of the prepared tablets was deter-mined using hardness tester (Monsanto) and the fri-ability was assessed using Roche friabilator(FT1020, LABINDIA, Navi Mumbai, India). Fordetermining friability, 20 pre-weighed tablets weretaken and rotated at 25 rpm for 4 min in friabilatorand then reweighed after removal of fines. Fromthis, percentage reduction in weight indicating fri-ability was calculated. For determining wettingtime, 5 round pieces of tissue paper (0.45 µm poresize, Hi-media) were placed in a Petri dish.Appropriate quantity of water containing a water-

soluble dye (eosin 0.01%), was placed on the tissuepaper. A tablet was kept on the surface of the tissuepaper and time required for water to reach the uppersurface of the tablet was determined. For disintegra-tion time, a modified method was used, in which 6mL of Sorensonís buffer (pH 6.8) at 37 ± 0.5OC wasplaced in a 10 mL cylindrical glass vessel, so that 2mL of the media remained below the sieve and 4 mLabove. Single tablet was placed on the sieve andentire assembly was mounted on a high precisionwater bath shaker (Narang Scientific Works, NewDelhi, India). The time taken for all the drug parti-cles to pass through the sieve was noted which wasconsidered as disintegration time. Six tablets wereselected randomly from each batch and were testedfor disintegration time and the mean value was cal-culated (30-32). In vitro dissolution studies of opti-mized fast dissolving tablet and commercial tabletwere performed in triplicate using dissolution appa-ratus (DS 8000, LABINDIA, Navi Mumbai, India)in Sorensonís buffer (pH 6.8), 900 mL, at 37 ± 0.5OCusing USP type II apparatus at 100 rpm.


The choice of a particular method/carrier in thepreparation of solid dispersion significantly affects

Figure 4. FTIR of olmesartan medoxomil pure drug, poloxamer 188, modified locust bean gum and solid dispersion prepared with polox-amer 188 and modified locust bean gum using lyophilization method


the pharmaceutical behavior of the prepared prod-uct. Owing to this, in the present study, solid disper-sions were prepared using different methods inmerger with different carriers in order to determinethe effect of individual method or carrier on the finalformulation. The locust bean gum used as carrierwas modified in order to reduce its viscosity withoutsignificantly altering other parameters and theresults are shown in Table 1. The viscosity wasdetermined at different shear rate and the viscosityprofile of locust bean gum and modified locust beangum at a shear rate of 20 s-1 and 40 s-1 is shown inFigure 1. There is a prominent difference in viscos-ity with a value of 1251 ± 13 and 137.2 ± 1.5 cps atshear rate of 40 s-1 for locust bean gum and modifiedlocust bean gum, respectively, which showed 9-folddecrease in viscosity of the carrier on modification.Solid dispersions were prepared in different drug tocarrier ratios ranging from 1 : 1 to 1 : 5 using modi-fied locust bean gum (natural) and poloxamer 188(synthetic) as carriers. Similarly, two differentmethods viz. modified solvent evaporation andlyophilization were also evaluated for the prepara-tion of solid dispersions. The composition of all theprepared solid dispersions of OLM prepared usingboth methods in unification with both carriers aregiven in Table 2.

Drug content of all the formulations was foundin the range 94.8 ± 1.4% to 99.5 ± 0.8%. To deter-mine the effectiveness of prepared solid dispersions,all formulations were subjected to solubility testingand the results are shown in Table 2. It was observedthat as the drug to carrier ratio was increased, thesolubility of the drug also increased in a linear man-ner with no significant difference observed after 1 :4. So, the drug to carrier ratio of 1 : 4 was optimizedin all cases. It was also found that the solubility ofdrug increased to higher value using lyophilizationtechnique as compared to modified solvent evapora-

tion which is due to the formation of porous andfluffy product by lyophilization technique whichincreases the surface area and, in turn, the surfacefree energy, resulting in higher solubility and disso-lution (26). The lower is the particle size of soliddispersion; higher is the solubility which is alsoproved here in this present study. Among all thesolid dispersions at drug to polymer ratio of 1 : 4,particle size of two solid dispersions (OLM 9 andOLM 19) prepared using lyophilization techniqueshowed greater variability with elevated solubilityas compared to the dispersions prepared with modi-fied solvent evaporation method as shown in Table2. The particle size of these two optimized solid dis-persions was found to be 514.3 ± 2.5 nm and 58.8 ±1.8 nm when prepared with poloxamer 188 (OLM 9)and modified locust bean gum (OLM19), respec-tively, as shown in Figure 2. Whereas, it was foundto be 877.3 ± 4.1 nm and 238.6 ± 3.1 nm for polox-amer 188 and modified locust bean gum, respective-ly, for solid dispersion prepared using modified sol-vent evaporation method.

The results of particle size and solubility analy-sis proved the supremacy of lyophilization methodover modified solvent evaporation method. Even theresults showed a great difference in the solubilityand particle size of solid dispersions prepared withboth carriers also. However, this fact of carriereffect was further determined by subjecting all for-mulations prepared with both techniques to dissolu-tion and nanocharacterization studies to strengthenthe findings.

In vitro dissolution studies

The result of dissolution studies of preparedsolid dispersions of OLM are shown in Figure 3.The percentage release was found to be better incase of solid dispersions prepared by lyophilizationtechnique as compared to modified solvent evapora-

Table 5. Composition of factorial batches.

Ingredients (mg) OFDT1 OFDT2 OFDT3 OFDT4 OFDT5 OFDT6 OFDT7 OFDT8 OFDT9

Solid dispersion 200 200 200 200 200 200 200 200 200

Camphor 6 6 6 12 12 12 18 18 18

Crospovidone 3 6 9 3 6 9 3 6 9

Avicel Ph 102 31 31 31 31 31 31 31 31 31

Lactopress 23 23 23 23 23 23 23 23 23

Mannitol 22 19 16 16 13 10 10 7 4

Talc 6 6 6 6 6 6 6 6 6

Mag. sterate 9 9 9 9 9 9 9 9 9


tion method, whereas, it was highest when modifiedlocust bean gum was used as carrier. In lieu to this,solid dispersions prepared using poloxamer 188 andmodified locust bean gum with lyophilization tech-nique showed dissolution efficiency (DE120) of70.59% and 84.39% at 120 min, indicating higherdissolution efficiency using modified locust beangum as shown in Table 3. This enhanced dissolutioneffect with modified locust bean gum is due to itsswelling nature which results in increased surfacearea during dissolution resulting in higher dissolu-tion rate of the drug (25). Moreover, modified locustbean gum is a natural polymer and the use of naturalcarrier for the preparation of solid dispersions ismore beneficial because of their low cost, biocom-patibility, and biodegradability. These findings withboth carriers were further supported by carrying outvarious characterizations of two optimized solid dis-persions showing enhanced solubility, dissolutionefficiency and reduced particle size.

Nanocharacterization of solid dispersion

Figure 4 shows FTIR spectrum of OLM,poloxamer 188, modified locust bean gum and soliddispersions prepared using both carriers. FTIR spec-tra of pure OLM shows characteristic peaks at3284.90 cm-1 (broad intermolecular hydrogen bond,

O-H stretch), 2965.68 cm-1 (aliphatic C-H stretch),1706.01 cm-1 (C=O of carboxylic group), 1476.52cm-1 (C-N stretch), 1394.67 cm-1 (in plane O-H bend)and 1051.17 cm-1 (ring C-O-C stretch). The soliddispersions prepared using both carriers exhibitcharacteristic bands of the drug and do not exhibitdisappearance of any peak. This indicates nonexis-tence of any interaction between the drug and thecarriers used.

Determination of amorphization with DSC andXRD

DSC curves of pure drug, poloxamer 188 andsolid dispersion prepared with poloxamer 188 areshown in Figure 5. For pure OLM, a sharpendothermic peak is observed at 183.05OC, charac-terizing melting point of OLM which indicates thatthe pure drug was in crystalline form. Poloxamer188 showed a melting endothermic peak at 58.71OC.Upon formation of solid dispersion of drug withpoloxamer 188, the melting endotherm of drug dis-appeared. On the other hand, no sharp endothermicpeak was observed in the DSC curve for solid dis-persion prepared with modified locust bean gum asshown in Figure 6, while it showed broadening andslight shifting of OLM peak indicating reduction incrystallinity of drug or dispersibility of drug in this

Table 6. Blueprint of 32 full factorial design.

Variable levels in Disintegration Batch Codecoded form time

% Friability

X1 X2 DT (s) ± SD F (%) ± SD

OFDT1 -1 -1 80 ± 1.60 0.611 ± 0.017

OFDT2 -1 0 65 ± 1.84 0.483 ± 0.013

OFDT3 -1 1 54 ± 1.53 0.281 ± 0.009

OFDT4 0 -1 62 ± 1.21 0.657 ± 0.017

OFDT5 0 0 43 ± 0.84 0.538 ± 0.015

OFDT6 0 1 31 ± 0.89 0.342 ± 0.008

OFDT7 1 -1 57 ± 1.63 0.729 ± 0.021

OFDT8 1 0 35 ± 1.34 0.632 ± 0.017

OFDT9 1 1 20 ± 1.59 0.443 ± 0.014

OCP 0.99 0.18 31.99 0.599

Coded ValuesActual values (mg)

X1 X2

-1 6 3

0 12 6

1 18 9

* X1 indicates amount of camphor (mg); X2, amount of crospovidone (mg); DT, disintegration time; and F, fri-ability. OCP used as checks point and optimized batch


carrier. In DSC analysis of both solid dispersions,there was disappearance of the drug meltingendotherm in the solid dispersion prepared withpoloxamer 188, which could be due to the presence ofthe amorphous form of OLM in the solid dispersion.But the sharp peak corresponding to carrier remainedand was at slightly lower temperature than that ofpure poloxamer 188. It might be due to the reason thatdrug molecules get dispersed in the poloxmer 188matrix of the solid dispersion and the thermal proper-ty was changed or it might be due to the formation ofeutectic mixtures in solid dispersions leading to thedepression of melting point. As in both solid disper-sions drug to carrier ratios were the same, but therewas incongruity in these results, so in order to deeproot these findings, XRD of pure drug and its soliddispersions was carried out (33, 34).

XRD patterns of pure drug, carriers and soliddispersions are shown in Figure 7. The XRD of pureOLM showed characteristic peaks with high intensi-ty at various 20 values which were intense andsharp, indicating its crystalline nature. These highintensity peaks diminished in case of solid disper-sions prepared using poloxamer 188 whereas these

characteristic peaks disappeared in case of solid dis-persions prepared using modified locust bean gumindicating reduction in crystallinity of drug via for-mation of amorphous solid dispersion. The degreeof crystallinity was decreased to maximum extent incase of solid dispersion prepared using modifiedlocust bean gum by lyophilization technique. Boththe above discussed parameters indicate amorphiza-tion of OLM by the formation of solid dispersionswhich is responsible for substantial enhancement insolubility.

Scanning electron microscopyThe photomicrographs of OLM, solid disper-

sions prepared with modified locust bean gum andpoloxamer 188 are shown in Figure 8. Pure drugappeared as crystals whereas both carriers and soliddispersions revealed amorphous particles. Therewas reduction in crystallinity of drug on formationof solid dispersion; moreover, solid dispersions pre-pared with modified locust bean gum showedreduced particle size in nanometric range as indicat-ed in Figure 8D, which were clearly visible only athigher magnification. The SEM studies further con-

Figure 5. DSC of olmesartan medoxomil pure drug, poloxamer 188 and solid dispersion prepared with poloxamer 188 using lyophilizationmethod


Figure 6. DSC of olmesartan medoxomil pure drug, modified locust bean gum and solid dispersion prepared with modified locust beangum using lyophilization method

Figure 7. XRD of olmesartan medoxomil pure drug, poloxamer 188, modified locust bean gum and solid dispersion prepared with polox-amer 188 and modified locust bean gum using lyophilization method


Table 7. Regression analysis data of olmesartan medoxomil.

ResponseDisintegration time (sec) Friability (%)

FM RM FM RM

b0 43.33333 49.66667 0.53933 0.52400

b1 -14.50000 -14.50000 0.071500 0.071500

b2 -15.66667 -15.66667 -0.15517 -0.15517

b11 6.50000 - 0.017500 -

b22 3.00000 - -0.0405000 -

b12 -2.75000 - 0.011000 -

* FM = Full model, RM = Reduced model

Table 8. Results of ANOVA of full models and reduced model for dependent variables.

Full modelFor disintegration time

df SS MS f R2

Regression 5 2866.92 573.38 1587.83 0.9996

Residual 3 1.08 0.36 - -

Reduced model

Regression 2 2734.17 1367.08 61.29 0.9533

Residual 6 133.83 22.31 - -

Full modelFor friability

df SS MS f R2

Regression 5 0.18 0.036 2275.49 0.9997

Residual 3 0.000047 0.000015 - -

Reduced model

Regression 2 0.18 0.088 118.75 0.9754

Residual 6 0.0044 0.00073 - -

* df = degree of freedom, SS = sum of squares, MS = mean of squares, f = Fischer's ratio, R = Regression coefficient.

firmed the result of particle size analysis, dissolutionstudies and XRD.

Transmission electron microscopyIt was carried out in order to determine the

morphology (particle shape and particle size) of thesolid dispersions. The particle size of solid disper-sion prepared with modified locust bean gum wasfound to be 36.59 nm whereas that prepared withpoloxamer 188 was 505.68 nm as shown in Figure 9,indicating smallest particle size and higher solubili-ty with modified locust bean gum. Also, solid dis-persion prepared with modified locust bean gumrevealed regular spherical shape, due to which it hasshown acceptable flow properties. The reduced par-ticle size is responsible for its improved solubilityand dissolution rate as compared to solid dispersionprepared with poloxamer 188 and pure OLM.

Besides cost effective, natural, biocompatibleand biodegradable polymer, modified locust beanhas also proved to be a potential carrier for improv-ing the solubility and reducing particle size of OLM.It revealed better results as compared to poloxamer188. Similarly, lyophilization method has proved tobe the most appropriate method for preparation ofsolid dispersions. Based on these results, OLM19prepared with lyophilization technique along withmodified locust bean gum as carrier showing high-est solubility, reduced particle size, spherical andamorphous particles and reduced crystallinity wasoptimized for preparation of fast dissolving tablets.

Preparation of drug free tablets

The compositions of drug free tablets areshown in Table 4. Different superdisintegrants andsublimating agents were used in order to determine


their effect to optimize better one. All preparedtablets showed uniformity in weight and hardness.Crospovidone decreased the disintegration timemore effectively as compared to Ac-Di-Sol whenused in equal concentration, wherein at a concentra-tion of 3% w/w it was found to be 41 ± 1.48 and 60± 1.15 s for crospovidone and Ac-Di-Sol, respective-ly. No significant difference in disintegration timewas observed after this concentration. The same wasobserved in the case of sublimating agent where

camphor showed prominent results. But on increas-ing the concentration of camphor beyond 3%, fri-ability of tablets started increasing although the dis-integration time was decreased. It is stated that thesuperdisintegrant act through strain recovery, wick-ing and capillary action (35) and sublimating agentgenerate porosity in the tablet which increases wateruptake and, in turn, facilitate the wicking action ofsuperdisintegrant (36). Both crospovidone and cam-phor when used in combination showed better result

Figure 8. SEM of pure olmesartan medoxomil (A), modified locust bean gum (B), poloxamer 188 (C), solid dispersion prepared with mod-ified locust bean gum (D), solid dispersion prepared with poloxamer 188 (E)


Figure 9. TEM of solid dispersion prepared by poloxamer 188 (A) and modified locust bean gum (B)

Figure 10. Response surface plot of disintegration time and % friability (A), contour plot of disintegration time and % friability (B),response surface prediction plot (C)


as compared to those when used alone (37). So, onthe basis of disintegration time, crospovidone andcamphor were selected as superdisintegrant and sub-limating agent to be used in combination in the con-centration of 1-3% for the preparation of tablets.

Experimental design of fast dissolving tablets

A 3 factorial design was used in order to opti-mize the amount of both subliming agent and super-disintegrant to obtain the desired disintegration timeand friability. The amount of camphor (X1) andcrospovidone (X2) were chosen as independent vari-ables. A statistical model incorporating both interac-tive and polynomial terms was used to estimate theresponse by using equationY = b0 + b1X1 + b2X2 + b12X1X2 + b11X1X1 + b22X2X2; (2)

Y is dependent variable (disintegration timeand friability), b0 is arithmetic mean response of theall 9 runs, and both b1 and b2 are estimated coeffi-cients for X1 and X2, respectively. Here X1 and X2

provide the average result on varying a single factorat one time, whereas X1X2 is the interaction termwhich illustrates how the response changes when 2factors are changed simultaneously. Both polynomi-al terms i.e., X1X1 and X2X2 are included to deter-mine nonlinearity. The composition of all preparednine formulations is shown in Table 5.

There was a significant difference in disinte-gration time from 80 ± 1.60 to 20 ± 1.59 s where %friability also showed a wide variation from 0.611 ±0.017% to 0.443 ± 0.014% in all the preparedbatches. It is clearly depicted from Table 6 that boththe chosen independent variables have significant

effect on disintegration time and % friability. Thefitted equations (full and reduced models) relatingdifferent responses, disintegration time, and % fri-ability to the transformed factor are revealed inTable 7.

The polynomial equations can be utilized todraw conclusions from the magnitude of coefficientand positive or negative sign. The results of theanalysis of variance (ANOVA) as shown in Table 8,were executed to identify the insignificant factors.The value of correlation coefficient was near to 1 forboth disintegration time or percentage friability,thereby indicating a good fit for all dependent vari-ables. Among both independent variables (disinte-gration time and friability), regression analysis indi-cate that coefficients b11, b22, b12 (p ≤ 0.05) wereinsignificant in predicting disintegration time andfriability. Hence, these terms were omitted from fullmodel to generate reduced model.

Both coefficients b1 and b2 bear a negative signas shown in multiple linear regression analysis(reduced model) which indicate that on increasing theconcentration of either camphor or crospovidone, thedisintegration time decreases. This is due to the factthat higher percentage of camphor induces higherporosity facilitating higher water uptake which leadsto reduced disintegration time. On the contrary, thisincrease in the concentration of camphor increasedfriability as the coefficient b1 bears a positive sign.However, on the other hand, negative sign of thecoefficient b2 indicated that on increasing the concen-tration of crospovidone, the friability decreased andmechanically strong tablets were produced

Figure 11. Dissolution profile of optimized fast dissolving tablet and marketed tablet


Optimization of formulation variables

The optimization of tablet components (cam-phor and crospovidone) was undergone to target thedisintegration time and % friability of 30 s and 0.6%respectively. The optimized amount determinedwith the help of software is depicted in surfaceresponse curves as shown in Figure 10.

A checkpoint batch (OCP) was prepared at X1

= 0.99 level, and X2 = 0.18 level at which disinte-gration time and % friability was 31.99 s and0.599%, respectively. The desirability of the opti-mized batch was 0.980 which is almost near to 1.From the reduced model, it was found that the disin-tegration time of the check point batch (OCP) was31 ± 1.06 s and friability was 0.602 ± 0.013%. Theoptimized batch (OCP) depicted the expectedresults, therefore this model is statistically valid. Itwas found that the observed responses of the pre-pared optimized preparation were similar to the pre-dicted values indicating the feasibility of this opti-mization process. The optimized batch producingfast dissolving tablets was prepared and comparedwith commercial tablet and the results are shown inFigure 11. The prepared tablets revealed fast andimproved dissolution as compared to commercialtablet within a time period of only 5 min which indi-cates better effect of prepared formulation for imme-diate treatment of hypertension.

CONCLUSION

Highly soluble solid dispersions of OLM withenhanced efficacy were resulted from the use of anatural carrier (modified locust bean gum), bypass-ing the utilization of any surfactant or solubilizingagent. This optimized solid dispersion prepared withmodified locust bean gum in union with lyophiliza-tion technique showed the smallest particle size of58.8 ± 1.8 nm and higher solubility of 1097 ± 15.92µg/mL. This is due to the fact that lyophilizationmethod results in porous and fluffy product whichincreases the surface area and in turn the surface freeenergy, resulting in higher solubility; while modifiedlocust bean gum possess swelling nature which alsoresults in increased extensive surface area during dis-solution leading to higher dissolution rate of thedrug. It was found that the particle size and solubili-ty of the dispersion is attributed to the type of carrierand method used. The solid dispersion prepared withlyophilization technique along with modified locustbean gum as carrier showing superior properties wasoptimized and was formulated into fast dissolvingtablet to attain quick action which can be used as asuccessful alternative for already available marketed

tablets of OLM to triumph over its drawback of lowsolubility and low bioavailability.


The authors confirm that this article contenthas no conflicts.

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Received: 19. 09. 2016


Increasing incidence of cancer is a globaldilemma involving multiple environmental andphysiological factors. The chemotherapy is an effec-tive approach to treat different types of cancer, suchas breast cancers, and prostate cancers (1, 2).However, lack of targetability and specificity to thetumor cell, the emergence of multiple side effectsand multidrug resistance, toxicities and uncontrolledtherapeutic levels hindered therapeutic success.Doxorubicin hydrochloride (DOX) is a chemothera-peutic agent that is classified as class I non-selectiveanthracycline antibiotics that were firstly obtainedfrom Streptomyces peucetius (3). Chemically, DOXis (7S, 9S)-9-hydroxyacetyl-4-methoxy-7,8,9,10-tetra-

hydro-6,7,9,ll-tetrahydroxy-7-O-(2í3í6í-trideoxy-3í-amino-α-L-lyxohexopyranosyl)-5,12-naphthace-nedione), as represented in Figure 1. It consists of asugar group (daunosamine) attached to an aglyconecomponent, comprising of a tetracyclic ring withquinine-hydroquinone functional groups. Methoxysubstituent is attached to a carbonyl group that pro-vides the site for further interaction with other com-ponents (4).

Mechanistically, DOX affects the DNA repli-cation process by interacting with the enzymestopoisomerase I and II that are responsible for theuncoiling of double stranded DNA. The hindrancein the replication process results in programmed cell

FORMULATION AND COMPATIBILITY ASSESSMENT OF PLGA/LECITHINBASED LIPID-POLYMER HYBRID NANOPARTICLES

CONTAINING DOXORUBICIN

NAYAB TAHIR1, ASADULLAH MADNI1*, PRINCE MUHAMMAD KASHIF1, MUBASHAR REHMAN1, AHMED RAZA1, MUHAMMAD IMRAN KHAN2,

MUHAMMAD ABDUR RAHIM1 and ABDUL JABAR1

1Department of Pharmacy, The Islamia University Bahawalpur, Pakistan2Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan

Abstract: In the world of materials, many individual components donít meet the need of drug delivery systemfor optimum therapeutic outcomes. However, the combination of materials provides this opportunity. The suc-cess of such combinatorial system is based on the compatibility of different formulation components with eachother. The presence of any physical or chemical interaction affect the outcomes of the system in terms of sta-bility, bioavailability, and entrapment of the active ingredient. ATR-FTIR spectrometry have been extensivelyemployed in the investigation of compatibility among the different pharmaceutical ingredients. The presentstudy was planned to study any possible interaction among the doxorubicin, PLGA and lecithin in resultingphysical mixtures and lipid polymer hybrid nanoparticles. All physical mixtures were prepared in equimolarratios by simple blending, while nanoparticles with modified single step nanoprecipitation method combinedwith self-assembly. The particle size range from 178 to 197 nm with polydispersity index (PDI) of 0.14 to 0.25,while the zeta potential varied from -17 to -24.5 mV. The spectra of doxorubicin indicate the characteristicpeaks at 3320 cm-1 (N-H stretching), 1730 cm-1 (carbonyl group) and at 1615 cm-1 and 1580 cm-1 for amide I andaide II groups. The IR spectra of PLGA indicate the ester carbonyl group symmetric stretching (ñC=O) at 1747cm-1, deformational vibration of -C-H group at 1452 cm-1 and terminal hydroxyl group at 3501 cm-1; whilelecithin at 2922 cm-1 (stretching vibration of methylene group), 1229 cm-1 (PO2), and 1065 cm-1 (P-O-C stretch-ing). The slight shift of the peaks of doxorubicin at 3270 cm-1 (N-H stretching) indicate the successful encap-sulation of drug in nanoparticulate formulations. The results suggested no significant interaction of doxorubicinwith the PLGA and lecithin, when combined in the physical mixtures and the lipid polymer hybrid nanoparti-cles.

Keywords: compatibility studies, doxorubicin, ATR-FTIR, lipid-polymer hybrid nanoparticles, physical mix-ture

1563

* Corresponding author: e-mail: [email protected]; phone: +9203006821979, fax: +92629255565

1564 NAYAB TAHIR et al.

death (5, 6). It is recommended in different malig-nancies and carcinomas such as solid tumors, lym-phomas, leukemia, breast and lung carcinomas bythe Food and Drug Administration, USA (7, 8).

In the past few decades, nanotechnology hasbeen considered a forefront area of research todevelop multifunctional nano-carriers for the deliv-ery of anti-cancer drugs. The present study has beendesigned to formulate these nano-carriers from dif-ferent natural and synthetic polymers to get sus-tained, targeted, and delivery of an effective con-centration of chemotherapeutic (9, 10). Combiningof polymeric nanoparticles and liposomal drug car-riers as lipid-polymer hybrid nanoparticles haveoffered distinct advantages of improved solubility,controlled release, enhanced residence time due toless opsonization and targeted delivery with fewerside effects and enhanced patient compliance. (11).These systems have merged the advantages of bothindividual carrier systems and addressed their short-

comings (12). Other advantages of this system arethe biocompatibility, biodegradability and highencapsulation of therapeutic moieties while the coat-ed lipid potentially acts as a barrier to prevent thedrug leakage and degradation of the inner polymer-ic core (13).

In the process of drug design and preformula-tion studies, any chemical and/or physical incom-patibility is considered a major hindrance for obtain-ing the desired therapeutic objectives. Therefore, thecompatibility analysis of the active pharmaceuticalingredient (API) and other formulation excipients isa determinant factor in the pre and post formulationinvestigations (14). Most of the excipients lackdirect pharmacological effects but it may involve insome inadvertent processes such as polymer degra-dation and formation of newer compounds. Thus,the chemical nature, formulation stability, andbioavailability of the API is directly influenced bythe excipients (15, 16). A limited data are availablefor the compatibility studies of hybrid nanoparticlesystems. The studies have been conducted by differ-ential scanning calorimetry (DSC), thermogravime-try (TG), and isothermal titration calorimetry where-as, the non-thermal compatibility studies involvedifferent spectroscopic methods, including Fouriertransform infrared (FTIR) spectroscopy, diffusereflectance spectroscopy (DRS), X-rays diffraction(XRD) and nuclear magnetic resonance (NMR) (17-19).

In the present study, attenuated total reflectionFourier transform infrared (ATR-FTIR) spec-troscopy has been employed as a reliable tool for theprompt identification of different API, excipientsand their physical or chemical interaction based onthe shifting of the bands due to presence of bonding

Table 1. Coding for the pure chemicals and equivalent ratio physical mixtures for ATR-FTIR analysis.

Sample code Equivalent ratios Chemical description

A 1 Doxorubicin

B 1 PLGA 50 : 50

C 1 PLGA 75 : 25

D 1 Lecithin

PM1 1 : 1 Doxorubicin + Lecithin

PM2 1 : 1 Doxorubicin + PLGA 50 : 50

PM3 1 : 1 Doxorubicin + PLGA 75 : 25

PM4 1 : 1 PLGA 75 : 25 + Lecithin

PM5 1 : 1 PLGA 50 : 50 + Lecithin

PM6 1 : 1 : 1 Doxorubicin + PLGA 50 : 50 + Lecithin

PM7 1 : 1 : 1 Doxorubicin + PLGA 75 : 25 + Lecithin

Figure 1. Structure of doxorubicin

Formulation and compatibility assessment of PLGA/lecithin... 1565

between functional groups (20). ATR-FTIR tech-nique can be employed for qualitative and quantita-tive analysis of samples and is therefore consideredas an important technique to find out the interactionor incompatibility by producing the unique peaks orband shifted from their normal positions (21). Thesample of liquid, solid or gas is analyzed by theattenuated total reflection mode and is reflected by abroadening of existing peaks, appearance of newpeaks and overlapping with higher intensities are thefundamental evidence to explain the possible inter-actions (22, 23).

Therefore, the present study has been under-taken to develop lipid-polymer hybrid nanoparticles,binary and tertiary physical mixtures of differentstructural components. The compatibility studiesbetween doxorubicin and lecithin, and/or poly lac-tic-co-glycolic acid (PLGA). Drug-lipid and drug-polymer interactions were analyzed in the formula-tions and all physical mixtures by using ATR-FTIRspectroscopy technique.

EXPERIMENTAL

Materials

Doxorubicin (C27H29NO11, PubChem CID:31703) was purchased from Beijing MesochemTechnology Co., Ltd. (China). PLGA (poly (D. L-lactic-co-glycolic acid, 75 : 25 and 50 : 50)([C3H4O2]x[C2H2O2]y, PubChem CID: 23111554)RESOMERÆ was purchased from Evonik Chemi-cals, Lecithin (C35H66NO7P, PubChem CID:57369748) from Xiían Rongsheng BiotechnologyCo. Ltd. (China) and all other reagents and sol-vents used in the present study were of analyticalgrade.

Preparation of physical mixtures

The physical mixtures of drug, polymer andlipid were prepared by taking equimolar amounts (1: 1 w/w) through physical mixing approach. All thecomponents were mixed in mortar with pestle for 15min to ensure the uniform mixing and obtaining a

Table 2. Composition of blank and drug-loaded LPHNPs.

Formulation Doxorubicin PLGA 75 : 25 PLGA 50 : 50 Lecithin code (mg) (mg) (mg) (mg)

Fb - 60 - 09

F1 10 60 - 09

F2 10 - 60 09

Figure 2. SEM micrographs of (A) porous surface of Fb, (B) blank nanoparticles (Fb), (C) DOX-loaded LPHNPs (F1), and (D) DOX-loadedLPHNPs (F2)


uniform blend (24). The physical mixtures combina-tions were coded and analyzed as given in Table 1.

Fabrication of lipid-polymer hybrid nanoparti-

cles

A single step modified nanoprecipitationmethod in combination with the self-assembly wasemployed to fabricate the lipid-polymer hybridnanoparticles (LPHNPs) of PLGA (poly (D, L-lac-tic-co-glycolic acid) and lecithin, as described byZhang et al. (25). A suitable amount of PLGA wasdissolved in acetonitrile to form 2 mg/mL and 3mg/mL solutions. Lecithin (15%) of the total poly-mer weight was added to the 4% hydroalcoholicsolution and heated at 65OC to form a uniform solu-tion. Then, the polymer solution was added at a rateof 1 mL/min into the preheated lecithin solutionwith constant stirring at 65OC and vigorously vor-texed for 5 min and subjected to gentle stirring at850 rpm for 2 h for the self-assembling of the HNPs.The organic solvent was evaporated and HNPs werecollected.

DOX-loaded HNPs were prepared by the samemethod. The drug (doxorubicin) was added into 4%ethanolic aqueous solution along with the lecithin inthe properly calculated dose before mixing with theorganic phase as given in Table 2. The collected HNPswere washed three times using ultracentrifuge Sigma1-14 (Sigma Laborzentrifugen GmbH, Germany) toremove the unentrapped drug and lyophilized(CHRIST Alpha 1-2 LD plus, UK) for 24 h.

Characterization of the LPHNPs

Determination of particle size, size distribution

and zeta potential

The particle size and the particle size distribu-tion (polydispersity index) of blanks and DOXloaded LPHNPs were analyzed by the dynamic lightscattering (DLS). Whereas, the surface charge (zetapotential) was measured on the electrophoreticmobility basis by using the Malvern zeta Sizer NanoZS instrument (Malvern Instruments Ltd, UK). Thesamples 25 µL of nanoparticle were diluted with the975 µL with distilled water prior to analysis.

Surface morphology of the LPHNPs

The morphology of the nanoparticles was ana-lyzed by using the scanning electron microscope(SEM) (Quanta 250 FEG, FEI, America). Thisanalysis was performed by sprinkling tests throughsticky tape stuck on aluminum stub and all the pho-tomicrographs were recorded at 5 kV with differentamplification powers.

Attenuated total reflectance infrared Fourier

transform spectroscopy (ATR-FTIR)

ATR-FTIR spectroscopy was employed toidentify the physical or chemical interactionbetween the drug and other formulation compo-nents. The FTIR spectra of doxorubicin, polymeror lipid alone, binary mixtures of the drug withpolymer and lipid, tertiary blends of drug, lipid,and polymer and the lipid-polymer hybridnanoparticle formulations (blank and drug-loaded)were obtained by using Bruker tensor 27 series(Germany) FTIR spectrometer (26). The instru-ment utilized pike single bounced attenuated totalreflectance method containing Zinc-Selenium(ZnSe) single crystal standard sample cell. Thesamples were placed at the interface of directinfrared light and internal reflection element(ZnSe) (27). All the spectra were obtained byscanning the sample over a range of 4000-400 cm-1

with 16 scans per sample at an average resolutionof 4 cm-1.


Particle size, size distribution and zeta potential

The particle size was found to be in the sizerange of 178 to 197 nm with particle size distribution(PDI) from 0.14 to 0.25, whereas, zeta potentialvaries from -17 to -24.5 mV (Table 3). The blanknanoparticles were smaller in size (178 nm) as com-pared to the DOX-loaded nanoparticles (F1 and F2).The amount of drug encapsulated in the PLGA coremight be responsible for this increase in size, which isin accordance with many previous studies (28, 31-33).

Table 3. Particle size, polydispersity index and zeta potential of LPHNPs formulations.

Formulation Particle size* Polydispersity index Zeta potentialcode (nmsa) (PDI) (mV)

Fb 178 ± 0.9 0.14 ± 0.01 -24.5 ± 0.90

F1 197 ± 3.0 0.22 ± 0.019 -17.1 ± 2.20

F2 190 ± 2.3 0.25 ± 0.031 -17.3 ± 0.35

*All the results are triplicate and represented with ± SD (standard deviation).


Surface morphology of the LPHNPs

Scanning electron microscopy was employed forthe morphological analysis (shape and surface) of thenanoparticles. The nanoparticles were generallyspherical shape particles that confirmed the successfulformulation of LPHNPs as shown in Figure 2. Theirregular shaped particles and micron size particles arethe self-assembled lipid particles that remain after thecentrifugation in the formulation dispersion. However,for the analysis of distinct layers of the polymeric core

and lipid shell, high resolution transmission electronmicroscopy (HR-TEM) might be required.

FT-IR spectroscopic analysis

Attenuated total reflection Fourier transforminfrared spectroscopy (ATR-FTIR) is successfullyemployed in the pharmaceutical, chemical andbiotechnological industries for compatibility studiesof active and/or inert ingredients of various formula-tions (27, 29). The FTIR spectra of individual com-

Figure 3. FTIR spectra of individual ingredients (a) doxorubicin, (b) PLGA 75 : 25, (c) PLGA 50 : 50, (d) lecithin


ponents, binary and tertiary physical blends andlipid-polymer hybrid nanoparticle formulations wereobtained and depicted for any possible interaction.

FT-IR spectra of individual ingredients

The FTIR spectra bearing the characteristicpeaks of doxorubicin, PLGA 75 : 25, PLGA 50 : 50

and lecithin have been shown in Figure 3. Theimportant bands and their respective functionalgroups of all these entities have also been summa-rized in Table 4. FTIR fingerprints of doxorubicin inFigure 3 (a), indicated the primary band of N-Hasymmetric stretching at 3320 cm-1 and peak at 2935cm-1 specifying the C-H stretching vibration (30).

Table 4. Functional groups and characteristic peaks of individual ingredients.

Individual Functional Characteristiccomponents groups peaks

References

N-H, C-H, C=O, N-H (amide I, II),3320 cm-1, 2935 cm-1, 1730 cm-1, 1615 cm-1,

DoxorubicinC-C, C-O-C, & C-O

1580 cm-1, 1413 cm-1, 1211 cm-1, 1071 cm-1 (9, 32, 33)and 1004 cm-1

PLGA ñC-H, ñC=O, -C-H, -O-CH2-, 2995 cm-1 and 2946 cm-1, 1747 cm-1, 1452 cm-1,

75 : 25 -CñO, -C-H, -C-O-C, -C-C, -CH2-S1423 cm-1, 1382 cm-1, 1268 cm-1, 1182 cm-1, (34, 35)

1047 cm-1, 866 cm-1, 749 cm-1, 709 cm-1

PLGATerminal -OH, ñC-H, ñC=O, C-H, 3501 cm-1, 2947 cm-1, 1748 cm-1, 1452 cm-1,

50 : 50 -O-CH2-, -CñO, -C-H, -C-O-C, -C-C, 1423 cm-1, 1383 cm-1, 1270 cm-1, 1165 cm-1, (36, 37)

-CH2-S (thioether) 868 cm-1, 749 cm-1 and 709 cm-1

Lecithin C-H, -CH2, C=O, C-H, P=O, P-O-C, 2922 cm-1, 2852 cm-1, 1736 cm-1, 1455 cm-1,

(38)N+(CH3)3 1229 cm-1, 1065 cm-1, 970 cm-1

Table 5. Functional groups and characteristic peaks of physical mixtures.

Physical Functional Characteristicmixtures groups peaks

PM1 N-H, C-H, C=O, N-H (amide I, II), 3317 cm-1, 2925 cm-1, 1730 cm-1, 1616 cm-1 and 1580 cm-1,

C-H, P=O, P-O-C, N+(CH3)3 1284 cm-1, 1235 cm-1, 1072 cm-1 and 969 cm-1

PM2-OH, N-H, C=O, N-H, -C-O, C-O-C, 3526 cm-1, 3322 cm-1, 1730 cm-1, 1615 cm-1, 1376 cm-1,

C-C, -CH2-S 1072 cm-1, 1004 cm-1, 869 cm-1, 709 cm-1

PM3N-H, C=O, C-C, C-H, C-O-C, 3323 cm-1, 1730 cm-1, 1413 cm-1, 1283 cm-1, 1071 cm-1,

-C-O 1004 cm-1

PM4 -CH2, C=O, C-H, P=O, C-O-C, N+(CH3)3 2852 cm-1, 1737 cm-1, 1454 cm-1, 1230 cm-1, 1066 cm-1, 971 cm-1

PM5C=O, C-H, -C-O, P=O, C-C, P-O-C, 1740 cm-1, 1454 cm-1, 1395 cm-1, 1228 cm-1, 1168 cm-1,

N+(CH3)3 1066 cm-1, 970 cm-1

PM6 N-H, C-H, C=O, C-H, C-C, P=O, -C-O 3322 cm-1, 2923 cm-1, 1730 cm-1, 1462 cm-1, 1413 cm-1,

1234 cm-1, 1004 cm-1

PM7N-H, C-H, C=O, N-H, P=O, C-O-C, 3324 cm-1, 2923 cm-1, 1730 cm-1, 1617 cm-1, 1580 cm-1,

N+(CH3)3 1234 cm-1, 1072 cm-1, 870 cm-1

Table 6. Functional groups and characteristic peaks of lipid-polymer hybrid nanoparticle formulations.

Formulations Functional Characteristiccodes groups peaks

Fb

C-H, C=O, N-H (amide I, II), 2919 cm-1, 1753 cm-1, 1626 cm-1, 1527 cm-1, 1385 cm-1, -C-O, C-O-C, P=O 1183 cm-1, 1088 cm-1

F1 N-H, C=O, N-H (amide I, II), 3268 cm-1, 1748 cm-1, 1624 cm-1, 1525 cm-1, 1385 cm-1,

-C-O, C-C, C-O-C 1186 cm-1, 1081 cm-1

F2 N-H, C=O, N-H (amide I, II), 3270 cm-1, 1752 cm-1, 1625 cm-1, 1524 cm-1, 1386 cm-1,

-C-O, C-C, C-O-C 1187 cm-1, 1084 cm-1


Figure 4. FTIR spectra of physical mixture (PM1-PM7)


The peak at 1730 cm-1 showed the presence of the C-O group, whereas, sharp peaks at 1615 cm-1 and1580 cm-1 have been considered as characteristicpeaks for bending vibration of (N-H) amide I andamide II groups, that overlap with the carbonylgroups of the anthracene ring (31). The peaks at1413 cm-1, 1211 cm-1, 1071 cm-1 and 1004 cm-1 veri-fied the C-C stretching, CñOñC asymmetric stretch-ing vibration and C-O stretching, respectively (32).

The IR spectral analysis of polymer PLGA 50 :50 and PLGA 75 : 25 are shown in Figure 3 (b) and(c). The band at 2995 cm-1 and 2946 cm-1 indicated theñC-H symmetrical stretching at CH and methyl (CH3)group. These bonds and stretching vibrations havebeen associated with the lactic acid monomer of thePLGA polymer. A sharp peak at 1747 cm-1 corre-sponds to the ester carbonyl group symmetric stretch-ing (ñC=O) found at a similar position as described bythe Pirooznia (34). The distinct peaks located at 1452cm-1, and 1423 cm-1 ensure the presence of a glycolicacid fraction of the polymer. These signals revealedthe deformational vibration of the bonds between car-bon and hydrogen atoms (C-H) of O-CH2-group. Thesignal at 1382 cm-1 was attributed to the saturated C-Hbending in the ñCH3 (methyl) group (34, 39). The

bands present at 1268 cm-1 disclose the C-O-C stretch-ing while the peaks at 1182 cm-1 and 709 cm-1 corre-sponded to the symmetrical stretching of an ethergroup (-C-O-C) and thioether (-CH2-S) group, respec-tively. The vibrational bands at 866 cm-1, and 749 cm-1

were associated with the stretching of C-C bonds.However, in the FTIR spectra of PLGA 50 : 50 indi-cate a peak at 3501.78 cm-1 disclose the presence ofterminal hydroxyl (-OH) group (36).

In the IR spectra of lecithin [Fig. 3 (d)] are thecharacteristic peaks of C-H (stretching vibration ofmethylene group) at 2922 cm-1 and symmetricalstretching of ñCH2 of alkyl chain at 2852 cm-1 (40).The stretching vibrations of the C=O group(between the hydrophilic head and lipophilic tail)exhibited at 1736 cm-1 and C-H deformation andscissoring vibrations were observed at wavenumber1455 cm-1. The asymmetric stretch of P=O (over-lapped with P-O), P-O-C stretching and N+(CH3)3

stretch were depicted at 1229 cm-1, 1065 cm-1, and970 cm-1, respectively (41, 42).

FTIR spectra of physical mixture

The physical mixtures of drug, polymer, andlipid were prepared in equimolar ratios and analyzed

Figure 5. FTIR spectra of blank and doxorubicin loaded lipid-polymer hybrid nanoparticles

Wawenumber cm-1


for any possible physical/chemical incompatibilityby using their IR spectra (Fig. 3).

The characteristic peaks of all the individualcomponents (Fig. 3) were compared with the IRspectra of all physical mixtures (PM1-PM7) andhave been summarized in Table 5. The binary mix-ture of doxorubicin with the lecithin (PM1) andpolymer (PM2 and PM3) indicated the presence ofcharacteristic peaks of C=O at 1730 cm-1 and N-H(amide I and amide II) group at 1616 cm-1 and 1580cm-1. Similarly, the IR spectra of a tertiary mixturecontaining doxorubicin, lecithin and PLGA 50 :50/PLGA 75 : 25 (PM6 and PM7) also indicated thepresence of characteristic peaks of these compo-nents without any prominent shift. These resultsdemonstrate the absence of any interaction betweenthe drug and other formulation ingredients.

FTIR analysis of LPHNP formulations

The lipid-polymer hybrid nanoparticles pre-pared by modified nanoprecipitation method com-bined with the self-assembling process with differ-ent concentrations of PLGA and lipid have been pre-sented in Table 6. The IR spectrum of blanknanoparticles (Fig. 5-Fb) indicated the intense sig-nals of carbonyl (C=O) group with a slight shift inwave number at 1753 cm-1, which might be due tothe overlapping of identical groups in lecithin andPLGA (43). Similarly, the characteristic peaks of theC-O-C group were identified in the region of 1047cm-1 in the PLGA (Fig. 3) while the group P-O-C inlecithin indicate the stretching vibration at 1065 cm-1.In hybrid nanoparticle formulations, a broader bandwas observed at 1081 cm-1 (1080-1090 cm-1) result-ed from the slight shifting of above-mentionedpeaks indicating the presence of both the compo-nents in the formulation.

The peak at 3268 and 3270 cm-1 indicating theN-H stretching of doxorubicin in drug-loaded formu-lations (Figs. 5-F1 and 4-F2) was not observed in the IRspectra of the blank formulation (Fig. 5-Fb), whichillustrated the successful entrapment of the drug with-out any chemical interaction. It was confirmed that thecharacteristic peaks pertaining to lecithin and PLGAwere well conserved in the IR spectra of physical mix-ture and formulations with slight variation in the wavenumber. Considering all the data analyzed, it was sug-gested that no physicochemical interaction wasobserved in the physical mixtures and preparedLPHNPs formulations (44, 45).

Conclusion and future prospective

Lipid-polymer hybrid nanoparticles rangingfrom 178 to 197 nm were successfully fabricated with

modified single step nanoprecipitation method com-bined with self-assembly. The non-destructiveapproach using ATR-FTIR spectrometry analysisconfirmed the absence of physical or chemicalincompatibility between doxorubicin and the respec-tive formulation components. The absorption spec-trum of doxorubicin exhibited no significant variationwith PLGA, lecithin, their physical mixture, and informulation system. A slight shift in the characteristicpeaks of DOX was observed in the hybrid nanopar-ticulate system, which indicated the successful encap-sulation in the polymer-lipid system and absence ofany interaction. However, this study revealed the sta-ble nature of doxorubicin, which predicted its possi-bility for the preparation of PLGA-lecithin hybridnanoparticles. However, the compatibility and stabil-ity of such novel nano-systems will be further inves-tigated by other techniques that might help in the clin-ical application of such delivery systems.

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37. Palacios J., Albano C., Gonz·lez G., CastilloR.V., Karam A. et al.: Polym. Eng. Sci. 53,1414 (2013).

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39. Jana U., Mohanty A.K., Pal S.L., Manna P.K.,Mohanta G.P.: Nano Convergence. 1, 1 (2014).

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44. Hu Y., Zhao Z., Ehrich M., Fuhrman K., ZhangC.: Polymer 80, 171 (2015).

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Received: 5. 12. 2016


Therapeutic benefit of large number ofchemotherapeutic drugs administered in a tradition-al way is sometimes limited due to poor biopharma-ceutical physicochemical properties and issues ofdrug toxicity. Most of these chemotherapeutic drugshave poor aqueous solubility and dissolution thateffect the formulation process and also limit theirtherapeutic benefit (1). Poor aqueous solubility notonly hamper the uptake, transport and distributionbut also effect bioavailability of a drug. Severaltechniques have been used to increase drug solubil-ity including complexation with physically andchemically stable cyclodextrin macromolecules (2,3). Due to superior complexing properties of HP-β-CD, it is most preferred for complexation (4, 5).These starch derivatives collaborates via dynamiccomplexation in a manner that hides undesired prop-erties of multiple drugs.

5-Fluorouracil, an antimetabolite is used wide-ly in chemotherapy regimens. It is distributed andeliminated rapidly after oral intravenous injectionwith half-life of 8-20 min (6). It shows poor absorp-tion with variable bioavailability. This propertymakes it suitable candidate for microencapsulation.In order to improve its circulation time and achievesustained drug release, use of biodegradable andnon-biodegradable polymers and incorporation of 5-FU into particulate carrier has already beenemployed (7-11).

The ability of polymeric based microspheres tomodify release behavior of drug is well known.Microspheres have gained great deal of attentiondue to their control release behavior and are verysuitable to gain sequential and simultaneous deliv-ery of anticancer agents with reduced side effects.

In order to modify release of drug, differentcombinations of crosslinked polymeric microcarriersare prepared for sustained delivery of 5-FU with ulti-mate aim to improve erratic and oral bioavailability.This study describes the use of hydroxypropyl-β-cyclodextrin to investigate the possibility of com-plexation with 5-FU for improving solubility, disso-lution, therapeutic efficacy and bioavailability ofmodel drug. Polymeric formulations selected on thebasis of in vitro results have been further studied forbiological evaluation in rabbits in order to authenti-cate their effectiveness up to a considerable extent.

EXPERIMENTAL

HPLC analysis

A sensitive, rapid and simple HPLC methodhas been validated successfully for determination of5-FU in rabbit plasma. In vivo performance ofselected drug loaded microcarriers has been con-ducted on the basis of HPLC results, which investi-gates potential of developed microspheres andmicrobeads.

PHARMACOLOGY

PHARMACOKINETIC STUDIES OF 5-FLUOROURACIL IN RABBIT PLASMA

BUSHRA NASIR1*, NAZAR MUHAMMAD RANJHA1, MUHAMMAD HANIF1, SAIRA NASIR2, GHULAM ABBAS1, 3, SAMINA AFZAL1, SHEIKH ABDUR RASHID4 and FURQAN IQBAL1

1Faculty of Pharmacy Bahauddin Zakariya University Multan, Pakistan2The Women University Multan, Pakistan

3Faculty of Pharmaceutical Sciences Government College University Faisalabad, Pakistan4Faculty of Pharmacy Gomal University Dera Ismail Khan, Pakistan.

Abstract: New polymeric complex loaded microspheres and microbeads of hydroxypropyl-β-cyclodextrinpolyvinyl alcohol and sodium alginate had been proposed for (5-fluorouracil) 5-FU delivery. In-vitro and in vivoexperiments indicated that drug-loaded microspheres and microbeads presented, a sustained release of 5-FUcompared to the 5-FU oral drug solution and that the area under curve (AUC) was increased sufficiently withpromising formulation for site-specific delivery of 5-FU to the colon with no change either in physical appear-ance, drug content or dissolution pattern at 40OC for six months.

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* Corresponding author: e-mail:[email protected]

1574 BUSHRA NASIR et al.

Pharmacokinetic analysis

Thirty six healthy albino rabbits obtained fromanimal house of Faculty of Pharmacy BahauddinZakariya University, Multan, Pakistan of weight 2.0to 2.5 kg were selected to investigate the effect offormulations on pharmacokinetics of 5-FU obtainedas a gift sample from Roche PharmaceuticalsKarachi, Pakistan. 5-FU is a popular antineoplasticagent normally used to cure carcinomas of colon.Rabbits were divided into three groups (1, 2 and 3)at random. Standard diet was given to animals,housed in a well maintained room at 25 ± 1OC.Animals were fasted for 24 h before starting theexperiment but allowed a free access to water. Adrug solution of 10 mg/kg was given orally to rab-bits of group 1, through a feeding tube followed byrinsing with water. In the second phase of study, 10mg/kg drug loaded microbeads were administered torabbits of group 2. Third phase of study deals withthe administration of 10 mg/kg drug loaded micros-pheres to group 3 of rabbits. All animals weretagged properly and retained in the wooden boxesduring sampling procedure.

Blood samples of 3 mL were taken from thejugular vein of each rabbit and collected in a citrat-ed tubes at 10, 15, 20, 25, 30, 40, 50 min and up to1 h from a control group1 and up to 24 h from group2 and group 3 after successive intervals of 0.5, 1,1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h by administratingmicrobeads and microspheres. Plasma samples werefrozen and maintained at -70OC until analysis aftercentrifugation. Protocols of the study were approvedby Research and Ethics Committee of Faculty ofPharmacy, BZU Multan.

Pharmacokinetic profiling and quantification of

plasma concentration

After administration of oral drug solution, 5-FU loaded microbeads and 5-FU loaded micros-pheres, plasma drug concentration of administeredpure drug and drug loaded microbeads and micros-pheres was determined from peak area by usingMicrosoft Excel 2010 program.

Pharmacokinetic parameters such as maximumplasma drug concentration (Cmax), time of maximumdrug concentration in plasma (Tmax), area under theplasma concentration curve (AUC), area under thefirst moment curve (AUMC), elimination half-life(t1/2 kel), absorption half-life (t1/2 ka), rate of absorp-tion (Ka), rate of elimination (Ke), mean residencetime (MRT), volume of distribution (Vd) and clear-ance (CL) were calculated using Kinetica ver 4.4.1and are mentioned in tables 6.4 to 6.6.


Statistical analysis was performed by usingone-way ANOVA in order to determine statisticalsignificant/non-significant interpretation.

RESULTS AND DISSCUSION

HPLC analysis

In vivo performance was done to achieve sepa-ration by using (methanol : water, 20 : 80 v/v) as amobile phase with pH adjusted at 3.2 using perchlo-ric acid solution, maintained at a flow rate of1mL/min. Extraction of drug was obtained by spiking500 µL of plasma with 50 µL of drug. Perchloric acidsolution (10% v/v) was then added to spiked plasma

Figure 1. Plasma concentration vs. time (Mean ± SE) plot of 5-FU (10 mg/kg) administered oral drug solution to healthy rabbits of Group 1

Pharmacokinetic studies of 5-fluorouracil in rabbit plasma 1575

sample and vortex mixed up to 10 min. Further cen-trifugation of sample was done at 3500 rpm for 15min. A clear supernatant (10 µL) layer was separatedand injected into HPLC system for analysis.

Pharmacokinetic analysis

Group 1

The concentrations of pure drug in plasma of12 rabbits (group 1) after oral administration of drugare presented (Fig. 1) which illustrates (mean ± SD)of plasma drug concentration versus time. The phar-macokinetic parameters are presented in Table 1.

A constant variation in Tmax and Cmax has beenreported in different studies within a similar

patients. Variation of 0-80% in the bioavailability of5-fluorouracil has been observed via oral route (12-13). Variations in pharmacokinetic parameters of 5-FU are highly dependent on dosage and route ofdrug administration (14). When a dose of 0.5 g wasadministered to eleven patients of cancer by intra-venous route, only a single patient received 1.0 gpaired oral and intravenous drug doses. It wasobserved that elimination half-life changes from 6.5to 13.9 (min). AUC shows an increase from 807 ±32.8 to 1537 ± 124.4 as the dose of drug increasesfrom 0.5 g to 1 g, respectively. The values of phar-macokinetic parameters (Mean ± SD) obtained afteradministration of oral drug solution are comparable

Figure 2. Plasma concentration vs. time (Mean ± SE) plot of microbeads administered to healthy rabbits of Group 2

Table 1. Pharmacokinetic parameters of 5-FU administered as oral drug solution in rabbits of Group 1, 2 and 3.

Pharmacokinetics Drug Solution (Mean ± (SE)No.

parameters Group 1 Group 2 Group 3

1 Cmax (µg/mL) 193.069 ± 0.892 84.458 ± 0.284 84.446 ± 2.415

2 Tmax (min) 18.54 ± 0.001 194.52 ± 0.0123 193.62 ± 0.0615

3 AUCtot (µg.h/mL) 192.92 ± 0.712 744.97 ± 4.310 733.481 ± 12.350

4 AUMCtot (µg.h2/mL) 57.355 ± 15.911 10163.725 ± 15.77 9261.823 ± 216.6

5 Elimination t1/2 (min) 13.2 ± 0.003 290.88 ± 1.358 400.26

6 Absorption t1/2 (min) 12.3 ± 0.002 129.96 ± 0.021 127.758

7 Kel (min-1) 0.0561 ± 0.05 0.0049 ± 0.0013 0.0050 ± 0.109

8 Ka (min-1) 0.513 ± 0.047 0.0053 ± 0.0017 0.0054 ± 0.117

9 MRT (h) 0.499 ± 0.002 12.365 ± 1.310 10.381 ± 1.829

10 ClT (L/min) 0.0615 ± 0.002 0.01335 ± 7.929 0.0135 ± 0.0024

11 Vd (L) 0.0198 ± 0.0002 0.0451 ± 0.0024 0.0453 ± 0.0011


with the previous reported studies in which similardrug has been administered via parenteral route (14-16). Similar work has been performed by Minhas(17) indicating elimination half-life of oral drugsolution to be 14.607 ± 2.8509 (min), eliminationrate constant to be 0.049 ± 0.010 min-1 and values forCmax and AUC to be 302.387 ± 8.446 (µg/mL) and156.536 ± 11.891 (µg.h/mL), respectively.

Group 2

The concentrations of 5-fluorouracil determinedin plasma of 12 rabbits (group 2) after oral adminis-tration of formulated microbeads are presented inFigure 2 which illustrates (mean ± SD) of plasmaconcentration versus time profile of 5-FU. The phar-macokinetic parameters are represented in Table 1.

Increase in the value of Cmax was in the agree-ment with the reported results of Ciftci et al. (18).The mean clearance and volume of distribution formicrobeads are 0.01335 ± 7.929 (L/min) and 0.0451± 0.002 (L). The elimination half-life and absorptionhalf-life are 290.88 ± 2.220 (min) and 129.96 ±0.021 (min). Elimination half-life was increased upto 290.96 min, producing a sustained action in orderto maintain target concentration in body for anextensive time period. The increase in drug half-lifeis consistent with nanoparticles of 5-FU preparedfrom PEG (19). Similar pattern of 5-fluorouracilrelease was observed by Sastre et al. (10) where therelease of 5-FU was evaluated from poly (D,L-lac-tide) and poly (D, L-lactide-co-glycolide) polymeric

microspheres, showed a significant increase in AUC(up to 50 times) and MRT (up to 196 times) wasobserved after injecting microspheres subcutaneous-ly into Wistar rats.

Group 3

The concentration of 5-fluorouracil determinedin the plasma of 12 rabbits (group 3) after oraladministration of microspheres are presented inFigure 3 which illustrates (mean ± SD) of plasmaconcentration versus time profile of 5-FU. The phar-macokinetic parameters are represented in Table 1.

The mean values of Tmax and AUC were nearlysimilar when compared with group 2. Similarly thevalues of mean clearance and volume of distributionare 0.0135 ± 0.0024 (L/min) and 0.0453 ± 0.0011(L). The previous study carried out by Li et al. (19)showed an increase in apparent volume of distribu-tion resulting in more effective anticancer results of5-Fu/PEG nanoparticles. The sustained release char-acteristics of microspheres and microbeads arereflected also in MRT of 5-fluorouracil in the body.It has been observed that MRT considerably increas-es after oral administration in prepared microcarriersi.e., microbeads and microspheres up to 10.381 ±1.829 (h) and 12.365 ± 1.310 (h) compared to oraldrug solution with a value of 0.499 ± 0.002 (h).

Desired and sustained drug release profile wasachieved by formulating microbeads and micros-pheres which shows an increase in mean residencetime (MRT) and area under the plasma concentra-

Figure 3. Plasma concentration vs. time (Mean ± SE) Plot of 10 mg/kg) microspheres administered to rabbits of Group-3

Pharmacokinetic studies of 5-fluorouracil in rabbit plasma 1577

tion time curve (AUC) following microbeads andmicrospheres administration, when compared withoral drug solution. The same pattern of study wasconducted by Zinutti et al. (20) following oral micros-pheres administration. Similarly increased area underthe curve, 12.53 ± 1.65mg/L h vs. 7.80 ± 0.83 mg/L hand 5.82 ± 0.83 mg/L g, longer elimination half lifehaving a value 15.43 ± 2.12 hours vs. 2.25 ± 0.22 and1.43±0.18 hours and MRT of 7.65±0.97 hours vs.3.61±0.41 and 2.34±0.35 hours was observed in 5-Fu-loaded hollow microspheres (21).

In addition it has been observed that afteradministration of oral drug solution, 2 out of 12 rab-bits were found dead, few hours after the experi-ment. However, death has not been observed aftermicroencapsulation of 5-FU in microbeads andmicrospheres showing decreased toxic effects.Improved efficacy and enhanced therapeutic effectof 5-FU-loaded microspheres has also been reportedby Zankhana et al. (22). Similarly, efficacy of guargum was also evaluated for colon targeting contain-ing 5-FU by fabricating it into microspheres (23).

In the present study, cyclodextrin and 5-FUcomplex loaded microcarriers are prepared by utiliz-ing dual approach that successfully involved twosteps approach in a single delivery device. Firstly,the 5-FU is complexed with HP-β-CD and secondly,is encapsulation of complexed drug into polymericcarrier. Developed formulations shows promisingproperties of increased loading capacity, increasedentrapment efficacy and provides sustained and con-trolled release of drug. The addition of HP-β-CD, amicrobial activated biodegradable polymer addvalue to the formulated carriers with better ADMEproperties, rare toxicity and are patient friendly.

Typical characteristics of inclusion containingformulations includes faster dissolution rate and moreeffective absorption which translates into improvedoral bioavailability of drugs with reduction in drugdosages and increased biological activity (24, 25).

Paclitaxel when complexed with cyclodextrinand loaded into nanoparticles of poly (anhydride)resulted in remarkable improvement in bioavailabili-

ty of drug upto 80% (26). Bilensoy et al. (27) devel-oped a vaginal gel loaded with 5-FU complexed withcyclodextrin to ensure long residence at the infectionsite with favorable drug release profile and reducedside effects. Lie et al. (28) investigated the pharma-cokinetics behavior of β-cyclodextrin-oridonin com-plex in rats. Significant increase in drug bioavailabil-ity with enhanced solubility has been observed.

Mognetti et al. (29) prepared nanosponges con-taining paclitaxel-β-cyclodextrin complex thatshowed complete release of drug within 2 hourswithout causing recrystallization of drug withenhanced pharmacological activity. Dhule et al. (30)successfully evaluated liposomal curcuminís abilityagainst cancer. Complex of 2HP-β-CD and curcum-in-liposome showed effective anticancer results bothin vitro and in vivo.

At present, various approaches are being usedfor oral administration of 5-FU including pH sensi-tive polymers, enzyme dependent delivery system,prodrug approach and drug release by colonicmicroflora (30). Gastro-intestinal microflora is a keyfactor to drug delivery devices that are formulated totreat GI disorders. These microflora metabolizeabout 400 different types of microorganisms andseveral chemicals (30). A small number of microor-ganisms are present in stomach and upper intestineof rabbit and human as rabbits provides comparablegastrointestinal physiology and conditions withhuman GIT in terms of pH in different parts of GIT.So rabbits are used in this study to evaluate in vivoperformance of formulated microcarriers for sus-tained delivery of 5-FU to treat colon disorders.

Pharmacokinetic profiling and quantification of

plasma concentration

High variability in 5-FU parameters results ininstability in the concentration of drugs even afteradministration of same drug dose. Oral use of thisagent however avoid excessive side effects especial-ly in GIT cancer. Various formulations of micros-pheres and microbeads were evaluated in animalswhich indicates suitable pharmacokinetic outcomes.

Table 2. Results of analysis of variance (ANOVA).

ANOVA

Pharmacokinetic SS df MS F p-value F-crit Statistical resultsparameter

Cmax 34.2905 11 3.117318 3.144897 0.009151 2.2163092 Highly significant

Tmax 1.281609 11 0.11651 5.349254 0.000296 2.216309 Highly significant

AUC 742.9287 11 67.5389 0.000479 1 2.216309 Highly insignificant



Analysis of variance showed p value less than0.05 considered to be a significant difference inresults that are mentioned in Table 2.

CONCLUSION

Various limiting factors have been observed ina conventional colon specific drug delivery systemsresulting in adverse effects, incomplete release ofdrug at the target site and local irritation such asdiarrhea. For this variety of delivery strategies anddevices have been proposed for colonic drug target-ing. The use of cyclodextrins and chemically modi-fied derivatives, HP-β-CD as colon targeting carrieris an interesting approach in this regard since theseoligosaccharides are neither absorbed norhydrolyzed in the small intestine and stomach butare absorbed in large intestine. Increase in thebioavailability, expressed as change in Tmax, AUCand Cmax demonstrates that CD improves effective-ness of a given dose of therapeutic drug.

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Received: 8. 09. 2016


Alkylating cytostatics belonging to the groupof oxazaphosphorines (cyclophosphamide ñ CP,ifosfamide ñ IF) are broadly used in chemotherapyof cancer (both solid tumors and proliferative condi-tions of the hematopoietic system), and in pharma-cotherapy of chronic inflammatory diseases andautoimmune conditions (primary systemic vasculi-tis, visceral lupus, periarteritis nodosa) (1, 2).General adverse effects of those agents include nau-sea and vomiting, alopecia, hypersensitivity reac-tions and myelosuppression (3). Long-term adminis-tration of CP/IF is associated with a toxic effect ongonads and sterility, and with a paradoxically

increased risk of many types of cancer (prostatic,pulmonary, cystic, breast, endometrial, thyroid,hepatic, gastric) (4). Moreover, chronic use of largeCP/IF doses (on the level of 60 mg/kg a day) result-ed in development of myocarditis, increasedarrhythmogenesis and congestive heart failure, andwith interstitial pulmonary fibrosis (4, 5). In case ofifosfamide, a neurotoxic effect was also reported,manifested with signs of encephalopathy, usuallydisappearing following completion of the treatment(6, 7).

One of the most significant adverse drug reac-tions (ADR) associated with oxazaphosphorines,

NEPHROTOXICITY OF A SINGLE DOSE OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE IN RATS

£UKASZ DOBREK1*, BEATA SKOWRON1, AGNIESZKA BARANOWSKA1, KATARZYNA CIESIELCZYK1, MARTA KOPA—SKA2 and PIOTR THOR1, 2

1Chair and Department of Pathophysiology, Jagiellonian University Medical College, Czysta 18, 31-121 KrakÛw, Poland

2Department of Human Physiology, Faculty of Medicine, University of RzeszÛw, W. Kopisto 2a, 35-959 Rzeszow, Poland

Abstract: Oxazaphosphorines (cyclophosphamide ñ CP, ifosfamide ñ IF) are alkylating cytostatics used inchemotherapy of cancer and autoimmune diseases. They have numerous adverse effects, including uro- andnephrotoxic, dependent of the type of drug, time of therapy and presence of any coexistent nephrotoxic factorsin a treated patient. Purpose of this study was to estimate the renal function and the level of urinary bladder dys-function occurring in rats following administration of a single, large CP/IF dose. The experiment involved 30rats who were administered a single intraperitoneal dose of 150 mg/kg b.w. of CP (group 1) or IF (group 2) ornormal saline (group 3 ñ control), respectively. Following the administration animals were placed in individualmetabolic cages. 24 h later rats were sacrificed, blood collected and nephrectomy and cystectomy performed inorder to prepare specimens for histopathological analysis. Circadian diuresis was also assessed, along with aqualitative urine assessment with strip tests and laboratory renal function parameters in plasma and urine: sodi-um, urea, creatinine and uric acid levels, and circadian elimination of sodium, potassium, urea, creatinine, uricacid and protein. Creatinine clearance, urea clearance and fractionated sodium elimination (FENa) and renalfailure index (RFI) were also calculated. An increased diuresis and acidification of urine with reduced circadi-an elimination of potassium and a significant proteinuria, as well as increased plasma levels of creatinine andurea were found in the group of rats that received a single dose of CP, compared to control animals. Rats treat-ed with IF also demonstrated acidification of urine, reduced circadian potassium elimination, a significant pro-teinuria and increased plasma creatinine and urea levels, but their diuresis was comparable to that observed incontrol animals. IF-treated animals were also characterized by reduced urea clearance, FENa and RFI.Histopathological analysis confirmed presence of inflammatory changes in urinary bladders in both groups 1and 2, and absence of any significant morphological disorders in kidneys. Obtained results suggest a dysfunc-tion of distal tubules and collective tubules developing as a result of administration of a single nephrotoxic doseof IF/CP. FENa and RFI results indicate also a higher nephrotoxic potential of IF administered as a single dose.

Keywords: cyclophosphamide, ifosfamide, nephrotoxicity, rats

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1580 £UKASZ DOBREK et al.

and particularly with cyclophosphamide, is hemor-rhagic cystitis initiated by urotoxic acrolein released incourse of CP/IF metabolism. The endogenous, hepaticbio-transformation of cyclophosphamide is basedmostly on cytochromes CYP2B6, CYP2C9 andCYP3A4 (8). Metabolism of cyclophosphamide withthose cytochromes leads to final production of a 4-hydroxy derivative, that undergoes conversion toaldophosphamide releasing nitrogen mustard ñ a com-pound attributed the main cytostatic and alkylatingproperties of cyclophosphamide (8). Acrolein is alsoformed in course of those transformations. It is a high-ly reactive, α,β-unsaturated aldehyde that causesreduction of the intracellular level of glutathione and ofother compounds containing thiol groups, and combin-ing with nucleophilic compounds. Acrolein is a stronginducer of lipid peroxidation, and changing the intra-cellular redox balance leads to dysregulation ofcytokines that participate in inflammatory processes,proliferation and apoptosis (9). In the urinary bladderthe compound induced a cascade of inflammatorychanges, release of numerous proinflammatory media-tors and free radicals that damage the urothelium. Thisdisturbance is characteristic particularly to cyclophos-phamide, as its chemical structure makes it specifical-ly prone to acrolein generation. A detailed pathophysi-ological description of oxazaphosphorine-triggeredcystitis may be found in review articles (3, 10-13),including the one published by us (14). Similarly,another significant ADR characteristic for CP/IF isnephrotoxicity. Its pathogenesis is also associated witheffects dependent on acrolein, synthesized withcytochromes CYP2B6 (15, 16) and CYP2C9 (17),present also in extrahepatic tissues, e.g., in kidneys.

Chloroacetic aldehyde, released along withacrolein as a result of biotransformation of oxaza-phosphorines in course of renal injury, is nephrotox-ic, especially for proximal and collecting renaltubules. The compound intensifies the unfavorableeffect of acrolein causing impairment of function ofcells in renal tubules by further reduction of glu-tathione, acetyl-coenzyme A and ATP levels, andblock of the enzymatic complex of NADHubiquinone oxidoreductase ñ a component of themitochondrial respiratory chain (3). Considering thefact that during its bio-transformation ifosfamide gen-erates 40-times more acetic aldehyde than the similardose of cyclophosphamide, it is believed that nephro-toxicity is a principal, although not sole, adverseeffect of that oxazaphosphorine derivative (18).

A clinical manifestation of nephrotoxic effect ofoxazaphosphorines covers a broad spectrum of disor-ders dependent on the type of drug (CP/IF), dose,administration route, time of therapy and some addi-

tional factors (patient age, coexistence of othernephrotoxic factors). Literature data indicate develop-ment of both glomerulopathy and tubulopathy, andeven of renal failure in CP/IF-treated patients (19, 20).

Considering a variable character of renal dys-functions and the fact that ñ as mentioned above ñnephrotoxicity is a common ADR observed for bothagents (although more characteristic for IF, as cysti-tis is the dominating adverse effect of CP), the aimof the study was to estimate the renal function andthe level of urinary bladder injury in rats receiving asingle, large dose of CP/IF.

EXPERIMENTAL

The medical experiment described in this paperwas approved by the 1st local Ethical Commissionin Krakow (No. 1/2016). The experiment was con-sistent with the EU Directive 2010/63 on the protec-tion of animals used for scientific purposes and withthe Polish legislation ñ The Law of 15 January 2015on the protection of animals used for scientific oreducational purposes (Journal of Laws, 26 February2015, item 266).

Test groups of animals

Ten-week old (mature) albino Wistar rats ofboth sexes in equal proportions, with initial bodyweight of 243.3 g were used in the experiment.Animals were obtained from the Central AnimalHouse of the Pharmaceutical Faculty of the UJCM.After being transported to the local Animal House ofthe Chair of Pathophysiology UJCM, animals wereacclimatized for 7 days. During that time they werekept in collective cages of five animals of the samesex. Duration of acclimatization was consistent withliterature data recommending the period of 5-15days (21). Animals were handled and clinicallyobserved during that period. On the day 8, animalswere transferred to a collective pen and randomizedinto study groups (of 10 animals each, with an equalproportion of both sexes):

Group 1 ñ rats receiving cyclophosphamide atthe single dose of 150 mg/kg b.w.Group 2 ñ rats receiving ifosfamide at the sin-gle dose of 150 mg/kg b.w.Group 3 ñ control ñ rats receiving normalsalineAll animals completed the study. Therefore, 30

animals participated in the experiment.

Plan of the experiment

The principal part of the experiment was com-pleted using metabolic cages allowing measurement

Nephrotoxicity of a single dose of cyclophosphamide and... 1581

of circadian diuresis and consumption of feed, in anair-conditioned pen at constant temperature of 22OCand relative humidity of 60%, with a 12/12 hday/night cycle (light phase 8 a.m. ñ 8 p.m; darkphase 8 p.m. ñ 8 a.m.). Following intraperitonealadministration of CP/IF/normal saline, respectively,to animals in groups 1, 2 and 3, study animals wereplaced in individual metabolic cages for 24 hours,with unlimited but monitored access to water andstandard feed (Labofeed, Kcynia, Poland). Beforeadministration of a drug/normal saline animals wereweighed using a standard laboratory scales in orderto determine the required CP/IF dose and placed ina tamer for rectal measurement of body temperatureusing a digital thermometer for rodents (Vivari,UK). Selection of the dose of 150 mg/kg b.w. for theapplied nephrotoxic CP dose was consistent with lit-erature data, recommending a single administrationof 100 mg/kg b.w. (22), 150 mg/kg b.w. (23, 24), or200 mg/kg b.w. (25, 26) as the amount causing asub-lethal toxic effect in rats. On the same basis, therecommended single nephrotoxic dose of IF used forexperimental purposes ranges between 60-240 inrabbits (27) to as much as 400 mg/kg b.w. in mice(28). Adoption of that dosage regimen results withacute urinary bladder injury developing within 4 ñ24 h after CP/IF administration. Both CP and IFwere purchased from Sigma Aldrich in crystallineform. Individual doses were prepared by reconstitu-tion in 0.5 mL of normal saline and mixing with avortex (directly before administration) of an analyt-ically weighed dose calculated according to thebody weight of a given animal. Animals in the con-trol group received an intraperitoneal injection withpure normal saline at the same volume of 0.5 mL, asthe volume for animals in groups 1 and 2. Keepinganimals is metabolic cages allowed the assessmentof the following parameters within 24 h after admin-istration of CP/IF/normal saline: body weight [g](determined by subsequent weighing on an analyti-cal scales), body temperature [OC] (as indicated bysubsequent rectal measurement), circadian waterconsumption [mL/24 h], circadian feed consumption[g/24 h] and circadian diuresis [mL/24 h]. Afterdaily urine output was measured, urine was ana-lyzed using strip tests. Urine was subsequently cen-trifuged (Heraeus Instruments Megafuge 1.0 R; 2000rpm ñ 719 g for 5 min), and divided into portions.Resulting samples were stored at -20OC until labora-tory determinations completed with the ADVIA1200 SIEMENS analyzer. Sodium [mM/L], potassi-um [mM/L], creatinine (Cr) [mM/L], uric acid[µM/L] and protein [g/L] levels were determined.Having results of circadian diuresis, circadian elimi-

nation of sodium, potassium, urea [mM/24 h], uricacid, creatinine [µM/24 h] and protein [mg/24 h]with urine was calculated. Creatinine clearance wasalso estimated, according to the formula:

CLcr = Cr urine [µM/L] ◊ diuresis [mL/min] /Cr plasma [µM/L]

Urea clearance was calculated according to theformula:

CLurea = urea urine [mM/L] ◊ diuresis [mL/min] / urea plasma [mM/L]Fractional excretion of sodium (FENa) was cal-

culated using the formula:FENa = [(Na urine [mM/L] ◊ Cr plasma [µM/L]) ◊

100] / [(Na plasma [mM/L] ◊ Cr urine [µM/L])]The renal failure index (RFI) was calculated

with the formula:RFI = [(Na urine [mM/L] ◊ Cr plasma [µM/L])] /

Cr urine [µM/L]After the period of monitoring in metabolic

cages animals were deeply anesthetized by intraperi-toneal administration of 60 mg/kg b.w. of sodiumpentobarbital (Morbital; Biowet, Pu≥awy). Undergeneral anesthesia blood was collected from theheart in amount allowing completion of all plannedlaboratory plasma determinations, and study ratswere sacrificed by repeated intraperitoneal adminis-tration of lethal dose of sodium pentobarbital (200mg/kg b.w.). Similarly to urine, collected blood wascentrifuged and resulting plasma samples were keptfrozen for the time of laboratory determinations.Sodium, potassium, urea [mM/L] and creatinine(Cr) and uric acid [µM/L] levels were determined inplasma with the same analyzer as the one used forurine samples. Protein was not assayed in plasma.After definitive cease of vital signs, cystectomy andnephrectomy was performed in study animals inorder to obtain tissue necessary for subsequenthistopathological assessment of kidneys and urinarybladders.

Urine analysis with strip tests

Strip tests (Insight, ACON Laboratories),allowing pH and specific gravity (SG) assessment,glucose level, presence of blood, leukocytes,nitrites, urobilinogen, ketones, bilirubin and protein,were used for qualitative and semi-quantitativeassessment of urine obtained from study animals. Toperform a test, strips were placed in previouslymixed urine samples, excess was filtered out andstrips left for 60 s (according to the manufacturerísinstructions after that time the result of blood andprotein presence could be considered reliable). Afterthat time strips were compared to the scale printedon the package and results were recorded.


Histological assessment of collected urinary blad-

ders and kidneys

The histological analysis was performed atthe Department of Anatomo-pathology of the Non-public Healthcare Institution ÑProsmedî inKrakÛw. Kidneys and bladders collected in courseof the post-mortem examination were washed innormal saline and fixed for 24 hours in 8% formalin phosphate buffer solution (PBS pH 7.4).Collected specimens were subsequently washedunder running water for 2 h, and dehydrated inethanol (condensations increasing from 50 to100%). Before embedding in paraffin, specimenswere passed through xylene solution in order to becleared. From xylene, specimens were transferredto a 1 : 1 xylene and paraffin mixture and incubat-ed at 37oC for 2 h. Then, individual tissue frag-ments were transferred twice into pure paraffin andincubated at 62OC. After 2 h, specimens wereembedded into paraffin blocks. After setting downblocks were sliced with a microtome, and resultingsections were dried on a glass slide at 37OC.Specimens were stained with hematoxilin-eosin(HE) in order to facilitate the histological assess-ment of inflammation. Specimens of urinary blad-ders and kidneys from animals receiving normal

saline (control group 3) were compared to thosefrom animals treated with CP/IF.

The histological analysis was performed usingan optical (light) microscope (Delta Optical) at amagnification of 40 × (urinary bladders) and 100 ×(kidneys). The microscopic images were taken usinga DLT-Cam Basic 2MP microscopic camera andDLTCamViewer software.


Results of qualitative and semi-quantitativeurine analysis completed with strip tests were notsubject to statistical assessment. Obtained results ofvital signs and laboratory determinations of urineand plasma were initially assessed using theShapiro-Wilk test, in order to verify normality ofdistribution. With verified normality of distribution,the essential statistical analysis of differencesbetween groups (group 1 vs. group 3, and group 2vs. group 3) was performed using the t-Student test,with the accepted statistical significance level of p ≤0.05. In those cases when results verified by the ini-tial Shapiro-Wilk test failed to meet criteria of a nor-mal distribution, differences were tested with theMann-Whitney test, also with the statistical signifi-cance level of p ≤ 0.05.

Table 1. Metabolic cage measurement - results (mean ± SD) of the living parameters of the animals used in the experiment.

Group 1 Group 2 Group 3Statistical analysis

CP-treated rats IF-treated rats Control rats(p value)

1-3 2-3

Body weight[g] 248.50 ± 32.30 257.00 ± 10.89 226.6 ± 44.89 NS NS

Body temperature[OC] 36.23 ± 0.90 35.50 ± 0.25 37.43 ± 0.93 0.04 0.01

Daily water intake[mL/24h] 22.50 ± 11.47 13.50 ± 7.19 26.34 ± 4.07 NS 0.02

Daily feed intake[g/24h] 1.98 ± 1.37 4.53 ± 3.72 23.75 ± 6.16 < 0.001 < 0.001

NS - Non-significant

Table 2. The results of the estimated plasma parameters (mean ± SD) in study rats.



1-3 2-3

Na [mM/L] 141.95 ± 1.26 144.75 ± 1.95 142.06 ± 1.79 NS 0.04

Urea [mM/L] 8.97 ± 1.54 10.93 ± 2.12 6.34 ± 0.75 0.03 < 0.001

Creatinine [µM/L] 38.45 ± 7.96 34.67 ± 7.48 28.85 ± 1.77 0.05 0.05

Uric acid [µM/L] 165.82 ± 45.77 178.38 ± 21.88 157.96 ± 35.14 NS NS

NS - Non-significant


RESULTS

Vital signs

After the end of monitoring in metabolic cages,body weight of study animals was not significantlydifferent. 24 h after administration of a drug, ani-mals receiving CP or IF had a significantly lowerbody temperature compared to control animals.Similarly, a statistically significant reduction of cir-cadian feed consumption was found in both groups1 and 2 compared to the control, which was proba-bly a result of unfavorable systemic effect of CP/IF,particularly on gastrointestinal mucosa. Moreover,rats receiving IF consumed a significantly loweramount of water within 24 hours post the drugadministration, compared to the control.

Detailed results of measurement of vital signsare presented in Table 1. Considering the fact thatmeasurements were characterized by a normal dis-tribution, the statistical analysis of intra-group dif-ferences was performed using the t-Student test.

Qualitative and semi-quantitative urinalysis with

strip tests

Values of pH and specific gravity are describedbelow, along with description of parameters meas-ured in urine. A minor leukocyte count (estimated at

15 (±); n = 7) and a minor proteinuria estimated at(+) (30 [mg/dL]; n = 5) were demonstrated in con-trol animals. Absence of other measured compo-nents was confirmed in their urine.

Urine from animals receiving CP was charac-terized by leukocytosis in 25% of cases estimated at+ (70 [leu/µL]), in 25% at ++ (125 [leu/µL]), and inthe remaining half of cases at +++ (500 [leu/µL]).Presence of blood was confirmed in all urine sam-ples, estimated at +++. Similarly, proteinuria wasfound in all animals in the group 1, estimated at +++(300 [mg/dL]). Nitrites, urobilinogen, ketones,bilirubin or glucose were found in urine from noneof CP-treated animals.

Strip urinalysis of rats treated with IF gavesimilar results: no urobilinogen, ketones, bilirubin orglucose were found, but presence of nitrites wasdemonstrated in 25% of cases. Leukocytosis waslower than that measured in the group 1: in 25% ofcases it was estimated at +/- (15 [leu/µL]), in 25% at+ (70 [leu/µL]), and no leukocytes were found inurine in the remaining half of cases. Presence ofblood estimated at +++ was demonstrated in half ofcases; but no hematuria was found in the other half.Similarly to CP-treated animals, rats treated with IFdemonstrated proteinuria, estimated at +++ (300[mg/dL]) in all cases.

Table 3. The results of the estimated urine parameters (mean ± SD) in study rats.



1-3 2-3

Diuresis[mL/24 h] 16.70 ± 8.31 8.95 ± 7.49 5.85 ± 2.37 0.04 NS

Diuresis[mL/min] 0.012 ± 0.006 0.006 ± 0.005 0.004 ± 0.002 0.04 NS

pH 6.75 ± 0.29 6.13 ± 0.75 8.94 ± 0.18 < 0.001 0.002

SG [g/cm3] 1.014 ± 0.003 1.023 ± 0.006 1.011 ± 0.002 NS 0.007

Na [mM/L] 46.00 ± 10.94 80.83 ± 36.08 127.58 ± 34.67 0.004 NS

K [mM/L] 68.10 ± 27.31 159.32 ± 81.20 352.96 ± 64.66 0.004 0.004

Urea [mM/L] 414.35 ± 115.32 949.25 ± 532.44 1003.96 ± 188.91 0.004 NS

Creatinine [mM/L] 3.35 ± 1.17 6.75 ± 4.34 6.10 ± 1.10 0.008 NS

Uric acid [µM/L] 826.18 ± 321.76 1378.10 ± 401.61 1286.54 ± 296.60 0.05 NS

Urine protein [g/L] 2.5 ± 0.92 3.91 ± 1.15 0.81 ± 0.51 0.008 0.03

Na [mM/24 h] 0.78 ± 0.44 0.55 ± 0.14 0.75 ± 0.37 NS NS

K [mM/24 h] 1.09 ± 0.49 1.03 ± 0.20 2.05 ± 0.86 0.05 0.02

Urea [mM/24 h] 6.36 ± 1.91 5.72 ± 0.61 5.80 ± 2.38 NS NS

Creatinine [µM/24 h] 50.49 ± 18.12 42.21 ± 13.76 35.19 ± 13.85 NS NS

Uric acid [µM/24 h] 13.53 ± 6.82 10.58 ± 5.36 7.49 ± 3.75 NS NS

Urine protein [mg/24 h] 38.70 ± 17.12 23.57 ± 14.83 5.27 ± 4.47 0.004 0.03

NS - Non-significant, SG - specific gravity


Parameters determined in plasma

Considering signs of hemolysis evident inmajority of plasma samples, results of plasma potas-sium levels had been discarded as non-reliable.

Values of plasma parameters assessed in CP-treated were not significantly different from controlanimals, except for a statistically significantincreased urea and creatinine levels. But IF-treatedanimals were characterized with a significant hyper-natremia, increased plasma urea and creatinine lev-els, with a statistically insignificant reduction of theplasma uric acid level. Detailed results of measure-ment of plasma parameters are presented in Table 2.Considering the fact that measurements had no nor-mal distribution, the statistical analysis of intra-group differences was performed using the Mann-Whitney test.

Parameters determined in urine

The circadian diuresis and the calculatedminute diuresis was significantly higher in CP-treat-ed animals. On the contrary, IF-treated rats demon-strated no significant difference in diuresis com-pared to control animals. In both groups of treatedanimals a significant acidosis of urine was found,compared to the control group. Specific gravity ofurine from rats receiving IF was significantly highercompared to the control.

Urine levels of all determined substances weresignificantly lower in the group 1 (CP treatment)

compared to the control, except for protein. Proteinlevels were statistically significantly higher in CP-treated animals. IF-treated rats were characterizedby significantly lower and higher of potassium andprotein levels, respectively, compared to the con-trol.

The analysis of circadian urine elimination oftested substances demonstrated in both groups oftreated animals a statistically significant reductionof potassium elimination and a significant protein-uria. Other urine components were eliminated inamounts similar to those observed in the controlgroup.

Detailed results of measurement of plasmaparameters are presented in Table 3. Considering thefact that measurements were characterized by a normaldistribution, the statistical analysis of intra-group dif-ferences was performed using the t-Student test.

Values of calculated parameters: creatinineclearance, urea clearance, fractionated sodium elim-ination and renal failure index were not significant-ly different in CP-treated rats compared to the con-trol. On the contrary, comparing IF-treated animalswith the control, a statistically significant reductionof urea clearance, FENa and RFI were found.Creatinine clearance was an exception.

The above mentioned relationships are illus-trated in Figures 1-4, below (graphs present medianvalues, quartiles and ranges for individual vari-ables).

Figure 1. Creatinine clearance in cyclophosphamide-treated (left) and ifosfamide-treated (right) rats (no statistically significant differencesrevealed in both comparisons)

1.6

1.4

1.2

1.0

0.8

0.6

0.4

1.6

1.4

1.2

1.0

0.8

0.6

0.4

Control CP Control IF

Median Quartiles Range Median Quartiles Range


Results of histopathological analysis of urinary

bladders and kidneys

Histopathological assessment of urinary blad-der specimens from rats receiving a single largedose of CP demonstrated congestion with inflamma-tory infiltration in stroma of the cystic mucosa, andepithelium with signs of regeneration. Moreover,

clusters of dilated tubules covered by a regular cubicepithelium and a low cylindrical epithelium werevisible in selected specimens. Urinary bladders ofanimals treated with a single large dose of IF pre-sented similar disturbances to those observed in CP-treated rats, with additional signs of exfoliation ofthe lining epithelium.

Figure 2. Urea clearance in cyclophosphamide-treated (left) and ifosfamide-treated (right) rats (p < 0.05 for Control-IF rats, non-signifi-cant for Control-CP ones)

Figure 3. Fractional excretion of sodium (FENa) in cyclophosphamide-treated (left) and ifosfamide-treated (right) rats (p < 0.05 forControl-IF rats, non-significant for Control-CP ones)

Control CP

Median Quartiles Range

Control IF


1.4

1.2

1.0

0.8

0.6

0.4

0.2

1.4

1.2

1.0

0.8

0.6

0.4

0.2

Control CP


Control IF


0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20


Histopathological analysis of kidneys demon-strated minor congestion in both CP and IF-treatedanimals, but besides that the presentation was with-in the normal range determined for control animals.The samples of microscopic images of the urinarybladders and kidneys obtained from control andCP/IF treated rats are shown in Figures 5 and 6,respectively.

DISCUSSION

The experiment demonstrated existence of thefollowing principal disorders:

1. Rats treated with a single dose of CP demon-strated reduced body temperature and reduced circa-dian feed consumption, with increased diuresis anddecrease of urine pH. That group presented areduced circadian elimination of potassium and asignificant proteinuria, as well as increased plasmacreatinine and urea levels, compared to control ani-mals.

2. Similarly to CP-treated rats, animals receiv-ing a single dose of IF were also characterized byreduced body temperature and reduced circadianfeed consumption, and by urine pH decrease.Contrary to CP-treated animals, that group present-ed diuresis comparable to that observed in controlanimals, with a lower daily water consumption.Administration of a single dose of IF resulted in

reduced 24-hour elimination of potassium and a sig-nificant proteinuria, just like in case of a single doseof CP. Similarly to animals treated with CP, admin-istration of IF resulted in increased creatinine andurea plasma levels. Moreover, IF-treated animalspresented reduced urea clearance values, fractionat-ed sodium elimination and acute renal failure index.

Although being statistically significant,reduced urine levels of all parameters determined inCP-treated animals compared to the control group,were rather a result of increased diuresis in thatgroup of rats, and not a result of a real increase ofurine elimination of those substances. Particularly,the analysis of circadian elimination of electrolytesand nitrogen compounds demonstrated a reducedurine elimination of potassium only, with a signifi-cant proteinuria in CP-treated animals (and in IF-treated, as well). Considering those laboratory resultsglobally, it should be stated that renal dysfunctionsobserved after administration of both CP and IF wereof functional character and were not accompanied byany morphological injury of kidneys evident in thehistopathological assessment. Therefore, a singleexposure to oxazaphosphorines was sufficient tocause a functional disorder of kidneys, manifestedprincipally by impaired tubular transport in terms ofsecretion and reuptake of potassium, and ñ in case ofCP-treated animals ñ by impaired condensation ofurine. Decreased urine potassium elimination may be

Figure 4. Renal failure index (RFI) in cyclophosphamide-treated (left) and ifosfamide-treated (right) rats (p < 0.05 for Control-IF rats, non-significant for Control-CP ones)

Control CP


Control IF


1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.8

0.7

0.6

0.5

0.4

0.3


associated with an expected retention of the elementin blood. That assumption could not be verifiedbecause of hemolysis of plasma samples. Regulationof plasma potassium levels (and consequently thedynamics of its elimination with urine) is basedmostly on exchange of the ion between intra- andextracellular fluid. Kidneys are a critical organengaged in regulation of systemic amount of potassi-um. Within a nephron, potassium ions undergoglomerular filtration, and are subsequently reuptakenin proximal tubules and in the loop of Henly. Thefinal urinary elimination of majority of potassiumions depends on the active secretion of the ion bycells of distal and collecting tubules, determined byincreased plasma levels of the electrolyte, increasedNa+- K+/H+ exchange with participation of thealdosterone-regulated ATPase in basal-lateral mem-brane and a direct secretion of the ion into urine (29,30). Considering a reduced circadian potassiumelimination observed in study animals, a thesis maybe formulated that both CP and IF contribute to dys-function of distal and collecting tubules.

Urine acidification is an additional argumentsupporting tubulopathy caused by CP/IF. Theprocess is also associated with potassium metabo-lism, as mentioned above (K+/H+ exchange). Ingeneral terms it may be stated that two processesoccur in nephrons: reuptake of alkalis and secretionof acids. The first one takes place in proximaltubules, and the other one in distal tubules (31).Secretion of H+ ions into urine takes place via thementioned above antiport K+/H+ ñ ATPase and theluminal pump H+ ñ ATPase, and a final acidifica-tion of urine depends also on phosphate and ammo-nia buffer (32, 33). It seems that CP and IF mayimpair those mechanisms, leading to final reductionof urine pH and increased potassium reuptake.

Moreover, CP-treated animals demonstratedpolyuria, which ñ in face of circadian water con-sumption comparable to that observed in the controlgroup ñ may constitute another argument in favor ofdysfunction of distal and collecting tubules.Reuptake of water and final condensation of urinedepends on aquaporins ñ integral proteins of water

Figure 5. Light microscopy of the urinary bladders ñ in control (left) and in the CP/IF (right) rats

Figure 6. Light microscopy of the kidneys ñ in control (left) and in the CP/IF (right) rats


channels induced in apical membranes of collectingtubules by antidiuretic hormone (34). Therefore,polyuria observed in the group of rats receiving anephrotoxic dose of CP may be a result of impair-ment of that mechanism. That observation is con-trary to literature reports indicating a possible devel-opment of Ñsyndrome of nephrogenic syndrome ofinappriopriate antidiuresisî induced by CP as aresult of increased expression and hyperactivity ofaquaporins in collecting tubules of rats followingadministration of the dose of 12-96 mg/kg b.w. (35)or 25-50 mg/kg b.w. of CP (36), in 12-72 h afteradministration of cyclophosphamide. The quotedstudies indicate also that the effect may be ADH-independent, and time- and dose-dependent, there-fore the effect of CP on the volume of diuresis hasto be more precisely defined.

A significant proteinuria was demonstrated inboth treated groups. Pathogenesis of that disorderseems complex and may be a result of above men-tioned dysfunction of renal tubules, or be a conse-quence of other disorders. According to the generalpathophysiological classification, proteinuria maybe of prerenal character (overload), renal character(glomerular or tubular) or extrarenal character (e.g.,inflammatory, neoplastic or obstructive disturbancesof the lower urinary tract) (37, 38). Therefore, pres-ence of increased urinary elimination of protein instudy rats may be a result of impairment of resorp-tive function of tubules, but considering presence ofoxazaphosphorine-induced inflammatory changes inthe urinary bladder, it may be also of extrarenalcharacter.

Reduced values of both FENa and RFI were alsodemonstrated in IF-treated rats. Fractional excretionof sodium is defined as the amount of sodium final-ly eliminated with final urine after the phase ofglomerular filtration (39, 40). The renal failureindex describes a relationship between the plasmacreatinine level and urine sodium and creatinine lev-els (41). Both FENa and RFI are currently regardedmodern laboratory parameters of acute kidney injury(AKI) ñ the term used currently in place of former-ly used ìacute renal failureî. The disorders is aresult of pre-, intra- and extrarenal processes leadingto development of acute tubular necrosis and finallyto irreversible failure of the organ (42, 43). Clinicalguidelines recommend the diagnosis of AKI, alongwith a possible pathophysiological differentiationinto ante- and intrarenal form, based on values of theabove mentioned values of FENa and RFI, that aredecreased in the prerenal form. Although clinicalobservations cannot be uncritically translated intoexperimental conditions, reduced values of both

FENa and RFI were observed in rats receiving a sin-gle nephrotoxic dose of IF, compared to correspon-ding values in control animals (Figs. 3 and 4).Reduced FENa in IF-treated animals was accompa-nied by reduced circadian urinary sodium elimina-tion [mM/24 h]. Although no statistical significancewas reached in comparison to control animals, atrend could be observed. No FENa or RFI reductionwas demonstrated in animals receiving a single,nephrotoxic dose of CP. Therefore, those resultsmay suggest development of functional AKI causedby IF, with normal diuresis comparable to thatobserved in the control, and a normal creatinineclearance, which does not exclude the presence ofAKI ñ the disorder may occur in oliguric form orwith a paradoxical normal diuresis (44).Confirmation of the pathophysiological prerenalcharacter of AKI in those animals (as evidenced byreduced FENa and RFI compared to the control), andparticularly of the potential mechanism responsiblefor prerenal functional kidney injury requires somemore detailed studies.

CONCLUSIONS

Multiple renal dysfunctions resulted fromoxazaphosphorines administration were reported ñ acombination of glomerular, proximal or distal tubu-lar damage, Fanconi syndrome and reversible AKIor a permanent chronic kidney disease wererevealed. These disturbances are characterized by aconsiderable variability in the onset, clinical mani-festation, severity and reversibility and dependenton the dose and time of the therapy (18-20).

Renal dysfunctions observed in study animalsare consistent with the above mentioned literaturedata. Described disorders developing as a result ofadministration of a single, nephrotoxic dose ofCP/IF also suggest a dysfunction of mostly distaland collecting tubules, with absence of morphologi-cal changes in kidneys. Proteinuria, urine acidifica-tion with reduced circadian elimination of potassiumand increased plasma levels of creatinine and ureawere demonstrated as a result of a single exposure tonephrotoxic doses of CP/IF. The commonly recog-nized lower nephrotoxicity of cyclophosphamidewas also confirmed in this study. No laboratory fea-tures of AKI (RFI, FENa) were demonstrated follow-ing administration of a single dose, contrary to IF-treated rats. However, a significant polyuria wasobserved in case of CP, in place of the literature-reported antidiuresis syndrome, which may be aresult of different methodology of the study, differ-ent dosage and observation time.


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30. Chamienia A.: Chor. Serca Nacz. 1, 97 (2004).31. Roth K.S.: Clin. Pediatr. 40, 533 (2001).32. Soriano J.R.: J. Am. Soc. Nephrol. 13, 2160

(2002).33. Goldfarb S.: Renal tubular acidosis, in Hospital

Physician Nephrology Board Review Manual.Turner White Communications Inc., Wayne PA2003.

34. Agarwal S.K., Gupta A.: Indian J. Nephrol. 18,95 (2008).

35. Kim S., Jo C.H., Park J.S., Han H.J., Kim G.H.:Electrolyte Blood Press. 9, 7 (2011).

36. Kim S., Choi H.J., Jo C.H., Park J.S., KwonT.H. et al.: Am. J. Physiol. Renal Physiol. 309,F474 (2015).

37. Venkat K.K.: South. Med. J. 97, 969 (2004).38. StompÛr T.: Forum Nefrol. 2, 50 (2009).39. Mutter W.P., Korzelius C.A.: Hosp. Med. Clin.

1, e338 (2012).40. Gotfried J., Wiesen J., Raina R., Nally J.V.:

Cleve Clin. J. Med. 79, 121 (2012).41. Zygner W., GÛjska-Zygner O., Weso≥owska A.,

WÍdrychowicz H.: Acta Parasitol. 58, 297(2013).

42. Hurtarte-Sandoval A.R., Carlos-Zamora R.:Surgery Curr. Res. 4, 155 (2014).

43. Varrier M., Fisher R., Ostermann M.: EMJNephrol. 3, 75 (2015).

44. Basile D.P., Anderson M.D., Sutton T.A.:Compr. Physiol. 2, 1303 (2012).

Received: 21. 09. 2016


The dangers of addiction are generally well-known, nevertheless people continue to start takingdrugs. Dependence on drugs and alcohol is assignedto the most common brain disorders. Almost 50% ofall persons who have applied for reliance on drugtherapy as the primary drug used opioids. Therefore,researchers are interested in naturally occurring psy-choactive alkaloid ibogaine, found in African plantTabernanthe iboga Baill. (Apocynaceae). Psycho-active properties of ibogaine have been known fordecades (1).

There are published data that ibogaine canfacilitates the symptoms of abstinence (2). In the late20th century, many scientific researches started to beconducted with an aim to find out the mechanism ofaction of ibogaine. The pharmacology of ibogaine is

quite complex, affecting many different neurotrans-mitter systems simultaneously. Non-clinical studieshave demonstrated that ibogaine reduces craving forcocaine and morphine, attenuates morphine with-drawal symptoms (3). However, the pharmacologi-cal targets underlying the physiological and psycho-logical actions of ibogaine are not completely under-stood (4). The identified antagonistic activity of ibo-gaine on N-methyl-D-aspartate receptors as well asits agonist activity on opioid receptors can beregarded as a possible mechanism of anti-addictiveaction (5).

Ibogaine is metabolized by cytochromeP4502D6 (CYP2D6) into active metabolite noribo-gaine (12-hydroxyibogamine) (6), which can condi-tion the long-term effect of ibogaine (7, 8).

INVESTIGATION INTO PHARMACOKINETIC PROPERTIES OF ACTIVEALKALOID IBOGAINE AND ITS METABOLITE NORIBOGAINE

ASTA KUBILIENE1*, AUDRIUS SVEIKATA2, ANDREJUS ZEVZIKOVAS1, ILONA SADAUSKIENE3 and LEONID IVANOV3

1Department of Analytical and Toxicological Chemistry, Lithuanian University of Health Sciences, Eiveniu 4, LT-50161 Kaunas, Lithuania

2Department of Theoretical and Clinical Pharmacology, Lithuanian University of Health Sciences, –iaurÎs pr. 57, LT-4926 Kaunas, Lithuania

3Neuroscience Institute, Medical Academy, Lithuanian University of Health Sciences, Eiveniu 4, LT-50161 Kaunas, Lithuania

Abstract: The interest in alkaloid ibogaine found in African plant Tabernanthe iboga Baill. has been persistingfor several decades. Behavioral, neuroendocrine and neurochemical effects of ibogaine and its active metabo-lite ñ noribogaine ñ were examined, however there are limited pharmacokinetic data in literature and existingdata is conflicting. We determined pharmacokinetic parameters of ibogaine and noribogaine not only in plas-ma, but in organs (heart, liver, spleen, brain, kidney and muscle) of mice after ibogaine and noribogaine intra-gastrical administration too, because there are no pharmacokinetic studies of internal organs. Circulating plas-ma levels of ibogaine and noribogaine peaked immediately at 30 min after ibogaine administration, whereaslevel of noribogaine after noribogaine administration increased slowly to plateau at 4 h. Shorter half-life (T1/2)and smaller volume of distribution (Vd) of ibogaine show the faster elimination from plasma, compared withnoribogaine. Largest Vd value in plasma calculated for noribogaine after noribogaine administration. Ourresults demonstrate that noribogaine reaches higher concentration in brain after ibogaine intragastrical admin-istration. Higher concentration and total systemic exposure (AUCtot) of noribogaine in brain could be more effi-cacious alternative to ibogaine as a medication for the treatment of various types of addiction. High values ofAUCtot in heart samples may determine the long elimination from this organ and can lead to cardiovascularabnormalities. Our findings also raise the hypothesis that ibogaine is metabolized not only in the liver, but alsoin kidney and brain too.

Keywords: ibogaine, noribogaine, pharmacokinetics, Tabernanthe iboga

1591


1592 ASTA KUBILIENE et al.

Although they share similar chemical structures,ibogaine and noribogaine display different bindingprofiles (9). Although both alkaloids increase extra-cellular 5-HT in the brain, noribogaine is morepotent in this respect (10). Previous study reports,that noribogaine is less toxic (11) and can be saferand causing less undesirable reactions as comparedwith parent drug (10). However, usually noribogaineis analyzed after administration of ibogaine whilewe investigated the pharmacokinetic parameters ofnoribogaine after administration of both ñ ibogaine,as well as noribogaine.

In previous study (12) we calculated theamounts of tested substances in organs (heart, liver,spleen, brain, kidney and muscle) of mice after ibo-gaine and noribogaine administration directly intothe mice stomach via the stomach tube. In presentstudy, we evaluated the main pharmacokineticparameters of ibogaine and noribogaine in plasmaand organs of mice.

EXPERIMENTAL

Animals

The experiments were conducted with non-lin-ear white laboratory mice (20ñ25 g) of the age of 4to 6 weeks in compliance with the Law on the Care,Keeping and Use of the Animals of the Republic ofLithuania. The animals were kept in compliancewith the requirements of the Good LaboratoryPractice (GLP). The experiments were performed incooperation with the Laboratory of MolecularNeurobiology of the Institute of Neurosciences of

the Medical Academy of the Lithuanian Universityof Health Sciences. Adult male mouse (n = 54)weighing 20-25 g were housed individually inrooms maintained at ~20OC, with a regular light-dark cycle. Water was available in the living cagesat all times. Permission for experiments withLaboratory Animals No. 0172 was issued by theState Veterinary Authority of Lithuania.

Chemicals and reagents

Ibogaine hydrochloride, gradient purity eluentsand fluorescein sodium salt (internal standard) pur-chased from manufacturer ìSigma-Aldrich Chemieî(Switzerland) were used for the researches.Noribogaine base was kindly supplied by ReformItalia (Endine, Italia). Ibogaine and noribogainewere stored protected from light. Water purified byMillipore water purification system (Millipore,Bedford, USA) was used for the experiments. OasisHLB cartridges (30 mg sorbent, size of particles ñ30 µm) were supplied by Waters (Milford, USA).Blank human plasma was received from theDivision of Haematology of the Clinics of theLithuanian University of Health Sciences. Heparinsodium salt was purchased from UAB ìLabochemaLTî.

Pharmacokinetic experiments

Mice were randomly assigned to three groups ñibogaine group, noribogaine group and controlgroup. Ibogaine and noribogaine solutions havebeen prepared ex tempore prior to each test by dis-solving accurately weighed quantities of the sub-

Figure 1. Chromatograms of blank plasma and blank plasma spiked with noribogaine (peak 1), ibogaine (peak 2) and fluoresceine (peak 3)

Investigation into pharmacokinetic properties of active alkaloid... 1593

stance in deionized water. The volume of solutionwas recalculated in accordance with the weight ofeach mouse (e.g., for the mouse of the weight of 25g ñ 0.25 mL of prepared solution). Single dose ofibogaine (26.3 mg/kg) and noribogaine (31.5 mg/kg)were administered intragastrically to mice via a spe-cially designed stomach tube. Control mice receivedthe same amount of saline. The same method ofadministration was used.

Animals were dislocated and decapitated 15min, 30 min, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h, 48 h afterthe intragastric administration of substance. Threemice were decapitated in each time interval. Theblood was collected to centrifugal tube with 25 µL

of heparin, centrifuged and collected blood plasmawas frozen in the temperature of -40OC. Preparedinternal organs (liver, kidney, brain, heart, spleenand muscle) were frozen in the temperature of -40OCuntil the time of assay by high performance liquidchromatography (HPLC).

Ibogaine and noribogaine concentrations weredeterminated by HPLC with fluorescence detector.Solid-phase extraction (SPE) was used for theremoval of proteins and interfering components.Analytical procedure from plasma and organs andchromatographic analysis were described in previ-ous study (12). The HPLC method validationparameters in our study were specificity, precision

Figure 2. Chromatograms obtained from liver (A), spleen (B), heart (C), kidney (D), brain (E), muscle (F) of tested and control mice. Peak1 ñ noribogaine, peak 2 ñ ibogaine, peak 3 -fluoresceine


(intermediate precision), linearity and lower limit ofquantitation. The specificity of the method wasinvestigated by analyzing three different batches ofblank human plasma samples. It shows, that peaks inblank plasma did not impede the identification of thetested materials (Fig. 1).

The same results were obtained analyzinginternal organs of tested and control mice (Fig. 2).

RSD were calculated: it was 5.1% for noribo-gaine and 6.9% for ibogaine. The LLOQs (lowerlimit of quantitation) were 1.4 ng/mL for ibogaineand 2.15 ng/mL for noribogaine. The correlationcoefficients (r) for calibration curves were equal toor better than 0.98 (12).

Pharmacokinetic parameters were calculatedby using pharmacokinetic program ìKineticaî (4.0version, USA). Calculated pharmacokinetic parame-ters include the following: volume of distribution(Vd), area under curve (AUC), elimination half-life(T1/2), maximum concentration (Cmax). Pharmacoki-netic (AUCtot, Cmax, Tmax, T1/2,) variables were ana-lyzed by non-compartmental model following atrapezoidal rule.

RESULTS

Pharmacokinetic profiles in plasma

Table 1 summarizes the pharmacokineticparameters for ibogaine and noribogaine after intra-gastrically administration of ibogaine (26.3 mg//kg)or noribogaine (31.5 mg/kg). Noribogaine wasdetected in plasma and organs after administrationof either ibogaine or noribogaine. The metabolitewas detected at the earliest time point (15 min), con-sistent with first pass metabolism of the parent drug(13).

Figure 3 illustrates the time-concentration pro-files for ibogaine and noribogaine in blood plasma.Following intragastric administration circulatinglevels of ibogaine and noribogaine peaked at 30 min(Tmax) after ibogaine administration. NoribogaineCmax (185 ± 0.02 ng/mL) was much less than that ofibogaine (475 ± 0.05 ng/mL), inconsistent with pre-vious reports (14), giving a noribogaine-to-ibogaineCmax ratio of 0.39. These data show, a much smallerfraction of ibogaine is metabolically converted tonoribogaine when ibogaine is administered intragas-

Figure 3. Time-concentration profiles for ibogaine (IBO) and noribogaine (NOR) in plasma of mice after intragastric administration ofibogaine (26.3 mg/kg) or noribogaine (31.5 mg/kg) (n = 3)

Table 1. Basic pharmacokinetic parameters of ibogaine and noribogaine in plasma of mice.

Pharmacokinetic Ibogaine Noribogaine Noribogaineparameters after ibogaine after ibogaine after noribogaine

Tmax, (h) 0.5 ± 0.015 0.5 ± 0.01 4 ± 0.02

Peak plasma concentration Cmax (ng/mL) 475 ± 0.05 185 ± 0.02 150 ± 0.02

Elimination half-life T1/2 (h) 1.95 ± 0.11 4.42 ± 0.09 6.14 ± 0.21

Volume of distribution Vd (mL/mg) 0.04 ± 0.01 0.20 ± 0.01 0.37 ± 0.05

Clearance CL (mL/h/mg) 0.015 ± 0.00 0.03 ± 0.001 0.04 ± 0.001

Data represent the mean ± SD values from individual animals (n = 3) assayed in duplicate


trically. Plasma levels of ibogaine were undetectable24 h, but plasma levels of noribogaine were 2.5 ±0.01 ng/mL after intragastrically administration ofibogaine. The mean Vd of ibogaine in plasma was0.04 ± 0.01 mL/mg body weight and the mean T1/2

was 1.95 ± 0.11 h in agreement with previousreports (10, 15). The mean Vd of noribogaine afteribogaine administration was 0.20 ± 0.01 mL/mg andT1/2 was 4.42 ± 0.09 h.

Circulating levels of noribogaine increasedslowly to a peak (150 ± 0.02 ng/mL) at 4 h (Tmax)after intragastric administration of noribogaine.Plasma levels of noribogaine decreased rapidly butit was 2.5 ± 0.01 ng/mL 24 h after noribogaineadministration. The mean Vd of noribogaine was0.37 ± 0.05 mL/mg body weight and the mean T1/2

was 6.14 ± 0.21 h.

Pharmacokinetic profiles in organs

The main data of pharmacokinetic parametersfor ibogaine and noribogaine in organs of mice arepresented in Table 2. The results demonstrate thatibogaine and noribogaine are rapidly detected inorgans following intragastrically administration ofibogaine or noribogaine. Four hours after intragas-trically administration to mice, ibogaine was foundin high concentration in spleen (4.88 ± 0.11 ng/mg)and the lower in brain (0.3 ± 0.02 ng/mg). Thelongest total systemic exposure (AUCtot) was tospleen (134.54 ± 34.49 ng◊h/mg) and the smallest ñ

to muscle (2.75 ± 0.40 ng◊h/mg) and brain (3.55 ±0.31 ng◊h/mg).

The highest concentration of noribogaine wasfound in the spleen too (15.49 ± 0.08 ng/mg) afteribogaine administration and in the liver (15.55 ±0.09 ng/mg) after administration of noribogaine.The lowest levels of noribogaine were in heart afteribogaine or noribogaine administration. The longesttotal exposure of noribogaine calculated to spleen(97.23 ± 0.16 ng◊h/mg) after ibogaine administra-tion and to liver (97.92 ± 1.44 ng◊h/mg) afteradministration of noribogaine. Whereas the smallestwere to muscle (9.28 ± 0.54 ng◊h/mg), brain (9.48 ±0.29 ng◊h/mg) and heart (9.57 ± 1.34 ng◊h/mg)after ibogaine and to muscle (14.05 ± 0.41 ng◊h/mg)and spleen (15.29 ± 0.57 ng◊h/mg) after noribo-gaine administration.

DISCUSSION AND CONCLUSIONS

It is known that administration of ibogaine toprimates leads to formation of metabolite ñ noribo-gaine (7, 8). The circulating time and concentrationof metabolite noribogaine depend on route ofadministration of ibogaine. It is estimated that a sub-stantial fraction of ibogaine is metabolically con-verted to noribogaine when ibogaine is given via thei.p. route and much smaller fraction of ibogaine isconverted to noribogaine after i.v. administration ofibogaine (10). The present results demonstrate, that

Table 2. Basic pharmacokinetic parameters (n = 3) of ibogaine and noribogaine in organs of mice (mean ± SD).

Ibogaine after ibogaine

Spleen Liver Heart Kidney Brain Muscle

Tmax, (h) 4 ± 0.02 0.5 ± 0.015 0.25 ± 0.00 2.0 ±0.1 4.0 ± 0.06 4.0 ± 0.2

Cmax (ng/mg) 4.88 ± 0.11 2.04 ± 0.11 1.47 ± 0.08 1.25 ± 0.09 0.3 ± 0.02 0.39 ± 0.07

AUCtot (ng◊h/mg) 134.54 ± 34.49 10.16 ± 0.64 38.85 ± 5.67 40.37 ± 2.07 3.55 ± 0.31 2.75 ± 0.40

Noribogaineafter ibogaine


Tmax, (h) 4 ± 0.015 0.5 ± 0.00 4 ± 0.2 2 ± 0.01 0.5 ± 0.15 2 ± 0.1

Cmax (ng/mg) 15.49 ± 0.08 12.05 ± 0.10 0.60 ± 0.08 7.32 ± 0.12 1.95 ± 0.14 1.71 ± 0.09

AUCtot (ng◊h/mg) 97.23 ± 0.16 35.55 ± 0.08 9.57 ± 1.34 40.33 ± 0.53 9.48 ± 0.29 9.28 ± 0.54

Noribogaine after noribogaine


Tmax, (h) 0.5 ± 0.03 2 ± 0.015 4 ± 0.5 0.5 ± 0.00 2.0 ± 0.15 2.0 ± 0.1

Cmax (ng/mg) 2.23 ± 0.10 15.55 ± 0.09 0.91 ± 0.04 6.04 ± 0.10 4.92 ± 0.11 3.05 ± 0.11

AUCtot (ng◊h/mg) 15.29 ± 0.57 97.92 ± 1.44 26.03 ± 1.92 30.26 ± 0.54 24.27 ± 0.58 14.05 ± 0.41

Data represent the mean ± SD values from individual animals (n = 3) assayed in duplicate.


noribogaine Cmax was lower than that of ibogaineafter intragastrically administration of ibogaine,inconsistent with previous reports (14, 16). Previousstudies have shown that the ratio of noribogaine toibogaine in the bloodstream is much higher whenibogaine is injected by the i.p. route rather than i.v.route (10). We observed that the ratio of noribogaineto ibogaine after intragastrically ibogaine adminis-tration is higher rather after i.v. injection but lowerrather i.p. injection. Comparing our results with lit-erature data, we can conclude that the peak concen-tration of noribogaine is achieved faster (Tmax 0.5 h)when ibogaine is administrated intragastricallyrather than the i.p. route (Tmax 2.4 h) or i.v. route (Tmax

2.2 h) (10).Our results demonstrate that volume of distri-

bution (Vd) of noribogaine reaches a higher valuesthan ibogaine after intragastric administration ofeither ibogaine or noribogaine. Low values of half-life and Vd of ibogaine show the faster eliminationfrom plasma in agreement with previous reports (17)indicating that long-lasting effect of ibogaine attrib-utable to metabolite (7, 18).

However, a major aim of the present study wasto compare the pharmacokinetic properties of nor-ibogaine after administrations of ibogaine and nor-ibogaine. Our results demonstrate that noribogaineachieves higher concentration in a shorter period oftime in plasma after administration of ibogaine ratherthan noribogaine itself inconsistent with report ofZubaran et al. (14). However, peak concentrationoccurs at 2-3 h and slow elimination of noribogainewas established in previous reports too (19).Noribogaine is less distributed and elimination isfaster after administration of ibogaine. Thus, noribo-gaine may be more potent in the treatment of drugdependence when noribogaine is administered itselfrather than ibogaine althouth its absorbtion is longer.

Data presented in the literature show that phar-macokinetic parameters depend on different route ofadministration and dose of substance. After i.p. ands.c. injection of ibogaine in rats, the highest level ofthe substance is achieved in brain and adipose tissueone hour after administration (18). Whereas presentresults demonstrate high concentration of ibogainein spleen 4 hours, and in liver 30 min after intragas-trical administration.

Present data in brain tissue show that ibogaineand noribogaine penetrate the blood-brain barrier inagreement with previous reports (10, 13, 14).Noribogaine achieves higher concentration in braintissue compared with ibogaine after ibogaine admin-istration. However, the concentration of noribogainein brain are greather after administration of noribo-

gaine than of ibogaine. The concentrations of ibo-gaine and noribogaine have been measured in ratbrain following both oral and i.p. administrations(40 mg/kg i.p., 50 mg/kg per os) (13, 14, 16). Theresults for concentrations of noribogaine in lowerbrain regions (cerebellum and brainstem) afteradministration of ibogaine were lower, compared toibogaine. However, the concentrations of noribo-gaine in the higher regions of the brain (cortex andstriatum) were higher after intragastric administra-tion of either ibogaine or noribogaine. The concen-trations of noribogaine in all regions of brain weremuch greater after administration of noribogainethan after ibogaine (13, 14).

Literature data show, that ibogaine can lead toserious cardiac-rhythm abnormalities (20-24). So,we compared total systemic exposure in heart to ibo-gaine and noribogaine. High values of AUCtot allowto agree with these statements. Furthermore, highvalue of AUCtot of noribogaine suggests, that nori-bogaine can lead this abnormalities too. Unfor-tunately, additional experiments are needed to deter-mine effects of noribogaine on the cardiovascularsystem. Although it is believed that noribogaineconstitutes the major cardiac risk after ibogaineintake (17).

Interestingly, concentration of ibogaine andnoribogaine increases in spleen, liver and brain,while concentration in blood plasma is alreadydeclining. Ibogaine is sequestered in fat (18).Another depot might be the platelets or other bloodcomponents, as concentrations of ibogaine werehigher in the whole blood than in plasma (25). Itcould affect the resultss of our study, whereas wetested plasma of mice (which does not contain theplatelets), while spleen and liver are tissues of high-er blood perfusion (i.e., platelets can affect theresults of ibogaine and noribogaine concentrations).Partitioning of the parent drug into brain lipid mayserve as a slow release storage ìëdepotî (13). It canlead to concentration of ibogain eincreasing in thebrain even when the concentration in plasma alreadydeclines.

We observed that noribogaine Cmax exceed thatof ibogaine in liver, kidney and brain of mice afteribogaine administration. This finding is consistentwith the conversion of ibogaine to noribogaine viafirst-pass metabolism in the liver, as previouslyreported (6, 10, 26). Noribogaine Cmax (7.32 ± 0.12ng/mg in kidney, 1.95 ± 0.14 ng/mg in brain)exceeded that of ibogaine (1.25 ± 0.09 ng/mg in kid-ney, 0.3 ± 0.02 ng/mg in brain ) to yield a noribo-gaine-to-ibogaine Cmax ratio of 5.86 in kidney and6.5 in brain. These results confirm the hypothesis


that ibogaine is o-demethylated to noribogaine inbrain (13) and possibly in kidney. However, morefurther studies are needed to estimate this.

The present results demonstrate a widespreaddistribution of ibogaine and noribogaine throughoutthe body after ibogaine and noribogaine administra-tion. However, some of the results are difficult toexplain, for example low values of SD for Cmax orlong absorption of noribogaine after administrationof noribogaine. It may be affected by our chosennon-compartment model and low number of ani-mals. Although an increasing number of pharmaco-kinetic studies of ibogaine and noribogaine are inscientific databases (16, 18, 19, 26, 27), but differentresults lead us to extend studies using two-compart-ment or even multi-compartment model and com-pare the results.

High concentration and AUCtot of noribogainein brain could be more efficacious alternative to ibo-gaine as a medication for the treatment of addiction.High values of AUCtot in heart samples may deter-mine the long elimination from this organ. So, fur-ther studies, can noribogaine as ibogaine lead to car-diovascular abnormalities, are needed. NoribogaineCmax ratio with ibogaine Cmax in kidney and brainafter ibogaine administration may also result inmetabolism of alkaloid in these organs.

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Received: 7. 12. 2015


Under the Pharmaceutical Law Act of 6September 2001 (1) (Ph. Law Act) pharmacy is ahealthcare protection facility in which entitled per-sons provide specified pharmaceutical services (Art.86). Community pharmacy is intended to supply acertain population in medicinal products, magistralmedicines and other products catalogued in the Art.86 (8) in conjunction with the Art. 72 (5) of the Ph.Law Act.

On the basis of the above-mentioned, pharma-ceutical services include dispense of medicinalproducts and medical devices, preparation of magis-tral medicines and information to be provided aboutthe medicinal products and medical devices (Art. 86(2)). Pharmacy therefore under the Ph. Law Act isnot equated with a place of drug sales but a place ofdrug dispensing. On the other hand, reimbursementmargin pertains to a concrete drug or medicinalproduct not to the pharmaceutical service. Non-reimbursement margin does not include pharmaceu-tical service as well, which, to be mentioned, is notvaluated at all. The cost of pharmaceutical service is

not comprised into the drug price except magistralmedicines, which are prepared based on thepatientsí prescription. Therefore, pricing of a magis-tral medicine include the so called ìtaxa laborumîunderstood as the cost of this customization (2).

In theory, the pharmaceutical sector may beidentified by two extreme market models: central, inwhich the whole market is managed and controlledby the governmental institutions and model based onthe market mechanism, which functions basing on acompetition of a wide range of manufacturers andbuyers. Polish pharmaceutical market is an exampleof model in which some factors of a central modelenact together with those classified to the marketmechanism. Therefore, a community pharmacy inPolish system of health care is to be placed in a verydistinctive way. Retail sale is carried mostly underthe market-driven rules ñ what can be illustrated bydemonopolization and a possibility of running apharmacy by a various entities, non-necessary witha pharmaceutical diploma and the freedom of choos-ing a form of this business activity. At the same

GENERAL

LAW AND ECONOMICS OF BRANDED-TO-GENERIC, GENERIC-TO-GENERIC AND GENERIC-TO-BRANDED SUBSTITUTION

PROCESS IN POLAND

KATARZYNA ANNA GRUCHA£A and AGNIESZKA ZIMMERMANN*

Department of Medical and Pharmacy Law, Faculty of Health Sciences with Subfaculty of Nursing andInstitute of Maritime and Tropical Medicine, Medical University of Gdansk,

Tuwima 15 St., 80-210 GdaÒsk, Poland

Abstract: Polish setting is an example of two extreme-sides models of a market in pharmaceutical backgroundñ market and central mechanism. Unfortunately, absence of vital institutional and social infrastructure under-stood as law that serves the goal of system efficiency, leads to leak of prognostic conflicts solving, lack of con-trol over the public funds and lack of sustainable development. Substitution is a process that influences patients,pharmacists, marketing authorization holders and healthcare system in general. Pharmacy under Polish law is aplace of drug dispensing not yet a place, where adequate healthcare protection (pharmaceutical) services arecarried. The intention of the paper is to objectively understand the law effects in this background and raise voic-es ñ authors take the narrative view and do not advocate for any stakeholder of the system. Therefore, throughan international discussion, respecting the trend of globalization and consolidation of legacy systems into glob-al drug safety system, the goal of influencing the long-term shape of an institutional and social harmony with-in the process of drug substitution in Poland is to be stressed through this paper.

Keywords: community pharmacy, drug substitution, economics, legislation, reimbursement, pharmacy law

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1600 KATARZYNA ANNA GRUCHA£A and AGNIESZKA ZIMMERMANN

time, pharmacy is a subject of certain limitations, forexample, the number density ban or legally obligedto work in defined hours and heavily controlled bythe governmental institutions (2). Although suchscenario is present in many countries, where gov-ernments do apply limits for the market mechanismin health care, some of those countries also addi-tionally support this specific sector for example byguidelines aiming at patient security, expendituresrationalization and minimization of misunderstand-ing the applied legal rules.

According to IMS Health, at the end of the year2015 there were 14569 pharmacies in Poland, out ofwhich 5400 operating in chains. Analyzing thedemographical data, the average pharmacy inPoland covers 2734 citizens, in comparison Ger-many 3958, Spain 2203, the United Kingdom 4452,The Netherlands 8467 and Czech Republic 4351. Inthe year 2013 in the pharmacistsí opinion, as theIMS Health claims, 52% of pharmacies highlightedthe fallout of financial situation, moreover, 21%fallout of ability to pay its suppliers on time. As keypoints threatening the pharmacies situation in thefollowing year the rising market share of pharmaciesin chains, unclear, incoherent law and competitionwere indicated. The same source points out that 78%of closed pharmacies in early 2014 were the indi-vidual ones. Almost 30% of pharmacies are branchpharmacies, which in fact, generate over 44% ofmarket sales ñ such trend shall continue to grow (3).IMS Health indicates that pharmacies in middlebranches (15-29 and 30-49) operate most effective-ly ñ meaning high sales, low prices and efficientinventory turnover.

Branded-to-generic, generic-to-generic and

generic-to-branded substitution

Substitution is a process of switching the pre-scribed pharmaceutical product into its availableequivalent. Such process arises in pharmacybetween a pharmacist or a pharmacy technician anda patient, with an indirect participation of a physi-cian and, in terms of reimbursement pharmaceuti-cals, commonly a public payer. Substitution occursboth in branded (branded product understood byAuthors as innovative, reference product) andgeneric scope. A generic drug, as World HealthOrganization (WHO) claims, is a pharmaceuticalproduct in principal intended to be switched, whatmust be highlighted, with an innovative product,launched after innovativesí patent expire. WHO rec-ommends trade of generics to be carried out by aninternational nonproprietary name (INN) rather thana proper name (4). The Art. 15 of the Ph. Law Act

defines the equivalent (so the generic) drug as aproduct with the same qualitative and quantitativecomposition, the same pharmaceutical form as thereference drug, and which bioequivalence with thereference product has been confirmed by thebioavailability tests. Salts, esters, ethers, isomers,mixtures of isomers, complexes or derivatives of anauthorized active substance are to be the same activesubstance unless differ remarkably from this activesubstance with its characteristics in terms of safetyor efficacy. If so, the marketing authorization hold-er encloses documentation confirming the safety orefficacy of salts, esters, ethers, isomers, mixtures ofisomers, complexes or derivatives of an authorizedactive substance. Reference product (the innova-tive), unlike the generic, is authorized after pre-clin-ical then clinical trials. Generic drugs are subjectonly of bioequivalence verification with the refer-ence product. Therefore, it is stated that a similarprofile of biologically active compound changes inblood a base to assume that both drugs indicate sim-ilar efficacy and safety. The rules of bioequivalencetests on the European market arise from theEuropean Medicines Agency (EMA) guidelines. Itis stated that bioequivalence tests are carried out onhealthy volunteers. Each volunteer obtains bothinnovative and generic drug usually in a randomorder (5). Permissible percentage of absorbeddosage, max blood concentration level and timeafter which it will appear may differ. Commonlypracticed range of biological equivalence is between80%-125%. The Orange Book ñ Approved DrugProducts with Therapeutic Equivalence Evaluationsissued by the Food and Drug Administration (6) ñFDA divides medicines into two categories ñ A,which classifies therapeutic equivalent medicineswith the reference ones and B, which classifies med-icines not therapeutically equivalent with the refer-ence medicines, due to differences in bioequiva-lence, which most commonly arise from specificpharmaceutical form or specific pharmacokinetics(7). Going further, in category A there are two sub-categories ñ first, in which biological equivalence isnot an issue, and second in which biological equiv-alence with the reference drug is confirmed by the invivo tests. In the U.S. FDA is responsible for super-vision of drug registration and directly points whichdrug is a reference one in the Reference Listed Drug(RLD) list. Accordingly, consecutive generic prod-ucts being the subject of an authorization (byAbbreviated New Drug Application, ANDA) referto the same reference product (6). Furthermore, onlyreference drug occurs under the brand, registeredname, generics under generic names ñ INN, as

Law and economics of branded-to-generic, generic-to-generic and... 1601

WHO recommends. Polish factual data differ fromabove-mentioned model. Guideline on theInvestigation of Bioequivalence, CPMP/EWP/QWP/1401/98 Rev. 1 (European Medicines Agen-cy) contains two criteria, which determine the rulesof choosing a reference product in respect to thegeneric product being authorized on the Europeanmarket ñ those are actual register in the EuropeanUnion and marketing authorization based on the fulldocumentation agreeable with the Directive2001/83/EC of the European Parliament and of theCouncil on the Community code relating to medici-nal products for human use. The content of activesubstance in series of tested and reference productmay vary maximum by 5%. What is meant byabove, the selection of a reference product lies onthe applicant unlike the U.S. market, where suchproduct is issued by an marketing authorizationholder.

Reimbursement policy

Current pharmaceutical law (not only inPoland) indicates tendency for applying substitu-tion/switching solutions due to rational savings inhealth care system. There are scientific proves ofìprosubstitutionî solutions under the pharmaceuti-cal law, which aim to increase the share of genericdrugs on the reimbursement drug market (8, 9). For

substitution to arise legally in Poland, the followingcriteria must be fulfilled (according to Art. 44 of thelaw of 12 May 2011 on the reimbursement of medi-cines, food products of special nutritional purposeand medical devices ñ Reimb. Act):1. Both pharmaceutical products must have the

same international name, dose and pharmaceuti-cal form, which does not give rise to therapeuticdifferences and has the same therapeutic indica-tion;

2. Retail price of which does not exceed the publicfund financing limit;

3. A pharmacy is obliged to ensure that such medi-cine is available (10).

The criteria mentioned above concern alsomedical devices and foodstuffs for particular nutri-tional uses. It is possible to switch medicine not onlyto less expensive one but, since summer 2016, alsoto the one having the same price, more expensivedrug or a drug, which is not reimbursed ñ 100%patient surcharge (11). Such act of change is to be atop-down step forward to the sustainable develop-ment and ease to an individual approach to a patientundergoing a substitution process. Situations (forpatient) of not receiving a prescribed drug, whichwas the cheapest one in a single limit group, becauseof its lack in retail and wholesale sale are, prospec-tively, no longer to appear. Since autumn 2016,

Figure 1. Example of a single limit group calculation (in PLN)


patients over 75 years old are to receive medicinesfree of charge (12).

Under the Polish law, it is absolutely intolera-ble to perform therapeutic substitution, understoodas switching medicines with similar therapeuticeffect but with different active ingredient. It is alsoabsolutely intolerable to switch medicine, which hasbeen prescribed by an authorized person with theappropriate note ìdispense as writtenî. Under theReimb. Act (Art. 44) one and only possibility of dis-pensing equivalent of prescribed medicine is evidentand categorical patientsí claim after being informedby a pharmacist/technician about available equiva-lent. Setting prices and margins for the reimbursedmedicines are strictly defined in the Reimb. Act. Atfirst, manufacturers negotiate prices with theMinistry of Health (official price) ñ a new reim-bursement list is launched every two months (how-ever, a drug is placed on a reimbursement list for atleast two years). Wholesalersí margin is set on thelevel of 5% and is calculated from the official price.Retail margin is calculated from the wholesalersíprice of a medicine being a base in a certain limitgroup as stated in the Art. 7 (4) of the Reimb. Act(see Fig. 1).

According to the Reimb. Act (Art. 14) there arefour payment categories of a reimbursed drug: freedispensing, flat-rate, 30% and 50%. All those entitleto a public financial limit and with eventual patientsurcharge as an effect of a difference between retailprice and the level of a public financial limit. Publicfinancial limit is directly colligated with a price of amedicine, foodstuffs intended for particular nutri-tional uses and medical devices, which is a limitbase in each limit group. The amount of pay differ-ential between the official retail price and the pub-licly financed sum of a certain medicine is apatientís surcharge (Fig. 1). In consequence, forexample, for a medicine with free dispensing it isnot univocal that a patient will receive it without anycharges.

DISCUSSION

Law and economics, as defined by Posner, isthe ìapplication of the theories and empirical meth-ods of economics to the central institutions of thelegal systemî (13). Authors do take issue with thefar-reaching interpretations that efficiency should bethe goal of law and do agree with the Hansonís con-clusions defining the field as understanding the laweffects itself and how those laws might be altered tobetter serve the goal of efficiency (13). Efficiencyitself aims at minimizing the waste of resources,

including energy, time and materials while success-fully completing the desired gain. System of healthcare faces different, sometimes contrary challengesñ a well-being of a patient from one hand, profits forthe commercial entities on the other hand, includingpharmacies and sources for maintaining its workers.For a necessary harmonization it is needed to workfor solutions, which do protect patients but also donot conflict in business activities of entities deliver-ing health care services to patients. In terms of phar-macy, facing competition may go hand in hand withits social, even servant profile. Globally inevitablephenomenon of pharmacies connecting into chainsraises a question: does a purely economic under-standing of effectiveness of chain pharmacies has afree space for efficient fulfillment of public healthprotection acts (for example by a pharmaceuticalservice). Does market acts and competition resultingfrom legal capacity of a pharmacy, which in fact, isan enterprise, shall give way to its social, even ser-vant obligations. Therefore, should an issue of phar-macy operating effectively be widen to its superiorpurpose. Does the problem of market manipulation,understood as the ìutilization of cognitive biases toinfluence peoplesí perceptions and, in turn, behav-iorî is to be acknowledged as possible in currentreality (14). Pharmaceutical services need to be dis-tinguished from the health services, which are actsaiming at health remaining, saving, recover orimprovement and other medical acts arising fromthe treatment process or other regulations definingthe rules of its execution (12). Pharmaceutical serv-ice is, without a doubt, a service, which aims atremaining, saving, recovering or improvement ofhealth but it is a subject undefined and not includedin any legal act as the health service.

The acknowledgement of a drug to be bioequiv-alent does not guarantee therapeutic equivalence tothe reference drug. Therefore, it is a subject of a con-stant discussion, does proven bioequivalence is a suf-ficient argument to substitute pharmaceutical prod-ucts or maybe it should be determined by the clinicaltrials, which result whether a generic drug is thera-peutically equivalent with an innovative one (7). Itshould be highlighted that bioequivalence test is per-formed always in comparison with the referenceproduct. Those tests are not held between two gener-ic products, therefore the substitution, which takesplace in a pharmacy, between two generic products isnot confirmed by any knowledge of their bioequiva-lence towards each other. Each of those genericscould differ by 80%-125% from the reference drug.Therefore, it is hard to confirm the exact differencebetween two generic pharmaceutical products.


The availability of generic drugs on the marketdrives the competition, mostly between pharmaceu-tical companies, which in the end, magnifies theaccessibility of low-priced offered medicines topatients and public payer. Generic products domi-nate the pharmaceutical market in Poland. In termsof sales share by volume they stand for 84% of totaland, in terms of value 62% (year 2014) (15).According to Deloitte, the U. S. generic drugsaccount for around 70% of the U. S. drug market byvolume. In Europe around 50%, however the pro-portion differs significantly by country (16). What isinteresting, Deloitte highlights ìin the U. S. genericuse is almost 90% within the off-patent market butin many European countries potential savings arenot fully exploited due to lower utilization of gener-ics in key therapy areasî. As already has been indi-cated in the literature (5, 17-19) in terms of narrowtherapeutic index (NTI) drugs ìroutine switchingbetween different manufacturers of antiepilepticdrugs should be avoidedî.

In Polish factual data substitution does notmean switching only to a generic product. Someoriginal pharmaceuticals, which are not a subject ofreimbursement, are cheaper than their reimbursedequivalents. In factual data, substitution arises bothin reimbursed and payable in full medicines.

In factual data a pharmacy as an entity, in thebeginning is obliged to buy certain medicines, applycertain retail margins and when a patient arrives

with a certain prescription, for example of free dis-pensing, dispense this medicine according to its cat-egory. Under the Reimb. Act twice a month a phar-macy is obliged to send to a public payer (NationalHealth Fund) reports about reimbursement it hasmade during 14-day-period and only then willreceive invested earlier money (see Fig. 2). The INNprescribing is of high matter for the harmonic sub-stitution process (see Fig. 3). A top-down approachand number of reforms, including physician incen-tives, are needed to successfully change prescribinghabits. INN prescribing increases the role of phar-macist, who can then perform a pharmaceuticalservice to best suit individual a medicinal product,and do not conflict with the pharmacy as a businessentity. Frequent changes in reimbursement list inPolish setting ñ every two months ñ make an inven-tory turnover difficult to perform effectively. InFrance for example, ambulatory care physiciansagreed to prescribe by INN in return for higher fees(20).

A tendency of legal authorities aiming at look-ing for cost savings seems likely to lead to anincreased use of generic drugs in key therapy areas,where it is safe to occur. A priori substitutionprocess leads to cost savings. Therefore, it is impor-tant to support pharmacies with substantial legalìtoolsî of such process in various therapeutic areas(different for example cardiovasculars and AEDs)symbiotic with pharmacy being a business entity.

Figure 2. Example of a possible scenarios of receiving a margin (in PLN) for a medicine ketoprofen 1 in accordance with theReimbursement of Medicines, Foodstuffs Intended for Particular Nutritional Uses and Medical Devices law of 12 May 2011


Each person undergoing a substitution should beassessed carefully. An electronic view of medicalhistory of a patient, where a pharmacist may seewhat medicines this particular patient is currentlytaking and which has already been substituted in thepast, may eliminate potential mistakes of not ade-quately enacted (due to low information provided)process of substitution. The U. S. has provided itsmedical professionals with the Orange Book andclearly defined processes of a drug being the refer-ence one, therefore the U. S. market may benefitfrom the savings it has made during a decade ñ $1.2trillion (60% of savings in nervous and cardiovascu-lar systems) (21).

It seems likely that the next few years willundergo a wider discussion on systemic sustainabledevelopment of a substitution process from thebeginning, so medicine being registered till its dis-pensing and control of an enacted process. As it maybe the INN prescribing, as in the United Kingdom,will be raised on the merits. Percentage of INN pre-scribing is the highest (over 80%) in the UK, theUSA and China, which, growing fast, is an interest-ing example ñ its Government ensured 90% healthcoverage for its citizens (2009-2011) (22).

CONCLUSIONS

Nontransparent drug registering methods, non-transparent side effects monitoring (as well as

access to such base), frequent changes in reimburse-ment drug list and lack of cooperation between var-ious medical professions (ex. lack of access topatientsí taken and prescribed drugs for pharma-cists) stay in conflict with the harmonic (for allstakeholders) process of drug substitution.Relevance of registering method of a generic drug isof high matter for the substitution process.Substitution, under law, should be held in accor-dance with the therapeutic areas in order to fullyexploit benefits of such process. Law has its othergoals than efficiency, however, it can be altered tobetter serve the goal of efficiency of a system as awhole. Pharmacy is a place of public health protec-tion ñ each country, which in fact, is in disposal ofthe instrument such as law, may control both expen-diture rationalization of members of health care sys-tem and provide them with essential, needful toolsfor sustainable development. Legislator, by includ-ing in pharmacy definition words ìpublic health pro-tectionî on one hand obligates this entity to fulfill itscrucial, overarching goal from the other, therefore,leaves to the market self-law. The INN prescribingshould be legally introduced as compulsory. Asevaluated by Deloitee ìthere is a strong trendtowards consolidation and globalization of legacysystems into single, global drug safety system withharmonized business processesî. Authors do believethat such trend will reach Poland visibly in upcom-ing years.

Figure 3. Harmonic substitution process


REFERENCES

1. The Pharmaceutical Law Act of 6 September2001 (Journal of Laws from 2008, No. 45, item271 as amended ñ in Polish).

2. Zimmermann A.: Acta Pol. Pharm. 70, 339(2013).

3. http://www.pgf.com.pl/uploads/IMS_Health.pdf [accessed 8.05.2016].

4. http://www.who.int/trade/glossary/story034/en/[accessed 9.05.2016].

5. Sznitowska M., Kaliszan R.: Biopharmacy,Elsevier Urban & Partner, Wroc≥aw 2014 [inPolish].

6. http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ucm079068.htm [accessed 01.08.2016].

7. WoroÒ J.: Selected Clinical Issues [WybraneProblemy Kliniczne], 4, 241 (2010).

8. Carroll N.V., Fincham J.E., Cox F.M.: Med.Care, 25, 1069 (1987).

9. Andersson K., Bergstrom G., Petzold M.G.,Carlsten A.: Health Policy 81, 376 (2007).

10. The law of 12 May 2011 on the reimbursementof medicines, food products of special nutrition-al purpose and medical devices (consolidatedtext Journal of Laws from 2016, item 1536 inPolish).

11. The law of 18 March 2016 amending the law onthe Publicly Funded Healthcare Services andother legal acts (Journal of Laws, item 652).

12. The law of 27 August 2004 on the PubliclyFunded Healthcare Services (consolidated textJournal of Laws from 2015, item 581 as amend-ed in Polish).

13. Hanson J.D., Hart M.R.: Law and Economics ina Companion to the Philosophy of Law andLegal Theory, D.M. Patterson Ed., 299,Blackwell, Oxford 1996.

14. Hanson J.D., Kysar D.A.: New York UniversityLaw Review 74, 3, 1999.

15. http://www.producencilekow.pl/images/pigul-ki/Pigulka_54.pdf [ accessed 24.10.2016].

16. Deloitte Report: Global life sciences outlook(2014).

17. Perucca E.: Lancet Neurol. 15, 343, 2016. 18. Privitera M.D., Welty T.E., Gidal B.E., Diaz

F.J., Krebill R., et al.: Lancet Neurol. 15, 365(2016).

19. Shin J-W., Chu K., Jung K-H., Lee S-T., MoonJ., et al.: Int. J. Clin. Pharmacol. Ther. 52, 1017(2014).

20. Godman B., Bishop I., Finlayson A.E.,Campbell S., Kwon H., et al.: Expert Rev.Pharmacoecon. Outcomes Res. 13, 469 (2013).

21. http://www.gphaonline.org/media/cms/2013_Savings_Study_12.19.2013_FINAL.pdf [aces-sed 18.05.2016].

22. Rana P., Roy V.: Fundam. Clin. Pharmacol. 29,529 (2015).

Received: 29. 08. 2016


Non-conventional formulations such as phar-maceutical drug delivery systems containing a deliv-ery base and an active agent have created new appli-cation rules for pharmacist and patients in therapies(1). Although the patientí adherence related to theapplication of medicinal patches (TransdermalTherapeutic Systems) is relatively high compared tothe other dosage forms (2, 3), the lack of patientsíknowledge about the correct application coulddecrease the effectiveness and safety of the therapy.The knowledge of standard application rules relatedto this non-conventional dosage form is essentialbelonging the maximum effectiveness of therapy (1,4, 5).

Health literacy concerns the knowledge andcompetences of persons to meet the complexdemands of health in modern society (e.g., personalcommunication skills or reading comprehensionskill (6)).

The health literacy and the pharmaceuticalcounseling (e.g., patient education with written

information) are closely related in community phar-macy (7). The evaluation of health literacy can con-tribute to the development of the adequate counsel-ing and services as well as in the necessary pharma-ceutical therapeutic interventions of different groupsof patients (8). The pharmacists have to recognizethe lack of health literacy in community pharmaciesand consequently to improve the patientís knowl-edge and comprehension with effective practice.There are several international formal assessmentsof health literacy (9, 10).

The aim of the patient education is to increasethe patientís adherence and the effectiveness of drugtherapy (11) with adequate and practical applica-tions in general practice settings (12), therefore thebarriers or facilitating factors and the health out-comes should be determined according to health lit-eracy (13).

Special health literacy studies were developedby researchers related to different therapies, dosagesforms or the different types of patientsí information

THE EFFICACY OF WRITTEN INFORMATION ABOUT THE APPLICATIONRULES OF TRANSDERMAL PATCHES ñ READING COMPREHENSION

QUESTIONNAIRE SURVEY IN HUNGARIAN COMMUNITY PHARMACIES

ORSOLYA SOMOGYI and ROM¡NA ZELK”*

Faculty of Pharmacy, University Pharmacy Department of Pharmacy Administration, Semmelweis University, Budapest

Hogyes E Street 7-9, H ñ 1092 Budapest, Hungary

Abstract: Although patientí adherence related to the application of transdermal patches is relatively high com-pared to the other dosage forms, the lack of the correct use could decrease the effectiveness of the therapy.Therefore, the primary aim of our study was to evaluate the efficacy of a Patient Information Leaflet (PIL) con-cerning the adequate use of medicinal patches. The questionnaire survey was developed to explore the level ofpatientsí reading comprehension in four Hungarian community pharmacies. The distribution of four separatedcomprehension categories was measured. For the hypothesis testing it was assumed that the reading compre-hension of ìpatch ever adoptersî is higher than ìpatch never adoptersî. 46% of participants (n = 163) has ìade-quateî, 40% has ìhigh sufficientî, 12% has ìlow sufficientî and 2% has ìinadequateî level of reading com-prehension based on responses. The hypothesis testing showed no significant difference (p = 0.428). Theinstructions of PIL are effective although the majority of participants (54%) has lack of complete understand-ing. Patients have not met these application rules, according to hypothesis testing. Written medicine informa-tion is useful in patient education, but PILs cannot replace the pharmacistsí obligation to provide verbal coun-seling.

Keywords: transdermal patch, counseling, community pharmacy, written information, reading comprehen-sion, questionnaire survey

1607

* Corresponding author: e-mail: [email protected]; tel: +36 208259621; fax: +3612170927

1608 ORSOLYA SOMOGYI and ROM¡NA ZELK”

(e.g., written, verbal or online information). Themeasuring of reading comprehension helps to eval-uate the efficacy of written information (e.g., textsof Patient Information Leaflets ñ PILs) and enablesthe development of understandable text for patients(with illustrations) in the serious therapeutic issuesand in different patientís groups (14-22).

Short, concise messages (consumers prefer thesee.g., in ìbubble formatî) (20) should be created withfamiliar words and recognizable icons (23). Beside themanufacturersí leaflets concerning the applicationrules of medicines, patients receive useful writteninformation about the new medications. Although thepatients can read the instructions, but the leafletsshould not replace the pharmacistís obligation to pro-vide verbal counseling in pharmacies (24).

Pharmacists and the community pharmaciesístaff need additional training regarding health litera-

cy and new services should be developed in focus ofpatientsí knowledge in order to enhance the effec-tiveness of home medicine use (e.g., easy-to-readprinted materials in pharmacy) (25-28).

The primary aim of the present study was toevaluate the efficacy of a self-developed PatientInformation Leaflet (PIL) concerning the adequateuse of transdermal patches and the consequent phar-macistsí intervention in Hungarian communitypharmacies in order to improve the patientsí knowl-edge about the correct application.

EXPERIMENTAL

The survey was administered in four commu-nity pharmacies in two different regions ofHungary (Central Hungary and West Hungary)from March of 2016 to April of 2016. The pharma-

Table 1. Tasks of reading comprehension test, the type of the questions and the corresponding scoring system of assessment.

Correct answer Reading comprehension tasks Type of question or valuable Points

answers

The small lesions on your skin surface might not affect the absorption of the drug, just the visible bigger lesions

True - false False 1

Each patch can be cut to modify the dose of the active agent True - false False 1

The well-selected place of patches is important on your skin for the adequate protection and tight adhesion and it contributes to True - false True 1the systematic exchange of patches

If you would like to use a patch on the same skin area again, you must let the affected surface be patch-free for at least 1 day

True - false False 1

So-called surfactants chemicals for example in soap, in oils andin creams can modify the transdermal absorption of the active True - false True 1substance

Circle the letter of the correct applications (You can mark Only d)more than one answer) and e) 3

marked

a) Before applying a medicinal patch you should clean the surface d) and e) +of skin with soap. other marked 2

answer (s)b) Before applying a medicinal patch you may not clean the surface of skin. Only d)

or 2c) Although it is necessary for reason of bad adhesion, depilation Multiple choice only e)is not allowed at the affected skin before applying a medicinal patch.

Only d) + other marked

d) It is not allowed to use a razor for the affected skin depilation answer(s)before applying a medicinal patch. or 1

only e) + e) After sticking you should press the transdermal patch to the skin other marked and you can fix it in another way (for example with other answer(s)adhesive-tape), if it is necessary.

Not markedf) The best decision is a medicinal patch dosage form for d) and e)

0the local treatment of small joint's pain.

The efficacy of written information about the application rules of... 1609

cies were a varied of low, medium and high trafficcommunity pharmacies. The regular procedure wasensured by staff of pharmacies (pharmacists andassistants). As each patient voluntarily participatedin the survey, men and women over 18 years of ageanswered the questions on paper in pharmacies.Answering the questionnaire took about fifteen totwenty minutes.

At first, the text of PIL was developed based onevidence (1, 4, 5) and counseling guidelines of ìhealthliteracy friendly pharmaciesî (29). The original textwas written in Hungarian avoiding the technicalwords (using familiar words), the most importantinstructions were in bullet points and the keywordswere highlighted in the text. The size of paper withtext was ìA5î or ìA4î for better readability who didnot see the small font size. The Appendix is theEnglish version of the self-developed PIL concern-ing the adequate use of transdermal patches.

The questionnaire survey basically organizedfrom three parts and one cover letter with introduc-tion and participation information, as follows:1. page: the cover letter with introduction and infor-

mation for participants;2. page: questions regarding demography and per-

sonal use of medicinal patches (most of the ques-tions are closed-type);

3. page: the developed text of PIL concerning themost important application rules of transdermalpatches;

4. page: reading comprehension test and a questionconcerning the additional request of the patients(only closed-type questions).

In the course of the answering of the part ofreading comprehension test it was allowed to use thePIL without personal assistance of the staff.

The reading comprehension test consisted offive true / false type questions and a multiple choicetask. The last question explored to public opinionabout demand of verbal counseling related to cor-rect use of non-conventional drug formulations.

Table 1 summarizes the content and type of readingcomprehension tasks and the scoring system ofassessment.

Four reading comprehension levels have beenseparated with an eight-point scale based on themaximum available eight points. The four cate-gories are as follows:◆ 0 ñ 2 points: ìinadequateî level of reading com-prehension;◆ 3 ñ 4 points: ìlow sufficientî level of readingcomprehension;◆ 5 ñ 6 points: ìhigh sufficientî level of readingcomprehension;◆ 7 ñ 8 points: ìadequateî level of reading compre-hension.

Statistical evaluation of the results

IBM SPSS statistics was applied for the evalu-ation of the results. For hypothesis testing and themore other descriptive statistical tests χ2≤ tests wereselected. Kruskall Wallis test was used for agegroups analysis in eight-point scale without separat-ed comprehension categories.

For the hypothesis testing it was assumed thatthe reading comprehension of ìpatch ever adoptersîis higher than ìpatch never adoptersî because ìpatchever adoptersî have known the corresponding appli-cation rules.

RESULTS

163 patients (113 women and 50 men) with theage distribution represented in Table 2 participated inthe survey. Based on the responses 82 participantshave been used transdermal patch at least once, 10participants are applying a patch at the time of surveyand 71 participants have never used medicinal patch.

According to indications most of the partici-pants (75 persons) have used the transdermal patchfor pain and inflammation relief (NSAIDs content ñOTC) and in cases of chronic therapies most of the

Table 2. Distribution of reading comprehension by age with rank numbers.

Age Frequency Mean(years) (persons) Rank

Reading 18-30 29 89.60comprehension 31-50 61 88.89

51-70 58 73.53

70- 13 58.12

Total 161*

*missing dates are in two questionnaires


patients (6 persons) have used this dosage form forpain relief of cancer diseases (fentanyl content).

74% of ìpatch ever adoptersî (92 persons)could name one product at least (the brand-name ofpatch was well written) related to the selected indi-cation of their therapy.

The results of the measured reading compre-hension is ìadequateî (7ñ8 points) in 46% of 163participants, 40% has ìhigh sufficientî (5ñ6 points),12% has ìlow sufficientî (3ñ4 points) and 2 percenthas ìinadequateî (0ñ2 points) level of reading com-prehension.

The hypothesis testing did not show a signifi-cant difference between the reading comprehensionof two groups related to transdermal patch use (p =0.428 with 95% confidence intervals).

The difference was significant, according todifficulty of reading comprehension tasks: p < 0.001and χ2 = 87.519 in six questions. The multiplechoice task was the most difficult for patients; in thiscase 37% of participants could answer without error(3 points).

The difference of reading comprehension isnearly significant by age (p = 0.051). Table 2 illus-trates the results of Kruskall Wallis test. The resultsindicate that the reading comprehension of 18ñ50year-old participants is better than the group of aged51 and over in practice.

The responses of the special request concern-ing the pharmacistsí information service show thedemand of personal pharmaceutical counselingrelated to non-conventional dosage forms in com-munity pharmacies. 80% would require this service,14% selected the ìI donít knowî choice and 6%would not require advices from pharmacist.


The self-developed PIL has been effective con-cerning the correct use of transdermal medicinalpatches because in most of cases (86%) the level ofreading comprehension was ìadequateî (46%) orìhigh sufficientî (40%).

The authorsí previous questionnaire surveywas developed to explore the level of patientsíknowledge of the correct use of transdermal patcheswithout counseling based on their own applyinghabits. It was administered in thirteen Hungariancommunity pharmacies from October of 2012 toMay of 2015. Most of the participants, includingmen and women over 18 years of age (n = 233), usedmajor analgesic patches (fentanyl); the remainderwere given nitroglycerin, NSAID analgesics patchesduring the survey. Based on patientsí applying

habits only 9 tests were flawless from 233 complet-ed questionnaires (30).

Results of reading comprehension study andour previous survey confirm that the text of PIL issufficiently readable and understandable for theimprovement of the patientsí knowledge related touse of transdermal patches at home. A similarAmerican survey was determined to evaluatewhether patients read non-manufacturer-developedleaflets and assess patientsí opinions concerning theunderstandability and usefulness of these leaflets(24). Approximately two-thirds of the patients sur-veyed reported that they least frequently read theleaflets provided with new medications. The major-ity (over 90%) reported the leaflets to be useful andeasy to understand (in our study it was measured:86%). Pharmacists should advocate reading theleaflet and promote it as a useful resource (24).

Although the written instructions have proveduseful, 54% of participants has lack of proper under-standing (ìhigh / low sufficient or inadequateî levelof reading comprehension), the PIL should notreplace the verbal counseling and patient educationrelated to this dosage form. This finding is similar tothe result of a study concerning hormone therapies.The assessment of the readability of written infor-mation of 31 hormone therapy products showed thatthe majority of the materials are written at a highreading level. These findings have implications forhealth literacy and counseling efforts when helpingwomen to understand their options for menopausalsymptom management (15). Pharmacists may needto supplement written information with additional,more understandable self-developed PILs orìpatient-friendlyî verbal instructions (15).

There is a definite demand (based on the requestsof 80% of respondents) for verbal counseling con-cerning the application rules of non-conventionaldosage forms mainly in cases of complex instructions.The multiple choices were the most difficult task inreading comprehension test (only 37% of responseswere correct) opposite the true / false type questions.Usually complex information caused difficulties forpatients, e.g., according to another study, patients ofall health-literacy levels had better understanding ofmedication warning labels that contained single-stepversus multiple-step instructions in primary care clin-ic at the Louisiana State University (23).

The hypothesis testing showed no significantdifference between reading comprehension ofìpatch ever adoptersî and ìpatch never adoptersî.The latter demonstrates that the patients were notfamiliar with these practical application rules oftransdermal patches. In community pharmacies the

The efficacy of written information about the application rules of... 1611

pharmacists should inform patients about the impor-tance of correct home medicine use. The self-writtenPIL along with the verbal counseling can effective-ly contribute to the successful drug therapy.

The self-developed PIL is useful to give thetransdermal patch adopters in community pharma-cies along with the point by point verbal explana-tions. The pharmacist should pay a special attentionto patients over 50 years of age in order to reach aneffective and safe home use of transdermal patches.

Finally, the pharmacists have essential role incommunity pharmacies to improve the health-litera-cy competencies of consumers or patients in gener-al. Although the majority of consumers had ade-quate skills, which was confirmed by several inter-national studies, in specified cases (e.g., complexinformation, advanced age, less formal education(26)) additional written and verbal informationinterventions are required by pharmacy staff.

Acknowledgments

The authors are grateful to the Patika 52Pharmacy, Patika Libra Pharmacy, Kiscelli Pharma-cy, Celli Szent M·rton Pharmacy and the Institute ofBehavioral Sciences Semmelweis University fortheir valuable contribution in the survey.

Ethical approval

Not required. Each pharmacy signed a declara-tion confirming the regular procedure. As eachpatient voluntarily participated and was accordinglyinformed in the survey, which is a non-intervention-al study, therefore no further ethical approval wasrequired.

Funding

None.

Competing interests

None declared.

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Received: 30. 08. 2016

APPENDIX

Patient Information Leaflet concerning the correct application rules of transdermal patches

Please, read the text concerning the application rules of medicinal patches.

✓ Before applying a medicinal patch you should never use a razor for skin depilation because a razor cancause small (micro) lesions on your skinís surface, which might affect the absorption of the drug. If nec-essary, use scissors for skin depilation (1)!

✓ The patch should be applied to clean, dry skin. You may cleanse the skin but use only clean water andthen wipe the skin, because any chemicals (for example soap) can modify the liberation and absorption theactive substance from the patch (1, 4).

✓ Avoid using oils and creams before applying the patch because they too can damage the effect of the drugand the adhesion of the patch (1, 4).

✓ The location of patch application should be alternated. If you would like to use a patch on the sameskin area again, you must let the affected surface be patch-free for at least 3 days (1, 4).

✓ In chronic illness therapies where the aim is the effects on the whole body (not one body partís pain andinflammation relief) it is necessary to selected a protected surface of skin from external effects but it is notnecessary to place the patch in the middle of your back because it is difficult to affix. You should look fora place on your body where you can see the patch well and do not forget to change the location, for exam-ple, shoulders, scapula, abdominal wall (1, 4, 5) etc.

✓ In applying a patch, ensure that it adheres tightly to the surface of your skin. Avoid the bodyís bights

or small joints (knees, wrist, underarm, elbows, etc.). The adequate dosage forms are the gels or creamsfor the treatment of these bights or small joints, not the medicinal patch. In applying exert pressure on the

patch on your skin for 30 s. If necessary, use another adhesive-tape to fix the medicinal patch (1, 4, 5)!

✓ Always read the patient information leaflet of manufacturers primarily related to information about

cutting. Although cutting can cause damage of the structure of some patches, you can cut other patches tomodify the dose of the active (for example sometimes it is allowed to cut in half) (4, 5).


Health screening programs in community phar-macies and their importance of identifying peoplewith chronic diseases such as diabetes or hyperten-sion are well known. The results of these servicescould initiate appropriate treatment and preventlong-term complications from chronic diseases.Diabetes mellitus and hypertension are major globalproblems. The number of people with diabetes hasrisen from 108 million in 1980 to 422 million in2014. Data from 2015 indicate that one in 11 adultshas diabetes. In 2040, about one in 10 adults will besick. Moreover, one in two adults has undiagnosed

diabetes (1). Globally, the overall prevalence ofincreased blood pressure in adults aged 25 and overwas about 40.0% in 2008. The number of peoplewith uncontrolled hypertension increased from 600million in 1980 to nearly 1 billion in 2008 (2). Thisdramatic numbers brought about many changes andactivities in health promotion around the world.

It is very important to perform health screen-ings in each population. Community pharmacies inmany countries have implemented screening servic-es which are very sensitive and effective. In theSwedish study, 6.9% elderly persons in community

EVALUATION OF OPINIONS ON COMMUNITY PHARMACY-BASEDHEALTH SCREENINGS FOR COMMON CHRONIC DISEASES

MAGDALENA WASZYK-NOWACZYK1*, ANNA LUCZAK2, DAWID MICHALAK2,

JOANNA KACZMARCZYK3, HALINA MYRDA3, MICHA£ MICHALAK4

and ANNA RATKA3

1Department of Pharmaceutical Technology, Pharmacy Practice Division, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 PoznaÒ, Poland

2Studentís Pharmaceutical Care Group, Department of Pharmaceutical Technology, Pharmacy Practice Division, Poznan University of Medical Sciences,

Grunwaldzka 6, 60-780 PoznaÒ, Poland3Department of Pharmaceutical Sciences, College of Pharmacy, Chicago State University,

9501 S. King Drive / 3070 Douglas Hall, Chicago, United States of America4Department of Computer Science and Statistics, Poznan University of Medical Sciences,

Dπbrowskiego 79, 60-529 PoznaÒ, Poland

Abstract: The prevalence of chronic diseases increases in the society. The health care system should be focusedon improved disease prevention and health promotion. The pharmacist is very often the first health profession-al contacted by the patient. It would be beneficial for society to provide health screening services in a commu-nity pharmacy. The aim of the study was to evaluate and compare opinions about health screenings for chron-ic diseases in community pharmacies located in PoznaÒ, Poland, and Chicago, USA. An anonymous simple 12-item questionnaire was developed in Polish and English to assess attitudes about screening services in a com-munity pharmacy. The questionnaire as a cross sectional study was carried out from March 2016 to June 2016within patients in community pharmacies in Poznan (n = 265, 37.0% men and 63.0% women) and in Chicago(n = 190, 42.6% men and 57.4% women). A majority of respondents, 72.1% in PoznaÒ and 77.0% in Chicago,confirmed that information about prevention of common chronic diseases was expected to be provided in acommunity pharmacy. Both groups were also interested in participation in health screenings for common chron-ic diseases in a pharmacy (66.8% Poles and 58.0% Americans). Respondents in Poznan, prefer to have suchservices paid by National Health Fund while in Chicago, by a community pharmacy (p < 0.001). These find-ings show general interest in participating in health screening services provided by pharmacists. Developmentof new screening services in community pharmacies may help to prevent or lower the risk of complicationsassociated with common chronic diseases.

Keywords: screening services, health promotion, community pharmacy, pharmacist, chronic diseases

1613

* Corresponding author: e-mail: [email protected]; phone: 61-854-66-84

1614 MAGDALENA WASZYK-NOWACZYK et al.

pharmacy were detected with suspicions for diabetestype 2 and 71.5% had at least two risks factors ofthis disease. In addition, 54.0% individuals showedelevated blood pressure and 16.3% had hypertension(3). The Thai Diabetes Prevention Program in com-munity pharmacies identified that half of the testedclients were at risk of diabetes and provided anopportunity for participants to learn about the pre-vention of diabetes. During a 3-month service for397 individuals, nearly half were at a high risk fordiabetes (4). These studies showed that it is veryimportant to implement a screening campaign forchronic diseases in a community pharmacy. Asequential screening in pharmacy practice maydetect up to 7% patient suspected for diabetes. Mostpeople visiting community pharmacy are open tocounselling about lifestyle. Health screenings arerecommended in asymptomatic adults, includingthose with smoking habits, high blood pressure, highcholesterol, and obesity (5).

Pharmacists working in community pharma-cies are respected and trusted by society and there-fore are in an excellent position to engage in screen-ing, monitoring and educating patient with potentialproblem of chronic diseases. Community-basedscreening programs can result in delay of complica-

tions of chronic diseases and improved quality oflife.

The aim of the study was to evaluate and com-pare opinions about health screening services incommunity pharmacies located in PoznaÒ, Poland,and Chicago, USA. The effect of age, gender andeducation was evaluated.


The survey as a cross sectional study was car-ried out from March 2016 to June 2016 in commu-nity pharmacies. It covered 265 respondents inPoznan (37.0% men and 63.0% women) and 190participants in Chicago (42.6% men and 57.4%women) selected at random. They were patients ofindependent community pharmacies in PoznaÒ andChicago that voluntary agreed to fill out the ques-tionnaire. The most numerous group of respondentsconsisted of Poles aged from 18 to 30 years old(32.4%) and Americans aged from 18 to 30 yearsold and from 41 to 50 years old (in both cases23.2%). The majority of the responding participantsin PoznaÒ and Chicago had graduate education(37.7% and 37.9%, respectively). Socio-economicdata included information about gender, age and

Table 1. Characteristics of study participants.

PoznaÒ Chicagon (%) n (%) n (%)

Gender

Female 167 (63.0) 109 (57.4)

Male 98 (37.0) 81 (42.6)

Total 265 (100.0) 190 (100.0)

Age(years)

18-30 86 (32.4) 44 (23.2)

31-40 34 (12.8) 36 (18.9)

41-50 60 (22.6) 44 (23.2)

51-60 55 (20.8) 29 (15.3)

61-70 19 (7.2) 28(14.7)

> 70 11 (4.2) 9 (4.7)

Total 265 (100.0) 190 (100.0)

Education

Primary 4 (1.5) 3 (1.6)

Vocational 32 (12.1) 17 (8.9)

High school 62 (23.4) 58 (30.5)

College 67 (25.3) 40 (21.1)Graduate 100 (37.7) 72 (37.9)

Total 265 (100.0) 190 (100.0)

Evaluation of opinions on community pharmacy-based health... 1615

education are presented in Table 1. The study wasapproved by the ethics review board of PoznanUniversity of Medical Sciences.

An anonymous questionnaire was developed inPolish and English by the authors of this study toassess opinions about health screening services in acommunity pharmacy among the patients.Translation from Polish to English was done by abilingual pharmacist to ensure that questionsaddressed identical concepts. The content of surveywas validated by licensed pharmacists in PoznaÒand in Chicago. The survey included also a shortdescription of this new service.

Statistical analysis of the results was per-formed using the Statistica 10.0 application(StatSoftÆ). The relationship between analyzednominal data was performed by chi-square test ofindependence (χ2). All statistical analyses were per-formed at p < 0.05.

RESULTS

The study confirmed that higher percentage ofrespondents reported suffering from diabetes andhypertension in Chicago compared with PoznaÒ(respectively p = 0.006, p = 0.004; Table 2) and hada wider knowledge about possibility of simple diag-nostic tests purchasing in community pharmacy (p <0.001; Table 3). Information about prevention ofcommon chronic diseases were expected by 72.1%of respondents in PoznaÒ and 77.0% in Chicago.Availability of information on common disease pre-vention was mainly expected by Polish andAmerican respondents in 18-30 and 61-70 year oldgroup (p = 0.049, p = 0.002, respectively) and byAmericans with vocational and graduate education(p = 0.011; Tab. 4.). Both groups were interested inscreening services participating concerning com-mon chronic diseases in community pharmacy

Table 2. Prevalence of diabetes and hypertension in study participants.

Question in questionnaire: Do you have diabetes/high blood pressure?

Disease:Anwers: PoznaÒ Chicago

n (%) n (%)p-value

Yes 12 (4.5) 10 (5.3) 0.006

Diabetes No 252 (95.5) 173 (91.0)

I don't know 0 (0.0) 7 (3.7)

Total 264 (100.0) 190 (100)

Yes 62 (23.4) 51 (26.8) 0.004

Hypertension No 203 (76.6) 132 (69.5)

I don't know 0 (0.0) 7 (3.7)

Total 265 (100.0) 190 (100.0)

p < 0.05

Table 3. Respondents' opinions concerning possibility of purchasing simple diagnostic tests in acommunity pharmacy.

Question in questionnaire: Do you know that you can purchase in a community pharmacy diagnostic tests (e.g.,to measure blood levels of glucose and cholesterol)?

Anwers:PoznaÒ Chicagon (%) n (%)

p-value

Yes 119 (44.9) 132 (71.0) < 0.001

No 144 (54.3) 54 (29.0)

I don't know 2 (0.8) 0 (0.0)

Total 265 (100.0) 186 (100.0)

p < 0.05


(66.8% Poles and 58.0% Americans, Fig. 1), butAmerican respondents indicated it more often fordiabetes and hypertension than Poles (p = 0.039, p <0.001, respectively; Table 5). Poles assumed thatsuch services should be paid by National HealthFund and Americans selected more often communi-ty pharmacy as a source of foundation (p < 0.001;

Table 6). Participants were also asked to valuatescreening service in pharmacy. Respondents inPoznan preferred to pay for these service about 23z≥otych while in Chicago 41 USD (157 z≥otych). InPoznan, the most interested in screenings were par-ticipants from age groups 18-30 and 51-60 yearsold, and with primary education (p < 0.001). In

Table 4. Effect of age and education on expectation of availability of information about prevention of common chronic diseases in a com-munity pharmacy.

Question in questionnaire: Would you like your community pharmacy provide information on prevention of most common public diseases?

Yes No I don't know Totalp-valuen (%) n (%) n (%) n (%)

POZNA—Age (years)

18-30 70 (81.4) 8 (9.3) 8 (9.3) 86 (100.0) 0.049

31-40 24 (70.6) 9 (26.5) 1 (2.9) 34 (100.0)

41-50 38 (63.3) 9 (15.0) 13 (21.7) 60 (100.0)

51-60 36 (65.5) 7 (12.7) 12 (21.8) 55 (100.0)

61-70 16 (84.2) 1(5.3) 2 (10.5) 19 (100.0)

> 70 7(63.6) 2 (18.2) 2 (18.2) 11 (100.0)

Total 191 (72.1) 36 (13.6) 38 (14.3) 265 (100.0)

Eduction

Primary 4 (100.0) 0 (0.0) 0 (0.0) 4 (100.0) 0.056

Vocational 23(71.9) 2(6.2) 7(21.9) 32 (100.0)

High school 39 (62.9) 10 (16.1) 13 (21.0) 62 (100.0)

College 57 (85.0) 5 (7.5) 5 (7.5) 67 (100.0)

Graduate 68 (68.0) 19 (19.0) 13 (13.0) 100 (100.0)

Total 191 (72.1) 36 (13.6) 38 (14.3) 265 (100.0)

CHICAGOAge (years)

18-30 38 (88.4) 5 (11.6) 0 (0.0) 43 (100.0) 0.002

31-40 26 (76.5) 7 (20.6) 1 (2.9) 34 (100.0)

41-50 34 (77.3) 6 (13.6) 4 (9.1) 44 (100.0)

51-60 21 (72.4) 3 (10.3) 5 (17.3) 29 (100.0)

61-70 23 (82.1) 4 (14.3) 1 (3.6) 28 (100.0)

> 70 2 (22.2) 4 (44.5) 3 (33.3) 9 (100.0)

Total 144 (77.0) 29 (15.5) 14 (7.5) 187 (100.0)

Education

Primary 1 (33.3) 1 (33.3) 1 (33.3) 3 (100.0) 0.011

Vocational 16 (94.1) 0 (0.0) 1 (5.9) 17 (100.0)

High school 42 (75.0) 9 (16.1) 5 (8.9) 56 (100.0)

College 23 (59.0) 10 (25.6) 6 (15.4) 39 (100.0)

Graduate 62 (86.1) 9 (12.5) 1 (1.4) 72 (100.0)

Total 144 (77.0) 29 (15.5) 14 (7.5) 187 (100.0)

p < 0.05


Table 5. Respondents' interest in participation in health screening services for diabetes and hypertension in a community pharmacy.

Question in questionnaire: Would you be interested in participating in health screenings in a community pharmacy? -No, -I do not know, -Yes, in screening for:-Hypertension, -Diabetes, -Other:ÖÖÖ(specify)

Patients' PoznaÒ Chicagodiseases:

Anwers:n (%) n (%)

p-value

Yes 137 (79.2) 89 (89.0) 0.039

Diabetes No 36 (20.8) 11 (11.0)

Total 173 (100.0) 100 (100.0)

Yes 118 (68.2) 96 (96.0) < 0.001

Hypertension No 55 (31.8) 4 (4.0)

Total 173 (100.0) 100 (100.0)

p < 0.05

Figure 1. Interest in participation in screening services for common chronic diseases in a community pharmacyQuestion in questionnaire: Would you be interested in participating in health screenings in a community pharmacy?

Table 6. Preferences for source of funding for health screening services in a community pharmacy.

Question in questionnaire: Health screenings for common diseases performed in a community pharmacy should be paid by: -Government/National Health Fund, -Insurance company, -Community pharmacy, -Other:ÖÖÖÖ(specify)

Anwers: PoznaÒ Chicagop-valuen (%) n (%)

Yes 225 (93.0) 84 (45.9) < 0.001National Health Fund /

No 17 (7.0) 99 (54.1)Goverment

Total 242 (100.0) 183 (100.0)

Yes 10 (4.1) 27 (14.7) < 0.001

Community pharmacy No 232 (95.9) 156 (85.3)

Total 242 (100.0) 183 (100.0)

p < 0.05

Chicago, the highest interest was in in age group 51-60 years old, with high school education (p < 0.001;Table 7) . Additional analysis of results by age, gen-der and education didnít achieve the level of statis-tical significance.


The need for implementation of screeningservices was confirmed on the basis of patientsíopinions in this study. American respondents indi-

POZNA— (n = 265) CHICAGO (n = 188)


cated it more often than Poles for diabetes andhypertension. Maybe it is because of the fact thatAmericans more frequently suggested that they weresuffering from diabetes and hypertension.International Diabetes Federation shows in turn thatthere is 39 million of diabetes in America and 52million in Europe (6). There is also about 40.0% ofEuropean and 36.0% of American population with ahigher blood pressure (2). Individuals participatingin community pharmacy screening services inPoland were more diverse; it is likely some of themcould have undiagnosed diseases. According to

global statistic there are about 33.1% of undiag-nosed diabetes in Poland and 27.1% in NorthAmerica (6).

Participants in this study were also interestedin education about prophylaxis and prevention ofcommon chronic diseases. Previous studies showedinterest to learn about these conditions and thatpharmacists were important part of health care sys-tem (7, 8). Based on the survey conducted amongthe pharmacists in 2011 after the screening cam-paign, the screening process took < 10 min for 3%of participants, 10ñ15 min for 27%, 15ñ20 min for

Table 7. Effect of age and education on preference for cost of health screening services in a community pharmacy.

Question in questionnaire: How much in your opinion should be the cost for screening on common diseases performed in a communitypharmacy?

Mean value Min value Max valuen(z≥oty)

SD(z≥oty) (z≥oty)

p-value

POZNANAge (years)

18-30 51 28.2 15.8 7 100 < 0.001

31-40 14 19.3 13.6 5 50

41-50 27 17.5 14.4 1 50

51-60 22 26.0 11.5 10 50

61-70 10 20.0 11.5 10 50

>70 6 19.2 16.8 0 50

Education

Primary 3 36.7 11.5 30 50 < 0.001

Vocational 10 18.0 8.6 5 30

High school 27 19.6 14.1 0 50

College 41 29.7 16.3 7 100

Graduate 49 21.0 17.5 1 100

CHICAGOAge (years)

18-30 14 89.1 (23.2 USD) 56.1 39 195 < 0.001

31-40 13 120.0 (31.3 USD) 49.0 39 234

41-50 15 130.0 (33.8 USD) 185.1 39 780

51-60 12 292.5 (76.2 USD) 649.3 0 2340

61-70 7 183.9 (47.9 USD) 107.3 39 390

> 70 1 195.0 (50.8 USD) - 195 195

Education

Primary 0 0.0 (0.0 USD) 0.0 0 0 < 0.001

Vocational 8 131.6 (34.3 USD) 65.7 39 195

High school 20 241.8 (62.9 USD) 519.5 39 2340

College 10 124.8 (32.5 USD) 106.9 39 390

Graduate 24 108.9 (28.4 USD) 66.0 39 390

p < 0.05


32%, 20ñ25 min for 20%, 25ñ30 min for 14%, and >30 min. for 4%. Pharmacists stated that nearly allparticipants were very satisfied with the screeningand the quality of information provided (9). In addi-tion, lifestyle modifications through diet alternationsand exercise improvement, education can delay orprevent progression from impaired glucose toleranceto type 2 diabetes or hypertension presence (10).Screening and counselling in community pharmaciesof individuals at risk for diabetes may results inchanges in lifestyle and body weight (11). Moreover,multidisciplinary screening and interventions bypharmacists and also nurses resulted in reducedblood pressure in patients with diabetes (12).

The sequential screening method for diabetesis reasonable in terms of cost and effectiveness(13). Diabetes and hypertension pharmacy-basedscreening was more costly, but the success rate forreferral was higher compared with a community-based service (14). In this study, Poles preferredthat such services should be paid by NationalHealth Fund while Americans preferred communitypharmacy to pay costs of screening services.Respondents in PoznaÒ determined the sum as 23z≥otych and in Chicago as 41 USD (157 z≥otych).The most interested in paying were Poles in the agegroups 18-30 and 51-60 years old, with primaryeducation. In Chicago, participants 51-60 years oldwith high school education were interested in pay-ing higher cost. Krass and colleagues reported thereal total cost of screening each person in a phar-macy was AUS $ 7.76 (about 23 zlotys) for the ticktest only and AUS $ 11.83 (about 35 z≥otych) forthe sequential screening ñ tick test only and finger-tip test for capillary blood glucose service and con-sisted of variable and fixed costs (13).

Screening programs for chronic conditionshave limitations, including substantial cost, limitedparticipation, false-positive cases, false reassurancefor negative cases, and potential for social inequity(15, 16). Community pharmacy screening servicescan help increase health awareness and identify newcases of disease (17-19). The limitation of conduct-ed study is the sample because it is not representa-tive of the general population in Poland and UnitedStates of America. The study sample consisted ofparticipants from PoznaÒ and Chicago. Thus, theresults may not be generalizable to populations otherthan the study sample. The respondentsí willingnessto pay for screening services was a subjective opin-ion on the amount they may agree to pay not an actu-al transaction data.

There is no data from Polish studies abouthealth screenings in community pharmacy so this

investigation provides first set of opinions aboutsuch a program in Poland. The increased respon-dentsí willingness to benefit from health screeningservices should encourage pharmacists to developand provide screenings in community pharmacies.The results of the study support the need for healthscreenings in a community pharmacy. Future inves-tigation is needed to understand how to effectivelyorganize and finance these community pharmacyservices in Poland.

Acknowledgment

This study was supported by the funding foryoung scientists from Poznan University of MedicalSciences (grant no. 502-14-03314429-09415).

REFERENCES

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Received: 26. 08. 2016

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EXAMPLES:

1. Gadzikowska M., Grynkiewicz G.: Acta Pol. Pharm. Drug Res.59, 149 (2002).

2. Gilbert A.M., Stack G.P., Nilakantan R., Kodah J., Tran M. etal.: Bioorg. Med. Chem. Lett. 14, 515 (2004).

3. Roberts S.M.: Molecular Recognition: Chemical andBiochemical Problems, Royal Society of Chemistry, Cambridge1989.

4. Salem I.I.: Clarithromycin, in Analytical Profiles of DrugSubstances And Excipients. Brittain H.G. Ed., pp. 45-85,Academic Press, San Diego 1996.

5. Homan R.W., Rosenberg H.C.: The Treatment of Epilepsy,Principles and Practices. p. 932 , Lea & Febiger, Philadelphia1993.

6. Balderssarini R.J.: in The Pharmacological Basis ofTherapeutics, 8th edn., Goodman L., Gilman A., Rall T.W., NiesA.S., Taylor P. Eds., Vol 1, p. 383, Pergamon Press, MaxwellMacmillan Publishing Corporation, New York 1985.

7. International Conference on Harmonization Guidelines,Validation of analytical procedures, Proceeding of theInternational Conference on Harmonisation (ICH), Commissionof the European Communities, Geneva 1996.

8. http://www.nhlbi.nih.gov/health/health-topics/topics/ms/(accessed on 03. 10. 2012).

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