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Journal of Pharmacy and Chemistry(An International Research Journal of Pharmaceutical and Chemical Sciences)

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January - March 2016 2 Journal ofPharmacyandChemistry •Vol.10 • Issue.1

ISSN 0973 – 9874 J.Pharm.Chem CODEN: JPCOCM

Journal of Pharmacy and Chemistry(An International Research Journal of Pharmaceutical and Chemical Sciences)

Volume 10 • Issue 1 • January – March 2016

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CONTENTS

Anti-Bacterial and Antioxidant Screening of Marine Algal Extracts of Hypnea Musciformis (Wulfen) Lamour ............................................................................................................................ 3SHIREESHA T, RAVINDRA REDDY K AND JAYAVEERA K N

Prophylactic And Curative Effects Of Mimosa Pudica Root Against Cisplatin InducedNephro Toxicity In Wistar Male Rats ........................................................................................................................... 10KARRA GEETHA AND N. RAMA RAO

Effect of Flavonoid Rich Fraction of Andrographis echioides in Streptozotocin-Induced Diabetic Rats ............ 16M.V. JYOTHI KUMAR, Y.K. PRABHAKAR, M. SARITHA, T. KRISHNA TILAK, S. ABDUL NABI,MD. SUBHAN ALI, K. PEDDANNA AND CH. APPA RAO

Effect of Mercury on Seed Germination and Seedling Growth of Albizia lebbeck (L.) ........................................ 21S.SOMASEKHAR, K.V. MADHUSUDHAN AND G. MEERA BAI

Strategies for enhanced production of Super oxide dismutase in Pichia fermentans .......................................... 25NETHRAVATHI. V , DR. ANISA AKHTHAR AND DR. K.N. JAYAVEERA

Evaluation of Angiogenic Potential of Nicorandil, a KATP Channel Agonist by Chorioallantoic Membrane Assay. (CAM) .................................................................................................................... 31CHANDANA KAMILI , K. RAVI SHANKAR, AND UMA MAHESHWARARAO VATTIKUTTI

Formulation and Characterization of Dried Nanosuspensions of Glipizide ............................................................ 36S. RAJA SHEKHAR AND P. VIJAYA LAKSHMI

Instruction to Authors

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ANTI-BACTERIAL AND ANTIOXIDANT SCREENING OF MARINE ALGAL EXTRACTS OF HYPNEA MUSCIFORMIS (WULFEN)

LAMOURSHIREESHA T*, RAVINDRA REDDY K$ AND JAYAVEERA K N#

*Dept.Pharmaceutical Biotechnology, Govt. Polytechnic for Women, Kadapa$Dept. of Biotechnology, Creative Educational Society College of Pharmacy, Kurnool.

#Dept. of Chemistry, VEMU Institute of Technology,P.Kothakota(Near Pakala),Chittoor Dt. A.P

ABSTRACT

In the present study, the seaweed marine algae Hypnea musciformis (Wulfen) Lamour was selected and subjected to Anti-bacterial and anti-oxidant activities using standard methods. Antioxdant activities were studied using Hydrogen peroxide assay and Total antioxidant capacity models using ascorbic acid as standard drug. The methanolic extract is more potent than ethanolic and ethyl acetate extract as shown by the low IC50 value of this extract, in these models as compared to the standard Ascorbic acid. The IC50 value of standard ascorbic acid in model was found to be 3.49 µg/ml, whereas for the extracts it ranged from 4.17 to 12.63 µg/ ml, with the methanolic extract showing lowest IC50 value of 4.17. The total anti-oxidant capacity (TAC) of the ethanolic extract of marine algae for 100g of the extract was found to range from 5.77 mg to 15.2 mg. The results of the present study indicated that the collected seaweeds; Hypnea musciformis successfully displayed anti-bacterial and antioxidant capacity which can be attributed to its phenolic and flavonoid content. Methanol extract of Hypnea musciformis had the highest antioxidant content. Our results in this study indicate that some compounds in Hypnea musciformis have antioxidant properties and may contribute in the therapeutic effect of these medicinal properties. However, there is a need of detailed scientific study on traditional medical practices to ensure that valuable therapeutic knowledge of some seaweed is preserved and also to provide scientific evidence for their efficacies.

Key words: Hypnea musciformis, antibacterial, hydrogen peroxide assay, IC50 and total antioxidant capacity.

Introduction Phenolic compounds can act as antioxidants by chelating metal ions, preventing radical formation and improving the antioxidant endogenous system. The term “phenolic compound” describes several hundred molecules found in edible plants that possess on their structure a benzenic ring substituted by, at least, one hydroxyl group.[1] Herbal Antioxidants use has increased in recent years in order to reduce the use of synthetic forms such as Butylated Hydroxyanisole (BHA) and Butylated Hydroxytoluene (BHT). Natural antioxidants from plant origin can react rapidly with these free radicals and retard or alleviate the extent of oxidative deterioration [1]. Furthermore, antioxidants from natural sources can also increase the shelf life of foods. Therefore, the consumption of antioxidant and/or addition of antioxidant to food materials could protect the body as well

as the foods against these events [5]. Tannins are defined as naturally occurring plant polyphenolic compounds and are widespread among terrestrial and marine plants (Haslam, 1989, Waterman and Mole, 1994) [11, 24]. Flavonoids, the largest group of phenolic compounds is known to contain a broad spectrum of chemical and biological activities including antioxidant and free radical scavenging properties (Kahkonen et al., 1999) [15].

Antioxidants act as free radical scavengers, reducing agents, quenchers of singlet oxygen molecule, and activators for antioxidative enzyme to suppress the damage induced by free radicals in biological system. Reactive oxygen species (ROS) such as hydroxyl, superoxide and peroxyl radicals are formed in human cells by endogenous factors and result in extensive oxidative damage which can lead to age related degenerative conditions, these include cancer, cardiovascular disease, artherosclerosis, hypertension, ischemia/re-perfusion, diabetes mellitus, hyperoxaluria,


neuro degenerative disease such as Alzheimer’s and Parkinson’s disease and ageing [4, 2, 12 & 10].The aim of the present study is to elucidate the anti-oxidant properties of the marine algae, Hypnea musciformis (Wulfen) Lamour as a part of exploring these marine species in the treatment of cancer, cardiovascular disease, artherosclerosis, hypertension, ischemia / re-perfusion, diabetes mellitus, hyperoxaluria, neuro degenerative disease such as Alzheimer’s and Parkinson’s disease and ageing.

Seaweeds serve as an important source of bioactive natural substances. Seaweeds are commonly classified into three main groups based on their pigmentation. Phaeophyta or brown seaweeds are predominantly brown due to the presence of the carotenoid fucoxanthin and the primary polysaccharides present include alginates, laminarins, fucans and cellulose [1, 2.] Chlorophyta, or green seaweeds, are dominated by chlorophyll a and b, with ulvan being the major polysaccharide component [3]. Seaweeds are considered as a source of bioactive compounds with cystostatic, antiviral, antihelminthic, antifungal and antibacterial activities. They have also been used to treat some diseases like cancer, arthritis etc. Seaweeds are naturally renewable sources which are also used as food, feed and fertilizer in many parts of the world. They have been screened extensively to isolate life saving drugs or biologically active substances all over the world [4]. The revolutionized therapy of infectious diseases by the use of antibacterial drugs has certain limitations due to changing patterns of resistance in pathogens and side effects they produced.

These limitations demand for improved pharmacokinetic properties which necessitate the continued research for new antibacterial compounds for the development of drugs [5,

6]. The extraction of major compounds from the different species of seaweeds was solvent dependent. There are a lot of reports from around the world related to that seaweed species were extracted using organic solvents [7-12]. In the present study we have focused our vision to investigate the potential ability of the seaweed, marine algae Hypnea musciformis (Wulfen) Lamour for Anti-bacterial and anti-oxidant activities using standard methods.

METHODSANTIBACTERIAL ASSAY

MEDIUM USED

Nutrient Agar medium and broth were used for the screening of antibacterial activity of the seaweed extracts.

PREPARATION OF INOCULUM

The stock cultures were maintained at 4ºC on the slant slopes of nutrient agar medium. Active cultures for experiments were prepared by transferring a loopful of cells from the stock cultures to test tubes of Mueller Hinton Broth (MHB) that were incubated without agitation for 24 hrs at 37º C. To 5 mL of MHB, 0.2 mL of culture was inoculated and incubated till it reached the turbidity equal to that of

the standard 0.5 Mc Farland solution at 600 nm which is equivalent to 106-108 CFU /mL.

TEST ORGANISMS

The test organisms were collected from the Department of Medical Microbiology, Vellore Institute of Technology, Vellore, Tamil Nadu, and India. The two gram positive: viz., Staphylococcus aureus and Bacillus subtilis, three gram negative: viz., Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia were used in the present study.

AGAR WELL DIFFUSION METHOD

The antibacterial activity of seaweed extracts of HYPNEA MUSCIFORMIS (WULFEN) LAMOUR Obtained with Chloroform, Ethyl acetate and Methanol were evaluated by the agar well diffusion method (Deena and Thoppil, 2000). The strains that had been incubated for 24 hrs for bacteria were used for the assay. A sterile cotton swab was dipped into the bacterial suspension and then evenly streaked over the entire surface of a sterile Mueller Hinton Agar plate to obtain uniform inoculums. The wells were punched on the seeded plates using a sterile borer (7 mm), and the plates were allowed to dry for 5 mins. The solvent extraction of hexane, ethyl acetate and methanol extracts (75 μL, 100 μL and 150 μL) were dispensed into each well using a sterie micropipette. The Dimethyl Sulfoxide (DMSO) was used as a negative control and Streptomycin (10 μL) was used as positive control. The plates were incubated overnight for bacteria at 37°C. The antibacterial activity was determined by measuring the diameter of zone of inhibition (mm).

HYDROGEN PEROXIDE SCAVENGING ASSAY The free radical scavenging activity of the extract was determined by Hydrogen peroxide assay (Gulcin et al., 2004). Hydrogen peroxide (10 mM) solution was prepared in phosphate buffered saline (0.1M, pH 7.4). 1ml of the extract (50, 75, 100, 150 and 200 µg/mL)) was rapidly mixed with 2 ml of hydrogen peroxide solution. The absorbance was measured at 230 nm in the UV spectrophotometer after 10 min of incubation at 37oC against a blank (without hydrogen peroxide).

The percentage of scavenging of hydrogen peroxide was calculated using the formula

% of scavenging = ((Ao-A1) / Ao) x 100

Where Ao is absorbance of control and A1 is absorbance of sample.

DETERMINATION OF TOTAL ANTIOXIDANT CAPACITY (TAC) Total antioxidant activity of seaweed extract was determined according to the method of (Mitsuda et al., 1996). 7.45 ml of sulphuric acid (0.6M), 0.99g of sodium sulfate


HYDROGEN PEROXIDE SCAVENGING ACTIVITY

H2O2 Activity –standard ascorbic acid Methanolic Extract

RESULTSHYDROGEN PEROXIDE SCAVENGING ACTIVITY

Std name Std. conc. (μg) Std.Volume (μl) Std. Abs. Control Abs. % of InhibitionSTD1 36 10 0.99 1.094 9.51STD 2 72 20 0.967 1.094 11.61STD 3 128 30 0.946 1.094 13.53STD 4 144 40 0.925 1.094 15.45STD 5 180 50 0.891 1.094 18.56

IC 50 (μg/ml) 3.49

Sample name Sample conc. Sample Volume(μl) Sample Abs. % of InhibitionME 1 36 10 1.056 3.47ME 2 72 20 1.038 5.12ME 3 128 30 1.011 7.59ME 4 144 40 0.989 9.60ME 5 180 50 0.939 14.17

IC 50 (μg/ml) 4.17

Sample name .Sample conc (Sample Volume(μl .Sample Abs of Inhibition %EE 1 36 10 0.528 1.73EE 2 72 20 0.519 2.56EE 3 128 30 0.504 3.95

(28mM) and 1.23g of ammonium molybdate (4mM) were mixed together in 250ml with distilled water and labeled as Total Antioxidant Capacity (TAC). 0.1 ml of the seaweed

extract (50, 75, 100, 150 and 200 µg/mL)) was dissolved in 1ml of TAC and the absorbance was read at 695 nm after 15 min. Ascorbic acid was used as standard.


EE 4 144 40 0.489 4.68EE 5 180 50 0.459 7.04

(IC 50 (μg/ml 8.25

Sample name .Sample conc (Sample Volume(μl .Sample Abs of Inhibition %EA 1 36 10 0.356 1.17EA 2 72 20 0.338 1.70EA 3 128 30 0.311 2.59EA 4 144 40 0.289 3.60EA 5 180 50 0.231 4.77

(IC 50 (μg/ml 12.63

TOTAL ANTIOXIDANT CAPACITY

ESTIMATION OF TAC –STANDARD-ASCORBIC ACIDSTANDARD VOLUME( µl) CONCENTRATION(µg) OPTICAL DENSITY

S1 10 10 0.118S2 20 20 0.289S3 30 30 0.477S4 40 40 0.647S5 50 50 0.807

ESTIMATION OF TAC-SAMPLEConcentration /100 g

SAMPLE VOLUME (µl) 0D CONCENTRATION (µg)ME 100 0.242 15.20 15.2mg

EE 100 0.154 9.67 9.67mg

EA 100 0.092 5.77 5.77mg

TABLE SHOWING ANTIBACTERIAL ACTIVITY OF THE EXTRACTS ON E.COLI AND STAPHYLOCOCUS.AUREUS

Inhibition zone measurement (mm)

Bacteria Strain-NCIM Control –Kana-mycin

Volume(µl)25 50 75

ME Equivalent Concentration(µg)90 180 270

S.aureus 2079 23 0 13 19E.coli 2065 17 3 5 15

EE Equivalent Concentration(µg)90 180 270

S.aureus 2079 23 0 2-3 3-4E.coli 2065 17 2 4 6

EA Equivalent Concentration(µg)90 180 270

S.aureus 2079 23 0 0 2-3E.coli 2065 17 0 0 0


TABLE SHOWING ANTIBACTERIAL ACTIVITY OF THE EXTRACTS ON B.SUBTILIS ANDKLEBSIELLA PNEUMONIAE

Inhibition zone measurement (mm)

Bacteria Strain-NCIM Control –Kanamycin

Volume(µl)25 50 75ME Equivalent Concentration(µg)90 180 270

B.subtilis 2063 22 0 12 20Klebsiella pneumoniae 9633 16 4 5 10

EE Equivalent Concentration(µg)90 180 270

B.subtilis 2063 22 0 2-3 3-4Klebsiella pneumoniae 9633 16 0 0 0

EA Equivalent Concentration(µg)90 180 270

B.subtilis 2063 22 0 0 2-3 Klebsiella pneumoniae 9633 16 0 0 0

ME- Methanolic extract EE- Ethanolic extract EA- Ethyl acetate extract Note: C – Control antibiotic kanamycin

DISCUSSION Antimicrobial activities found in seaweeds were considered to be an indication of synthesis of bioactive secondary metabolites. The marine macro-algae have an effective antibacterial activity against most of the human bacterial pathogens. It was reported that 151 species of macro-algal crude extracts showed inhibitory activities against pathogenic bacteria [21]. There have been a number of reports that demonstrate the antimicrobial activity of marine plants [22] marine algae or seaweeds [23]. Still, in India only limited information is available on marine algae.

In the present study, Hypnea musciformis seaweed, Methanolic extract (ME) showed maximum zone of inhibition against all the pathogens examined with maximum activity against B. subtilis 20 mm and minimum activity against K.pneumoniae (10 mm). Significant activity was observed with Staph.aureus-19 mm and E.coli - 15 mm. the methanolic extract showed significant zone of inhibition as compared to the standard drug Kanamycin.

The present results agreed with the findings of Rao and Parekh (1981) and Padma Kumar and Ayyakkannu (1997) that organic extract of Indian seaweed exhibit antimicrobial activity against gram negative and gram positive biomedical pathogens. In the present findings we have immense potential on the control of clinical pathogens, since the strains used in the study were collected from hospital sources and most of the strains appeared as multi drug resistant and cannot be controlled with commercially prescribed antibiotics [25]

Natural antioxidants are not limited to terrestrial sources and reports have revealed seaweeds are also rich

in natural antioxidant compounds [15]. The presence of phytoconsitutents, such as phenols, flavanoids and tannins in seaweeds and seagrasses indicated a possible role that its extracts may have antioxidant activity. This activity was believed to help in preventing a number of diseases through free-radical scavenging activity [16].

Higher total antioxidant activity of 1.52 ± 0.05 mg ascorbic acid equivalent/g of seaweed was observed methanolic extract of Hypnea musciformis (Wulfen) Lamouras, whereas the ethylacetate extract showed minimum activity.

The hydrogen peroxide scavenging activity of extract of Hypnea musciformis were assessed and methanolic extract was found be more potent than other two extracts. These extracts showed an IC50 value of 4.17 mcg, 8.25 mcg, and 12.63 mcg respectively for methanolic, ethanolic and ethyl acetate extracts. The present results are in agreement with Matsukawa et al. (1997), who found that the antioxidant activity of brown algae was superior to that of red or green groups.

CONCLUSION Overall results showed that Red algae, Hypnea musciformis exhibits maximum antibacterial and antioxidant activity. The antibacterial and antioxidant activities could be attributed to the presence of different secondary metabolites such as phenolic compounds, and carotenoids and the mechanism of action might be due to their individual or collective participation. The experimental findings envisaged Hypnea musciformis extracts as a good candidate in developing new antibacterial and antioxidant agents.


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D



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Prophylactic And Curative Effects Of Mimosa Pudica Root Against Cisplatin Induced Nephro Toxicity In Wistar Male Rats

KARRA GEETHA*1,2 AND N. RAMA RAO 2 1. CMR College of Pharmacy, Kandlakoya, Medchal, Hyderabad, Telangana, India

2. Chalapathi Institute of Pharmaceutical Sciences, Lam, Guntur, Andhra Pradesh, India

ABSTRACT

Cisplatin has been one of the commonly prescribed drugs in the treatment of cancer. Cisplatin mediated nephrotoxicity is remarkably documented by reactive oxygen species generation and decreased levels of antioxidant mechanisms in the body. In this study, we evaluated the prophylactic and curative effects of ethyl acetate extract of Mimosa pudica roots against cisplatin-induced nephrotoxicity in Wistar male rats. Single dose of cisplatin (5mg/kg i.p) caused a marked renal damage, characterized by a significant increase in serum creatinine, blood urea nitrogen (BUN), malondialdehyde (MDA), significant reduction in the kidney levels of CAT and GSH levels when compared to normal control. All the doses of plant extract (200 , 400 and 600 mg/kg) significantly decreased the increased levels of blood urea nitrogen (BUN), serum creatinine, lipid peroxidation (MDA) and increased in the glutathione (GSH), catalase (CAT) levels when compared to cisplatin control group. Our results indicated that ethyl acetate extract of Mimosa pudica roots could have a potent nephroprotective and nephrocurative effect by its anti-oxidant property.

Keywords: Cispltin, Mimosa pudica, Nephroprotective, Nephrocurative, Anti-oxidant enzymes.

Introduction Nephrotoxicity can be defined as renal disease or dysfunction that arises as direct or indirect result of exposure to medicines and industrial or environmental chemicals. Cisplatin (Cis diamine dichloroplatinum II) is a highly effective antineoplastic DNA alkylating agent used against a wide variety of cancers.1 The use of cisplatin is frequently limited by various significant side effects such as bone marrow suppression, peripheral neuropathy, ototoxicity, anaphylaxis, and nephrotoxicity. After a single dose of cisplatin (50–100 mg/m2), approximately one-third of the patients develop nephrotoxicity 2, 3.

Primary targets of cisplatin in kidney are proximal straight and distal convoluted tubules where it accumulates and promotes cellular damage, by multiple mechanisms including oxidative stress (ROS generation and further leading to stress and lipid peroxidation) DNA damage and apoptosis (Involves apoptosis or necrosis of renal cells).4

Mimosa pudica is a member of the Mimosaceae family. It is diffusely spreading, slightly woody annual or perennial herb. Mimosa pudica was reported to have

Alkaloids, Glycosides, Carbohydrates, Proteins, Flavonoids and Phenols. Leaves and stems reported to contain the alkaloid mimosine, leaves yield mucilage, the roots yield tannins, flavonoids and alkaloids.5

Traditionally Mimosa pudica is used for the gastrointestinal, respiratory, gynaecological, neurological, genito-urinary, inflammatory diseases, giddiness, headache and fever. Anti-oxidant property, hepatoprotective activity, anti-diabetic activity, wound healing activity, anti-asthmatic property, hypolipidemic activity were already reported.6-9 The present study was design to determine nephroprotective and nephrocurative activity of ethylacetate extract of Mimosa pudica roots against Cisplatin induced nephrotoxicity.

MATERIALS AND METHODSPlant Material

Mimosa pudica roots were collected from Tirupathi, Chittoor district and authenticated by botanist Dr.Madhavachetti, Department of botany, S.V.University, Tirupathi.

Preparation of plant extract

The dried roots were reduced to coarse powder and successively macerated by using solvents like n-hexane, ethyl acetate, ethanol and water.


Animals

Male wister rats weighing 180-200mg were housed under standard conditions. They were fed with standard pellet diet and water ad libitum. The experimental protocol was approved by Institutional Animal Ethics Committee as per the CPCSEA guidelines, CPCSEA/IAEC/CMRCP/Ph D-13/11-B. Government of India.

Estimation of Nephroprotective activity of ethyl acetate extract of Mimosa pudica in Cisplatin induced Nephrotoxicity:

Experimental design:

In the present study animals were divided into 7 groups and each group contains 6 rats. Group-I (Normal control): received vehicle (1ml/kg.p o). Group II (Disease control): received cisplatin (5mg/kg on 1st day i.p and vehicle from 5th – 15th day). Group III (Plant control): received plant extract ( for 10 days p o). Group IV: received 200mg/kg of plant extract (p.o) 1-10 days and cisplatin (5mg/kg i.p) on 11th day. Group V: received 400mg/kg of plant extract (p.o) 1-10 days and cisplatin (5mg/kg i.p) on 11th day. Group VI: received 600mg/kg of plant extract (p.o) 1-10 days and cisplatin (5mg/kg i.p) on 11th day. Group VII (Curative Group): received Cisplatin (5mg/kg i.p) on 1st day and best dose of the plant extract from the prophylactic treatment was selected and will be given from 5th day to 15th day.

Animals were sacrificed on 16th day of the study after collecting blood by retro orbital puncture and were centrifuged to separate the serum. It was used for estimation of creatinine, blood urea nitrogen and total proteins.10

Parameters assessed:

Body weight:The weight (in grams) of the animals was noted on the 1st day and last day of the study and the difference in body weight was noted.

Serum creatinine:Creatinine level in serum was estimated by alkaline picrate method using creatinine kit. The results were read absorbences at 520 nm .11

Blood urea Nitrogen (BUN): BUN level in serum was estimated by kit of Autospan pvt limited and read absorbences at 570nm.12

Total Proteins: Total proteins level in serum was estimated and turbidity was read at 578nm.13

GSH: GSH level in the kidney homogenate was estimated by the method of Ellmanet.et al., 1959.14

Catalase: Catalase activity in kidney tissue was determined by measuring the rate of decomposition of hydrogen peroxide at 240 nm, according to the method given by Aebi et al ., 1974. 15

Lipid peroxidation: The concentration of MDA in kidney homogenate as an index of lipid peroxidation was determined according to the method of Ohkawa et al., 1979. 16

Histopathalogical studies:

Kidney of sacrificed rats was carefully dissected out. After rinsing in normal saline the tissue was fixed in 10% formalin-saline dehydrated with 100% ethanol solution and embedded in paraffin. Then it was cut into 4-5µ thick sections stained with hematoxylin-eosin and observed under microscope.

Statistical analysis:Results were expressed as Mean+ SEM (standard error mean) by using ANOVA test, significanct difference between control and experimental groups was assessed by tukey test. The statistical analysis was performed by using graph pad prism 5.0 software.

RESULTS:Phytochemical screening:

Ethylacetate extract was treated with various reagents which showed the presence of various phytochemical constitutents like Flavaniods, Glycosides, Alkaloids, Carbohydrates, Tannins, Phenols, Saponins .

Effect of Mimosa pudica root ethyl acetate extract on Body Weight in Cisplatin induced nephrotoxicity:

Table 1 was shown that single dose of cisplatin (5mg/kg) induced significant weight loss in group III. However, body weight was significantly increased by Mimosa pudica in dose related manner in prophylactic and curative groups.

Ethylacetate extract of Mimosa pudica root (200mg/kg, 400mg/kg, 600mg/kg) reduced the increased serum creatinine, blood urea nitrogen,total protein significantly (p<0.05 respectively)in group IV to VI in dose dependent manner,in VII it was shown curative effect and single intraperitoneal administration of 5 mg/ kg cisplatin for one day was associated with significant (P < 0.05) elevations in the circulating levels of serum creatinine, total protein and blood urea in the group III rats.

Table 2 shows MDA levels were significantly increased in the cisplatin-treated animals compared to the normal group and plant extracts (200mg/kg, 400mg/kg, 600mg/kg) in prophylactic groups IV,V,VI and with a dose of 600mg/kg in curative group VII caused significant (p<0.05) decrease in cisplatin elevated activity of MDA in kidney.

GSH, SOD, Catalase levels were significantly (p<0.05) decreased after cisplatin administration when compared to the normal group. Pre-treatment of EARMP extract dose (200mg/kg,400mg/kg, 600mg/kg) caused significant increase in cisplatin diminished GSH, SOD, Catalase levels in kidney (p<0.05) in group IV,V,VI and with a dose of 600mg/kg in curative groupVII also diminished the cisplatin decreased effect in GSH.


TAB

LE

- 1

Eff

ect o

f M

imos

a pu

dica

roo

t eth

ylac

etat

e (E

AR

MP)

ext

ract

on

body

wei

ghts

, BU

N, S

Cr

and

tota

l pro

tein

s in

cisp

latin

indu

ced

neph

roto

xici

ty

S.

No.

Gro

upIn

itial

bod

y w

eigh

ts(g

ms)

Fina

l bod

y w

eigh

ts(g

ms)

Diff

eren

ce in

bo

dy w

eigh

ts

(gm

s)

BUN

(mg/

dl)

Seru

mcr

eati

nine

(mg/

dl)

Tota

lpr

otei

n(g

m/d

l)

1N

orm

al18

2. 1

±7.0

0820

1.6

±6.3

2919

.5 ±

3.58

213

.49

±0.3

840.

194

± 0.

002

5.40

1 ±

0.34

8

2Pl

ant c

ontr

ol(6

00 m

g/kg

; p.o

)17

2.6

±3.2

56 a

d18

9.5

±3.2

45 a

d16

.9 ±

3.12

7 ad

12.3

6 ±0

.256

ad

0.17

9 ±

0.00

1 ad

5.23

1 ±

0.23

5 ad

3D

isea

se c

ontr

ol(C

ispl

atin

5 m

g/kg

; i.p

)14

3.8

±2.3

42 b

139.

45 ±

5.22

0 b-4

.35

±3.1

42 b

48.0

0 ±

0.68

2 b1.

365

± 0.

005 b

17.2

08 ±

0.8

43 b

4Pr

ophy

lacti

c(2

00 m

g/kg

; p.o

)14

2.5

±4.5

92 c

150.

5 ±6

.532

c8.

0 ±1

.94 c

20.9

6 ±

0.44

8 c0.

927

± 0.

098 c

12.9

84 ±

0.7

58 c

5Pr

ophy

lacti

c(4

00 m

g/kg

; p.o

)15

5.5

±3.6

80 c

167.

1 ±6

.770

c11

.6 ±

3.09

c18

.85

± 0.

438 c

0.74

3 ±

0.03

2c10

.976

± 0

.658

c

6Pr

ophy

lacti

c(6

00 m

g/kg

; p.o

)16

0.5

±2.5

00 c

175.

1 ±6

.393

c14

.6 ±

3.89

3 c16

.65

± 0.

897 c

0.40

9 ±

0.00

4 c8.

354

± 0.

585 c

7Cu

rativ

e(6

00 m

g/kg

; p.o

)13

8.2

±1.1

27 c

151.

5 ±3

.228

c13

.3 ±

2.10

1 c15

.21

± 0.

328 c

0.33

1 ±

0.00

2 c9.

528

± 0.

453 c

All v

alue

s ar

e ex

pres

sed

as M

ean

± SE

M. b

indi

cate

s p<

0.05

whe

n co

mpa

red

cisp

latin

con

trol w

ith n

orm

al g

roup

; a a

nd c

indi

cate

s p<

0.05

w

hen

com

pare

d pl

ant c

ontro

l, te

st g

roup

s with

dis

ease

cont

rol r

espe

ctiv

ely;

d in

dica

tes p

<0.

05 w

hen

com

pare

d pl

ant c

ontro

l with

nor

mal

gro

up


Table: 2Effect of EARMP extract on MDA, GSH, SOD and catalase activity in cisplatin induced nephrotoxicity:

S. No. GROUPS SOD

(U/mg Tissue )CAT (µmoles of H2O2

consumed/min/mg protein)GSH

(µg/mg Tissue)MDA (nmoles/

mg protein)

1. Normal 17.23 ± 1.025 195.92 ± 6.765 0.555 ± 0.017 2.012 ± 0.01

2. Plant control(600 mg/kg; p.o) 16.25 ± 1.24 ad 190.02 ± 12.63 ad 0.407 ± 0.012 ad 2.002 ± 0.01 ad

3. Disease Control (Cisplatin 5mg/kg; i.p)

6.22 ± 0.22 b 150.18 ± 15.75 b 0.102 ± 0.012 b 3.95 ±1.96 b

4. Prophylactic (200 mg/kg; p.o) 12.92 ± 1.56 c 183.36 ± 14.95 c 0.331 ± 0.021 c 1.41 ± 0.053 c

5. Prophylactic (400 mg/kg; p.o) 15.1 ± 2.89 c 186.5 2 ± 15.12 c 0.423 ± 0.02 c 1.41 ± 0.01 c

6. Prophylactic (600 mg/kg; p.o) 15.51 ± 2.31 c 190.3 ± 15.24 c 0.395 ± 0.020 c 1.01 ± 0.033 c

7. Curative(600 mg/kg; p.o) 13.75 ± 1.96 c 189.8 ± 15.32 c 0.461 ± 0.018 c 1.23 ± 0.046 c

All values are expressed as Mean ± SEM. b indicates p<0.05 when compared cisplatin control with normal group. a and c indicates p<0.05 when compared plant control, test groups with disease control respectively. d indicates p<0.05 when compared plant control with normal group.

Histopathological examination of sections of rat kidney treated with Cisplatin (Group III, fig B) showed degenerating tubular structures with vacuolization, necrosis and loss of architecture of the tubules where as normal group (Group I, fig A) rats and group treated with plant extract (Group II, fig C) showed normal glomerulus and tubules with regular morphology. Group IV (fig D) showed the presence of large number of degenerating tubules. Group V (fig E) showed normal kidney morphology with only occasional degenerating tubules. Group VI (fig F) showed predominant normal kidney morphology . Group VII (fig G) showed normal kidney morphology with only occasional degenerating tubules.

DISCUSSION Cisplatin induces nephrotoxicity by the formation of ROS. It accumulates in renal cortex and causes renal damage, high doses of cisplatin causes decrese in renal blood flow, glomerular filtration rate and increases urea and creatinine level in blood.17 It also characterized by signs of injury such as changes in urine volume, bodyweight, increase the products of lipid peroxidation and change renal clearance.18

Kidneys have some antioxidant enzymes like superoxide dismutase, lipid peroxidase ,glutathione and catalase which protect kidney from free radicals like nitric oxide and superoxide etc. The cisplatin is inhibited the activity of

antioxidant enzymes in renal tissue like glutathione, SOD, GSH and catalase depletion and increase thiobarbuturic acid- reactive substances .19

There is continuing interest on the screening of medicinal plants with a view to determine new sources of natural antioxidants .20 Many types of antioxidants with different functions play their role in the defense network. It was also evident that the antioxidant supplementation helps in reducing the level of oxidative stress and in slowing or preventing the development of complications associated with diseases .21

In the present study Mimosa pudica was selected for nephroprotective activity. Biochemical parameters like Serum creatinine, BUN, Total proteins and enzymatic parameters like MDA,GSH and Catalase were studied. Urea and creatinine are considered as suitable prognostic indicators of renal dysfunction and kidney failure for any toxic compounds 22. Urea is the end-product of the protein metabolism, and it is synthesized in the liver from the ammonia produced in the catabolism of amino acids. It is transported by the blood to the kidney from where it is excreted 23. Increased blood urea nitrogen production in renal diseases may be accounted for by enhanced catabolism of both kidney and plasma proteins 24. Renal diseases induces elevation of the plasma levels of BUN and creatinine, which


Fig.1: Effects of the Mimosa pudica on cisplatin treated kidney histomorphology in rat (haematoxylin and eosin stain, 100 xs).

are considered significant markers of renal dysfunction. Creatinine is the catabolic products of creatinine phosphate which is used by the skeletal muscle. It is a metabolite of muscle creatine, whose amount in serum is proportional to the body’s muscle mass. The amount of creatinine is usually constant, so that elevated levels indicate diminished renal function only, since it is easily excreted by the kidneys 25.

In the present study, Nephrotoxicity was induced by single daily i.p injection of cisplatin (5mg/kg). It caused significant elevation of serum creatinine, total proteins, BUN and MDA levels and it also decreased SOD, GSH and catalase when compared to the normal group which was further confirmed by histopathological study. Cisplatin induced glomerular congestion, blood vessel congestion, and epithelial desquamation, accumulation of inflammatory cells and necrosis of the kidney cells were found to be reduced in the groups receiving ethyl acetate extracts of Mimosa pudica root along with cisplatin. The plant extract had more significant effect in prophylactic groups at 400mg/

kg, 600mg/kg when compared to 200mg/kg. Ethyl acetate extract of Mimosa pudica roots had significant curative effect when given at a dose of 600mg/kg.

CONCLUSION The results of the study disclosed that the ethyl acetate extract of Mimosa pudica roots has significant curative and significant dose dependent prophylactic effect against Cisplatin induced nephrotoxicity.

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10. A. Sreedevi, K. Bharathi, K.V.S.R.G. Prasad. Effect of alcoholic extracts of roots of dichrostachys cinerea wight &arn. Against Cisplatin-induced nephrotoxicity in Rats. Natural product radiance, 2009;8.(1),12-18.

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Analysis and Co-relation, Kaplan LA, Pesce AJ, Eds. Mosby CV, Toranto. 1: 1984;1268-1327.

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15. H.Aebi, S. R Wyss, B. Scherzeand, F. Skvaril : Heterogenecity of erythrocyte catalase II. Isolation and characterization of normal and variant erythrocyte catalase and their subunit. Enzyme.1974; 17(5):307-318.

16. H.Ohkawa N.Ohishi K. Yagi. Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Annals of Biochemistry. 1979; 351–358.

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D



*Address for correspondence: [email protected]

Effect of Flavonoid Rich Fraction of Andrographis echioides in Streptozotocin-Induced Diabetic Rats

M.V.JYOTHI KUMAR1, Y.K.PRABHAKAR2, M.SARITHA2, T. KRISHNA TILAK2, S.ABDUL NABI3, MD.SUBHAN ALI2, K. PEDDANNA2 AND CH. APPA RAO1*.

1 Department of Biotechnology, Sri Venkateswara University, Tirupati,AP, India1*, 2 Department of Biochemistry, Sri Venkateswara University, Tirupati,AP, India 3 Department of Biochemistry, University of Hyderabad, Hyderabad,TS, India

ABSTRACT

The present study was designed to investigate the antioxidant activity of total oligomeric flavonoid (AETOF) fraction from the leaves of Andrographis echioides (A. echioides) in streptozotocin (STZ) induced diabetic rats. Oral administration of the AETOF fraction at a dose of 50 mg /kg bw/day/ for 40 days showed significant decrease in hepatic and renal thiobarbituric acid reactive substances (TBARS) and activity of catalase (CAT). There was a significant improvement in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-s-transferase (GST) in liver and kidney of STZ induced diabetic rats after treatment with AETOF fraction, when compared with untreated diabetic rats. The results suggest that AETOF fraction has remedial effects on oxidative stress condition in STZ induced diabetic rats.

Keywords: AETOF fraction, Andrographis echioides, Streptozotocin, Antioxidant activity.

IntroductionDiabetes mellitus (DM) is a chronic endocrine

disorder, involving metabolic disorders of carbohydrate, fat, and protein. All forms of diabetes are characterized by a decrease in the circulating concentration of insulin (insulin deficiency) and a decrease in the response of peripheral tissues to insulin (insulin resistance) [1]. This disorder has reached epidemic levels and threatens as a worldwide epidemic. According to World Health Organization (WHO), diabetes is the 7th leading cause of deaths Worldwide [2-3].

In hyperglycemic condition, elevated free radical levels followed by generation of reactive oxygen species (ROS), lead to increased lipid peroxidation, altering the antioxidant defence mechanism and impaired glucose metabolism. These causes imbalance between radical generating and radical scavenging system [4]. Increased oxidative stress has been postulated in the diabetic state which coexists with a reduction in the antioxidant status [5]. Tissue antioxidant status has altered in diabetes resulting in increased oxidative damage of membranes and tissue injury [6-8]. Hence, cells have developed certain mechanisms to control free radicals through enzymatic manner by Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase which are directly involved in elimination of active oxygen species

[9]. Glutathione-S- transferase and glutathione reductase are involved in the detoxification of ROS [10]. In long term therapy currently available drugs associate with various undesirable side effects due to which the antidiabetic drug discovery shifted its focus to natural plant sources having minimal or no side effects. One of the etiologic factors implicated in the development of diabetes and its complications is the damage induced by free radicals and hence an antidiabetic compound with antioxidant properties would be more beneficial to treat ROS related complications during diabetic condition.

Plant derived phytoconstituents like glycosides, alkaloids, terpenoids, flavonoids etc. from the plants, are frequently implicated with antidiabetic effect with antioxidant properties [11].Flavonoids are polyphenolic constituents of medicinal plants present in fruits, vegetables, seeds, spices, herbs, etc. Flavonoids possess various biological activities like antioxidant, anti-inflammatory, antiallergic, antidiabetic, and vasodilatory properties [12]. Flavonoids contain phenolic group responsible for antioxidant activity which emphasizes potential health promoting and disease preventing nature [13].

A. echioides commonly called as “false water willow” belongs to the family Acanthaceae, reported for treatment of dysentery, jaundice [14], hepato protective and antioxidant activity [15]. Based on the earlier reports A. echioides is a


flavonoid rich plant [16-18]. Flavonoids play a vital role in biological system, including scavenging of reactive oxygen species. Phytoconstituents in A. echioides revealed the presence of various flavonoids [19], which makes our interest to isolate the total oligomeric flavonoid (AETOF) fraction from leaves of A. echioides and evaluate for its antioxidant activity in STZ-induced diabetic rats.

Materials and MethodsCollection of Plant material

A. echioides was collected from Seshachalam hill ranges of the Eastern Ghats, Tirumala, India. The plant material was authenticated by the taxonomist Dr. K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India (The voucher specimen (Herbarium accession number: SVUBH/589). The leaves of A. echioides were shade dried & powdered and the powder was used for the preparation of flavonoid fraction.

Preparation of AETOF fraction of A. echioides

The AETOF fraction was prepared by using the procedure described by Kilani et al., 2009 and Sunil et al., 2011 [20, 21].

Induction of Diabetes

Diabetes was induced in male Wistar albino rats aged 2-3 months (180-200 g body weight) by intraperitoneal administration of STZ (single dose of 50 mg/kg b.w) dissolved in freshly prepared 0.01M citrate buffer, pH 4.5. After 72 h rats with marked hyperglycemia (FBG 250 mg/dl) were selected and used for the study [22, 23]. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in polypropylene cages, as per the guidelines of the Institute Animals Ethics committee. This study was approved by the institutional animal ethics committee vide Resolution no: 35/2012-2013/(i)/a/CPCSEA/IAEC/ SVU/CAR-MVJK/dt. 01-07-2012.

Experimental design

The rats were divided into 5 groups and each group consisted of 6 rats as given below to evaluate the antioxidant activity of AETOF fraction in normal and STZ-induced diabetic rats.

Group 1: Normal untreated rats.Group 2: Normal rats treated with 50 mg AETOF/kg bw. Group 3: Diabetic untreated rats.Group 4: Diabetic rats treated with 50 mg AETOF/kg bw.Group 5: Diabetic rats treated with 0.02 g glibenclamide/kg bw.

AETOF fraction or glibenclamide was administered to the rats every day morning for 40 days by gastric intubation using oral gavage. All the five groups of rats were sacrificed on the 41st day after an overnight fast by cervical

dislocation followed by mild anesthetic ether. After sacrifice tissues of different experimental groups were collected and immediately frozen at ˉ20o C until the use, for enzyme activities.

Analytical procedures for In vivo Antioxidant Activity

The activities of all the antioxidant enzymes were estimated in Liver and Kidney tissue homogenates. The levels of TBARS in tissues were estimated by the method of Fraga et al., 1988 [24]. CAT activity was assayed following the method of Sinha, 1972 [25]. SOD activity was assessed according to the method of Kakkar et al., 1984 [26]. GPx activity was measured as described by Rotruck et al., 1973 [27]. GST activity was estimated according to the method of Habig et al., 1974 [28].

Statistical analysis

The results were expressed as mean ± S.D. The statistical analysis of results was carried out using one-way analysis (ANOVA) followed by DMRT.

Results

Table.1 and Table.2 Show the effect of long term treatment with the AETOF fraction on lipid peroxides, activities of CAT, SOD, GPX and GST.

Table.1 and Table.2 show the liver and kidney (respectively) levels of TBARS, activities of SOD, CAT, GPx and GST in the normal and experimental groups of rats. There was a significant increase in the levels of TBARS, CAT activity and a significant decrease in the activities of SOD, GPx and GST in both tissues of diabetic rats compared to those in normals. The treatment with AETOF fraction decreased the levels of TBARS, CAT activity and significantly increased activities of SOD, GPx and GST in the liver and kidney of diabetic rats. Treatment of the 5th group of rats with glibenclamide resulted in similar changes in the levels of lipid peroxides and antioxidant enzyme activities.

DiscussionDiabetes is one of the pathological processes known

to be related to an unbalanced production of ROS, such as hydroxyl radicals (HO), superoxide anions (O2

–) and H2O2. ROS play a key role in the development of diabetic complications as well as in a number of other disease states. As a safeguard against the accumulation of ROS, intracellular enzymatic antioxidant activities exist. ROS generated during metabolism can enter into reactions that, when uncontrolled, can affect certain processes leading to clinical manifestations. Oxidative stress induced by excessive production of superoxide and an imbalance in antioxidant enzymes has been linked to the development of diabetic complications. ROS are key participants in the damage caused by diabetic complications [29]. Therefore, cells must be protected from this oxidative injury by antioxidants. In this regard, the search for new antidiabetic


agents with antioxidant property has created a great deal of interest in phytochemicals. In particular, the flavonoids are known to have cytotoxic effects and interestingly flavonoids have been reported as antidiabetic agents [30]. Flavonoids possess biological and pharmacological properties like the anti-inflammatory, anticarcinogenic [31], antioxidant and antimutagenic properties [32].

Hypoinsulinaemia in diabetes increases the activity of the enzyme fatty acyl-coenzyme A oxidase, which initiates -oxidation of fatty acids, resulting in lipid peroxidation [33]. Increased lipid peroxidation impairs membrane function by decreasing membrane fluidity and changing the activity of membrane-bound enzymes and receptors [34]. The products of lipid peroxidation are harmful to most cells in the body and are associated with a variety of diseases, such as atherosclerosis and brain damage [35].

In recent years, there has been a resurgent interest in the herbal treatment of diabetes. The probable adverse reactions and undesirable side effects of synthetic drugs has led to the belief that natural products are safer, as herbal drugs are considered more harmonious with biological systems than the synthetically derived drugs [36]. It is believed that plants with antioxidant property can prevent or protect tissues against damaging effect of free radicals [37].

In the present study, we observed a significant increase in lipid peroxide levels (TBARS) in the plasma and tissues (liver and kidney) of diabetic rats compared to normal rats. Administration of AETOF fraction or glibenclamide decreased the levels of TBARS in the liver and kidney of diabetic treated rats. This shows that AETOF fraction might protect the tissues (liver and kidney) against the cytotoxic action and oxidative stress of Streptozotocin. Imbalance of antioxidant defence system results in alterations in the

Table.2Effect of long term treatment with AETOF fraction on TBARS levels and antioxidant enzyme activities in the

Kidney of different experimental groups

Groups Lipid Peroxides(nmoles MDA/ml)

Catalase(U/mg protein)

GlutathionePeroxidase

(U/mg protein)

SuperoxideDismutase

(U/ mg protein)

Glutathione-S- Transferase

(U/ mg protein)

1 0.127 ± 0.002 b 40.72 ± 2.35 b 0.198 ± 0.015 d 11.52 ± 0.67 d 12.62 ± 0.63 d

2 0.124 ± 0.003 a 38.47 ± 1.16 a 0.194 ± 0.011 d 11.77 ± 0.51 d 13.15 ± 0.52 d

3 0.247 ± 0.005 e 71.10 ± 1.93 e 0.0855 ± 0.007 a 4.29 ± 0.46 a 4.11 ± 0.67 a

4 0.173 ± 0.003 c 52.56 ± 1.52 c 0.154 ± 0.008 c 9.83 ± 0.77 c 8.22 ± 0.31 c

5 0.199 ± 0.004 d 60.49 ± 1.24 d 0.129 ± 0.010 b 6.59 ± 0.68 b 6.8 ± 0.52 b

Significance 0.000 0.000 0.000 0.000 0.000

Values are given as mean ± S.D from six rats in each group.Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT).

Table - 1Effect of long term treatment with AETOF fraction on TBARS levels andantioxidant enzyme activities in the liver of different experimental groups

GroupsLipid Peroxides

(nmolesMDA/ml)

Catalase(U/ mg

Protein)

GlutathionePeroxidase

(U/ mg protein)

SuperoxideDismutase

(U/ mg protein)

Glutathione-S- Transferase

(U/ mg protein)1 0.110 ± 0.006 a 60.40 ± 1.70 a 0.273 ± 0.005 c 12.33 ± 0.50 c 15.12 ± 1.36 d

2 0.109 ± 0.004 a 58.83 ± 1.48 a 0.277 ± 0.006 c 12.37 ± 1.54 c 16.19 ± 1.16 d

3 0.247 ± 0.003 c 109.64 ± 5.06 d 0168 ± 0.008 a 5.12 ± 0.91 a 6.99 ± 0.23 a

4 0.167 ± 0.004 b 86.68 ± 1.16 b 0.215 ± 0.005 b 11.56 ± 0.77 c 12.29 ± 0.65 c

5 0.170 ± 0.002 b 90.21 ± 2.25 b 0.212 ± 0.009 b 9.41 ± 0.27 b 10.15 ± 0.85 b

Significance 0.000 0.000 0.000 0.000 0.000Values are given as mean ± S.D from six rats in each group.Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT).


activity of antioxidant enzymes, such as CAT, SOD, GPx, and GST [38]. The STZ-induced diabetes disrupts actions of tissue (liver and kidney) antioxidant enzymes. The decreased activities of these enzymes may be due to the production of ROS such as superoxide (O2

−), hydrogen peroxide (H2O

2),

and hydroxyl radical (OH) that reduces the activity of these enzymes [39].

In our study, the activities of SOD, GPx and GST were decreased in diabetic rats compared to normal rats, which could be due to free radical-induced inactivation and glycation of the enzymes in diabetic state. Long-term treatment of diabetic rats with AETOF fraction had reversed the activities of these enzymatic antioxidants. This means that the AETOF fraction can reduce the potential glycation of enzymes or they may reduce the production of reactive oxygen free radicals and improve the activities of antioxidant enzymes.

In our study the activity of CAT was significantly increased in liver and kidney of diabetic untreated rats. The possible explanation for the increases in catalase activity is that it could be a compensatory mechanism to prevent tissue damage by the increased levels of H2O2 and decreased levels of GPx. In diabetes, it is thought that hypoinsulinemia increases the activity of the enzyme, fatty acyl coenzyme A oxidase, which initiates the oxidation of fatty acids, resulting in increased levels of H2O2. The CAT activity was restored to near normal in diabetic rats treated with AETOF fraction.

ConclusionThe overall results suggest that the AETOF fraction

pocesses natural antioxidant activity against oxidative stress in streptozotocin induced diabetic rats. Hence, AETOF fraction can be used in the management of oxidative stress related complications in diabetic condition.

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8. Lou MF. Redox regulation in the lens. Prog Retin Eye Res 2003; 22(5): 657-682.

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10. Ghosal S. Free radicals, Oxidation stress and Antioxidant defense. Phytomedica. 2000; 21: 1-8.

11. Marles RJ, Fransworth NR. Antidiabetic plants and their active constituents. Phytomedicine 1995; 2(2): 137-189.

12. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Rad. Biol. Med 1996; 20: 933-956.

13. Fernandez SP, Wasowski C, Loscalzo LM, Granger RE, Johnston GAR, Paladini AC, Marder M. Central nervous system depressant action of flavonoid glycosides. European J. Pharmacol 2006; 539 (3): 168-176.

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15. Basu SK, Rupeshkumar M, Kavitha K. Hepatoprotective and Antioxidant effect of Andrographis echioides N. against Acetaminophen induced Hepatotoxicity in Rats. J Biol Sci 2009; 9(4): 351-356.

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17. Jayaprakasam B, Damu AG, Gunasekar D, Blond A, Bodo B. Dihydro echioidinin, a flavanone from Andrographis echioides. Phytochemistry 1999; 52(5): 935-937.

18. Jayaprakasam B, Gunasekar D, Rao KV, Blond A, Bodo B. Androechin, a new chalcone glucoside from Andrographis echioides. J. Asian Nat. Prod. Res 2001; 3: 43-48.

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Skandrani I, Sghaier MB, Bouhlel I, Bhouri W, Mariotte AM, Ghedira K, Dijoux Franca MG, Chekir Ghedira L. Relationship correlation of antioxidant and antiproliferative capacity of Cyperus rotundus products towards K562 erythroleukemia cells. Chem. Biol Interact 2009; 181: 85-94.

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Effect of Mercury on Seed Germination and Seedling Growth of Albizia lebbeck (L.)

S. SOMASEKHAR1*, K.V. MADHUSUDHAN2 AND G. MEERA BAI3

1Department of Botany, SBSYM Degree College, Kurnool, AP, India2Department of Botany, GovernmentCollege for Men, Kurnool, AP, India

3Department of Botany, Rayalseema University, Kurnool, AP, India

ABSTRACT

Contamination of soils by heavy metals is of wide spread occurrence as a result of human, agriculture and industrial activities. A study was conducted to evaluate the effect of mercury on certain morphological and biochemical parameters of an important arid legume tree Albizia lebbeck, in relation to different mercury chlorideconcentrations (1 µM, 10 µM, 100 µM and 1mM) for a duration of eight days. The seeds germination and seedlinggrowth performance of responded differently to mercuric stresstreatments as compared to control.Results showed that the germination percentagedecreased with increasing mercury concentrations.The highest germination percentage was recorded at normal conditions. With the severity of mercury treatment, the values of root and shoot length and dry mass accumulation of root and shoot weredeclined.Root growth was more affected than shoot growth. The changes in theses morphological parameters were dependent on stress severity and duration. Thus these morphological traits may serve to determine suitable bio-indicators of mercury pollution.

Keywords: Albizia lebbeck , Mercury stress, Heavy metals, Growth.

INTRODUCTIONEnvironment Pollution is one of the severe problems

faced by the world today. Various efforts have been made for environmental conservation in India, but it still seems to be a formidable task.Urbanization and the industrial revolution put up severe issues caused by the accumulation of xenobiotics (Hg, Pb, Cd, SO2, etc.) in air, water and soil. Toxic metals are biologically magnified through the food chain. They infect the environment by affecting soil properties its fertility, biomass and crop yields, and ultimately human health. Heavy metal contamination of soil mainly results from anthropogenic factors such as mining and smelting procedures, industries, agriculture etc. [1]. Monitoring the endangerment of soil with heavy metals is of interest dueto their influence on ground water and surface water. Among the toxic elements released in the environment, mercury is considered highly toxic for the growth of plants. The effects of mercury on plants have been well documented. Mercury may enter plant foliage through two primary pathways: uptake of the oxidized from (Hg (II) or methyl mercury) and adsorbed onto soil particles and/or dissolved in soil water through roots [2]. Mercury is a silvery metal and is present in the environment in organic and inorganic forms. Mercury at low concentrations represents major hazards to living organism. Mercury used for eliminating various pests causes harmful effects on agricultural plants. They produce

toxic effects on the leaves where crucial functions such as photosynthesis and transpiration are carried out, cause morphological, anatomical and physiological changes, inhibits pollen germination and pollen tube formation and thus affected fruit production [3]. Albizia lebbeck (L.) Benth. is a leguminous tree in family Mimosaceae and it is cultivated around roadsides and industrial areas [4]. Metal toxicity is an important factor governing germination and growth of plants. Mercury chloride (Hg Cl2), the main representative of mercury compounds, is the target of numerous investigations, not only because of its intrinsic toxicity but also because it accounts for the toxicity of elemental mercury since the latter is converted to Hg+2by oxidation [5]. The concept that plants may be able to acclimatize with the incidences of pollution and contamination is now receiving greater attention, therefore present work has been carried out on the tolerance and adaptation of trees in metal polluted areas. It has been taken up to evaluate the effect of mercury on seed germination and seedling growth of an Albizia lebbeck in order to contribute it’s adaptation to environmental stress.

MATERIALS AND METHODSThe healthy and uniform seeds of Albizia lebbeck

were collected locally and surface sterilized with dilute concentration of sodium hypo-chloride for one minute to avoid any fungal contamination. Later the seeds were thoroughly washed with water several times. The seeds were washed with distilled water and transferred in petri dishes


(15cm diameter) and placed on Whatman filter paper No. 42 at room temperature (28°C ± 2). The petri dishes were kept under a light intensity of approximately 150 Wm-2 and at a temperature of 27 oC ± 3o. The experiment was conducted in completely randomized design with each treatment replicated five times. The studied mercuric chloride concentrations were 1 µM, 10 µM, 100 µM and 1mM. Solutions were daily changed. Treatment supplied with distilled water served as control. After induction of stress, the petri dishes were maintained for another 8 days and the experimental data were collected at different time intervals i.e. on day-4 and 8. On day-4 and 8, seed germination percentage, shoot and rootlength and Shoot and Root dry weight were noted. The Shoot and Root dry weight was determined by drying the plant materials in anoven at 80°C.The data were analyzed statistically using Duncan’s multiple range (DMR) test to drive significance [6].

RESULTS AND DISCUSSIONPlant growth and seed germination are greatly affected

by abnormal metabolic process due to contaminants of heavy metals.The response of plant growth to heavy metals treatment has become the subject of great interest due to their nature of toxicity to plants. Attention has been given, in many countries, about the effects of metal toxicities on plants growth. A little attention has been given on this species. In present investigation, the effect of mercuric chloride on seed germination, seedling growth, root and seedling dry weight of an important arid tree Albizia lebbeck was recorded. The seed germination and seedling growth responded differently to mercuric chloride treatment as compared to control. From the results (Table 1), it is clear that mercury treatment at all concentration decreased the percentage of seed germinationin all the days. However, on the day-4 and 8 it was more inhibited at 1mM HgCl2 treatment. It remained nearly constant in control plants throughout the experimentation. The present investigation revealed that germination of Albizia lebbeck was affected by mercury treatments, but the effect was more pronounced at higher concentrations than that of lower concentrations. These results agree with earlier reports [4]. High concentrations of heavy metals germination media caused tomato seed germination suppression is probably due to the more adverse effects of these metals on osmotic potential [7]. Heavy metals caused lower water uptake and transport in plants [8]. It may be pertained to the failure in water absorption at high metallic salt levels. There is another explanation for seed germination cessation which refers to the embryonic damages and even death under high metallic pollutant conditions [9]. Effect of different concentrations of Hg on the growth of Albizia lebbeck in terms of shoot and root length was measured and tabulated in table 1. At lower concentrations of Hg treatments i.e1 µM, 10 µM, root length was marginally inhibited, while the shoot growth was slightly inhibited. However, in 100 µM and 1mM HgCl2 treatments, both root growth and shoot length were severely inhibited. On day-8, after exposure to higher Hg concentration i.e.,1mM, the reduction in the root and shoot length was

11%, 28% respectively. Thus, the decrease in root growth was greater than shoot growth. The root and shoot growth was found to be dependent on concentration and duration of the stress.The decrease root and shoot growth in the heavy metal stressed plants was reported by several workers in Albizia sps. [4,5,10] and in other plants [7,8,9]. The root and shoot biomass in terms of dry weight was reduced in Hg-treated plants compared to controls at all stress levels on all days of sampling. Furthermore, the reduction was greater at higher Hg concentrations. The biomass reduction was more pronounced in roots than shoots. On day-4, the root and shoot biomass reduction was 47% and 59% respectively at 100 µM concentration of Hg, while the decrease was 18% and 42%respectively at 1mm Hg. Whereas on day-8, theroot and shoot biomass reduction was 18% and 42% at 100 µM concentration of Hg, while the decrease was 10% and 30% respectively at 1mm concentration of Hg. The magnitude of decline in seedling biomass accumulation was found to be dependent on the severity of stress. The decrease in dry mass of rootsand shoots was observed during toxic effect of heavy metals in several plant species, for eg., Albizia lebbeck [4,5,], in Albizia procera [10], in Shorea robusta [1]. Maximum reduction was caused on day-8 at 1mM of Hg treatment which showedreduction of 36% in seed germination, 11% in root length, 28% in shoot length, 17% in root biomass and30% in shoot biomass as compared to control.The reduction in plant growth during stress is due to low water potential, hampered nutrientuptake and secondary stress such as oxidative stress [11]. The effect of treatments was morepronounced in case of root length [1]. Roots are more prone to heavy metal toxicity relative toshoots.The higher impact of heavy metal was observed in the root growth as compared to shootleading to a reduction in its length and fresh weight [1]. The reductionin root length due to accumulation of metals within the root reduces the rate of mitosis inthe meristematic zones of roots,especially by blocking the metaphase in meristematic cells.Therefore, root showed reduction in length [1]. Root is capableof accumulating significant quantities of this heavy metal and simultaneously restrictsits translocation to the shoot. The decrease in shoot length andbiomass with increasing concentration of heavy metals may be due to the sensitivity of enzymes of the photosynthetic carbon reduction cycle [12]. Similar results in an experimentwhere 1, 5 and 10ppm of Cr, Ni and Hg affected the leaf area and root and shoot length of Albizia lebbek [13]. Our results are in agreement with the findings[13].In the present investigation, seedling growth performance of A. lebbeck gradually decreased with the increase in concentration of leadas compared to control.Similar results were found by applying lead treatments in the lab conditions at 10, 30, 50, 70 and 90 μmol/L concentrations which produced significant (p<0.05) effects on seed germination and seedling length of A. lebbeck while lead treatment at 50, 70 and 90 μmol/L significantly affected root growth and seedling drybiomass as compared to control [14].The results of this investigation have shown that mercury is more toxic to A. lebbeck root development than other growth parameters. The reduction


in root growth of A. lebbeck provides further evidence that the mercury in excess may be inhibitory to plant growth and development. The roots are normally considered in relation to their ability to supply water and nutrients to the plants. They are also required to produce hormones, which may regulate the growth and performance of both root and shoot. The significant decrease in seedling growth of A. lebbeck agrees with the conclusion that the excessive amount of toxic element usually caused reduction in plant growth.

CONCLUSIONSThe present investigation revealed thatmercury had

inhibitory effect on seed germination andgrowth parameters. Increase in the concentration of mercury at 1mM inthe medium, brought up different changes in the all growth parameter performance of A. lebbeck. Plantation of A. lebbeck in mercury-polluted area will help in reducing the burden ofmercury pollution. A. lebbeck can serve better in coordinating in landmanagement programs in metal

Table - 1Seed Germination, Root length, Shoot length, Fresh weight (FW) of root, Dry weight (DW) of root and Dry weight

(DW) of shoot control and different treatments of mercury in Albizialebbeck, on day-4 and 8

Parameters Day Control 1 µM 10 µM 100 µM 1mM

Seedgermination

(%)

04

08

79.60a± 0.9281.40a± 0.48

71.30b± 1.1877.12b± 1.35

68.58bc± 1.01

73.35bc± 1.01

58.42d± 0.9659.51d± 1.21

36.01e± 0.9636.48e± 0.98

Root length (cm plant-1)

04

08

3.01a± 0.72(100)4.82a± 0.48(100)

2.72a± 1.02(90.36)4.19b± 0.72(86.92)

2.59b± 0.38(86.04)2.80c± 1.16(58.09)

1.44c± 0.72(47.84)1.60d± 2.04(33.19)

0.66d± 0.91(21.92)0.54e± 1.92(11.20)

Shoot length (cm plant-1)

04

08

1.14a± 0.98(100)2.18a± 0.72(100)

1.08a± 0.72(94.73)1.94b± 1.53(88.99)

1.03b± 0.88(90.35)1.72c± 1.16(78.90)

0.85c± 1.74(74.56)1.26d± 2.04(57.79)

0.58d± 1.82(50.87)0.62e± 1.92(28.44)

DW of root(g plant-1)

04

08

0.0064a± 1.02(100)

0.0076a± 0.92(100)

0.0051b± 2.41(79.68)0.0060b± 0.98(78.94)

0.0044c± 1.35(68.75)0.0050c± 2.18(65.78)

0.0027d± 2.72(47.84)0.0029d± 2.04(38.15)

0.0012e± 1.98(18.75)0.0008e± 2.92(10.52)

DW of shoot(g plant-1)

04

08

0.0102a± 0.92(100)

0.0162a± 1.05(100)

0.0093a± 1.34(91.17)0.0141b± 1.94(87.03)

0.0082b± 1.41(80.36)0.0123c± 1.72(65.78)

0.0061c± 2.54(59.80)0.0077d± 2.48(47.53)

0.0043d± 2.32(42.16)0.0049e± 2.06(30.24)

The mean values (n=5) in a row followed by different letter for each plant species are significantly different (P≤0.05) according to Duncan’s multiple range (DMR) test. Figures in parentheses represent per cent of control.


contaminated areas.This can be viewed as an adaptive feature and forms one of the index to determine mercury tolerance in A. lebbeck.

REFERENCES[1] Pandey P, Tripathi AK. 2014; Ekológia (Bratislava)

33:116. [2] Boszke L, Kowalski A, Astel A, Barański A, Gworek,

B, Siepak J. 2008; Environ Geol, 55: 1075. [3] Gill HK, Garg H. 2014, Pesticides: Toxic Aspects, In

Tech, pp. 187. [4] Syed AR, Iqbal MZ, Athar M. 2011, AgricConspec

Sci. 76:109[5] Iqbal MZ, Athar M, Shafiq M. 2014 Advances in

Enviro Research, 3:207.

[6] Duncan M. 1955; Biometrics,42:1.[7] Jaja ET,Odoemena CSI. 2004; J ApplSci Environ

Manage 8: 51. [8] Becerril J, lez-MuruaCGl, Munoz-Rueda A, De Felipe

MR. 1989, Plant Physiol Biochem 27: 913.[9] Wierzbicka M, Obidzinska J. 1998. Plant Sci 137:155. [10] Pandey P, Tripathi AK. 2011. Int J Environ Sci, 5:1009. [11] Eun SO, Youn, HS, Lee Y. 2000. Physiol.Plant, 110:

357. [12] De Filippis LF, Ziegler H. 1993; J Plant Physiol, 142:

167. [13] Tripathi AK Tripathi S. 1998; J Environ Biol, 20:93. [14] Farooqi, Z.R., M.Z. Iqbal, M. Kabir and M. Shafiq.

2009. Pakistan J Bot, 41:27.

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Strategies for enhanced production of Super oxide dismutase inPichia fermentans

NETHRAVATHI. V1 , DR. ANISA AKHTHAR2 AND DR. K.N. JAYAVEERA3

1 PG Department of Microbiology, Governament Science College, Bangalore,2 PG Department of Microbiology, Governament Science College, Bangalore,

3 Dept. of ChemistryVEMU Institute of Technology,P.Kothakota(Near Pakala),Chittoor Dt.A.P

ABSTRACT

Wild type fungal strains were first classified according to their sensitivity to sodium azide and then were further used to generate corresponding mutants. P.fermentans were treated with sodium azide concentrations of 10, 50 and 100 ppm to categorize the samples in highly sensitive, moderately sensitive and tolerant groups. Altogether three mutants were generated by treating five moderately sensitive wild strains to 20, 30 and 40 ppm (sublethal concentration) of sodium azide. Mutant P.fermentans Pf4m is chosen for SOD production. The effect of Dissolved oxygen was evaluated on SOD production. The time cou rses of SOD production shows 2 maxima. 1st maxima was up to 48 hours. 2nd maxima was from 48 hours to 96 hours. The results demonstrated that the O2 level had a significant influence on SOD activity. Under high DO conditions higher levels of SOD were produced at 1st and 2nd maxima, reaching activities of about 100 and 110 U(mg protein)-1 respectively. The number of isoforms did not change under hyperoxic conditions when s mellis was exposed to an atmosphere of 20% O2. There was no significant change in Cu, Zn SOD activity, where as Mn SOD expression increased 10-12 fold by elevated DO. SO it appears that Mn-SOD is the primary defense in S mellis cells against O2 toxicity. The molecular mass of mutated P. fermentans Cu, Zn SOD was determined by matrix-assisted laser desorption ionization MS. The mass was calculated to be 22480.4 Da for the whole molecule and 14850.1 Da for the structural subunits. From these results it is suggested that the investigated enzyme is a dimer, composed of two identical structural subunits.

Key words: Mutant P.fermentans Pf4m, Sodium azide sensitivity, Dissolved Oxygen concentration Cu,Zn SOD, Mn SOD

Introduction Everyone requires oxygen to stay alive, and the same oxygen Contributes to the formation of deleterious oxygen free radicals. Many published research articles reveal that free radicals leads to the initiation of degenerative disease and also accelerated aging [1]. superoxide dismutase (SOD), catalase, and glutathione peroxidase are produce naturally by the cells of young human beings to protect against free radical damage during normal physiological conditions. SOD catalyses the conversion of free radicals to dioxygen and hydrogen peroxide. Finally, hydrogen peroxide will be converted in to water by the enzymatic action of catalase or peroxidase. Superoxide ions undergo sequential oxidative and reductive reaction by SOD’s neutralization activities [2] production of SOD and other antioxidant enzymes decrease

with age, and leads to age related degenerative diseases. Recent researches in nutritional science now advice adults to compensate the loss by powerful exogenous antioxidant enzymes nutritionally [3].

Most organisms, microorganisms, plants and animals have at least one superoxide dismutase enzymes [4]. The research has revealed that vegetarian diet rich such as fruits, vegetables, and green leaves decreases the problems of oxidative stress related disorder [5]. SODs were also produced efficiently by many microbial species. Seen to their continuous exposure to high oxidative stress during growth and metabolism, aerobic microorganisms represent an excellent source for production of superoxide dismutases. Of many aerobic microorganisms considered as a potent source of superoxide dismutase, Corynebacterium glutamicum, an


industrial relevant producer of amino acids and vitamins, is considered as an excellent candidate for this purpose seen to its high need of oxygen during amino acid production, nominating it to have a hyper antioxidant defense system including production of abundance superoxide dismutase enzyme [6,7].

Cloning techniques reported to be interesting to enhance superoxide dismutase production using cloning strategies. In addition other microbial species should also be considered for extraction of different superoxide dismutase types [8].

MATERIALS AND METHODSMutation induction by sodium azide treatment Categorization of yeast strain on the basis of sodium azide sensitivity Individual pure cultures were streaked on YPD plates containing varying concentrations (10, 50 and 100 ppm) of sodium azide, incubated at 300C for 10-12 days. Based on the complete inhibition of the growth of fungal strains, three categories were assigned respectively as (a) Highly sensitive (lethal concentration 10 ppm), (b) moderately sensitive (lethal concentration 50 ppm), and (c) Tolerant (lethal concentration 100 ppm).

Generation and selection of mutants

In this step moderately sensitive strains were streaked on the YPD plates containing 20, 30 and 40 ppm (sublethal concentrations) of sodium azide and incubated at 300C for 10-12 days. The resulting colonies were characterized morphologically. The colonies having morphology different from the wild type were considered as mutants.

Inoculum

80 ml seed medium was inoculated with 5 ml mutated P.fermantans spores suspension at a concentration of 2x108 spores’ ml-1.

Shake flask cultivation

Mutated P.fermantans inoculum taken in 500 ml Erlenmayer’s flasks and cultivation performed on a shaker (220 r.p.m.) at 30 °C for 24 h.

Bioreactor cultivation

160 ml of the seed culture was brought into the 3 l bioreactor, containing 1850 ml of the production medium. The cultures were grown at 30 °C for 120 h. Reactor cultivations were performed in a Eastbio GUCS-3 bioreactor (SIEMENS PLC,India). The fermenter was stirred by 2 rush ton turbines and was sparged using compressed air via a ring shaped sparger at the base of the vessel. The bioreactor was equipped with Inpro 6800 and Inpro 3030 (SIEMENS PLC) for the continuous measurement of pH foaming was controlled by the automatic addition of PPG 2000 (Cyanamid, Catania, Italy). Temperature was kept to 300C. The bioreactor was equipped with pH and automatic dissolved oxygen (DO)

monitoring equipment and a control system. The culture medium composed of sucrose 20gl-1, yeast extract 10gl-1; Ammonium orthosulphate 20gl-1; Autoclaved for 30min at 1100C. The fermentation parameters under DO-uncontrolled conditions were: impeller speed, 600 r.p.m.; air flow, 1 v.v.m. [1 vol. air (1 vol. liquid)-1 (min)-1]. In this case, only the changes in DO level during fermentation were measured. For the DO-controlled culture system, aeration and impeller speed were regulated in such a way as to produce 20%oxygen saturation in the liquid.

Preparation of cell free extracts Biomass was harvested by centrifugation (5000 X G for 10min at 40C), washed twice with PP buffer (potassium phosphate buffer) pH 5.5 and pH 6.0 50mm; EDTA, 0.1mM) and re suspended in the same buffer 1:1 W/V. Cells were disrupted for 30 min at 40C by 0.3mm glass beads in a vibration homogenizer at 1800 rpm. Whole cells and debris were removed by centrifugation at 13000xG for 15min at 40C and the supernatant was dialysed for 16h against PP buffer.

Quantitative Methods Nitro blue tetrazolium (NBT) reduction method of Beauchamp & Fridovich (1971) [9] is followed to measure total SOD enzyme activity. Enzyme activity of one unit SOD is defined as the amount of SOD required to inhibit the reduction of NBT by 50% (A560) and expressed as U (mg protein-1). Cu,Zn-SOD and Mn-SOD activities has to be distinguished, hence Cyanide (2 mM) is used. Cu,Zn-SOD is Cyanide-sensitive and Mn SOD is resistant to cyanide. Cu, Zn SOD isoenzyme activity is calculated as total enzyme activity minus the isoenzyme Cu,Zn-SOD activity in presence of 2 mM cyanide. This is followed by Protein estimation performed by the Lowry protocol and crystalline bovine serum albumin is used as standard. Somogyi Nelson method (1952) [10] was used to estimate soluble reducing sugars. Mycelial dry weight is measured after harvesting. The culture content is filtered with a Whatman no. 4 filter (Clifton, USA). The collected mycelia were washed twice with distilled water and dried at 105°C.Purification of mutated P.fermantans Cu, Zn SOD After filtration the mycelium was frozen till it is taken for purification. During purification the frozen mycelium is partially thawed and suspended in 4 volumes 25 mM potassium phosphate buffer, pH 7.8, containing 1 mM PMSF. The suspended material is disrupted at 40C for 30 min in a vibration homogenizer at 1800 rpm using 0.3mm glass beads. Homogenized mass is filtered to remove Cell debris by a Buchner funnel and the filtrate was clarified through centrifugation at 13000xG for 20 min at 4°C. The supernatant is separated and concentrated by a membrane ultrafiltration (Amicon PM-10). The concentrated crude extract is then subjected to fractionation by acetone, and totalextract is chilled to 20°C. To the fractionated solution, 0.4 volumes of Cooled acetone is added slowly with vigorous stirring for 30 min. This mixture is incubated at


20°C for 2 h, after precipitate it is collected and centrifuged at 13000xG for 20 min. The supernatant was collected and treated with 1.5 volumes of chilled acetone. The fungal Cu, Zn SOD mixture is allowed to stand for held for 12 h at 20°C. Then the precipitate is collected by centrifugation (13000xG for 20 min) and suspended in distilled H2O. After centrifugation clear viscous supernatant is collected carefully and loaded on a Sephadex G-75 column (2x50 cm; Pharmacia), which is previously equilibrated with 50 mM potassium phosphate buffer at pH 7.8 and the column is eluted in the same buffer. It is further purified by ion exchange chromatography on a DEAE cellulose-52 column (3.5x10 cm; Serva), equilibrated in 54 mM KH2PO4 buffer, pH 7.8. The Cu, Zn SOD is eluted with a NaCl gradient (0-0.1 M). For additional purification, the fractions are loaded onto an FPLC system, equipped with a 10/10 Mono Q anion exchange column. It is previously equilibrated with 50 mM potassium phosphate buffer, pH 7.8, and the elution is effected stepwise increases in NaCl (0-0.1 M). Fractions with 3200 U /(mg protein) are further purified and desalted on a Nucleosil RP C18 column (250-10 mm; Macherey-Nagel). This step will allow us to determine N-terminal sequence and amino acid composition. This is followed by HPLC separation. The below conditions are follwed for HPLC separation: eluant A, 0.058% trifluoroacetic acid (TFA); eluant B, 80% acetonitrile in A [11,12].

The gradient is run from 5 to 100% B within 60 min at a flow rate of 1 ml min-1

Characterization of mutated P.fermantans Cu, Zn SOD

Cu, Zn and Mn was performed on a Perkin-Elmer 400 S spectrometer- Flameless atomic absorption spectra (equipped with a HGA 76B graphite furnace) is taken for Cu, Zn-SOD isoenzyme and Mn-SOD isoenzyme. Purity of both isoenzyme is measured followed by molecular mass measurement on a 10% polyacrylamide gels (described by Laemmli (1970) [13]. Protien detection is carried out by comparing stained test sample with duplicate gels described by Misra & Fridovich, (1977) [14]. The following molecular mass standards are used: trypsinogen (24 kDa), egg albumin (45 kDa), bovine albumin (66 kDa) and Limulus Polyphemus haemocyanin (monomer, 70 kDa). Mass spectra is taken by matrix-assisted laser desorption ionization MS (MALDI 1-MS) (Kratos, MALDI III equipment; Shimadzu). The sample (10 pmol) is dissolved in 0.1% (v/v) TFA and

applied onto the target. Analysis is performed in a-cyano-4-hydroxycinnamic acid. Molecular mass is detected using 2 standards such as chicken egg ovalbumin (44400 kDa) and bovine serum albumi (66430 Da). were used to calibrate the molecular mass scale.Oorcinol/H2SO4 test is performed to analyse carbohydrates in protein [15].

Results and DiscussionInduced Mutations

Characterization of mutants

All wild type strains and their mutants were able to hydrolyse starch; tween 20 and casein utilize fructose and lactose. All were urease and catalase positive. All mutants were found to be different from their corresponding wild strains in colony color or in colony texture or in both (table 3.2).

Types of mutations in carbon metabolism

Mutants were characterized for monosaccharide (5c and 6c) and disaccharide sugar utilization. Altogether two mutants having negative mutation in loci of utilization of galactose or manose (loss-of-function LOF, single mutants) were observed (table 2) among mutants from Pf 1, Pf 4 and Pf 5. Pf 4 gave one gain-of-function (GOF) single mutant having positive mutation in the loci of utilization of sucrose disaccharide.

Table - 1Wild type strains and their corresponding mutants generated with sublethal concentration of sodium azide

Wild types Lethal concentrations Mutants

Pf 1 30ppm Pf 1mPf 4 40ppm Pf 4mPf 5 30ppm Pf 5m

Effect of DO concentration on Cu, Zn SOD production

It is well known that SOD expression is markedly enhanced by augmenting intracellular production of O¯

2 under conditions of increased levels of DO in the growth medium [16] . To produce SOD in large quantities, we examined growth, sucrose uptake and formation of SOD by P. fermantans in a bioreactor under DO-uncontrolled and

Table - 2Biochemical characterization of three moderately sensitive fungal strains to sodium azide and their

corresponding mutants

Wild typeColony color Colony

textureCarbohydrate Utilization Test

Upper Lower Ara Glu Man Gal Fru Lac SucPf 1m B B Bri - + + + + - + LOF (suc)Pf 4m B B Bri + + + + + - + GOF (Suc)Pf 5m B B Bri - - - - + + + LOF (Suc)


20% DO-controlled conditions (Fig.1, Fig. 2). Under DO-uncontrolled conditions, the culture showed a typical profile of fungal growth (Fig. 1). The biomass increased rapidly (exponential phase) when sucrose uptake was maximal, without a lag period (Fig. 2). The culture period from 36 to 84 h (stationary phase) showed decreased fungal growth and the carbohydrate source was almost exhausted. Moreover, the DO level decreased sharply and oscillated around zero. The sudden drop of oxygen concentration might be attributed to the oxygen required for cell growth and metabolism [17] .

Irrespective of accelerated sucrose uptake (Fig. 2), 20% DO-controlled conditions led to a reduction in duration of the stationary phase as well as to a decrease in the maximum level of biomass (Fig. 1) and intracellular protein content (data not shown). Petruccioli et al. (1995) [18] and Guidot et al. (1993) [19] demonstrated similar data for Penicillium variable and Saccharomyces cerevisiae, respectively, due to increasing DO level.

The effects of two different levels of DO (uncontrolled DO and DO at 20%) on SOD production were evaluated (Fig. 2). Increasing enzyme activity in P. fermatas cells, cultivated under both conditions, was detected in the early exponential phase and an active synthesis was observed during a period of intensive oxygen consumption. Moreover, the time courses of SOD production show two maxima. A similar phenomenon, a secondary increase in SOD activity during the late stationary phase, has been observed for SOD production by filamentous fungi and yeasts [20, 21]. It can be explained by an intensification of the process of O-

2 generation when the cells utilize endogenous sources of carbon and nitrogen (organic or amino acids).

The results demonstrated that the oxygen level had a significant influence on SOD activity. Under high DO conditions, higher levels of SOD were produced at 1st and 2nd maxima, reaching activities of about 100 and 110 U (mg protein)-1, respectively. Considering enzyme formation in both experiments, DO-controlled cultures produce approximately 1-7-fold higher SOD activity than

Table - 3

Comparison of SOD productivity at the activity maxima

Cultivation system¶

Cultivation time (h)

Wet cell weight (g/l)‡

Protein yield

(mg/l)†

Specific SOD activ-ity (U/mg protein)ξ

10-3x total SOD

activity (U/l)*

10-3x SOD productiv-ity (U/kg)$

Cu/Zn SOD (%)§

Uncontrolled DO1st Maximum2nd Maximum

4896

88.8066.20

472.30312.80

6260

29.2818.76

329.72283.38

87.0993.33

Uncontrolled DO1st Maximum2nd Maximum

4884

82.8046.20

296.2085.80

100110

29.629.43

357.72204.11

5749.09

Fig.1: Time courses of mycelia growth and dissolved oxygen concentration in mutated P.fermantans (Pf 4m), grown under DO-uncontrolled and 20% DO-controlled conditions.

Fig 2: Time courses of SOD production and Glucose consumption in mutated P.fermantans (Pf 4m), grown under DO-uncontrolled and 20% DO-controlled conditions.

DO-uncontrolled cultures. Exposure to high DO levels has been reported to cause both enhancement (Vercellone et al., 1990) [22] and decrease (Shilova et al., 1989) [23] in SOD activity for microbial cells.

The isoenzyme profiles of SOD, produced by mutated P. fermantans upon cultivation at different DO levels, are


illustrated in Fig. 3 and 4. The fungal strain produces two isoforms, namely Mn and Cu, Zn-containing SOD. Cu, Zn SOD activity was obtained as total activity minus the activity in the presence of 2 mM cyanide, and the conclusion from these results is that this isoenzyme was responsible for approximately 90% of total SOD activity in DO-uncontrolled cultures. The numbers of isoforms did not change under hyperoxic conditions when mutated P.fermantans was exposed to an atmosphere of 20% O2. There was no significant change in Cu, Zn SOD activity, whereas Mn SOD expression increased 10-12-fold by elevated DO. So, it appears that Mn SOD is the primary defense in mutated P. fermantans cells against oxygen toxicity. Similar findings about the lack of response of Cu, Zn SOD to oxidative stress have been reported for a variety of cells. The dependence of Cu, Zn SOD on oxygen was found to be related to the

availability of copper to mutated P.fermantans, yeasts (Howlett & Avery, 1999) [24] and mammalian cells.

The results of DO-controlled and DO-uncontrolled SOD production are summarized in Table 3. The highest efficiency of SOD biosynthesis was achieved at the 1st maximum of activity for both kinds of cultures.

Although specific SOD activity was higher at the 2nd maximum, total enzyme activity was only 77 and 33% of that of the first maximum for DO-uncontrolled and DO-controlled cultures, respectively. Because of the differences in the ratio of isoenzymes, the conditions for achieving the 1st maximum in DO-uncontrolled cultures appear to be the most appropriate for Cu, Zn SOD production by mutated P. fermantans. The productivity of mutated P.fermantans Cu, Zn SOD is comparable to the maximum enzyme productivity by micro-organisms [300x103 U (kg wet biomass)-1) [25].

Purification and characterization of mutated P. fermentans (P. f 4m) Cu, Zn SOD The results of the purification procedure are shown in Table 3.4. We found that fast performance anion exchange chromatography (FPLC) on a Mono Q 10/10 column is a highly efficient purification method for Cu/Zn SOD. After FPLC purification, the enzyme presents as a single band in PAGE with an activity of 2200 U/mg protein (Fig. 5, lane 1). The presence of 59 ng-atom copper and 52 ng-atom zinc (mg enzyme)-1 in the purified mutated P. fermentans Cu, Zn SOD was confirmed by atomic absorption spectrometry. No manganese or iron could be detected.

The molecular mass of mutated P. fermentans Cu, Zn SOD was determined by several methods. The sample was subjected to 12% SDS-PAGE, producing one band at 32 kDa (Fig. 5, lane 2). The same results were confirmed by means of matrix-assisted laser desorption ionization MS (Fig. 6.). The mass was calculated to be 22480.4 Da for the whole molecule and 14850.1 Da for the structural subunits. From these results it is suggested that the investigated enzyme is a dimer, composed of two identical structural subunits. The P. fermentans Cu, Zn SOD is a glycosylated enzyme,

Fig.3: IsoenzymeprofilesofSODproducedbymutatedP. fermantans (Pf 4m) upon cultivation at uncontrolled DO.

Fig.4:IsoenzymeprofilesofSODproducedbymutatedP.fermantans (Pf 4m) upon cultivation at controlled DO.

Table - 4

Purification steps for Cu, Zn SOD from mutated P. fermentans (Pf 4m)

Purification step Total protein(mg)

10-3x total SODactivity (U)

Specific SOD activity(U mg-1) Yield (%) Purification

(-fold)

Crude extract (after conc) 6680 735 110 100 1.0

Acetone fraction (0.4 vols) 4010 838 209 93 1.9

Acetone fraction (1.1 vols) 400 317 792 39 7.2

Sephadex G-75 100 176 1760 21 16.0

DEAE cellulose 52 60 118 1969 13 17.9

Mono Q 10/10 (FPLC) 36 80 2100 11 19.1


as determined by the orcinol-sulphuric acid assay, with a carbohydrate-binding site, Asn-Xxx-Ser/Thr, typical for N-glycosylation [26] .

Fig..5: PAGE (10%gel)ofpurifiedCu,ZnSOD.Lanes:1, gel stained for enzymic activity; 2, gel stained for protein; 3, standard mixture: a, trypsinogen (24 kDa); b, egg albumin (45 kDa); c, bovine albumin (66 kDa); d, Limulus polyphemus haemocyanin (monomer, 70 kDa).

Fig.6: Matrix-assisted laser desorption ionization mass spectrum of the functional unit of Cu, Zn SOD.The sample (10 pmol) was dissolved in 0.1% TFA and applied onto the target. Analysis was carried out in a-cyano-4-hydroxycinnamic acid. Solutions of chicken egg ovalbumin (44400 Da) and bovine serum albumin (66430 Da) were used to calibrate the mass scale.

References:1. Good fellow, M.; Williams, S.T; Mordarski, M.; The

Biology of Actinomycetes, 1st edition, Academic 2. Faulkner, D.J; Nat. Prod. Rep., 1996, 16, 155.3. Mehta, R.J.; Nisbet, L.J.; David, J.; Neuman;

Fermentation Technology – Prospects in India, 1984,

3, 52.4. Strohl, W.R.;Bioechnology of Antibiotics. 2nd Ed.,

Marcel Dekker, 1997, 128.5. Demain, A.L.; Applied Microbiology and

Biotechnology, 1999, 52, 455.6. Sharma, R.; Banerjee, U.C. ; CRIPS, 2001, 2(3), 2.7. Beauchamp, C.; Fridovich, I.; Anal Biochem, 1971,

44, 276.8. Saitoh, D.; Okada, Y.; Takahara, T.; Yamashita, H.;

Ohno, H.; Inoue, M.; Tohoku J. Exp. Med.,1994,174(1), 31.

10. Somogyi, M.; J Biol Chem.,1952,195,19.11. Rani, G.; Gigras, P.; Mohapatra, H. ; Goswami, V.K.;

Chauhan, B.; A biotechnological perspective Process Biochemis., 2000, 38, 1599.

12. Sivaramakrishnan, S.; Gangadharan, D.; K.M. Nampoothiri, K.M. ; Soccol, C.R. ; Pandey, A. ; Food Technol. Biotechnol., 2006, 44(2), 173.

13. Laemmli, U. K.; Nature, 1970, 227, 680.14. Misra, H. P.; Fridovich, I.; J Biol Chem., 1977, 252,

6421.15. Vorauer-Uhl, K.; Furnschlief, E.; Wagner, A.; Ferko,

B.; Katinger, H.; Eur J Pharm Sci, 2001, 14, 63.16. Corvo, M.L.; Jorge, J.C.; van’t Hof, R.; Cruz, M.E.;

Crommelin, D.J.; Storm, G.; Biochim Biophys Acta, 2002, 1564, 227.

17. Nishikawa, M.; Nagatomi, H; Nishijima, M.; Ohira, G.; Chang, B.J.; Sato, E.; Inoue, M.; Cancer Lett, 2001, 171, 133.

18. Petruccioli, M.; Fenice, M.; Piccioni, P.; Federici, F.; Enzyme Microb Technol, 1995, 17, 336.

19. Guidot, D. M.; McCord, J. M.; Wright, R. M.; Repine, J. E.; J Biol Chem ,1993, 268, 26699.

20. Pinheiro, R.; Belo, I.; Mota, M.; Appl Microbiol Biotechnol, 2002, 58:842.

21. Sturz, L.A.; Diekert, K.; Jensen, L.T.; Lill, R.; Cizewski Culotta, V.; J Biol Chem., 2001, 41, 38084.

22. Vercellone, P. A.; Smibert, R. M.; Krieg, N. R.; Can J Microbiol, 1990, 36, 449.

23. Shilova, N. K.; Matyashova, R. N.; Ilchenko, A. P.; Microbiologiya, 1989, 58, 430.

24. Howlett, N. G.; Avery, S. V.; (1999). FEMS Microbiol Lett., 1999, 176, 379.

25. Francois, C.; Marshall, R. D.; Neuberger, A.; Biochem J, 1962, 83, 335.

26. Bordo, D.; Djinovic, K.; Bolognesi, M.; J .Mol. Biol., 1994, 238, 366.

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Evaluation of Angiogenic Potential of Nicorandil, a KATP Channel Agonist By Chorioallantoic Membrane Assay. (CAM)

CHANDANA KAMILI *A, K. RAVI SHANKAR B, AND UMA MAHESHWARARAO VATTIKUTTI C

*a Department of Pharmacology, CMR College of Pharmacy, Hyderabad, India.b Department of Pharmacology, Sri Sai Aditya Institute of Pharmaceutical Sciences and Research, Surampalem. India.

cDepartment of Pharmacognosy, TRR College of Pharmacy, Hyderabad, India,

ABSTRACT

Objective: Angiogenesis is the development of new blood vessels. A wealthy number of ion channels are found on the endothelial cells. These ion channels play a vital action in cell proliferation and in related angiogenesis. We aimed to investigate the effects of Nicorandil (KATP channel activation).

Methods: The angiogenic activities of Nicorandil was investigated by measuring its effects on number of branches formed and angiogenic score by in-ovo (CAM, Chick Chorioallantoic membrane) method.

Results: The test and standard drug (Pyruvic acid) groups were compared with the control group using One-way ANOVA, followed by post hoc test, the Dennett’s test to compare mean of all the groups with the control mean. The results revealed that Nicorandil treatment led to significant cell proliferation and related angiogenesis in the dose dependent manner and were quite comparable with the standard angiogenic drug Pyruvic acid.

Conclusions: These scientific findings indicate the clinical benefits of Nicorandil in pathological situations involving excessive angiogenesis. Stimulation of cell cycle progression at various checkpoints may be the underlying molecular mechanisms for angiogenic action of the test drug.

Key Words: Nicorandil, Angiogenesis, Chick Chorioallantoic membrane, KATP channel activation.

INTRODUCTIONAngiogenesis is the generation of new blood vessels

from pre-existing vasculature [1]. It is also called as neovascularization. Angiogenesis has two faces, it plays vital role in maintenance of both health and diseases. Vascular system is the first organ system formed in the embryo and also forms the largest network in our body. But when deregulated, the formation of excess new blood vessels contributes to numerous malignant ischemic inflammatory infections and immune disorders. Historically the best known are cancer, psoriasis, arthritis, blindness, additional disorders such as obesity, asthma, arthrosclerosis and infectious diseases are included and the list is still growing. [2][3][4]. In the same way insufficient vasculature also results in many pathological conditions like stroke, coronary artery diseases, peripheral artery diseases, ulcers, chronic wounds etc. These diseases require neovascularization for their recovery. [5]

Angiogenesis is tightly controlled through a balance of pro-angiogenic and anti-angiogenic factors. Angiogenesis is stimulated by various pro-angiogenic factors including basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor (PDGF), Angiopoietins, Transforming Growth Factor (TGF), Tumor necrotic Factor- α, TNF-α, Interleukin-8, DLL4 etc.[17] Proangiogenic factors are counter balanced by a number of natural antiangiogenic molecules like thrombospondin-1 (TSP-1) and angiostatin/endostatin [6].

Endothelium is a multifunctional surface for signal transduction. Electrogenesis in EC is also potentiated by K+ channels, Ca2+ activated K+ channels and volume regulated anion channels (VRAC) [7]. Among the VRAC, Cl- channels counteract the depolarization of other channels and stabilize the Ca2+ influx. VRAC are also involved in the transport of organic molecules including amino acids, cell proliferation, EC permeation and cell- cell coupling. The abundance of ion channels in the plasma membrane of non-excitable cells has raised questions about their functional role in angiogenesis. [8].


Based on the above observations we have selected Nicorandil (KATP channel activation) to evaluate the anti-angiogenic effect at three different doses in Chick chorioallantoic membrane (CAM) assay (in ovo).

MATERIALS AND METHODS:Materials:

Chemical and lab wares:

Pure drug, Nicorandil was purchased from Sigma Aldrich Pvt. Ltd., All the chemicals used in the research are of AR grade.

Equipments:

Incubator, high resolution digital camera, trinoccular microscope.

Methods:

The study was approved by the Institutional animal ethical committee (IAEC), (Ref: CPCSEA /1657/ IAEC /CMRCP/PhD-14/30) CMR College of Pharmacy, Hyderabad, India. All the surgical procedures like extraction of albumin, preparation of window on egg shell, resection of CAM tissue were done by same investigator to increase the reproducibility of the process. After preparation of window on the shells, eggs were randomized to eliminate potential bias in the manual procedures within the different groups. [9, 10, 11]

Chick Chorioallantoic Membrane Assay:

Thirty fertile eggs were collected from a local hatchery on the Day 0. Checked for any damage, superficially cleaned with 70 % ethanol. Three concentrations (10-6M, 10-5M, 10-

M) of the test drug were selected based on the results of a previous study which have shown the concentration of 10-5M provides sub maximal efficacy of the drug. The eggs were incubated under constant humidity at 370C by placing them in a horizontal position. On the 3rd day a hole was made at the narrow end to withdraw 3 ml of albumin. The hole was sealed with surgical tape and the eggs were put back for incubation. On the 7th day of incubation a small window was cut opened on the shell. Eggs were randomly divided into 5 groups’. Sterile gel foam (3mm×3mm×1mm) piece was placed on the membrane. (Fig.1). The group I (normal control) was impregnated with sterile normal saline; group II was given with the standard angiogenic drug Pyruvic acid, the tests groups III, IV, V were impregnated with 10-6M, 10-5M, 10-4M /egg of Nicorandil respectively. Then eggs were incubated undisturbed till day 14. On the 14th day the CAM tissues were resected out [12, Christian P , Gerd D, 2004]. (Fig.2) Tissues were placed in 10 % formalin and stained with hematoxylin and eosin then examined under trinocular microscope. The number of vessel branch points in a unit square region were counted and anlyzed [13] [14]. Angiogenesis score 1-4 was given to each egg based on the number of branching points. If the no. of branching

Fig. 1: Chorioallantioc membrane (CAM) Assay

Fig. 1.1: Incubated eggs Fig. 1.2: Removal of albumin from eggs

Fig. 1.3: Windowing of the eggs Fig. 1.4: Instillation of test drugs in eggs


points is ≥35, the angiogenesis score is 4. If the branching points are 25-34, score is 3 and for 15-24, the score is 2. If the branching points are <15, the score is 1[17]. (For results refer Fig 2).

STATISTICAL ANALYSISThe statistical analysis was carried out by using graph

pad Prism 5. Results were presented as mean ± SEM. The differences between the groups were compared by one way ANOVA followed by post hoc Dunnett’s test. Results were considered statistically significant at p value<0.05.

RESULTSChick Chorioallantoic Membrane (CAM) Assay:

In CAM assay, the numbers of branching points were counted by observing under trinocular microscope in a unit square area under the gel sponge in the CAM tissue. Our results show significant reduction in the branching points in 10-5M and 10-4M Nicorandil.

Control

Pyruvic acid

Nicorandil(1

0-6M)

Nicorandil(1

0-5M)

Nicorandil(1

0-4M)0

1

2

3

4

5

Treatment

Angi

ogen

ic s

core

Table - 1

Effect of Nicorandil on no. of branching in CAM assay of angiogenesis

Group No. Treatment Dose/egg No. of branching points

1. Control - 2.170.753±

2. Standard (Pyruvic acid) 10-4M 4.000.0± ***

3. Nicorandil (low dose) 10-6M 2.000.632± ns

4. Nicorandil (medium dose) 10-5M 3.500.548± **

5. Nicorandil (high dose) 10-4M 3.670.516± ***

All the results were expressed as Mean ± SEM; n=6 The ANOVA test followed by Dunnett test was used to compare the data. Findings with p values <0.05 were considered statistically significant and ns as insignificant.

Fig 2: Graph showing the effect of Treatment groups on the no. of branching points in CAM Assay of angiogenesis

CONTROL STANDARD (Pyruvic acid)


DISCUSSION Angiogenesis plays a vital role in the pathological

progress such as growth and metamorphosis of tumor. Ion-channels are transmembrane minute opening proteins that regulates the flow of ions such as K+, Na+, Ca+2, Cl- etc. across the cell membrane and govern many physiological functions like skeletal muscle contraction, nerve conduction, cell volume regulation etc.[16] Defects in the ion-channels can results in many diseases like myriad disorder such as heart arrhythmia, cystic fibrosis and many [15].

Nicorandil is a nicotinamide nitrate used as an antianginal agent. It has two modes of action. Firstly, by opening adenosine triphosphate–dependent potassium channels, nicorandil increases transmembrane potassium conductance and relaxes peripheral and coronary arterioles. Secondly, with its nitrate moiety, nicorandil increases intracellular concentrations of cGMP, resulting in peripheral vein and coronary artery dilation. Nicorandil relaxes coronary vascular smooth muscle by stimulating guanylyl cyclase and increasing cyclic GMP (cGMP) levels (as shown first in our laboratory) as well as by the second mechanism resulting

in activation of K+ channels and hyperpolarization. Thus, because of its ability to dilate arteries and veins, nicorandil maximizes coronary flow while concomitantly reducing myocardial work through reductions in afterload. For these reasons, nicorandil has been successful in managing angina and hypertension. However, growing evidence suggests that this drug provides additional benefits that reach beyond its original therapeutic indications.

So this preconditioning would have initiated cardiac angiogenesis. It is evident from the present study Nicorandil at a dose of 10-5M and 10-4 has potent angiogenic action. Angiogenesis initiated may be due to triggering a molecular cascade resulting in increased vascular endothelial growth factor (VEGF), a proangiogenic factor, and B-cell lymphoma (Bcl)-2, an antiapoptotic factor. Although it is known that nicorandil increases Bcl-2, a connection between KATPchannel activators and increased VEGF expression has not been demonstrated earlier. The present research proves the angiogenic action by stimulation of VEGF expression. Moreover, the stimulation of capillary and arteriolar growth in the myocardium as a result of nicorandil administration has also been unexplored. The work by Xu et al entitled

NICORANDIL 10-6M NICORANDIL 10-5M

NICORANDIL 10-4MFig 3: Effect of test drugs on number of branches in CAM Assay angiogenesis


D

“Nicorandil promotes capillary and arteriolar growth in the failing heart of Dahl salt-sensitive hypertensive rats” demonstrates for the first time that KATP channel activation with nicorandil promotes coronary capillary and arteriolar growth. In addition, the Xu study demonstrated that two well-known proangiogenic factors, basic fibroblast growth factor (bFGF) and VEGF, could be upregulated and associated with nicorandil-mediated vascular growth.

These findings are interesting for several reasons. First, until now, there were questions regarding whether VEGF actions in angiogenesis were mediated by other angiogenic growth factors. By demonstrating VEGF and bFGF upregulation is associated with nicorandil-mediated vascularization, the Xu study has partially addressed this concern. Second, by establishing a connection between nicorandil and proangiogenic factors, this study has demonstrated a potential nonsurgical modality to enhance collateral coronary circulation in patients at high risk for coronary artery disease. Third, pharmacological upregulation of VEGF and bFGF by nicorandil provides an alternative to gene therapy. Finally, knowing that KATPchannel activators are associated with angiogenesis, KATP channel blockade may initially provide insight into the molecular basis governing tumor and neoplastic angiogenesis.

CONCLUSION Based on the scientific finds in the research, we report

here that Nicorandil (KATP channel activation) stimulates new vessel formation in all the in ovo model. These results suggest that Nicorandil may be useful in the therapy of angiogenic-insufficient disorders.

Conflicts of interest statement:

The authors declare no conflicts of interest.

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nels and transporters in tumor angiogenesis: An om-ics view. Biochim Biophys Acta. (2015 Oct); 1848(10 Pt B): 2647-56.

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Formulation and Characterization of Dried Nanosuspensions of Glipizide

S. RAJA SHEKHAR*1 AND P. VIJAYA LAKSHMI2

1. CMR College of Pharmacy, Kandlakoya (vil), Medchal Road, Hyderabad, Telangana-501401. 2. Siddhartha Institute of Pharmacy Korremula Road, Narapally, R.R District, Ghatkesar, Telangana 501301

ABSTRACT

Nanosuspensions are colloidal dispersion of nanosized drug particles stabilized by surfactants. They can also be defined as a biphasic system consisting of pure drug particles dispersed in an aqueous vehicle in which the diameter of the suspended particle is less than 1µm in size. Glipizide is an oral rapid- and short-acting anti-diabetic drug from the sulfonylurea class. It is classified as a second generation sulfonylurea, which means that it undergoes enterohepatic circulation. The aim of the present work is to formulate and evaluate Glipizide oral dried nanosuspension. Glipizide Nanosuspension was prepared by precipitation technique. After preparation of Nanosuspension various characterisation studies were done such as drug content, % yield, Scanning electron microscopy (SEM), Zeta potential, particle size determination and Invitro drug release. PVPK30 and polaxomer are used as stabilizers. From the dissolution study F4 formulation which contains PVP K30 as stabilizer was considered as optimised formulation. It showed maximum drug release at 30 min. FTIR and DSC studies revealed that good compatibility between drug and excipients. Zeta potential studies were revealed their good stability in dispersion.

Key words: Glipizide, PVP K30, Polaxomer 188, Nano suspension.

INTRODUCTION Nanosuspensions are colloidal dispersions of nanosized drug particles stabilized by surfactants. They can also be defined as a biphasic system consisting of pure drug particles dispersed in an aqueous vehicle in which the diameter of the suspended particle is less than 1μm in size [1]. Reduction of drug particles to nanometer range leads to an enhanced dissolution rate not only because of increased surface area but also because of saturation solubility. The increase in the saturation solubility and solution velocity of nanoparticle is due to increase of vapour pressure of the particles. More than 40 percent of the drugs coming from Highthroughput screening are poorly soluble in water [2]. Obviously poorly water-soluble drugs show many problems in formulating them in conventional dosage forms. One of the critical problems associated with poorly soluble drugs is too low bioavailability and or erratic absorption [3]. Nanosuspensions are promising strategy for the efficient delivery of hydrophobic drugs.

Advantages [4,5]:

• Oral route of drug administration have wide acceptance up to 50-60% of total dosage forms

• Ease of administration

• Solid dosage forms represent unit dosage forms in which one usual dose of the drug has been accurately placed

• Self medication

• Avoidance of pain

• Patient compliance

Disadvantages:

• Action of drugs is slower

• May cause nausea and vomiting

• Cannot be used for uncooperative patient

• Absorption of drugs may be variable and erratic

Glipizide comes under category of Sulfonylureas which likely bind to ATP-sensitive potassium-channel receptors on the pancreatic cell surface, reducing potassium conductance and causing depolarization of the membrane. Depolarization stimulates calcium ion influx through voltage-sensitive calcium channels, raising intracellular concentrations of calcium ions, which induces the secretion, or exocytosis, of insulin[6].


MATERIALS AND METHODSMATERIALS

The following materials available were used as supplied by the manufacturer without further purification or investigation. Glipizide was procured from Sura labs, Hyderabad as a gift sample, PVP, Polaxomer, Potassium dihydrogen phosphate, HCL and Sodium hydroxide pellets were procured from Merck specialities Pvt. Ltd.

MethodologyPreformulation studies

Solubility studies: Solubility of Glipizide was carried out in different solvents like- distilled water, methanol, ethanol, octanol, DMSO and Di methyl formamide(DMF).It is soluble in octanol, slightly soluble in methanol, very slightly soluble in water .Saturated solutions were prepared by adding excess drug to the vehicles and shaking on the shaker for 48 hr. at 25°C under constant vibration. Filtered samples (1ml) were diluted appropriately with 0.1N Hcl buffer and solubility of Glipizide was determined spectrophotometrically at 227nm.

Melting Point: Melting point of the drug was determined by capillary tube method and found to be 156-160oC.

Organoleptic properties: The colour, odour and taste of the drug were recorded using descriptive terminology and found to be white to off-white crystalline powder, tasteless and odourless.

Drug-Excipient Interactions Studies: The compatibility between the pure drug and excipients was detected by FTIR spectra obtained on Bruker FTIR Germany(Alpha T).The solid powder sample directly place on yellow crystal which was made up of ZnSe. The spectra were recorded over the wave number of 4000 to 400cm-1.[7]

Prepration of nanosuspensions: All the ingredients including drug, polymer and excipients were weighed accurately according to the batch formula (Table-1). The required amount of polymer (carrier) and stabilizer were accurately weighed and added to required measure of H2O in a beaker. The drug was dissolved in solvent (methanol) and added to the above mixture in a drop wise manner using

a syringe while on stirring Magnetic stirring for 1 hour and then ultrasonication for 2 hours.

Evaluation of Nanosuspension Rosuvastatin: The following evaluations were done to the formulated nanosuspensions:

A. Drug content uniformity: 10ml of each formulation was taken and dissolved in 10ml isotonic solution and kept overnight. 10 mg (similar as in formulation) of drug was taken and dilution was made to 10μg/ml. The dilutions were filtered and analyzed using UV for their content uniformity. The absorbance of the formulations were read using one cm cell in a UV-Visible spectrophotometer. The instrument was set at 227 nm. The drug content in each formulation was calculated based on the absorbance values of known standard solutions.

B. Entrapment efficacy: Entrapment efficacy was calculated by following formula:

%Entrapment efficiency= Drug content *100/Drug added in each formulation.[8]

C. pH measurement: The pH values were measured at 25 ◦C using a pH digital meter at 20 ± 1 ◦C. The formulation was brought in contact with the electrode of pH meter and equilibrated for 1 min. This method was done in triplicate and mean was calculated along with standard deviation.[9]

D. In vitro drug release study: This is carried out in USP dissolution test apparatus-II (Electrolab TDT-06N), employing paddle stirrer at 50 rpm and 200 ml of pH 6.8 phosphate buffer as dissolution medium. The release study is performed at 37 ±0.5oC. Samples of 5 ml are withdrawn at predetermined time intervals and replaced with fresh medium and analyzed for Glipizid after appropriate dilution by measuring the absorbance at 227 nm.

E. Entrapment efficacy: The entrapment efficacy of the formulated Nanosuspension was found to be in the range of 55.4%-96.7% respectively.

F. %Transmittance measurement: UV-Visible spectrum of pure Nanosuspension was recorded in range of 200-400 nm.

Table No.1Formulations F1 to F10

INGREDIENTS F1 F2 F3 F4 F5 F6 F7 F8 F9 F10Drug (mg) 500 500 500 500 500 500 500 500 500 500PVP K 30 (mg) 250 500 750 1000 1250 - - - - -Polaxomer 188 (mg) - - - - - 250 500 750 1000 1250RPM 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000Methanol(ml) 10 10 10 10 10 10 10 10 10 10Water(ml) 500 500 500 500 500 500 500 500 500 500


G. Zeta Potential: The measurement itself is a particle electrophoresis, the particle velocity is determined via the doppler shift of the laser light scattered by the moving particles. The field strength applied was 20 V/cm. The electrophoretic mobility was converted to the zeta potential in mV using the Helmholtz-Smoluchowski equation. At standard measuring conditions (room temperature of 25 °C, water) this equation can be simplified to the multiplication of the measured electrophoretic mobility (μm/cm per V/cm) by a factor of 12.8, yielding the ZP in mV.[10]

H. Scanning Electron Microscopy (SEM):

The surface morphology of the raw materials and of the ternary systems was examined by means of JSM‐6400 (Jeol, Japan) scanning electron microscope. The samples were previously fixed on a brass stub using double‐sided adhesive tape and were then made electrically conductive by coating with a thin layer of gold and palladium alloy (180‐200 Å) using a fine coat ion sputter (JEOL, fine coat ion sputter JFC‐1100). The pictures were taken at an excitation voltage of 20 kV and magnification in the range of 118 to 245X.[11,12]

Spray drying of Nanosuspension: Spray drying was carried out to get the dry nano size powder. An optimised batch of aqueous nanosuspension was transferred into nano size powder by a lab spray dryer LU-222 lab ultima. Spray dried powder was directly collected after the process. In this process, the spray dryer was set to the conditions given in following table no.2.

Table - 2. Spray dryer Parameter

Inlet Temperature 105-110oCOutlet Temperature 100oCCool Temperature 40oCAspirator flow rate nm3/hr 45

Feed pump flow rate ml/min 3Cycle time min 70

RESULTS AND DISCUSSION

Standard Graph of Glipizide in pH 6.8 phosphate buffer.

Fig.1: Standard graph of Glipizide at pH 6.8 phosphate buffer (227nm)

Drug–Excipient compatibility studies

Fig. 2 : Fourier Transform-Infrared Spectroscopy

DSC studies:

Fig.3: DSC Spectrum of Glipizide Pure drug and optimised formulation

Characterisation of NanosuspensionTable - 3

Drug content and % Yield of Nanosuspension

Formulation Code Drug content % Yield

F1 81.75±0.46 85.62±0.48F2 90.02±1.20 91.80±0.88F3 92.51±0.66 95.68±0.90


F4 95.80±0.65 97.05±1.05F5 95.83±0.82 97.67±1.17F6 73.58±1.50 81.80±0.72F7 81.33±1.17 85.24±1.45F8 93.33±0.86 96.75±1.40F9 94.16±1.32 94.46±0.70F10 95.00±0.70 96.81±0.94

Drug content: From the table 3, it was noted that increase the concentration of stabilizer obtaining more drug content. All the formulations drug content was found to be in the range of 73.52±1.50 to 95.46±0.82.

Percentage Yield: From the table 3, it was noted that increase the concentration of stabilizer obtaining more yield. The entire formulations percentage yield was found to be in the range of 81.80±0.72 to 97.67±1.17.

SEM Studies:

Fig. 4 : SEM Studies of Optimised formulation

Zeta potential:

Fig.5:ZetapotentialStudiesofOptimisedformulation

Fig. 6 : invitro drug release studies

From the dissolution studies, it was evident that the formulations prepared with PVP K30 were shown good drug release at 30min in concentration of 1000mg. Increase the concentration of stabilizer above 1000mg retards the release of drug.

Formulations prepared with Polaxomer 188 were shown good drug release at 45 min in the concentration of 1000 mg and 1250mg of stabilizer. Among all formulations F4 formulation was considered as optimised formulation which was shown maximum drug release at 30 min.

SUMMARY AND CONCLUSION In the present investigation Nanosuspension drug delivery system for Glipizide was done. Glipizide is a poorly water-soluble (BCS class II) antidiabetic drug. Due to the poor water solubility of this drug, its bioavailability is dissolution rate-limited. Nanosuspension was prepared by precipitation technique. The obtained nanosuspension was carried out for characterisations like Drug content which was found to be in the range of 73.52±1.50 to 95.46±0.82, Percentage Yield found to be in the range of 81.80±0.72 to 97.67±1.17.

Dissolution studies of the formulations prepared with PVP K30 were shown good drug release at 30min in concentration of 1000mg. Increase the concentration of stabilizer above 1000mg retards the release of drug. Formulations prepared with Polaxomer 188 were shown good drug release at 45 min in the concentration of 1000 mg and 1250mg of stabilizer. Among all formulations F4 formulation was considered as optimised formulation which was shown maximum drug release at 30 min.

Drug and Excipient Compatibility studies showed the compatibility between drug and excipients. From the DSC studies, it has shown drug melting point was 207.8oCel. After mixing with the polymer it has shown at 208.1oCel near to pure drug melting point. Hence DSC studies were


also shown good compatability. Optimised formulation has taken for SEM studies. It has shown that nano suspension particle size was 200nm and Zeta potential studies were revealed that good stability in dispersion of optimised nano suspension.

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for oral delivery using solid dispersions. Eur J Pharm Biopharm, 2000: 50(1), 47-60.

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3. Cornelia MK, Muller RH. Drug nanocrystals of poorly soluble drugs produced by high-pressure homogenisation. Eur J Pharm Biopharm. 2006: 62, 3–16.

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8. Van Eerdenbrugh, B., Van den Mooter, G., Augustijns, P. Top-down production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products. International Journal of Pharmaceutics.2008: 364, 64–75.

9. K Itoh, A Pongpeerapat, Y Tozuka, T Oguchi and K Yamamoto. Nanoparticle formation of poorly water soluble drugs from ternary ground mixtures with PVP and SDS. Chem Pharm Bull, 2003; 51: 171-4.

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Invitro drug release studies:

Tim

e((

Min Pure

Drug F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

0 0 0 0 0 0 0 0 0 0 0 0

5 0.96 ±0.06

13.48 ±1.08

14.41 ±1.23

20.97 ±0.52

53.74 ±1.12

20.23 ±1.13

11.24 ±1.39

13.14 ±0.67

19.22 ±0.62

24.11 ±0.53

21.20 ±0.33

10 2.42 ±0.08

28.56 ±1.61

28.87 ±0.92

44.10 ±0.49

78.36 ±1.12

41.70 ±0.54

22.69 ±1.71

27.83 ±1.26

39.47 ±0.73

37.31 ±0.78

42.93 ±0.58

15 4.11 ±0.26

40.98 ±0.99

45.32 ±0.82

62.26 ±0.72

96.11 ±0.47

58.55 ±0.76

36.87 ±0.65

39.58 ±0.38

52.36 ±1.18

50.33 ±0.66

52.07 ±0.53

20 7.28 ±0.39

54.12 ±0.61

56.78 ±0.41

72.89 ±0.94

68.68 ±1.24

47.46 ±0.96

50.87 ±0.16

64.08 ±0.1

63.29 ±0.85

64.90 ±0.89

30 9.64 ±0.28

65.02 ±0.74

68.92 ±0.79

84.34 ±1.12

80.13 ±1.28

59.90 ±0.51

63.16 ±0.73

76.46 ±0.91

77.60 ±1.33

80.03 ±1.01

45 13.52 ±0.31

76.44 1.76±

80.67 0.83±

95.17 0.38±

92.07 0.56±

70.55 0.75±

75.84 0.33±

88.12 0.46±

94.99 0.46±

94.53 0.54±

60 15.20 ±0.42

85.84 ±1.49

93.86 ±1.31

92.19 ±0.24

78.37 ±2.92

86.96 ±0.52

95.38 ±1.09

D

online : issn 2349-669x j.pharm.chem coden:jpcocm journal

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