universitÉ d’antananarivo

MÉMOIRE

présenté pour obtenir l’

HABILITATION À DIRIGER DES RECHERCHES

Volume I : Publications

Par

RASOANAIVO Herilala Léa

Soutenu le 14 Août 2015 devant le jury composé de :

Président : Pr. RAKOTOVAO Marcelle Directeur : Pr. RAHARISOLOLALAO Amélie Rapporteurs : Pr. WADOUACHI Anne : Pr. RANDIMBIVOLOLONA Fanantenanirainy Examinateurs : Pr. RAZANAMAHEFA Bakonirina Voninorosoa : Pr. LEMAIRE Marc

UNIVERSITÉ D’ANTANANARIVO

------------------- FACULTÉ DES SCIENCES

------------------- DÉPARTEMENT DE CHIMIE ORGANIQUE

Valorisation chimique de Chrysophyllum boivinianum et des sous-produits de transformation des fruits de

Mangifera indica Var.hiesy

L’apparition de l’homme, il y a 3 millions d’années marque le début de la connaissance

des plantes qu’elles soient comestibles ou toxiques, qu’elles permettent de tuer le gibier et

l’ennemi ou de soigner.

Les plantes constituent, à la fin du XXè siècle, la source principale des 60% des

médicaments dont nous disposons. Les 40% restants représentent les médicaments de

synthèse ; ils sont nés de la modification chimique de molécules ou de parties de molécules

naturelles prises comme têtes de série.

A l’heure actuelle, l’idée :" ce qui est naturel est bon, inoffensif contrairement aux produits

de synthèse (médicaments, plastique,..)" fait que le marché des produits bio (alimentaire,

phytomédicament, biocarburant,..) a considérablement augmenté pour répondre aux besoins

quotidiens même dans les pays industrialisés.

La plante est une des sources des produits bio. Ses propriétés sont dues aux métabolites

qu’elle contient. Les métabolites primaires (protéines, glucides, lipides) constituent la classe

des substances nécessaires à la vie de la cellule, les éléments de la membrane cellulaire ou les

substances de réserve. Les métabolites secondaires, substances de poids moléculaires peu

élevés, dérivent du métabolisme primaire (les alcaloïdes, les composés phénoliques, les

hétérosides et les terpénoides).

L’étude chimique des plantes permet de détecter, d’extraire, puis de séparer les différents

métabolites afin d’isoler et d’identifier dans le but de les valoriser.

Le volume 1 de ce mémoire présente des résultats de cette étude chimique des plantes

depuis 2007 à 2015 par les biais de dix-sept (17) publications dont dix (10) publications dans

les journaux scientifiques et les sept (07) sous forme de communications orales et affichés :

- sur les molécules bioactives des plantes médicinales : huit (8) publications dans divers

journaux (Journal of Pharmacognosy and Phytochemistry, Natural Product Research,

International Journal of Indigenous Medicinal Plants, International Journal of Chemical

Studies, Natural Product Communications, In Vivo ; une (1) communication orale présentée

lors du colloque GDRI en 2010 ; trois (3) communications affichées pendant le colloque de

BIOMAD III à Mahajanga en 2013.

-sur les extraits ou molécules des sous-produits de transformation des fruits, valorisables à

des fins non thérapeutiques : deux (2) publications dans le Journal of Pharmacognosy and

Phytochemistry ; trois (3) communications orales dont l’une lors du colloque "Chimie et

déchets végétaux" à l’académie des Sciences, Paris en 2013 et les deux (2) autres pendant le

colloque de BIOMAD III à Mahajanga en 2013.

Abstract. Fractionation of the cyclohexane extract from thestem bark powder of Zanthoxylum madagascariense led to theisolation of a new benzophenanthridine-type alkaloid,hydrochloride of 2,3-methylendioxy-8-hydroxy-7-methoxy-benzo[C]phenanthridine (Rutaceline), characterized on thebasis of its spectral data. Rutaceline was evaluated for itsantiproliferative capacity on the human colorectaladenocarcinoma (Caco-2) and the African green monkeykidney (Vero) cell lines. The 50% inhibition of cell growth(IC50) obtained after 24 h incubation was similar for both cellslines (110-115 Ìg/ml, i.e. 269-281 ÌM), but at 48 h the IC50value for the Caco-2 cells was lower than for the Vero cells (20Ìg/ml, i.e. 49 ÌM versus 90 Ìg/ml, i.e. 220 ÌM) indicating ahigher cell growth inhibitory effect on the colonadenocarcinoma cells. At the respective IC50 concentrations,Rutaceline did not significantly induce apoptosis but inducedcell cycle arrest in the G0/G1 phase, as well as a decrease ofcells in S phase. Rutaceline also induced DNA fragmentationin both cell lines, as revealed by agarose gel electrophoresis, anda dose-dependent clastogenic effect in both cell lines as revealedby the Comet assay.

Cancer is one of the leading causes of death in the worldthat claims more than seven million lives per year (1). Everyyear, thousands of children and teenagers under twentyyears of age are diagnosed with cancer in the United States.With the introduction of new therapeutic strategies duringthe past thirty years, survival for many diagnostic groups hasincreased tremendously. The surveillance, epidemiology andend results program estimate that the overall 5-year survivalrate in 1998 was 80% for the group of cancer patients undertwenty years old. Nevertheless, cancer remains the leadingmedical cause of death among children between one andnineteen in the United States (2).

Natural products have been the main anticancer pro-drugs for centuries and represent 50% of drugs usedclinically in developed countries, 25% of which are derivedfrom higher plants. The plant kingdom represents anenormous potential of molecules to be discovered, since itwas estimated one decade ago that more than 90% of plantspecies have not yet been exhaustively studied (3).

Recent investigations have been focused on the activityof the components found in Citrus species, includingflavonoids, carotenoids and limonoids, especially in termsof their anticarcinogenic effects. The antioxidantmicronutrients, carotenoids, vitamin C, vitamin E andpolyphenols, especially flavonoids, play an important role inthe prevention of cancer (4).

Although whole organic foods should be the first line ofdefense against cancer, certain supplements have beenshown to help in the prevention of certain types of cancer.For example, folic acid (B vitamin family member), found

417

Correspondence to: Marta Cascante, Department of Biochemistryand Molecular Biology, Faculty of Biology, University ofBarcelona, Av. Diagonal 645, 08028 Barcelona, Spain. Tel: +34934021593, Fax: +34 934021219, e-mail: [email protected]

Key Words: Rutaceline, benzophenanthridine, cancer.

in vivo 21: 417-422 (2007)

Anticancer Effect of a New Benzophenanthridine Isolated from Zanthoxylum madagascariense (Rutaceline)

G. PACHÓN1, H. RASOANAIVO2,5, A. AZQUETA3, J.C. RAKOTOZAFY2,5, A. RAHARISOLOLALAO2,5, A. LÓPEZ DE CERAIN3, J. DE LAPUENTE4, M. BORRAS4,

S. MOUKHA5, J.J. CENTELLES1, E.E. CREPPY5 and M. CASCANTE1

1Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain;

2Laboratoire de Chimie, Faculté des Sciences, Université d’Antananarivo, B.P. 906 Antananarivo 101, Madagascar;

3Department of Bromatology, Technology of Aliments and Toxicology, Faculty of Pharmacy, University of Navarra, Irunlarrea s/n, 31008 Pamplona;

4Unity of Experiment Toxicology and Ecotoxicology (UTOX-PCB), Parc Científic de Barcelona, Edifici modular, C/Josep Samitier 1-5, 08028 Barcelona, Spain;

5Laboratoire de Toxicologie et d’Hygiène Appliquée, Université de Bordeaux 2, Victor Ségalen, Faculté des Sciences Pharmaceutiques, 146, rue Léo-Saignat, 33076 Bordeaux, France

0258-851X/2007 $2.00+.40

in many vegetables, beans, fruits, whole grains, and fortifiedbreakfast cereals, may hold promise as an anticarcinogen incolon cancer. Deficient intake of folic acid, which plays animportant role in DNA synthesis and repair, has been linkedto an increased risk of colon cancer amongst others. Animalstudies have shown that diets lacking in folic acid areassociated with DNA strands breaks in the organs mostsusceptible to cancer, leading to the development of thedisease (5).

The search for novel natural drugs has resulted in theidentification of several benzophenanthridine alkaloidspresent in plants as promising anticancer agents. Forexample, there is evidence that sanguinarine, which derivesfrom the root of Sanguinaria canadensis, induces apoptosisin the A431 skin carcinoma cell line and is a potentialantiproliferative agent that could be developed forchemotherapy of skin cancer (6). This root was classicallyused in traditional medicine for the treatment of gingivitisand dermatitis (7). Sanguinarine and other quaternarybenzo[c]phenanthridine alkaloids (QBA) have a number ofinteresting pharmacological activities including antitumoral(8), antimicrobial (9), antifungal (10) and antioxidant (11)properties.

Other benzophenanthridine alkaloids have not beenstudied as much as sanguinarine. Among them, fagaronineand its synthetic derivative ethoxidine inhibit DNAtopoisomerase (12). This inhibition is also observed forchelerythrine, a benzophenanthridine which inhibits proteinkinase C (13) and causes mitotic disruption by interactionwith tubulin (14).

An extract of Zanthoxylum americanum (Rutaceae) hasshown cytotoxicity (15) and recently a new benzophe-nanthridine called Rutaceline has been identified inZanthoxylum madagascariense (Rutaceline). In the presentwork its antiproliferative effect, capacity to alter the cellcycle, induce apoptosis, DNA fragmentation and genotoxicdamage were examined in a human intestinal cancer cellline Caco-2 and Vero cells from monkey kidney.

Materials and Methods

Materials. Dubelcco’s modified Eagle’s medium (DMEM),antibiotics, penicillin, streptomycin, Dulbecco’s phosphate-bufferedsaline (PBS) and agarose low melting point (LMP) were providedby Gibco-BRL (Eggenstein, Germany). Fetal bovine serum (FBS)was purchased from Invitrogen (Carlsbad, CA, USA). TrypsinEDTA solution C (0.05% trypsin – 0.02% EDTA) was obtainedfrom Biological Industries (Kibbutz Beit Haemet, Israel). Tris(hydroxymethyl) aminomethane was purchased from Aldrich-Chemise (Steinheim, Germany), DNAse free RNAse from RocheDiagnostic (Mannheim, Germany) and Annexin V/FITC Kit fromBender System MedSystem. RPMI, 1% sodium N-lauryl-sarcosinate (w/v), Proteinase K, MTT (3-(4,5-dimethylthiazol–2yl)-2,5diphenyltetrazolium bromide) and other reagents were providedby Sigma Chemical Co (St. Louis, MO, USA).

Rutaceline extraction. The stem bark of Zanthoxylummadagascariense was collected from Fianarantsoa and wasauthentically identified at the herbarium of Botanical andZoological Park of Tsimbazaza, Antananarivo, Madagascar. Thedried stem bark powder (600 g) was soxhlet extracted withcyclohexane over seven hours to give a dark viscous extract (70 g).The powder defatted was extracted with 80% ethanol over onehour. The ethanolic extract was filtered and concentrated undervacuum giving a honey like viscous residue (20 g). This was treatedwith HCl aqueous 1 N and extracted with CHCl3. The aqueouslayer was made basic to pH 8 with NH4OH. The solution wasextracted with CHCl3, the CHCl3 dried with Na2SO4 was filteredand evaporated to yield 4 g of residue. This was chromatographiedon 160 g Al2O3 (activity III) using a gradient of hexane and AcOEtto give 120 fractions. The eluant hexane AcOEt 60/40 yielded 50mg of rutaceline, a yellow powder. The concerted use of one andtwo dimensional NMR methods 1H, 13C, (COSY, HMBC, HMQC,NOESY) and mass spectrum allowed the identification ofrutaceline as a hydrochloride of 2,3-methylenedioxy-8-hydroxy-7-methoxy-benzo[C]phenanthridine (MM=409).

Cell culture. The human colorectal adenocarcinoma cell line, Caco-2, and African green monkey kidney, Vero cells, were purchasedfrom ATCC. The Caco-2 cell line was cultured in DMEMsupplemented with 10% heat inactivated FBS and 0.1% antibiotic;and the Vero cell line was cultured in RPMI supplemented with10% heat inactivated FBS and 0.1% antibiotic and 1% glutamine.Both cell lines were cultured as a monolayer at 37ÆC in ahumidified incubator with 5% CO2.

MTT assay. The assay was performed using the method describedby Mossman (16) slightly modified. Samples containing 200 Ìl cellsuspension (2x104 cells/ml) were plated in 96-well flat-bottomedmicrotiter plates. After adherence of the cells within 24 h ofincubation at 37ÆC, Rutaceline at concentrations ranging from1 Ìg/ml to 100 Ìg/ml was added (3-4 wells per concentration).After additional incubation time (24 and 48 h) at 37ÆC in ahumidified incubator with 5% CO2, 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) dissolved in PBS andsterile filtered was added to all the wells at a finalconcentration of 1 mg/ml. Following 1 h of incubation, thegenerated formazan was dissolved with 100 ml dimethylsulphoxide(DMSO) per well. The optical density was measured using anenzyme-linked immunosorbent assay (ELISA) plate reader (MerckWhitehouse Station NJ USA, ELISA system MIOS version 3.2.) at550 nm. The Rutaceline concentration that caused 50% inhibitionof cell growth (IC50) was calculated for each cell line.

Cell cycle analysis. The cell cycle was assessed through flowcytometry by using a fluorescence-activated cell sorter (FACS).The cells were cultured in 6-well flat bottomed microtiter platescontaining 2 ml of cell suspension. The number of cells wasdetermined by calculation according to the number of cells/well inthe 96-well plates (3,500). After 24 h of incubation at 37ÆC with5% CO2, Rutaceline was added at the respective IC50concentration for each cell type. Following 24 and 48 h ofincubation, cells were harvested by mild trypsinization, collected bycentrifugation and stained in Tris-buffered saline (TBS) for 1 h at4ÆC. FACS analysis was carried out at 488 nm in an Epics XL flowcytometry (Coulter Corporation, Hialeah, FL, USA). Data from

in vivo 21: 417-422 (2007)

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12,000 cells were collected and analysed using a Multicycleprogram (Phoenix Flow Systems, San Diego, CA, USA). Allexperiments were performed in triplicate.

Assessment of apoptosis. Apoptosis was assessed using an AnnexinV-FITC kit binding assay and analyzed by FACS. Cell culture,treatment with Rutaceline and cell collection, were carried out asdescribed in the cell cycle analysis section. Thereafter, cells wereresuspended in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140mM NaCl, 2.5 mM CaCl2). The Annexin V-FITC was addedaccording to the product insert and incubated for 30 min at roomtemperature in the dark. One min before FACS analysis,propidium iodide (PI) was added at a concentration of 20 Ìg/ml.Approximately 500,000 viable cells were counted to assessapoptosis. Experiments were performed in triplicate.

DNA fragmentation assays. Cells were cultured (5x105 cell/ml) in25 cm2 flasks, total vol. 10 ml/flask at 37ÆC. Rutaceline was addedat concentrations corresponding to the IC50 concentration for eachcell type or omitted for the controls. After 24 h incubation, thecells were washed with PBS (5 ml) and the DNA was extracted asfollow: cells were harvested in 2 ml of 20 mM Tris-HCl, pH 8.0,containing 5 mM EDTA and 1% sodium N-lauryl-sarcosinate (w/v).After 5 min, proteinase K (final concentration 200 Ìg/ml) was addedand the mixture incubated overnight at 37ÆC. Subsequently, 1 vol.of 7.5 M ammonium acetate and 1 vol. of chloroform/phenol (1:2)were added. The mixture was shaken for 20 min, and centrifuged at2,200 xg for 5 min at 20ÆC. The upper phase was collected andcombined with one vol. of SEVAG (chloroform/isoamyl alcohol; 34:1, v/v) to eliminate all trace of phenol. Then, the mixture wasshaken for 20 min and centrifuged again at 2,200 xg for 5 min at20ÆC. The aqueous phase was collected and the RNA was digestedwith DNAse free RNAse A (final concentration 9.8 Ìg/ml) at 37ÆCfor 30 min. Then, 1 vol. of SEVAG was added followed bycentrifugation at 2,200 xg for 5 min at 20ÆC. The upper phase wascollected and the DNA was precipitated with two vol. of ice-coldabsolute ethanol for 2 h at –20ÆC. After centrifugation at 2,500 xgfor 45 min at 4ÆC, the pellet was washed with a solution of ice-cold 70% ethanol, the pellet was air dried and the DNA wasresuspended in 1 ml of 20 mM Tris-HCl, pH 8.0, containing 5mMof EDTA for quantification by UV-spectrophotometry at 254 nm,the purity of DNA was controlled by the ratio of absorbance at260 and 280 nm, respectively. The acceptable ratio was set at 1.8.

Under such conditions, 10-20 Ìg of DNA was loaded onto a 1%agarose gel for electrophoresis (70 V, 30 mA for 1 h approximately).The gel was stained with ethidium bromide and photographedunder UV irradiation.

Single cell gel electrophoresis (SCGE). Cells were cultured in 6-well flat-bottomed microtiter plates containing 2 ml of cellsuspension (200,000 cells approximately). After 24 h ofincubation at 37ÆC with 5% CO2, Rutaceline was added at itsrespective IC20 concentration for each cell type. Methioninemethanosulfonate (MMS) at 300 ÌM was used as positivecontrol. Following 24 h of incubation, Caco-2 and Vero cellswere detached from the well with 0.05% trypsin solution andcollected by centrifugation. Cells were embedded in 0.6% LMPagarose prepared in MilliQ water (18 Mø) and layered oncommercial pre-coated slides (Trevigen®, Gaithersburg MD,Maryland, USA). The slides were placed in lysing buffer (2.5M

NaCl, 100 mM Na2EDTA, 10 mM Tris pH 10, N-lauryl-sarcosine1% (w/v)) with 1% Triton X-100 for 1 h at 4ÆC. The DNA of thenuclei in the agarose gels was unwound for 40 min inelectrophoresis buffer (1 mM Na2EDTA and 300 mM NaOH,pH>13). The SCGE slides were then electrophoresed for 30 minat 25V and 300 mA at 4ÆC. After neutralization with 400 mMTris buffer (pH 7.5), the slides were dried at room temperature.For image analysis the slides were hydrated and stained with 10Ìl 4,6-diamidino-2-phenylindole (DAPI).

Statistical analysis. Results are expressed as Tail Moment (migratedDNA x tail length). The data are presented as mean±SEM andanalyzed using the Mann-Whitney U-test and Student’s t-test. Alimit of p=0.05 was accepted for significant differences.

Results

Antiproliferative effect. The IC50 concentrations were obtainedby the MTT test. Rutaceline concentrations were plottedagainst the percentage of cell proliferation after 24 and 48 hof incubation. The IC50 values obtained in the Caco-2 cellswere: 110±4 Ìg/ml (269 ÌM) for 24 h and 20±2 Ìg/ml (49ÌM) for 48 h (Figure 1A). The IC50 values obtained in theVero cells were: 115±2 Ìg/ml (281 ÌM) for 24 h and 90±2Ìg/ml (220 ÌM) for 48 h (Figure 1B).

Cell cycle analysis and apoptosis. Cell cycle analysis of theCaco-2 and Vero cells was performed after 24 and 48 h ofincubation with Rutaceline at the respective IC50concentrations. Compared to the untreated cells, Rutacelineinduced cell cycle arrest in the G0/G1 phase as well as adecrease of cells undergoing the S phase, after 24 and 48 hon both cells lines (Figure 2). The student’s t-test was usedto analyze the differences observed between the controlsand the Rutaceline treated samples.

The assessment of apoptosis in Caco-2 and Vero cells wasperformed after 24 and 48 h of incubation with Rutaceline, atthe same concentrations mentioned above for the analysis ofthe cell cycle. FACS analysis was used to differentiate viablecells (Annexin V– and PI–), early apoptotic cells (Annexin V+

and PI–), late apoptotic/ necrotic cells (Annexin V+ and PI+)and necrotic cells (Annexin V– and PI+). The results showedthat Rutaceline did not induce apoptosis in either the Caco-2cells or the Vero cells at IC50 concentrations following 24 or48 h of incubation (Figure 3).

DNA damage. The results indicated that Rutaceline inducedDNA fragmentation in the Caco-2 cells and in the Verocells as shown by a diffuse pattern characteristic of cellulardeath by necrosis (Figure 4 A and B).

The results of the comet assay (expressed as TailMoment) are shown in Figure 5 for each cell line, for all thedifferent treatments with or without Rutaceline, (p<0.05).

The comet test results showed a concentration-dependentclastogenic effect of Rutaceline in both cell lines and at

Pachón et al: Anticancer Effect of Rutaceline

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IC20, was slightly higher than that produced by MMS. In ourexperiment, the Vero cells showed more sensitivity to thegenotoxic compound than the Caco-2 cells.

The percentage of apoptotic cells (identified as nucleishowing a characteristic "hedgehog" image) was lower than3% in both the Caco-2 and Vero Rutaceline treated cells atIC10 and IC20 and these were not significantly different fromthat of untreated cells.

Discussion

The present experiments were designed to study theantiproliferative effect of Rutaceline, a new benzophe-nanthridine isolated from Zanthoxylum madagascariense. Thedata clearly demonstrate that Rutaceline inhibited cellproliferation in the Caco-2 and Vero cell lines with IC50 in theÌM range (48 ÌM - 220 ÌM). Preliminary studies on the Caco-

2 cell line showed that Rutaceline inhibited the synthesis ofDNA (data not shown). The present data corroborated thisfact since a disruption in G0/G1 phase of the cell cycle of thiscell line was observed preventing cells from entering into the Sphase of the cell cycle, and preventing cell proliferation. Thisindicates that Rutaceline has similar effects as Sanguinarineand Chelerythrine regarding cytotoxic capacity (10, 11).

The fact that the IC50 concentrations decreased from theincubation time of 24 h to 48 h may be explained by abiotransformation of Rutaceline into a more toxic derivativein both cell lines or by its accumulation inside the cells.Both hypothesis need to be further investigated.

The antitumor effect of a drug can be due to cytostaticand/or cytotoxic effects. A substance is cytostatic if itprevents cell growth and/or produces a disruption in anyone of the three phases of the cell cycle. On the other hand,a cytotoxic substance causes cellular death by apoptosis or

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Figure 1. Effect of Rutaceline on cell proliferation of Caco-2 cells (A) and Vero cells (B), after 24 and 48 h of incubation. The relative percentage of cellproliferation was calculated considering that untreated cells at 24 and 48 h show 100% cell proliferation. Each point represents the mean of triplicateexperiments.

Figure 2. Distribution of percentage in different phases of the cell cycle of untreated and treated Caco-2 cells (A), and Vero cells (B), after 24 and 48 hincubation with Rutaceline, at respective IC50 concentrations. Data shown are mean value of three independent experiments. * p<0.05. CT: control(untreated).

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Figure 3. Percentage of early and late apoptotic and necrotic cells assessed by flow cytometry analysis of Annexin V-FITC staining and PI accumulationafter exposure of Caco-2 cells (A) and Vero cells (B) to Rutaceline at respective IC50 concentrations after 24 and 48 h of incubation. Values are expressedas means±SEM of three independent experiments. CT: control.

Figure 4. Agarose gel electrophoresis analysis of DNA from Caco-2 cells (A) and Vero cells (B). Cells were incubated in the absence of Rutaceline (line1) treated with Rutaceline at IC50 concentrations for 24 h (line 2) M:DNA size marker.

Figure 5. Results of comet assay on (A) Caco-2 cells (CCT: Control Caco-2; C10: Caco-2 IC10; C20: Caco-2 IC20; MMSC: Caco-2 with MethionineMethanosulfonate (MMS) positive control) and (B) Vero cells (VCT: Control Vero; V10: Vero IC10; V20: Vero IC20; MMSV: Vero positive control MMS.

necrosis. Rutaceline inhibits cell proliferation and inducescell cycle disruption in the G0/G1 phase accompanied by aslight increment of apoptotic cells after 48 h of treatmentand DNA fragmentation at IC50 concentrations in both celllines. This apoptotic cell death may be increased withincreasing Rutaceline concentrations in the culture medium.

In general, the Vero cells showed more sensitivity to thegenotoxic compound than Caco-2 cells. From the percentageof "hedgehog" comets observed, the increase in tail momentvalues could not be attributed to apoptosis (17, 18).

Caco-2 cells have been shown by Berger et al. (19) to bearthe multidrug resistance protein-2, (MRP-2), which confers onthem some resistance to several antitumor drugs. Since theyare very sensitive to Rutaceline and since the IC50 evendecreases after 48 h incubation indicating a higher sensitivityto this drug, it may be hypothesized that Rutaceline is notrefluxed from Caco-2 cells by MRP-2. This is an additionalinteresting indication for further applications.

Acknowledgements

This work has received financial support from the government ofCatalunya (ITT program of the Work Community of Pyrenees,2005SGR00204 and 2004XT-00089) and from the Spanish Ministryof Science and Technology (PPQ2003-06602-C04-01,-03,AGL2006-12210-C03-02).

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Received November 3, 2006Revised December 21, 2006

Accepted January 2, 2007

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PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain [email protected]

PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy [email protected]

PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China [email protected]

PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan [email protected]

PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia [email protected]

PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA [email protected]

PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA [email protected]

PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan [email protected]

PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK [email protected]

ADVISORY BOARD Prof. Berhanu M. Abegaz Gaborone, Botswana

Prof. Viqar Uddin Ahmad Karachi, Pakistan

Prof. Øyvind M. Andersen Bergen, Norway

Prof. Giovanni Appendino Novara, Italy

Prof. Yoshinori Asakawa Tokushima, Japan

Prof. Lee Banting Portsmouth, U.K.

Prof. Julie Banerji Kolkata, India

Prof. Anna R. Bilia Florence, Italy

Prof. Maurizio Bruno Palermo, Italy

Prof. César A. N. Catalán Tucumán, Argentina

Prof. Josep Coll Barcelona, Spain

Prof. Geoffrey Cordell Chicago, IL, USA

Prof. Ana Cristina Figueiredo Lisbon, Portugal

Prof. Cristina Gracia-Viguera Murcia, Spain

Prof. Duvvuru Gunasekar Tirupati, India

Prof. Kurt Hostettmann Lausanne, Switzerland

Prof. Martin A. Iglesias Arteaga Mexico, D. F, Mexico

Prof. Leopold Jirovetz Vienna, Austria

Prof. Vladimir I Kalinin Vladivostok, Russia

Prof. Niel A. Koorbanally Durban, South Africa

Prof. Karsten Krohn Paderborn, Germany

Prof. Chiaki Kuroda Tokyo, Japan

Prof. Hartmut Laatsch Gottingen, Germany

Prof. Marie Lacaille-Dubois Dijon, France

Prof. Shoei-Sheng Lee Taipei, Taiwan

Prof. Francisco Macias Cadiz, Spain

Prof. Imre Mathe Szeged, Hungary

Prof. Ermino Murano Trieste, Italy

Prof. M. Soledade C. Pedras Saskatoon, Canada

Prof. Luc Pieters Antwerp, Belgium

Prof. Peter Proksch Düsseldorf, Germany

Prof. Phila Raharivelomanana Tahiti, French Polynesia

Prof. Luca Rastrelli Fisciano, Italy

Prof. Monique Simmonds Richmond, UK

Dr. Bikram Singh Palampur, India

Prof. John L. Sorensen Manitoba, Canada

Prof. Valentin Stonik Vladivostok, Russia

Prof. Winston F. Tinto Barbados, West Indies

Prof. Sylvia Urban Melbourne, Australia

Prof. Karen Valant-Vetschera Vienna, Austria

HONORARY EDITOR

PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

[email protected]

Pyranocoumarin and Triterpene from Millettia richardiana Manitriniaina Rajemiarimirahoa,b,h*, Jean Théophile Banzouzib,c, Stéphane Richard Rakotonandrasanaa, Pierre Chalardd, Françoise Benoit-Vicale,f, Léa Herilala Rasoanaivog, Amélie Raharisololalaog and Roger Randrianjah

aCentre National d’Application des Recherches Pharmaceutiques (CNARP), BP 702 Antananarivo 101, Madagascar bCentre d’Etude et de Recherche Médecins d’Afrique (CERMA), 43, rue des Glycines, 91600 Savigny sur Orge, France cInstitut de Chimie des Substances Naturelles (ICSN-CNRS), Bâtiment 27, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France dLaboratoire de Chimie des Hétérocycles et Glucides (LCHG), Ecole Nationale Supérieure de Chimie de Clermont Ferrand (ENSCCF), Ensemble Scientifique des Cézeaux, 24 av. des Landais, BP 10187, 63174 Aubiere Cedex, France eCNRS/LCC (Laboratoire de Chimie de Coordination) UPR8241; 205, route de Narbonne, BP 44099 -31077 Toulouse Cedex 4, France fService de Parasitologie-Mycologie, CHU Toulouse Rangueil - 1 av. Jean Poulhès, TSA 50032, 31059 Toulouse Cedex 9, France gLaboratoire de Chimie des Substances Naturelles et de Chimie Organique, Département de Chimie Organique, Faculté des Sciences de l’Université d’Antananarivo, BP 906 Antananarivo 101, Madagascar hEcole Supérieure Polytechnique d’Antananarivo (ESPA), BP 1500 Antananarivo 101, Madagascar

[email protected]

Received: August 14th, 2012; Accepted: May 5th, 2013

From the stem bark of a Madagascan endemic plant, Millettia richardiana Baill., lonchocarpenin and betulinic acid were isolated and their structures established by spectroscopic methods. The analysis of dichloromethane fractions suggested the presence of β-amyrin, lupeol, palmitic acid, linoleic acid and stearic acid. Except for β-amyrin and lupeol, these compounds are described for the first time for the Millettia genus.

Keywords: Millettia richardiana, Pyranocoumarin, Triterpene, Lonchocarpenin, Betulinic acid. Millettia is a large genus of Fabaceae (about 100 species). Eight species are represented in Madagascar, of which M. richardiana Baill. is used for making furniture [1]. However, the African species showed antiparasitic [2a], anti-inflammatory [2b], molluscicidal [2c], leishmanicidal [2d] and antiplasmodial activities [2e]. Chemical investigation of Millettia species has led to the isolation of isoflavonoids [3a], steroids [3b], triterpenes [3c], coumarins [3d], and alkaloids [3e]. Pyridine metabolism in leaves of M. pinnata has also been studied [4]. In this paper we report the isolation and identification of a pyranocoumarin, lonchocarpenin (1), betulinic acid (2), β-amyrin, lupeol, palmitic acid, linoleic acid and stearic acid from the methanolic extract of the stem bark of M. richardiana.

Compound 1, a yellow powder, showed a molecular pseudo ion at m/z 449 suggesting an atomic mass of 448 and the molecular formula C27H28O6. The 1H NMR spectrum showed typical dimethylpyran ring signals at δH 6.50 (H-4’, d, J= 10 Hz), 5.76 (H-3’, d, J= 10 Hz), and 1.46 (2’-Me2, s) and 1,4-disubstitued aromatic ring signals at δH 7.47 (H-6" and H-2", d, J= 8.8 Hz) and 6.95 (H-5" and H-3", d, J= 8.8 Hz). The 1H NMR spectrum also revealed the presence of a C-prenyl group from the resonances at δH 3.47 (H2-1"’, d, J= 7.4 Hz), 5.21 (H-2"’, t, J= 7.3 Hz), 1.83 (H3-4"’, s) and 1.66 (H3-5"’, s), two methoxyl groups (δH 3.93 and 3.81) and the characteristic OH-4 resonance at δH 10.03 [5]. The HMBC correlations of H2-1"’ to C-7, C-8, C-8a, C-2"’, C-3"’ and the proton of a hydroxyl group to C-3, C-4 and C-4a indicated the location of the prenyl group at C-8 on the A ring and confirmed the location of the OH at C-4 on ring B. The correlations of H3 at δH 3.93 to C-5 and H3 at δH 3.81 to C-4" permitted the location of the two methoxyl groups at C-5 and C-4", respectively. The correlations of H-4’ to C-5, C-6, C-7, C-2’ and H-2"/H-6" to C-3

Lonchocarpenin (1) Betulinic acid (2) Table 1: NMR spectroscopic data of compound 1 (1H, 13C 400 MHz, CDCl3).

Position δC δH (H, m, J) COSY HMBC (H => C) 2 162.68 3 103.93 4 160.64 4a 101.71 5 150.18 6 110.58 7 154.67 8 114.95 8a 151.52 2’ 77.46 3’ 131.39 5.76, 1H, d (10) 4’ 2’, 5’, 6 4’ 115.65 6.50, 1H, d (10) 3’ 2’, 5, 6, 7 5’ 28.10 1.46, 6H, s 2’, 3’, 5’ 1" 123.82

2", 6" 132.01 7.47, 2H, d (8.8) 3" 3, 4" 3", 5" 113.72 6.95, 2H, d (8.8) 2" 1", 5", 4"

4" 158.84 1’" 22.06 3.47, 2H, d (7.4) 2’" 7, 8, 8a, 2’", 3’" 2’" 121.37 5.21, 1H, t (7.4) 1’" 3’" 132.53 4’" 25.98 1.66, s 2’", 3’", 5’" 5’" 18.27 1.83, s 2’", 3’", 4’"

4-OH 10.03, s 3, 4, 4a 5-OMe 64.55 3.93, s 5 4"-OMe 55.47 3.81, s 4"

a Carbon number to which the proton is correlated.

and C-4" suggested that the chromene ring was connected to C-6 and C-7 on ring A and the aromatic ring to C-3 on the B ring. This compound is lonchocarpenin (1) isolated for the first time in 1969 [6]. The proton (δH) and carbon (δC) chemical shifts of 1 are

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1100 Natural Product Communications Vol. 8 (8) 2013 Rajemiarimiraho et al.

reported here for the first time. By concerted use of one and two-dimensional NMR spectroscopy, compound 2 was identified as betulinic acid [7a,b]. However, the assignments of carbons 15 and 21 were not similar to those previously reported [7b]. Therefore, we revised the chemical shifts for C-15 (δC 30.26) and C-21 (δC 31.03) of betulinic acid. Further components identified were β-amyrin (4) [7c], lupeol (5) [8], palmitic acid (6), linoleic acid (7) and stearic acid (8) [9].

Experimental

General: NMR, BRÜKER Avance AC400; MS, Waters 2995/2996-Micromass Q-Tof micro spectrometer (ES+-MS) and Agilent 5975 spectrometer (EI-MS).

Plant materials: Millettia richardiana Baill. (Fabaceae) air-dried stem bark, collected in Sahafary, DIANA’s Region, Madagascar, was identified by botanists at the Centre National d'Application des Recherches Pharmaceutiques, Antananarivo, Madagascar, where a voucher specimen has been deposited in the Herbarium. Extraction and isolation: Dried stem bark (1 kg) was extracted with MeOH (3 x 4 L) at room temperature for 3 days each time. After concentration under reduced pressure, the MeOH extract (80 g) was suspended in H2O (500 mL) and partitioned sequentially using CH2Cl2, AcOEt and n-BuOH (500 mL x 3) furnishing dichloromethane (21.8 g), ethyl acetate (3.3g), n-butanol (18.7 g) and aqueous (30.0 g) extracts, respectively. The CH2Cl2 extract (18.0 g) was chromatographed over a silica gel column (160 g), eluting successively with CH2Cl2 and mixtures of CH2Cl2-CH3OH (97:3, 95:5 and 90:10), to give 6 fractions, E2A (CH2Cl2, 3.8 g), E2B (CH2Cl2, 1.1 g), E2C (CH2Cl2, 1.6 g), E2DE [CH2Cl2-CH3OH (97:3) 8.5 g], E2F [CH2Cl2-CH3OH (95:5),1.6 g] and E2G [CH2Cl2-CH3OH (90:10), 1.5 g]. Fraction E2B (1.1 g) was submitted to CC over silica gel (25 g), eluted successively with a gradient solvent system of n-hexane-CH2Cl2 (100:0 0:100) and a mixture of CH2Cl2-CH3OH (90:10), to give fractions E2B1- E2B6. Fraction E2B4 [n-hexane-CH2Cl2 (50:50), 207 mg] was subjected to a silica gel column (20 g), eluting with a gradient solvent system of

cyclohexane-EtOAc (100:0 85:15), to obtain compound 1 (85:15, 23.8 mg).

Fraction E2C (1.4g) was chromatographed over a silica gel column (33g), eluting successively with a gradient solvent system of n-hexane-CH2Cl2 (75:25 0:100) and mixtures of CH2Cl2-CH3OH (94:6 and 80:20), to provide fractions E2C1-E2C6. Fraction E2C1 [n-hexane-CH2Cl2 (75:25), (310 mg)] was submitted to flash CC (Easy VarioFlashD17, 10 g) and eluted with a gradient solvent system of cyclohexane-EtOAc (99:1 95:5) to afford a white solid (98:2, 70.3mg) identified as a mixture of compounds 3 and 4. Fraction E2C5 [CH2Cl2-CH3OH (94:6), 490 mg] was subjected to flash CC (RediSep, 12g) and eluted with a gradient solvent system of cyclohexane-EtOAc (89:11 50:50) to give compound 2 (81:19, 79.7 mg).

Fraction E2DE (9 g) was submitted to CC over silica gel (120 g) and eluted successively with a gradient solvent system of n-hexane- CH2Cl2 (100:0 0:100) and mixtures of CH2Cl2-CH3OH (94:16 and 80:20) to obtain fractions E2DE1-E2DE7. Fraction E2DE7 [CH2Cl2-CH3OH (80:20), 3 g] was chromatographed over silica gel (90 g), eluting with mixtures of CH2Cl2-CH3OH (100:0, 90:10 and 80:20) to give fractions E2DE7A-E2DE7C. Fraction E2DE7A (100:0, 760mg) was subjected to flash CC (Chromabond, 15 g) and eluted with a gradient solvent system of cyclohexane-EtOAc (100:0 15:85) to yield a yellow oil (97:3, 40.0 mg) identified as a mixture of compounds 5-7.

Acknowledgments - We gratefully acknowledge the SCAC of the embassy of French in Madagascar for financial support. We thank the team of the LCHG who have welcomed us heartily within its group. We wish to thank Mr Régis Egrot of ENSC, Clermont Ferrand, for the realization of the proton experiments. We wish to thank also Mr Bertrand Légeret, Ingénieur d'Etudes CNRS, laboratory SEESIB, UMR6504, and Doctor Gille Figueredo, responsible for the laboratory LEXVA Analytique, ENSC Clermont Ferrand, for the realization of the mass spectra.

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Natural Product Communications Vol. 8 (8) 2013 Published online (www.naturalproduct.us)

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Natural Product Communications 2013

Volume 8, Number 8

Contents

Original Paper Page

Natural Clovanes from the Gorgonian Coral Rumphella antipathies Hsu-Ming Chung, Wei-Hsien Wang, Tsong-Long Hwang, Yang-Chang Wu and Ping-Jyun Sung 1037

Microbial Biotransformation of 16α,17-Epoxy-ent-kaurane-19-oic acid by Beauveria sulfurescens ATCC 7159-F Ricardo A. Furtado, G. M. Kamal B. Gunaherath, Jairo K. Bastos and A. A. Leslie Gunatilaka 1041

Establishment of In vitro Adventitious Root Cultures and Analysis of Andrographolide in Andrographis paniculata Shiv Narayan Sharma, Zenu Jha and Rakesh Kumar Sinha 1045

New Cycloartane-type Triterpenes from Marcetia latifolia (Melastomataceae) and in silico Studies on Candida parapsilosis Protease Tonny C. C. Leite, Franco H. A. Leite, Ivo J. C. Vieira, Raimundo Braz Filho and Alexsandro Branco 1049

Triterpene Glycosides from the Sea Cucumber Eupentacta fraudatrix. Structure and Biological Action of Cucumariosides I1, I3, I4, Three New Minor Disulfated Pentaosides Alexandra S. Silchenko, Anatoly I. Kalinovsky, Sergey A. Avilov, Pelageya V. Andryjaschenko, Pavel S. Dmitrenok, Ekaterina A. Martyyas and Vladimir I. Kalinin 1053

Improved Extraction and Complete Mass Spectral Characterization of Steroidal Alkaloids from Veratrum californicum Christopher M. Chandler, Jeffrey W. Habig, Ashley A. Fisher, Katherine V. Ambrose, Susana T. Jiménez and Owen M. McDougal 1059

TLC-Image Analysis of Non-Chromophoric Tuberostemonine Alkaloid Derivatives in Stemona Species Sumet Kongkiatpaiboon, Vichien Keeratinijakal and Wandee Gritsanapan 1065

Two New Compounds from Gorgonian-associated Fungus Aspergillus sp. Xin-Ya Xu, Xiao-Yong Zhang, Fei He, Jiang Peng, Xu-Hua Nong and Shu-Hua Qi 1069

New Metabolites from the Algal Associated Marine-derived Fungus Aspergillus carneus Olesya I. Zhuravleva, Shamil Sh. Afiyatullov, Ekaterina A. Yurchenko, Vladimir A. Denisenko, Natalya N. Kirichuk and Pavel S. Dmitrenok 1071

Chiroptical Studies of Flavanone Marcelo A. Muñoz, María A. Bucio and Pedro Joseph-Nathan 1075

Flavonoids with Anti-HSV Activity from the Root Bark of Artocarpus lakoocha Boonchoo Sritularak, Kullasap Tantrakarnsakul, Vimolmas Lipipun and Kittisak Likhitwitayawuid 1079

Orphan Flavonoids and Dihydrochalcones from Primula Exudates Tshering Doma Bhutia, Karin M. Valant-Vetschera and Lothar Brecker 1081

First Identification of -Glucosidase Inhibitors from Okra (Abelmoschus esculentus) Seeds Wannisa Thanakosai and Preecha Phuwapraisirisan 1085

In vitro Anti-proliferative Effect of Naturally Occurring Oxyprenylated Chalcones Serena Fiorito, Francesco Epifano, Celine Bruyère, Robert Kiss and Salvatore Genovese 1089

Kaempferol 3,7,4´-glycosides from the Flowers of Clematis Cultivars Keisuke Sakaguchi, Junichi Kitajima and Tsukasa Iwashina 1093

7-O-Methylpelargonidin Glycosides from the Pale Red Flowers of Catharanthus roseus Fumi Tatsuzawa 1095

Pyranocoumarin and Triterpene from Millettia richardiana Manitriniaina Rajemiarimiraho, Jean Théophile Banzouzi, Stéphane Richard Rakotonandrasana, Pierre Chalard, Françoise Benoit-Vical, Léa Herilala Rasoanaivo, Amélie Raharisololalao and Roger Randrianja 1099

A Concise and Efficient Total Synthesis of α-Mangostin and β-Mangostin from Garcinia mangostana Dandan Xu, Ying Nie, Xizhou Liang, Ling Ji, Songyuan Hu, Qidong You, Fan Wang, Hongchun Ye and Jinxin Wang 1101

Variation in the Contents of Neochlorogenic Acid, Chlorogenic Acid and Three Quercetin Glycosides in Leaves and Fruits of Rowan (Sorbus) Species and Varieties from Collections in Lithuania Kristina Gaivelyte, Valdas Jakstas, Almantas Razukas and Valdimaras Janulis 1105

A New Cytotoxic Phenolic Derivative from the Roots of Antidesma acidum Sutin Kaennakam, Jirapast Sichaem, Pongpun Siripong and Santi Tip-pyang 1111

A New Nervogenic Acid Glycoside with Pro-coagulant Activity from Liparis nervosa Qin Song, Qingyao Shou, Xiaojun Gou, Fengzhen Chen, Jing Leng and Weifeng Yang 1115

Biologically Active Secondary Metabolites from Asphodelus microcarpus Mohammed M. Ghoneim, Guoyi Ma, Atef A. El-Hela, Abd-Elsalam I. Mohammad, Saeid Kottob, Sayed El-Ghaly, Stephen J. Cutler and Samir A. Ross 1117

Dibenzylbutane Lignans from the Stems of Schisandra bicolor Yinning Chen, Na Li, Yuehui Zhu, Cuilan Zhang, Xiaofei Jiang, Jianxiang Yang, Zhifang Xu, Samuel X. Qiu and Riming Huang 1121

Absolute Configuration of Falcarinol (9Z-heptadeca-1,9-diene-4,6-diyn-3-ol) from Pastinaca sativa Mireia Corell, Emile Sheehy, Paul Evans, Nigel Brunton and Juan Valverde 1123

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Journal of Pharmacognosy and Phytochemistry 2014; 2 (5): 98-105 ISSN 2278-4136 JPP 2014; 2 (5): 98-105 Received: 21-12-2013 Accepted: 04-01-2014 Volasoa Herilalaina Victorine Rambeloson Department of Chemistry, Faculty of Science, University of Fianarantsoa, Madagascar, P.O. Box 1264, Fianarantsoa 301, Madagascar Email:[email protected] Léa Herilala Rasoanaivo Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101, Madagascar Email: [email protected] Anne Wadouachi Laboratory of Antimicrobial Glycochemistry and Agroressources FRE CNRS 3517 University of Picardie Amiens France Email: [email protected] Rivoarison Randrianasolo Institute for Food Toxicology and Analytical Chemistry, Analytical Chemistry and Endocrinology, University of Veterinary Medicine, Bischofsholer Damm 15/123, D-30173 Hannover, Germany Email: [email protected] Hans Christoph Krebs Institute for Food Toxicology and Analytical Chemistry, Analytical Chemistry and Endocrinology, University of Veterinary Medicine, Bischofsholer Damm 15/123, D-30173 Hannover, Germany Amelie Raharisololalao Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101, Madagascar Email: [email protected]

Correspondence Volasoa Herilalaina Victorine Rambeloson Department of Chemistry, Faculty of Science, University of Fianarantsoa, Madagascar, P.O. Box 1264, Fianarantsoa 301, Madagascar Email:[email protected] Tel: +261324327885

Two new Xanthones from Garcinia chapelieri: Chapexanthone A; chapexanthone B

Volasoa Herilalaina Victorine Rambeloson, Léa Herilala Rasoanaivo, Anne Wadouachi, Rivoarison Randrianasolo, Hans Christoph Krebs and Amelie Raharisololalao

Abstract

From the stem bark hexanic extract of Garcinia chapelieri, chapexanthone A, B were isolated using combinations of column and thin-layer chromatographic methods. Their structures were elucidated by NMR spectroscopic methods, mainly 1D and 2D NMR.

Keywords: Garcinia chapelieri, Clusiaceae, Xanthones, Chapexanthone A, Chapexanthone B.

1. Introduction Garcinia chapelieri is an endemic plant of Madagascar. The decoction of steam with leaves of Garcinia chapelieri is used to treat yellow fever, irritations on the skin, stomachache and also toothache. Extensive phytochemical studies have shown that Garcinia species are rich in a variety of oxygenated and prenylated xanthones [2]. Some of these exhibit a wide range of biological and pharmacological activities [3, 6]: antifungal [4, 7], anti-thrombotic, vasorelaxant, agregation inhibitory platelet [8], anti-HIV [ 5, 9 ] and antimalarial [10]. According to available literature, no phytochemical research work has been carried out on this plant. Further investigation on the hexanic extract of the bark of the Garcinia chapelieri has resulted in the isolation of two xanthone. This paper describes structural elucidation of xanthone isolated from this plant. 2. Materials and Methods 2.1 General: 1D (1H, 13C, DEPT) and 2D (HSQC, HMBC) NMR spectra were measured on Varian Unity 400 and Varian Innova 500 spectrometers. Chemical shifts are shown in devalues (ppm) with tetramethylsilane (TMS) as internal standard. HREIMS were determined on VG ZAB 2SEQ mass spectrometer. Column chromatography (CC) was carried out on silica gel F254 (Merck) in glass blades. Thin layer chromatography (TLC) was performed on precoated Si Gel plates (Merck Kiesel gel 60GF254) and detection wavelength (254 and 365 nm) was used. 2.2. Plant material: Garcinia chapelieri H.Perr. (Clusiaceae) was collected in July 2012 in Farafangana Manombo, Vatovavy Fitovinany’s Region Madagascar and was identified by botanists at the Parc National Botanic and Zoologique Tsimbazaza, Antananarivo, Madagascar where a voucher specimen has been deposited in the Herbarium. 2.3 Extraction and isolation: The stem bark of Garcinia chapelieri were dried ground, reduced on powder. The powder was macerated successively with hexane, ethyl acetate and MeOH. The solvents were evaporated under reduced pressure to obtain crude residue: green, green and red solid gum (1.3 g; 7.75g; 12.45 g). The hexanic extract was subjected to column chromatography (silicagel, 2x60 cm) and was eluted with a gradient profil of hexane and acetone to give 95 mains fractions.

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Journal of Pharmacognosy and Phytochemistry

The fractions 5-6 were obtained after 100 mL and the fractions 7-8 were obtained after 120 mL and after elution with hexane and acetone (95:5). The purification of fractions 5-6 and the 7- 8 were carried out by column chromatography on Sephadex LH-20 with CDCl3-MeOH (80:20). 3. Result and Discussion: Reported in tables 1 and 2 The dried and ground stem bark of Garcinia chapelieri collected from Farafangana (Madagscar) was extracted with n hexanes, ethyl acetate and methanol. The hexanic was repeatedly chromatographied over Si gel and Sephadex LH-20 to gives 2 xanthones (1-2) Xanthone A, chapexanthone A (1) was obtained as a yellow powder. Its molecular formula C28H36O7 was deduced by HREIMS mass spectroscopy (m/z 484.25). The 1H NMR spectrum shows five aromatic protons, one of which couple ortho at δ 7.28 ppm (H-5) and 7.87 (H-6) , one of which couple meta at δ7.28 ppm (H-5) and at δ 7.06 ppm (H-7), at δ 6.64 ppm (H-2) and (H-4) and implying that the compound 1 is tri-O-substituted xanthone. In addition to the 1H NMR spectra show the presence of protons in the region of the aliphatic protons, indicating the presence of a linear chain, showed signal in the 0.7-3.90 ppm range. The presence of long chain moiety in the molecule was indicated by the peaks in the region of weak field. The 13C Broadband spectra showed 28 carbon atom signals indicating 13 correspond to the carbon atoms of skeleton xanthone there is no symmetry in the molecule. The presence of linear chain was indicated by fifteen peaks between 8 and 80 ppm. The 13C Broadband spectra showed too five shielded aromatic methine groups at δ 104.09 ppm (C-4); δ 107.12 ppm (C-2); 119.83(C-5); δ 114.57 ppm (C-8) and δ125.11 ppm (C-6) respectively. Among the eight quaternary carbon atom signals noted in the HSQC spectra, three corresponded to O-linked aromatic carbon atom δ 154.65 ppm (C-7); δ 162.01 ppm (C-3) and δ 165.00 ppm (C-1), while the peak at 183.29 ppm indicated the presence of carbonyl function C=O group assigned to C-9 [1]. The signal at δ 207.32 ppm is attributed to the carbon at position C-8’. The remaining signals correspond to the carbons of the methyl, methylene and methine confirms the presence of a linear chain. The 2D NMR experiments clearly showed that there were three aromatics protons on the A-ring. The substitute pattern of B-ring was clearly indicated by one singlet at δ 6.64 ppm (H-2) and (H-4) because the two protons are isochronous. According to the HMBC correlation the proton H-2 at δ 6.64 ppm correlates with the carbon atoms at δ 107.12 ppm (C-2) and δ 162.01 ppm (C- 3). Moreover, the 7-dihydroxylation pattern of A-ring was confirmed by the reciprocal shielding of C-7 at δ 154.65 ppm. The signal at δ 3.80 ppm in 1H NMR and δ 56.40 ppm in 13C Broadband spectra suggested the presence of alcohol function (-CH2OH). Finally, the cross peaks noted in the HMBC experiment between H-2’ δ 2.42 ppm and the carbon atoms δ 104.09 ppm (C-4), δ 162.01 ppm (C-3) indicated unambiguously the linkage between the chain linear moiety and the xanthone part. Thus xanthone 1 was identified to 1, 7-dihydroxy-3-O-[(1’,3’,4’,5’,6’,7’-hexamethyloctanal) (2’’hydroxymethylene)] xanthone named chapexanthone A.

Xanthone 2, chapexanthone B was obtained as a yellow needles and it molecular formula was assigned as C18H18O6 by the molecular ion peak at m/z 327.0911 in the HREIMS. NMR data suggested that 2 also had a xanthone skeleton. A comparison of the 1H and 13C NMR data of 2 with those of 1 revealed that the difference was the substituent at C-3. The linear chain in 1 replaced by the δ 78.84 ppm (C-1’), δ 55.50 ppm (C-2’) and δ 60.32 ppm (C-3’) in 2. Therefore, chapexanthone B can be assigned as 1, 7-dihydroxy-3-O-[(1’, 2’-dimethylpropan-3’-ol] xanthone. Data NMR of compound 2 and 1 is given respectively in table 1 and table 2.

Table 1: 1H and 13C NMR spectral data for compound 1

Position 1H 13C1 - 165.00 2 6,64 (s) 107.12 3 - 162.01 4 6,64 (s) 104.09 4a - 156.58 4b - 141.05 5 7.28 (s) 121.47 6 7,87 (d, 9.3Hz) 128.12 7 - 152.95 8 7,06 (d, 9.3Hz) 114.57 8a - 109.25 8b - 123.22 9 - 183.29 1’ 3.90 (d) 71.81 2’ 2.39 (d) 55.55 3’ 1.28 (s) 29.70 4’ 0.72 (s) 50.80 5’ 0.70 (s) 42.50 6’ 0.90 (s) 31.38 7’ 2.23 (s) 51.26 8’ 12.87 (s) 207.32

1’-CH3 1.28(s) 21.19 2’’-CH2OH 3.80, 3.90 (s) 60.33

3’-CH3 1.60 (s) 20.55 4’-CH3 1.03 (s) 12.47 5’-CH3 0.99 (s) 18.80 6’-CH3 0.85 (s) 9.60 7’-CH3 0.83(s) 8.46

a Assignments were confirmed by 2D experiments

Table 2: 1H and 13C NMR spectral data for compound 2a

Position 1H 13C1 163.77 2 6.79 (s) 106.12 3 - 162.79 4 6.97 (s) 103.66 4a - 162.37 4b - 152.62 5 7.14 (d,2.6 Hz) 115.20 6 8.06 (s) 128.11 7 - 156.41 8 8.26 (d,9.3Hz) 113.07 8a - 108.89 8b - 122.58 9 - 182.99 1’ 3.87 (d) 78.84 2’ 2.50 (t) 55.50 3’ 3.87 ; 3.76 (d) 60.32

1’-CH3 2.11 (s) 8.68 2’-CH3 2.35 (s) 8.96

a Experiments were carried out at 500 MHz for 1H and 400 MHz for 13C CDCL3

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Fig 1: Long-range heteronuclear correlation observed for 1 (chapexanthone A)

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Fig 2: Structure of chapexanthone B

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4. Conclusion Two new xanthones: chapexanthone A and chapexanthone B were isolated from the hexanic extract from the steam bark of Garcinia chapelieri. The structures of the isolated new compounds were identified as chapexanthone A and B on the basis of spectroscopic methods. The complete 1H and Broadband NMR spectral assignments of the one isolated compounds were made based on HSQC, HMBC spectroscopic data. 5. Acknowledgement The authors are grateful to the financial support of Project PARRUR. Authors are thankful to Laboratory carbohydrates University Jules Verne Amiens, France and Organic Chemistry Faculty of the University of Bielefeld, Germany for EIMS and NMR spectra. 6. References

1. Breitmayer E, Voelter W. Carbon -13 NMR spectroscopy Weinheim: VCH; 1989.

2. Chanmahasathien W, Li Y, Satake M, Oshima Y, Ishibashi M, Ruangrungsi N et al. Prenylated xanthones from Garcinia xanthochymus. Chemical and pharmaceutical bulletin 2003; 11(1):1332-1334.

3. Fotie J, Bohle DS. Pharmacological and biological activities of xanthones. AntiInfective Agents, Medicinal Chemistry 2006; 5(1):15-31.

4. Gopalakrishnan G, Banumathi B, Suresh G. Evaluation of the antifungal activity of natural xanthones from Garcinia

mangostana and their synthetic derivatives. Journal of Natural Products 1997; 60(1):519-524.

5. Groweiss A, Cardellina JH, Boyd MR. HIV-inhibitory prenylated xanthones and flavones from Maclura tinctoria. Journal of Natural Products 2000; 63(1):1537-1539.

6. Hostettmann K, Hostettmann M. Xanthones, Methods in plant biochemistry. Vol. 1, Plant phenolics. Dey PM, Harborne JB (edi). London: Academic Press, 1989, 493-508.

7. Ioset JR, Marston A, Gupta MP, Hostettmann K. Antifungal xanthones from roots of Marila laxiflora. Pharmaceutical Biology 1998; 36(1):103-106.

8. Jiang DJ, Dai Z, Li YJ. Pharmacological effects of xanthones as cardiovascular protective agents. Cardiovascular Drug Reviews 2004; 22(1):91-102.

9. Reutrakul V, Chanakul W, Pohmakotr M, Jaipetch T, Yoosook C, Kasisit J et al. Anti-HIV-1 constituents from leaves and twigs of Cratoxylum arborescens. Planta Medica 2006; 72(1):1433-1435.

10. Winter RW, Riscoe MK, Hinrichs DJ. Preparation of xanthone derivatives for treating infectious diseases and complexation of heme and porphyrins. Patent 2001, 2000-US42543 2001041773.

ISSN: 2321‐4902 Volume 1 Issue 5

Online Available at www.chemijournal.com

International Journal of Chemical Studies

Vol. 1 No. 5 2014 www.chemijournal.com Page | 42

A new triterpene and stigmasterol from Anthostema madagascariense (Euphorbiaceae)

Volasoa Herilalaina Victorine Rambeloson 1*, Léa Herilala Rasoanaivo 2, Anne Wadouachi 3, Amelie

Raharisololalao 4

1. UG 1 Department of Chemistry, Faculty of Science, University of Fianarantsoa, Madagascar, P.O. Box 1264, Fianarantsoa 301, Madagascar, [Email: [email protected]; +261324327885]

2. Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101, Madagascar, [Email: [email protected]; +261324022036]

3. Laboratory of Antimicrobial Glycochemistry and Agroressources FRE CNRS 3517 University of Picardie Amiens France, [Email: [email protected]]

4. Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101, Madagascar, [Email: [email protected]; +261320257754]

Purification of the ethyl acetate fraction from the stem bark of Anthostema madagascariense (Euphorbiaceae) resulted in the isolation of triterpene namely 3-acetoxy-olean-9(11)-en-28- oic acid and a sterol namely Stigmasterol. The structures of the two products isolated compounds were characterized on the basis of extensive spectral data NMR (1D and 2D) and in the comparison with the literature data. The compounds are reported for the first time from this plant. Keyword: Anthostema madagascariense, Triterpene, Sterol, Euphorbiaceae.

1. Introduction Anthostema madagascariense is a flowering plant species of the Family Euphorbiaceae. Anthostema is a small genus with 3 species of which 2 in continental Africa and 1 in Madagascar: Anthostema madagascariense is endemic of Madagascar. Evergreen, monoecious shrub to medium-sized tree up to 30 m tall, with abundant white latex in all parts; bole branchless for up to 12 m or more, generally straight and regular, up to 70 cm in diameter; bark surface densely fissured, reddish to blackish [4, 5, 9, 10, 11, 12]. A decoction of the stem bark of Anthostema madagascariense is used to treat fever, cough and liver. According to available literature, no phytochemical research work has been carried out on this plant. We now report the isolation of triterpene and sterol on the ethyl acetate extract

from the stem bark of the Anthostema madagascariense. 2. Materials and Methods 2.1 General 1D(1H, 13C, DEPT) and 2D (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC) NMR spectra were recorded on a Bruker Varian 300 NMR and 600 NMR operating respectively at 300.15/100.6 MHz and 600.15/100.6 MHz using CDCl3 and CD3OD as solvent and TMS as an internal standard. Column chromatography (CC) was carried out on silica gel 60F254 (Merck) in glass blades. Thin layer chromatography (TLC) was performed on precoated TLC plates (Merck, silica 60F254) and detection wavelength (254 and 365 nm) was used. Mass spectra were measured with waters 2995/2975-Micromass Q-Tof micro



spectrometer (ES+-MS) and Agilent 5975 spectrometer (EI-MS). 2.2. Plant material Anthostema madagascariense Baill. (Euphorbiaceae) was collected in July 2012 in Farafangana Manombo, Vatovavy Fitovinany’s Region Madagascar and was identified by botanists at the Parc National Botanic and Zoologique Tsimbazaza, Antananarivo, Madagascar where a voucher specimen has been deposited in the Herbarium. 2.3 Extraction and isolation: The stem barks of Anthostema madagascariense Baill. were dried, ground, reduced on powder. The powder was macerated successively with hexane, ethyl acetate and methanol. The solvents were evaporated under reduced pressure to obtain crude residue: green, green and red solid gum (2.05 g; 7.02 g; 7.27 g). The ethyl acetate extract was subjected to column chromatography (silicagel, 3 x 80 cm) and was eluted with a gradient profil of hexane and acetone to give 280 main fractions. The fractions 31- 32 were obtained after 660 mL elution with hexane and acetone (90:10), and the fractions 119- 138 were obtained after 2700 mL and after elution with hexane and acetone (40:60). The purification of fractions 31-32 (compound1) and the 119-138 (compound 2) were carried out by column chromatography on Sephadex LH-20 with CDCl3-MeOH (80:20). 3. Result and discussion: reported in tables 1 and 2 The dried and ground stem bark of Anthostema madagascariense was extracted successively with hexane, ethyl acetate and methanol. The ethyl acetate extract was repeatedly chromatography over Si gel and purified over Sephadex LH-20 to give two compounds a triterpene (1) and a sterol (2). Compound (1) was obtained as a white cottony needle; it gave a positive Liebermann-Burchard test. Its HREIMS spectrum showed a molecular peak at m/z 498.37 and 13C NMR exhibited thirty

two signals in accordance with the molecular formula C32H50O4. The 1H-NMR (Table 1) showed eight methyls, as singlets respectively at δ 0.76, 0.80, 0.83, 0.85, 0.87, 0.88, 1.18 and 2.00 ppm. A signal at δ 3.96 ppm attributable to a proton which is carried by a carbon bonded to a heteroatom. In the spectrum, there were also present an olefinic proton at δ 5.44 ppm. The presence of the singulet (3H) at δ 2.00 ppm indicated that the molecule contained a acetyle group. So that it consisted to a double bond. It was confirmed by the presence of the signal at 171.00 ppm [3] (DEPT). Further, in compound (1) there was a signal at 183.86 ppm consisting to acid group COOH [2] (DEPT). The DEPT spectrum showed thirty two carbon atoms which belong to different groups, carbonyl carbon of carbon acetate at δ 171.00 ppm (C-1’) , an acid at δ 183.86 ppm (C-28) and two olefinic carbons at δ 160.56 ppm (C-9) and δ 116.62 ppm (C-11). The signal δ 80.72 ppm a carbonyl signal is attributed to the carbon at position C-3. Thirty carbon atoms correspond to the basic skeleton triterpene where acetate is fixed: 10 methylene carbons, 8 methyl carbons, 5 methynique carbons and 9 quaternary carbons of which an acid. As is observed the existence of 32 carbon atoms of Broadband spectrum the triterpene structure was confirmed and by collecting all the NMR spectral data of the compound 1, we deduced to the conclusion that compound 1 is the 3-acetoxy-olean-9(11)-en-28-oic acid. It is a new pentacyclic triterpene. Compound (2) was obtained as a white solid and its molecular formula was assigned as C29H48O by the molecular ion peak at m/z 412.37 in the HREIMS. The mass spectral data of the compound gave a molecular formula C29H48O, which was supported by the 13C NMR spectral data. In the 1H NMR spectrum of compound 2 varied between 0.71 to 5.30 ppm, This spectrum showed the presence of 6 high intensity peaks indicating the presence of six methyl groups at δ 0.71 , 0.86, 0.92, 1.00, 1.18 and 1.36 ppm. The proton corresponding to the H-3 of a sterol moiety was appeared as a triplet of doublet of doublet at δ



3.17 ppm. At δ 5.24 ppm and at δ 5.30 ppm corresponds to a peak in the form of a single in the region of the ethylene protons suggesting the presence of three protons corresponding to that of a trisubstituted and a disubstituted olefin bond. The signal at δ 0.68 ppm and δ 1.18 ppm corresponds to H-18 and H-19 protons respectively of Stigmasterol.

In the NMR DEPT 135 ° spectrum showed 29 carbon atoms which is a steroid including quaternary carbon. In the twenty nine carbon atoms including 6 methyl carbons, 9 methylene carbons, 11 methynique carbons and three quaternary carbons .

Table 1: 1H and 13C NMR spectral dataa for compound 1

Position δ 1H (ppm) δ 13C (ppm)

Experimental Value δ 13C (ppm) [6]

Literature Value 1 1.53 ; 1.85 37.93 39.0 2 1.53 ; 1.85 23.47 28.2 3 3.85 80.72 78.1 4 - 39.03 39.4 5 1.37 55.59 55.8 6 1.18 ; 1.53 17.31 18.8 7 1.18 ; 1.53 33.32 33.3 8 - 37.32 39.8 9 - 160.56 48.2 10 - 51.46 37.4 11 5.44 116.82 23.7 12 1.85;2.10 27.96 122.6 13 - 37.68 144.8 14 1.37 40.76 42.2 15 1.37 ; 1.53 30.71 28.4 16 1.85 ; 2.28 29.30 23.8 17 - 49.07 46.7 18 1.37 ; 1.53 37.39 42.0 19 1.18 ; 1.53 41.42 46.5 20 - 30.71 31.0 21 1.37 ; 1.53 35.34 34.3 22 1.85, 2.28 33.78 33.2 23 0.83 15.64 28.8 24 0.85 16.60 16.6 25 0.87 26.19 15.6 26 0.88 27.96 17.5 27 0.88 22.45 26.2 28 - 183.86 180.2 29 0.80 28.66 33.3 30 0.76 28.66 23.8 1’ - 171.00 2’ 2.00 21.34



Table 2: 1H and 13C NMR spectral data for compound 2a

Position δ 1H (ppm) δ 13C(ppm)

Experimental Value δ 13C(ppm) [7]

Literature Value 1 1.32 ; 1.56 38.81 37.3 2 0.80 ; 2.10 33.06 31.6 3 3.17 m 79.07 71.8 4 1.84 ; 2.76 41.08 42.3 5 - 143.41 140.8 6 5.30 br s 122.75 121.7 7 1.62 ; 2.34 32.43 31.9 8 1.34 32.93 31.9 9 2.10 52.44 51.2

10 - 38.41 36.5 11 0.84 ; 1.84 23.29 21.1 12 0.95 ; 1.34 38.60 39.7 13 - 47.87 42.3 14 1.07 55.21 56.9 15 153 ; 2.18 24.15 24.4 16 1.53 ; 2.18 30.60 28.4 17 1.56 53.91 56.1 18 0.68 s 39.04 40.5 19 1.18 s 28.00 21.2 20 5.24 138.37 138.3 21 5.24 125.05 129.3 22 1,53 (d,7.5) 47.54 51.2 23 1.44 27.17 31.9 24 1.00 16.95 12.1 25 1.84 28.18 31.9 26 0.92 (d,6.5) 23.57 25.4 27 0.86 (d,6.5) 21.17 19 28 0 .71 (t,7.5) 17.05 11 29 1.36 28.66 21.2

The spectrum showed some recognizable signals at δ 143.41 ppm, δ 138.37 ppm, δ 125.05 ppm and δ 122.75 ppm which is assignable to the double bond at C-5, C-20, C-21 and C-6 [1]. The carbon at δ 143.41 ppm C-5 is a ethylene quaternary carbon. The δ value observed at 79.07 ppm is due to C-3 is a carbon hydroxyl group [8]. Additional, the signal observed at 28.00 ppm correspond to angular carbon at C-19. The protons at δ 1.00 ppm, at δ 2.18 ppm and δ 3.17 ppm are carried by the carbon at δ 16.95 ppm, at δ 52.44 ppm and δ 79.07 ppm respectively.

In the COSY experiment, irradiation of H-8 (δ 1.34 ppm) gave use to the enchancement of the H- 9 (δ 2.10 ppm). A proton geminate H-7 at δ 1.62 ppm and at δ 2.34 ppm. In the HMBC spectrum (Table 2), the protons resonanting at δ 0.80 ppm showed longe range heteronuclear connectivities with C-3 (79.07 ppm), C-9 (δ 52.44 ppm) and C-1 (δ 38.83 ppm). The H-18 protons, resonanting at 1.18 ppm, exhibited HMBC interactions with C-22 (δ 47.54 ppm), C-21 (125.05 ppm) and C-20 (δ 138.37 ppm).



Thus, the structure of compound 2 was assigned as the know compound Stigmasterol.

Fig 1: Structure of 3-acetoxy Olean-9(11)-en-28-oic acid

Fig 2: Long-range heteronuclear correlation observed for 2 (stigmasterol)



4. Acknowledgement

The authors are grateful to the financial support of Project PARRUR

Authors are thankful to Laboratory carbohydrates University Jules Verne Amiens, France for EIMS and NMR spectra.



5. Conclusion Stigmasterol and acetoxy myrtifolic acid were isolated from the ethyl acetate extract from the stem bark of Anthostema madagascariense. The structures of the sterol and triterpene compounds were identified as 3-acetoxy-olean-9(11)-en-28-oic acid and stigmastérol on the basis of spectroscopic methods. The complete 1H, NMR DEPT 135° and Broadband spectral assignments of the one isolated 2 compounds were made based on HSQC, HMBC spectroscopic data. 6. References

1. Agrawal PK, Jain DC, Gupta RK, Thakur RS. Carbon -13 NMR Spectroscopy of steroidal sapogenins and steroidal saponins. Phytochemistry Res 1985; 24(11):2476-96.

2. Ikuta A, Kamiya K, Satake T, Saiki Y. Triterpenoids from callus tissue cultures of Paeonia species. Phytochemistry 1995; 38(5):1203-1207.

3. Brachopérez JC, Best CR, Lanes F. Triterpènoïde pentacyclique. Rev Soc Quim Peru 2009; 75(4).

4. Capuron R. Studies on forest trees of Madagascar. Mandravoky (Anthostema madagascariense Baillon - Euphorbiaceae). CTFT, Antananarivo, Madagascar. 1966, 5.

5. Guéneau P, Bedel J, Thiel J. Wood and Malagasy species. Center Technical Tropical forest, Nogent-sur-Marne. France 1970–1975; 150.

6. Gohari AR, Saeidnia S, Hadjiakhoondi A, Abdoullahi M, Nezafati M. Isolation and Quantificative Analysis of Oleanolic Acid from Satureja mutica Fisch. & C. A. Mey. Journal of Medicinal Plants 2009; 8(5).

7. Pateh UU, Haruna AK, Garba M, Iliya I, Sule IM, Abubakar M, Ambi AA. Isolation of β-Stigmasterol, β-Sitosterol and 2-hydroxyhexadecanoic acid methyl ester from the rhizomes of Stylochiton Lacifolius Pyer and Kotchy (Araceae). Nigeria journal of pharmaceutical Sciences 2009; 8:19-25.

8. Pretsch EB, Affolter A. Structure determination of organic compounds Table of spectra data. SpringerVerlag. Berlin Heidelberg 2000; 71-150.

9. Rakotovao G, Rabevohitra R, Gerard J, Détienne P. Collas of Chatelperron, P., in preparation. Atlas wood Madagascar. FOFIFA-DRFP Antananarivo, Madagascar.

10. Sallenave P. Physical and mechanical properties of tropical wood. supplement. Center Technical Tropical forest, Nogent-sur-Marne, France. 1971; 128.

11. Takahashi A. Compilation of data on the mechanical properties of foreign woods (part 3) Africa. Shimane University, Matsue, Japan, 1978; 248. Anthostema madagascariense Baill. http://www.prota4u.info/protav8.asp?fr=1&g=pe&p=Anthostema+madagascariense+Baill; 2011.

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Journal of Pharmacognosy and Phytochemistry 2014; 3 (1): 68-72 ISSN 2278-4136 JPP 2014; 3 (1): 68-72 Received: 19-03-2014 Accepted: 12-04-2014 Léa Herilala Rasoanaivo Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique (LCSN/COB), Faculté des Sciences, Université d’Antananarivo, BP 906 Ankatso, Antananarivo 101, Madagascar Anne Wadouachi Laboratoire de Glycochimie des Antimicrobiens et des Agro-ressources. CNRS FRE 3517, Université de Picardie Jules Verne, Amiens France. Tianarilalaina Tantely Andriamampianina

Laboratoire de Pharmacologie Générale, de Pharmacocinétiques et de Cosmétologie(LPGPC), Faculté des Sciences, Université d’Antananarivo, BP 906 Ankatso, Antananarivo 101, Madagascar Solofoniaina Gabriel Andriamalala Laboratoire de Pharmacologie Générale, de Pharmacocinétiques et de Cosmétologie(LPGPC), Faculté des Sciences, Université d’Antananarivo, BP 906 Ankatso, Antananarivo 101, Madagascar Ernest Jeannot Bako Razafindrakoto Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique (LCSN/COB), Faculté des Sciences, Université d’Antananarivo, BP 906 Ankatso, Antananarivo 101, Madagascar Amélie Raharisololalao Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique (LCSN/COB), Faculté des Sciences, Université d’Antananarivo, BP 906 Ankatso, Antananarivo 101, Madagascar Fanantenanirainy Randimbivololona Laboratoire de Pharmacologie Générale, de Pharmacocinétiques et de Cosmétologie(LPGPC), Faculté des Sciences, Université d’Antananarivo, BP 906 Ankatso, Antananarivo 101, Madagascar Correspondence: Léa Herilala Rasoanaivo Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique (LCSN/COB), Faculté des Sciences, Université d’Antananarivo, BP 906 Ankatso, Antananarivo 101, Madagascar Email: [email protected]

Triterpenes and steroids from the stem bark of Gambeya

boiviniana Pierre

Léa Herilala Rasoanaivo, Anne Wadouachi, Tianarilalaina Tantely Andriamampianina, Solofoniaina Gabriel Andriamalala, Ernest Jeannot Bako Razafindrakoto, Amélie Raharisololalao, Fanantenanirainy Randimbivololona

ABSTRACT A chemical study was done on the stem bark of Gambeya boiviniana Pierre. This plant has been used in traditional medicine for treatment of different kinds of inflammation disorders. In the present study, anti- inflammatory activities of ethanolic, dichloromethane, ethyl acetate and butanolic extracts were assayed in mice using carrageenan-induced paw edema. Ethyl acetate extract was found to possess the most significant anti-inflammatory effect (67, 78%). These results are in accordance with the folk use of this plant. However, more research is needed for its use in clinical studies. The separations of the chemical compounds of ethyl acetate extract were carried out by different chromatographic technics and their structures were elucidated by spectroscopic method including nuclear magnetic, mass spectrometry, IR spectrometry, GC/MS. Nine compounds were identified during this investigation. There are lupeol acetate 3, β amyrin acetate 4, α amyrin acetate 6, taraxasterol acetate 5, fatty acid ester of lupeol 2 and fatty acid ester of β-amyrin 1, chondrillasterol 7, β-sitosterol 8, β-sitosterol-3-O-glucoside 9. Keywords: Gambeya boiviniana, Sapotaceae, anti-inflammatory, Triterpenes, steroids, stem bark. 1. Introduction Gambeya, syn Chrysophyllum Linn is a large genus of Sapotaceae about 80 species [1]. Ten species of Chrysophyllum have been represented in Madagascar [1, 2]. Of these Gambeya boiviniana Pierre, syn. Chrysophyllum boivinianum Baehni, Gambeya madagascariensis Lecomte known as vernacular name "famelona" with biggest leaves is distributed mainly in Comore and in the east of Madagascar. It is used for interior and exterior joinery, furniture, molding, paneling, flooring, light scales and in shipbuilding because of its elasticity [1]. In Madagascar, the leaves of Gambeya boiviniana provide one of the ingredients in herbal mixtures used to relieve the symptoms of malaria, fatigue, muscle pain, treat poisoning and heals the child subject to simple febrile seizure [1, 2].

The African species of Gambeya showed interesting pharmacological properties as anti-tumoral, antimicrobial, an anti-inflammatory [1, 2, 3, 4]. The chemical constituents of some Gambeya species of Africa have been studied such as steroids, steroids glycosides, pentacyclic triterpenoids, and fatty acid esters of triterpenoids have been identified [1, 2]. In this paper we study the acute anti-inflammatory activity of the extracts and the isolation and identification of steroids, triterpenoids and fatty acid esters of triterpenoids from the AcOEt extract of the stem bark of Gambeya boiviniana. Nine compounds were also identified as fatty acid ester of β-amyrin 1, fatty acid ester of lupeol 2, lupeol acetate 3, β amyrin acetate 4, α amyrin acetate 6, taraxasterol acetate 5, and chondrillasterol 7, β-sitosterol 8, β-sitosterol-3-O-glycoside 9. This is the first chemical and biological study of the stem bark of this plant to the best of our knowledge. 2. Materials and Methods 2.1 General experimental procedures 1D (1H, 13C, DEPT) and 2D (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC) NMR spectra were recorded on a Bruker Varian 300 NMR and 600 NMR operating at 300.15/100.6 MHz and 600MHz using CDCl3, CD3OD or DMSO-d6 as solvent and TMS as an internal standard. Column chromatography (CC) was carried out on silica gel F254 (Merck) in glass blades. Thin

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layer chromatography was performed on precoated TLC plates (Merck, silica 60F254) and visualized by UV light and by spraying with vanillin in H2SO4. Mass spectra were measured with Waters 2995/2996-Micromass Q-Tof micro spectrometer (ES+-MS) and Agilent 5975 spectrometer (EI-MS). 2.2 Plant Gambeya boiviniana Pierre (Sapotaceae), stem bark collected in mountain Analabe, rural district of Andapa, SAVA’s Region Madagascar in Jun 2011, were compared with 11 herbarium Tan identified by Desiré Ravelonarivo et al. at the Parc National Botanic and Zoologique Tsimbazaza, Antananarivo, Madagascar.A voucher specimen has been deposited in the “Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique”. 2.3 Animals Under a protocol approved by the Animal Care and Use Committee, mice swiss (25–30 g) of either sex were used for the pharmacological activities. All the animals were acclimatized to the laboratory conditions for a week before use. They were fed with standard animal feed and water ad libitum. 2.4 Extraction The air-dried powder stem bark (400 g) of Gambeya boiviniana was extracted by maceration with 80% ethanol. Crude ethanol extract (15,85 g), after removal solvent under reduced pressure, was suspended in water and partitioned successively with hexane, dichloromethane, ethyl acetate and n-butanol to give respectively hexane extract (0,01 g), dichloromethane extract (0,28 g), ethyl acetate extract (1,78 g) and n-butanol extract (6 g). The ethyl acetate extract was submitted to silica gel column. Gradient elution was carried out using cyclohexane and increasing the polarity with dichloromethane and then with dichloromethane increasing with ethyl acetate and methanol. 2.5 Antiinflammatory activity Carrageenan – induced paw edema The method of Winter et al. 1962 was employed in this experiment [1]. The mice were divided into six groups of six animals each (4 tests groups, reference group and vehicle control group). The volume of the right hind paw of each mouse was measured with a plethysmometer (model 7140, Ugo Basile) [1]. The ethanolic, dichloromethane, ethyl acetate, aqueous extracts of Gambeya boiviniana Pierre and the standard drug (phenylbutazone) were suspended in mixture of Tween 80 - distilled water (10:90) as solvent and administered orally at the dose of 250 mg/kg for all extracts and 100 mg/kg for phenylbutazone. The control group received the vehicle only (10 ml/kg, p.o.). Thirty minutes after the administration, acute paw edema was induced in the right hind paw by subplantar injection of 0.05 ml of 1% Carrageenan dissolved in physiological saline. The paw volume was measured just after carrageenan injection and at 30, 60 and 120 minutes [1]. The difference between the volume of the paw before and after carrageenan injection indicated the severity of edema. The percentage inhibition of the inflammatory reaction was determined for each animal by comparing with controls and calculated by the formula:

% inhibition= Vc-Vt x 100 Vc

Where, Vt means increase in paw volume in rats treated with test compounds. Vc means increase in paw volume in control group of mice [1]. Statistical analysis The experimental data were expressed as the mean ± standard error of the mean (SEM). The statistical analysis was carried out using unpaired Student’s ‘t ' test. P values <0.05 were considered as significant. 2.6 Isolation procedure The ethyl acetate extract (1,5 g) was subjected to column chromatography over silica gel (60 g silica gel 60, 80x2 cm). A total of 540 fractions were eluted with mixtures of cyclohexane/ methylene chloride (from 100:0 to 0:100), then with mixture methylene chloride/ethyl acetate (from 100:0 to 0:100) and finally with ethyl acetate/ methanol (from 100:0 to 0:100). The elutes were monitored using TLC and viewed under UV light (254 and 365 nm) and by spraying with 1% vanillin/5% H2SO4/EtOH reagent followed by heating at 100 °C. The fractions were combined on the basis TLC profiles and purified with MeOH. Further chromatography of the combined fractions [35-40] (10 mg) over silica gel 60 (20 mg) eluted with cyclohexane-CH2Cl2 (9:1) showed one spot containing esters of β-amyrin 1 with fatty acids (5 mg). Combined fractions [70-90] (25 mg) from MeOH exhibited one TLC spot containing esters of lupeol 2 with fatty acids (20 mg). The fraction 131 (12 mg) eluted with cyclohexane-CH2Cl2 (8:2) yielded one spot for a mixture (β-amyrin acetate 4, lupeol acetate 3, taraxasterol acetate 5). α-amyrin 6 (5 mg) was obtained in fraction 132. The combined fractions [390-400] (20 mg) eluted with AcOEt/MeOH (1:9) furnished chondrillasterol 7 (8 mg). The combined fractions [540-549] (10 mg) eluted with AcOEt/MeOH (1:9) yielded β-sitosterol 8 (8 mg). Fractions [575-589] (40 mg) eluted with AcOEt/MeOH (2:8) furnished β-sitosterol glucoside 9 (38 mg). Acid hydrolysis of sterol glycoside Compound 9 (9 mg) was heated at reflux with 10 ml of 2N HCl-MeOH (1:1) for 2 h. The MeOH was evaporated and the aqueous solution was extracted with CH2Cl2. The aglycone in the dichloromethane layer was analysed by TLC [CH2Cl2-MeOH 19:1]. The remaining aqueous phase was evaporated to dryness and monosaccharide glucose was identified by comparison with authentic samples on TLC. 2.7 Physical and spectroscopic data Fatty acid esters of β-amyrin 1: white powders (MeOH) 1H NMR (CDCl3, 600 MHz) δ (ppm):5.13 (1H,t,H-12), 1.13 (3H,s,CH3-27), 0.962 (3H,s,CH3-26), 0.96 (3H,s,CH3-25), 0.87 (6H,s, CH3-29 and CH3-30), 0.86 (3H,s,CH3-24), 0.83 (3H,s,CH3-23), 0.82 (3H,s,CH3-28) Long chain: 2.3(2H, H-2’), 1.25((CH2)n), 0.90(3H, CH3ter) 13C NMR (CDCl3, 100 MHz) δ (ppm); 145.38 (C-13), 121.53 (C-12),80.64(C-3), 55.3(C-5), 47.6(C-9), 47.3(C-18), 46.89 (C-19), 41.8(C-14), 39.90 (C-8), 38.3 (C-1), 37.2 (C-4), 37.1 (C-22), 36.90 (C-10), 34.8 (C-21) , 33.3 (C-29), 32.6(C-7), 32.5(C-17), 31.2 (C-20), 28.4 (C-24),26.2 (C-16), 25.92( C-27), 26.2 (C-15), 23.40 (C-28), 22.27 (C-2), 23.5(C-11), 16.78(C-23), 18.71(C-6 ), 15.51(C-25). Long chain 173.9 (C-1’), 34.75 (C-2’), 30.0 (CH2)n, 13.90 (CH3 ter) Fatty acid esters of Lupeol 2: white powders (MeOH) 1H NMR (CDCl3, 600 MHz) δ(ppm): 4.67(H-29β), 4.65(H-29α), 4.46(H-3),

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2.36(H-19), 1.69(H-30), 1.03(H-26), 0.94(H-27), 0.85(H-25), 0.84(H-24), 0.83(H-23), 0.78(H-28) Long chain: 2.28(H-3’), 1.61(H-4’), 1.25(CH2)n, O.87(CH3 ter) 13C NMR (CDCl3, 600 MHz) δ (ppm); 151.02 (C-20), 109.36 (C-29), 80.1 (C-3), 55.5 (C-5), 50.34 (C-9), 48.11 (C-18), 48.00 (C-19), 43.19 (C-17), 42.96 (C-14), 40.85 (C-8), 40.00 (C-22), 38.44 (C-1), 38.05 (C-13), 37.90 (C-4), 37.09 (C-10), 34.87 (C-16), 34.22 (C-7), 29.9 (C-21), 27.87 (C-24), 27.44 (C-15), 25.18 (C-12), 23.75 (C-2), 20.95 (C-11), 19.04 (C-30), 18.22 (C-6), 18.01 (C-28), 16.04 (C-26), 16.00 (C-23), 15.98 (C-25), 14.45 (C-27) Long chain: 174.01 (C-1’), 35.58 (C-2’), 25.10 (C-3’), 30.0 (CH2)n, 22.80 (CH2- CH3 ter), 14.03 (CH3 ter) Lupeol acetate 3: white powder1H NMR (CDCl3, 600 MHz) δ(ppm): 4.68(H-29β), 4.56(H-29α), 4.48(H-3), 2.37(H-19), 2.03(CH3-COO), 1.69(H-30), 1.03(H-26), 0.94(H-27), 0.85(H-25), 0.84(H-24), 0.83(H-23), 0.78(H-28) 13C NMR (CDCl3, 100 MHz) δ (ppm); 151.19 (C-20), 109.78 (C-29), 81.22 (C-3), 55.40 (C-5), 50.49 (C-9), 48.43 (C-19), 48.00 (C-18), 43.17 (C-17), 42.97 (C-14), 40.96 (C-8), 40.20 (C-22), 38.50 (C-1), 38.10 (C-13), 37.86 (C-4), 37.23 (C-10), 35.72 (C-16), 34.26 (C-7), 29.86 (C-21), 28.09 (C-24), 27.58 (C-15), 25.01 (C-12), 23.86 (C-2), 21.07 (C-11), 19.47 (C-30), 18.39 (C-6), 18.05 (C-28), 16.04 (C-26), 16.55 (C-23), 16.33 (C-25), 14.50 (C-27) Acetate 171.02 (C-1’), 21.42 (CH3) α-amyrine acetate 6: 13C NMR (CDCl3, 100 MHz) δ (ppm); 139.5 (C-13), 124.2 (C-12), 80.8 (C-3), 59.00 (C-18),55.40 (C-5), 47.67 (C-9), 39.6 (C-19), 38.07 (C-18), 33.7 (C-17), 42.0 (C-14), 39.6 (C-8), 41.5 (C-22), 38.40 (C-1), 37.94 (C-4), 36.6 (C-10), 32.89 (C-7), 31.2 (C-21), 28.09 (C-24), 27.58 (C-15), 26.72 (C-16), 25.01 (C-12), 23.86 (C-2), 21.07 (C-11), 19.47 (C-30), 19.32 (C-27), 18.39 (C-6), 18.05 (C-28), 16.04 (C-26), 14.53 (C-23), 16.33(C-25). Acetate 171.02 (C-1’), 21.42 (CH3) Chondrillasterol 7: 1H NMR (CDCl3, 400 MHz) δ (ppm):5.08 (1H,t, H-7), 5.05 (1H,t, H-22), 4.96 (1H,t, H-23), 3.59 (1H,t, H-3), 1.02 (3H,d,H-21), 0.85 (6H,d,H-26, H-27), 0.79 (3H,d,H-19), 0.74 (3H,d,H-29), 0.54 (3H,d,H-18), 13C NMR (CDCl3, 100 MHz) δ

(ppm); 139.5 (C-8), 138.0 (C-22), 129.5 (C-23), 117.32 (C-7), 70.8 (C-3 ), 55.94(C-14), 54.83(C-17 ), 50.83 (C-24), 49.18 (C-9), 42.96 (C-13), 40.53(C-20), 39.90 (C-5), 39.24 (C-12), 37.48 (C-1), 33.78 (C-10), 31.55(C-25), 31.20 (C-2), 29.90 (C-6), 29.80 (C-16), 25.16 (C-28), 23.90 (C-11), 23.80 (C-15), 21.08 (C-21),20.76 (C-26), 20.76 (C-27), 18.75 (C-19), 12.33 (C-29), 11.84 (C-18). β-sitosterol 8: 1H NMR (CDCl3, 400 MHz) δ (ppm); 5.33 (1H, H-6),1.01(3H, s, H-19), 0.92 (3H, s, H-21), 0.84 (3H, d, H-26), 0.82 (3H, d, H-27), 0.80 (3H, d, H-29), 0 .68 (3H, s, H-18) 13C NMR (C5D5N, 100 MHz) δ (ppm); 140.0 (C-5), 121.4(C-6), 71.62 (C-3), 56.5(C-14), 55.89 (C-17), 49.94(C-9), 45.65(C-24), 42.3 (C-4), 42.10(C-13), 39.20 (C-12), 37.4(C-1), 36.5(C-20 ), 35.82(C-10), 34.00(C-22), 32.00(C-7), 31.93(C-8), 31.72(C-2), 29.00(C-25), 28.3(C-16), 26.02 (C-23), 24.6 (C-15), 23.06 (C-28), 21.12 (C-11), 19.34(C-19), 19.34(C-21), 19.02(C-26), 18.75(C-27), 11.72 (C-29), 11.63(C-18). β- Sitosterol-3- β_-D-glucoside 9: 1H NMR (C5D5N, 400 MHz) δ (ppm); 5.33 (H-6,), 5.03 (Glc H-1’, d, J = 7.6 Hz), 0.97 (3H, d, J = 6.8 Hz, H-21), 0.92 (3H, s, H-19), 0.85 (3H, t, J = 7.6 Hz, H-26), 0.83 (3H, d, J = 6.8 Hz, H-27), 0.84 (3H, t, J = 6.8 Hz, H-29), 0.65 (3H, s, H-18) and 13C NMR (C5D5N, 100 MHz) δ (ppm); 141.80 (C-5), 122.80 (C-6), 103.47 (C-1’), 79.50 (CH sucre), 79.39 (2C, CH sucre, C-3), 76.2 (CH sucre), 72.5 (CH sucre), 63.7 (C-6’), 57.74 (C-14), 57.15 (C-17), 51.25 (C-9), 46.96(C-24), 43.39 (C-13), 40.86 (C-4), 40.24 (C-12), 38.3 (C-1), 37.3 (C-20), 35.1 (C-22), 33.09 (C-7), 32.96 (C-8), 31.16 (C-2), 30.03 (C-25), 27.27 (C-16), 25.4 (C-23), 24.3 (C-28), 22.2 (C-11), 21.1 1 (C-26), 21.1 (C-27), 20.9 (C-19), 19.9 (C-21), 13.08 (C-29), 12.89 (C-18 ); Q-tof premier UPCL/MS [M+Na]:599 3. Results and Discussion The results of anti-inflammatory activity studies of Gambeya boiviniana Pierre show that the ethanolic and ethyl acetate extracts at doses of 250 mg/kg reduced significantly the paw edema (52.82% and 67.78%; p<0.05) 2h after carrageenan injection when compared with control but it was not as strong as phenylbutazone (87,22%; p<0.05). Dichloromethane and butanolic extracts didn’t show a significant effect (Table I).

Table 1: Oral anti-inflammatory activity of extracts of the bark of Gambeya boiviniana Pierre on acute model of inflammation by

carrageenan-induced edema in left hind paw of mice (mean ± SEM; n=6. *Statistically significant from control P<0,05).

Treatments Doses

(mg/kg)

Volume in ml of the left hind paw of mice before and after carrageenan injection± SEM

0 2 h (% of

inhibition)Control 0,27±0,006 0,58±0,003

ethanolic extract

250 0,28±0,016 0,41±0,005 52,82*

CH2Cl2 extract 250 0,21±0,02 0,52±0,02 17,90 ethyl acetate

extract 250 0,29±0,01 0,35±0,008 67,78*

butanolic extract

250 0,25±0,01 0,55±0,01 10,55

Phenylbutazone 100 0,23±0,07 0,27±0,01 87,22* It is well known that carrageenan induced paw edema is characterized by biphasic event with involvement of different inflammatory mediators. In the first phase (during the first 2 h after carrageenan injection), chemical mediators such as histamine

and serotonin play role, while in Second phase (3–4 h after carrageenan injection) kinin and prostaglandins are involved [2]. Our results revealed that administration of ethanolic and ethyl acetate extract inhibited the

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edema starting from the second hours of inflammation, which is probably may be due to the inhibition of different aspects and chemical mediators of inflammation. The ethyl acetate extract of the stem bark of Gambeya boiviniana Pierre yielded triterpenoids and steroids by silica gel chromatography. Assignments of the 1H and 13C-NMR of these compounds were accomplished from 1H-1H COSY, 1H-13C HSQC and HMBC experiments. These compounds were identified by comparison of their spectral data with those of β-amyrin fatty acid ester 1 [2, 3], α-amyrin acetate 6, β-amyrin acetate 4 [2], lupeol fatty acid ester 2 [2], lupeol acetate 3 [2], taraxasterol acetate 5, chondrillasterol 7 [2], β-sitosterol 8 [2], and β-sitosterol glucoside 9 [2] (figure 1) reported in the literature. Otherwise the nature of the sugar contents in compound 8 was identified by TLC comparison with authentic sample produced through acid hydrolysis reaction.

These compounds isolated from ethyl acetate extract of Gambeya boiviniana Pierre found in many species have never been isolated before from species Gambeya other chondrillasterol [11, 12]. Although bioassays were not conducted on the isolated compounds from active ethyl acetate extract, there were previous studies reported on their biological activities. Acetylated alpha- and beta-amyrin presents sedative, anxiolytic, analgesic and anticonvulsant properties [2]. β-Sitosterol shows antiinflammatory, antiprostatic, anti-pyretic, antiarthritic, anti-ulcer, insulin releasing and oestrogenic effects and inhibition of spermatogenesis. It reduces risk of cancer and prevention of oxidative damage through its antioxidant activity [2]. Long-chain fatty acid esters of lupeol extracted from an African plant, Holarrhena floribunda (Apocynaceae), were shown to have strong antimalarial activity [2]. Lupeol acetate exhibited antinociceptive and anti-inflammatory activity [2].

Fig 1: Structure of triterpenes and steroids from the stem bark of Gambeya boiviniana Pierre. Presence of triterpenoids and steroids as the major compounds in ethyl acetate extract can approximately explain antiinflammatory activity of this extract to have strong effect. Triterpenoids have been linked with analgesic, antiinflammatory and antipyretic activities, not surprising therefore to observe activities in ethyl acetate extract since it contained triterpenoids as a major constituent [2]. The relative reduction in the activities observed with the ethanolic extract as compared with ethyl acetate extract is might be due to interaction between the other components. More studies are needed to show the mechanisms of the anti-inflammatory effects of these agents found in Gambeya boiviniana Pierre. 4. Conclusion The results of the present study showed that Gambeya boiviniana Pierre has anti-inflammatory properties and it justifies the

traditional use of this plant in the treatment of various types of pains and inflammation. 5. Acknowledgments We thank Professeur José Kovensky Director of the « Laboratoire de Glycochimie des Antimicrobiens et des Agro-ressources. CNRS FRE 3517”, Amiens, to have welcomed us heartily with his group of research. We wish to thank Mr Dominique Cailleu, Ingénieur d'Etudes CNRS technician NMR at the plate forme to have welcomed us for training on the use of NMR equipment, and Mr David Lesur, Ingénieur d'Etudes CNRS, “Laboratoire de Glycochimie des Antimicrobiens et des Agro-ressources. CNRS FRE 3517”, Amiens, for the realization of the mass spectra. Financially assistance from SCAC of the Embassy of France in Madagascar is gratefully acknowledged.

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Journal of Pharmacognosy and Phytochemistry 2014; 3(4): 225-233 E-ISSN: 2278-4136 P-ISSN: 2349-8196 JPP 2014; 3(4): 225-233 Received: 19-09-2014 Accepted: 02-10-2014

Léa Herilala Rasoanaivo (a)Laboratoire International Associé Antananarivo-Lyon1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. (b) Laboratoire International Associé Antananarivo-Lyon1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. Florian Albrieux Centre Commun de Spectrométrie de Masse, ICBMS - UMR5246 – Université Claude Bernard Lyon 1, France. Marc Lemaire (a)Laboratoire International Associé Antananarivo-Lyon1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. (b)Laboratoire CASYEN-ICBMS- UMR5246 - Université Claude Bernard Lyon 1, France Correspondence: Léa Herilala Rasoanaivo (a)Laboratoire International Associé Antananarivo-Lyon1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar (b) Laboratoire International Associé Antananarivo-Lyon1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar.

Chemical constituents of peels, kernels and hulls of fruits of Mangifera indica Var. Hiesy and their

potential valorization. Léa Herilala Rasoanaivo, Florian Albrieux, Marc Lemaire Abstract In the aim of valorization of the by-products of fruit processing of Mangifera indica Var. Hiesy, separation and phytochemical study were performed. Fatty acids and phytosterols of kernel oil of Mangifera indica Var. Hiesy are characterized by Gas Chromatography coupled with mass spectrometry or flame-ionization detector. Separation method is based on ethanolic extract and then organic –aqueous partition extract using organic solvent in increasing polarity. Purification of gallic acid and its esters was made by precipitation with ethanol 99 °C. Polyphenolic compounds such phenolic acids; gallotannins and flavonoid are identified and quantified in ethanolic extract of by-products (wastes) of fruits processing of Mangifera indica Var. Hiesy. Identification of polyphenol compounds has been established by high-performance liquid-chromatography-electrospray ionisation mass spectrometry and nuclear magnetic resonance spectrometry. Content in phenolic compound and tannin are given. The potential utilizations of metabolite separated are discussed. Saponification then acidification of extract rich in gallotannins gives gallic acid using in synthetic chemistry. Keywords: Mangifera indica L. Var. Hiesy / Anacardiaceae / Madagascar / gallotannins / oil / phenolic acids / valorization. 1. Introduction Mangifera indica L. (Anacardiaceae), fruit tree origins of the East of India and Birmane had over 500 varieties in the world [1]. This species is also represented in Madagascar by 46 varieties [2] with 205000 tons products each year [3]. In Madagascar, fruit of Mangifera indica L. is popularly known as “manga” followed by variety name such “hiesy” or “vato” or “Zanzibar”. According to our ethnobotanical investigations led with population and traditional healers in the region, manga kernel is traditionally used to treat parasite. After consumption and industrial processing of the fruits of manga, considerable amounts of manga peels, seeds are discarded as wastes. View the amount of peels and seeds of mangoes after their transformation into juice industry, chemistry and processing of these materials are the subject for several studies in the recent years. Peels, kernels recovery in various varieties of Mangifera indica allow for polyphenolic compounds [4]: phenolic acids, xanthones, flavonoids, and tannins [2]. Tannins extract of kernel have antimicrobial [6, 7, 8], antioxidant, anti-snake venom, hepatoprotective, anti-tyrosinase, inhibitory activity [9, 10, 11, 12]. Oils of kernel in many varieties of Mangifera indica L. are characterized [13, 14, 15]. Several valorizations of metabolite from kernel and peel were proposed [16]: starch of kernel to biopolymer [17]; peel and kernel to additive of biscuit [4], kernel oil to cocoa butter in cosmetic [19], proteins and starches for feeding the animals. Madagascar is an island rich in tropical fruits, the valorization of by-product of a fruit processing may be an economical source. In this study, the identification and quantification of kernel oil and polyphenolic compounds in kernel, hull and peel of fruit of Mangifera indica L. var. Hiesy, known as vernacular name “manga hiesy” or “manga hoesy” in Madagascar, are reported. The objective was to evaluate a possible utilization of separated fractions of these by-products of fruits processing.

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2. Materials and methods 2.1. Plant material Fruits of “manga hiesy” with a green peels were collected by Hervé Andriamadio in september 2013 in Antsohihy district on the road RN6 in North of Madagascar. 2.2. Chemicals and General experimental procedures Solvents used were purchased from VWR (Darmstadt, Germany) and were of gradient grade or analytical. Deionised water was prepared by a milli-Q water purification system (Millipore, France). Gallic acid and reagents were obtained from Sigma (Steinheim, Germany). Skin pre-chromed powder was furnished with SCRD group “Compagnie Française des extraits” (LeHavre, France). Thin layer chromatography was performed on precoated TLC plates (Merck, silica 60F254) and visualized by UV light and by spraying with vanillin in H2SO4 or with FeCl3 in CH3OH. 1D (1H, 13C, DEPT) and 2D (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC) NMR spectra were recorded on a Bruker Varian 400 NMR operating at 400.15/125.15MHz using, CD3OD as solvent and TMS as an internal standard. HPLC/MS analysis was carried with Agilent HPLC G1312A binary gradient pump series system equipped with HYSTAR software, a model G1329A autosampler. The HPLC system was connected in series with a Micro TOF-Q Bruker Daltonics (Bremen, Germany) mass spectrometer fitted with ESI source. Negative ions mass spectra of the column eluate were recorded in the range m/z 50-2000 (Resolution 10 000). Nitrogen was used as the drying gas at a flow rate 8L/min. The nebulizer temperature was set at 200 °C and the pressure is 2 bars. The gas chromatograph (GC–MS) was performed by using a Focus GC instrument (Thermo Electron Corporation, Bremen, Germany) equipped with a DB-5 MS capillary column (30 m, 0.25 mm inner diameter, 0.25 mm film thickness) and the mass analyzer was a simple quadrupole with an electron ionization system was used with an electron energy of 70 eV. The carrier gas was He with a flow rate of 1 mL/min. The column temperature was initially 70 °C for 2 min, which gradually increased to 310 °C at 15 °C/min and was finally kept at 310 °C for 10 min. The injector temperature was 220 °C, and the transfer line temperature was 280 °C. The gas chromatograph (GC-MS) was used for separation of methyl ester (or sterol sylilated). The fatty acids methyl esters were identified by comparison of their corresponding mass in MS library. Percent relative fatty acid (or phytosterol) was calculated based on the peak area of a fatty acid (or phytosterol) species to the total peak of all the fatty acids (or phytosterol) in the oil sample using GC-2025 Shimadzu equipped with flame-ionisation detector (GC/FID) and a polar capillary column (ZB-5MS) 0.25 mm internal diameter, 30 m length and 0.25 μm film thickness. Before injection, solutions of product or extract were filtered with 0.45 µm nylon 66 membrane filter (Supelco). 2.3. Extraction and isolation of the constituent Pulp, peel, kernel and hull of fruits (2250 g) are separated. The waste 50.5% w/w (1136.25 g; peels, kernels, hulls) of the fruits of “manga hiesy” was kept at room temperature (25-30 °C) for air drying (3 weeks). The air-dried powder 221.1 g (9.8%, w/w) of peels; 241.9 g (10.8%, w/w) of hulls and 173.4 g (7.7%, w/w) of kernels were used as material.

2.3.1 Oil kernel extract 20.0 g of powder kernels were extracted with hexane by soxhlet at 80 °C (3 x 200 ml, 8 h each). The solvent was dried over anhydrous sodium sulphate. The filtered solvent was evaporated under vacuum to afford a lipid pale yellow semisolid (1.4 g, 6.1% w/w) and a defatted kernel (18.3 g, 91.6% w/w). 2.3.1.1. Transesterification of fatty ester 25.0 mg of oil were heated with 1ml BF3-methanol and 1 ml 2, 2-dimethoxypropane reagent in reflux at 60 °C for 15 min [4]. The esters were extracted with hexane. The extracts were dried over anhydrous sodium sulphate, filtered and the solvent was removed to get (20.4 mg, 81.6% w/w) of the methyl esters of fatty acids. 2.3.1.2 Unsaponifiable extraction 423.8 mg of fat was refluxed with 15 ml of 0.5 N ethanolic potassium hydroxide during 4 hours for saponification. After filtration, the ethanol was evaporated and then water and diethyl ether were added. The upper ethereal layer was dried over anhydrous sodium sulphate and distilled of to get (12.8 mg, 3% w/w) unsaponifiable matter. The unsaponifiable fraction is introduced into a test tube in which silylation reagent (0.5 ml of pyridine, 0.1 ml of hexamethyldisilazane and 0.04 ml of trimethylchlorosilane) are successively introduced [21]. After maceration in oil bath at 60 °C for 5 min, the mixture was decanted. The supernatant is diluted with hexane then filtered before injection into the column. 2.3.2 Ethanolic extract in peel, hull and defatted kernel 40.0 g of peels, 24.3 g of hulls and 53.0 g of defatted kernels were extracted respectively three times in ultrasonic apparatus at 30 °C with mixture of ethanol water 80:20 (3 x 200 ml, 30 min each). The filtered solvent was evaporated under vacuum to obtain respectively 12.3 g (30.6%, w/w) green, 5.8 g (23.9%, w/w) green and 12.5 g (23.6%, w/w) red solid gum. The identification of constituents is obtained after purification of ethanolic extract. 2.3.2.1 Separation of ethanolic extracts 893.3 mg of ethanolic extract of the kernel was treated with ethanol 99 °C to give precipitates 67.2 mg (7. 4% w/w) rich in tannins noted product P. Product P is used for analysis in 1D NMR and 2D NMR and LC/MS ESI of tannin constituents of kernel in “manga hiesy”. Residue ethanolic of peel (12.3 g), hull (5.8 g) and kernel defatted (6.2 g) was suspended respectively in hot water (40 °C) and filtered in warm to remove the chlorophyll. The obtained extractive solution was respectively partitioned by successive extractions three times with different solvents of increasing polarity to yield hexanic (peel 3.4 g: 28.0%; defatted kernel 0.0012 g: 0.02% ; hull 0.09 g: 1.7% ), methylene chloride (peel 0.36 g: 2.94%; defatted kernel 0.012 g: 0.20%; hull 0.013 g: 0.22% ), ethyl acetate (peel 4.44 g: 36.27%; defatted kernel 2.23 g: 36.00%; hull 0.96 g: 16.55%) and aqueous (peel 4.00 g: 32.6%; defatted kernel 3.90 g: 62.75%; hull 4.63 g: 79.84%) fractions. AcOEt extracts of peel and hull are used respectively for identification of polyphenolic by comparison of retention time and mass with kernel constituent. Gallotannins in ethyl acetate extracts of peel, hull and kernel were saponified with NaOH 10 N then acidified with H2SO4 to

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give gallic acid. 2.3.2.2 Analysis of polyphenol in fractions of crude ethanolic extracts of “manga hiesy” by HPLC with MS ESI. Chromatography was carried out on an EC 125/3 NUCLEODOR C18 Polar Tec column (125 mm x 2mm, 3 µm particle size) from Macherey-Nagel (Germany), at room temperature using solvent A (0.01% v/v formic acid in water) and solvent B (acetonitrile) with a gradient program: 10% B to 17.5% B (25 min), 17.5% B to 20% B (25 min), 20% B to 40% B (20 min) and equilibration in 2 min. Flow rate and injection volume were 0.3 ml/min and 4 µl, respectively. Precipitates of ethanolic extract of kernel and ethyl acetate fractions of peel and hull were obtained from “manga hiesy” according to the protocol described in part 2.3.2.1. Each extract (4 mg/ml) was dissolved in methanol: water (20:80) for qualitative determination of constituents. The selectivity of the method was determined by analysis of standard compound and samples. The peak of gallic acid was identified by comparing its retention time with those of the standard and the mass spectrum. 2.3.3 Aqueous acetone extract of peel, hull and defatted kernel To evaluate the polyphenolic content, peel ( 2.5 g), hull ( 3.2 g ) and defatted kernel ( 5.0 g ) of “manga hiesy” were extracted respectively with acetone / water 70/30 in ultrasonic for 30 minutes ( 3 times ) to give aqueous acetone extracts of kernel ( 1.7672 g, 35,34%w/w) , peel (0.6225 g, 24.9%w/w), hull (0.5439 g, 21,7%) and a residue of kernel (3.2328 g, 64.656% w/w) , peel (2.5775 g, 80.54% w/w) , hull (1.9561 g, 78,2% w/w). 2.3.4 Determination of total polyphenol content in aqueous acetone and ethanol extracts of by-product of fruit processing of “manga hiesy” Total polyphenol content was measured by the Folin–Ciocalteu method with slight modification [22]. An aliquot of 0.5 ml of sample solution (with appropriate dilution to obtain

absorbance in the range of the prepared calibration curve) was mixed with 2.5 ml of Folin–Ciocalteu reagent (10 times dilution). After two minutes, 2.0 ml of saturated Na2CO3 solution (145 g/L) was added. The mixture was allowed to react at 50 °C for 5 min, then to cool and stand 5 min before the reading of absorbance of the reaction mixture at 760 nm. A calibration curve of gallic acid (ranging from 0.01 to 0.10 mg/ml) was prepared, and total polyphenolic content was standardised against gallic acid and expressed as mg gallic acid equivalent per gram of sample on a dry weight basis. 2.3.5 Quantification of hydrolysable tannin in aqueous acetone extract of “manga hiesy” The tannin content was determined by the official hide-powder method [23]. Aqueous solutions of concentration 4g / l (1 g/ 250 ml) were prepared from these samples, using hot water (40 ° C). 10 ml of each extract were evaporated and then dried in an oven at 100 °C until a constant weight to afford a mass of extract A. Twenty milliliter of each extract was filtered on filter paper three times until a clear solution. Ten milliliter of the filtrate were evaporated and dried in the same condition to give the mass of soluble extract B. Hundred milliliter of each sample are introduced into a 250 ml Erlenmeyer flask containing 3.5 g of skin powder pre-chromed then magnetically stirred for ten minutes. After filtration on Buchner, 10 ml of the limpid filtrate were evaporated and dried under the same conditions as above to give a mass of untannin solution C. The difference between the soluble extract mass (B) and the mass of extract untannin (C) gives the tannin content of the extract. 3. Results and discussion 3.1. Oil kernel The oil content of “manga hiesy” kernel (6, 1%) is low compared to literature data; 31% of oil kernel was found using a mixture of chloroform methanol as solvent [9]. Variation in oil yield may be due to the difference cultivation climate, ripening stage, the harvesting time of the seeds kernel and the extraction method used. The GC/MS of oil kernel of “manga hiesy” is given in figure 1.

RT: 0.00 - 28.03

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22.9017.32 25.46 25.9019.4215.8210.062.65 7.15 9.466.23 10.73

NL:1.59E7TIC F: MS DSQ131112-01

Fig 1: Separation of fatty acids and sterols in manga hiesy kernel oil by GC. Peak assignment: tr= 13.36: palmitic acid; tr=14.50: linoleic acid; tr=14.51:oleic acid, tr=14.64:stearic acid, tr=21.02:campesterol, tr=21.25:stigmasterol, tr =21.83 β-sitosterol.

3.2 Fatty acid composition of kernel oil The major unsaturated fatty acids in “manga hiesy” kernel oil

(6.1% of kernel) were oleic (46.22%) and linoleic (7.33%) acids and the main saturated fatty acids are stearic (8.72%),

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palmitic (37.73%). 3.3 Phytosterol composition of kernel oil The three major phytosterol (3% of kernel) detected in “manga hiesy” kernel (Figure 2) are campesterol (19.5%), stigmasterol (25.0%) and β-sitosterol (55.5%). The constituents of oil

kernel in “manga hiesy” are consistent with literature data (Gaydou et al., 1984). This result showed that oil kernel of “manga hiesy” can be used in cosmetic industry and it is a source of phytosterol used as a dietary supplement to prevent cardiovascular disease [25].

Fig 2: Phytosterol in kernel oil of “manga hiesy”.

3.2 Constituents of peel, hull and kernel of “manga hiesy” 3.2.1. Tannin and phenolic acid composition of ethanolic extract of kernel A fraction noted P (1.74%) of “manga hiesy” kernel powder

was prepared by precipitation with ethanol 99° of ethanolic extract. The separation by HPLC of this fraction from kernel is shown in Figure 3.

Fig 3: Separation of phenolic acids and gallotannins in “manga hiesy” kernel by HPLC. Peak assignment: (1) sodium gallate, (2) methyl gallate,

(3) tri-O-galloyl-glucoside, (4, 5, 6, 7, 8) five isomers of tetra-O-galloyl-glucoside, (9) penta-O-galloyl-glucoside, (10) hexa-O-galloyl-glucoside, (11) hepta-O-galloyl-glucoside, (12) octa-O-galloyl-glucoside.

Phenolic acids Three phenolic acids were detected in the ethanolic fraction of kernel. Compound 1 showed a [M-H]- ion of m/z 191 identified as sodium gallate by ESI mass spectrometry. Compound 2 showed a [M-H]- ion of m/z 183 identified as methyl gallate. The third compound had a [M-H]- ion of m/z 197 and identified as ethyl gallate is a compound 4. It is a major product of P fraction. Ten milligrams of ethyl gallate (total yield 5%) had been isolated from ethyl acetate fraction of kernel. The signal characteristics of galloyl moiety (G5 and G4 of phenolic acids in table 3), ethoxy group (1.32 ppm /18.3 ppm; 4.24 ppm /58.24 ppm) and methoxy group (51.6 ppm / 3.89 ppm) were observed in NMR spectra of fraction P [26]. Gallotannins Ten gallotannins were identified in kernel using mass spectra. They are consisted of glucose and three to nine gallic acids moieties. The NMR spectra of kernel fraction exhibited many

peaks for gallotannins [27, 28]. Data NMR of glucose and galloyl moiety in gallotannins of kernel fraction is presented in tables 1 and 2 respectively. Table 1: Data NMR of glucose moiety in gallotannins of the kernel of

manga hiesy in CD3OD

Glucose moiety δ1H (ppm) δ 13C (ppm)

CH 5.75 93.8 CH 3.77 73.28 CH 4.1 74.37 CH 3.63 71.39 CH 3,83 75.9

4CH2 3.25, 3.41, 3.50, 3.51

63.25, 62.39, 60.85, 60.84

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Table 2: Data NMR of galloyl moiety of hydrolysable tannins and esters of gallic acid in ethanolic extract of kernel of “manga hiesy” in CD3OD (δ ppm)

Position δ (13C)ppm

G1a δ (13C) ppm

G2a δ (13C) ppm

G3a δ (13C) ppm

G4a δ (13C) ppm

G5a

δ (13C) ppm G6a

-O-C=O 165.9 (1C) 165.5(1C) 165.4(1C) 164.7(1C) 167.6 (1C) 166.5 (1C)

1 118.8(1C) 118.7(1C) 118.5(1C) 118.7(1C) 119.9(1C) 119.5(1C)

2, 6 6.92b(2H) 108.8(2C)

6.97b(2H) 108.8(2C)

6.99b(2H) 108.9(2C)

7.06b(2H) 108.0(2C)

7.07b(2H) 107.1(2C)

7.13b(2H) 108.7(2C)

3 144.7(1C) 144.8(1C) 144.9(1C) 145.1(1C) 145.1(1C) 144.9(1C) 4 138.6(1C) 138.6(1C) 139.0(1C) 139.2(1C) 138.3(1C) 138.7 (1C) 5 144.7(1C) 144.8(1C) 144.9(1C) 145.1(1C) 145.1(1C) 144.9 (1C)

a galloyl moiety group b chemical shift of proton in galloyl moiety

Eight types of galloyl moiety for gallotannins and phenolic acids are observed in kernel fraction. Examples of polyphenol

structure detected in ethanolic extract of “manga hiesy” kernel are presented in Figure 4.

Fig 4: Examples of polyphenol structure in “manga hiesy” kernel.

Phenolic acids and gallotannins identified in kernel are given in table 3.

Table 3: Characteristic ions of phenolic acids and gallotannins from defatted kernel extract of Mangifera indica L. Var. Hiesy

N° peak Retention times (mn) Identified compounds [M-H]-

1 9.7 Sodium gallate 191 2 13.7 Methyl gallate 183 3 15.0 Tri-O-galloyl-glucoside 635 4 17.2 Tetra-O-galloyl-glucoside 787

5 to 8 17.2-18.2 (18.2) four isomers of tetra- O-galloyl-glucoside 787 9 19.1 Penta- O-galloyl-glucoside 939 10 20.3 Hexa- O-galloyl-glucoside 1091 11 21.1 Hepta- O-galloyl-glucoside 1243 12 21.6 Octa- O-galloyl-glucoside 1395

The fraction containing mainly gallic acid and ester represented 7.4% of ethanolic extract ie 1.74% of kernel.

3.2.2. HPLC/MS ESI analysis of ethyl acetate extract of “manga hiesy” peel The separation of AcOEt extract from “manga hiesy” peel is shown in figure 5.

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Fig 5: Separation of phenolic acids, flavonoid and gallotannins in “manga hiesy” peel by HPLC. Peak assignment: (1) gallic acid, (2) di-O-

galloyl-glucoside, (3) catechin (4, 5) two isomers of tri-O-galloyl-glucoside, (6) ethyl gallate, (7) three isomers of tetra-O-galloyl-glucoside, (8) penta-O-galloyl-glucoside, (9) hexa-O-galloyl-glucoside, (10) hepta-O-galloyl-glucoside, (11) nona-O-galloyl-glucoside.

Phenolic compounds are identified as phenolic acid, flavonoid and gallotannins by their mass and compared of constituents of

kernel, which are given in table 4.

Table 4: Characteristic ions of phenolic acid, flavonoid and gallotannin from peel extract of Mangifera indica L. Var. Hiesy

N° pics Retention time (mn) Identified tannin [M-H]-

1 7.8 Gallic acid 169 2 11.3 nd 443 3 13.8 Di-O-galloyl-glucoside 483 4 14.5 catechin 289 5 14.9 Tri-O-galloyl-glucoside 635 16.1 Tri-O-galloyl-glucoside 635

6 17.0 Ethyl gallate 197 7 18.2 Tetra- O-galloyl-glucoside (four isomers) 787 8 19.1 Penta- O-galloyl-glucoside 939 9 20.0 Hexa- O-galloyl-glucoside 1091 10 20.8 Hepta- O-galloyl-glucoside 1243 11 22.2 Nona- O-galloyl-glucoside 1548

Two phenolic acids are identified: gallic acid 1 and ethyl gallate 6. Based on the identification above compounds 3 and 5-11 could be attributed as di, tri-, hexa-, hepta-, octa- and nona-O-galloyl-glucoside respectively. The fraction (ethyl acetate extract of peel) containing mainly gallic acid and esters represented 36.28% of ethanolic extract

ie 11.10% of the peel. 3.2.3. HPLC/MS ESI analysis of ethyl acetate extract of “manga hiesy” hull The separation of AcOEt extract from “manga hiesy” hull is shown in Figure 6.

Fig 6: Separation of phenolic acids, flavonoid and gallotannins in “manga hiesy” hull by HPLC. Peak assignment: (1) gallic acid, (2) di-O-galloyl-glucoside, (3) catechin (4, 5) tri-O-galloyl-glucoside,(6) ethyl gallate, (7) three isomers of tetra-O-galloyl-glucoside, (8, 9) penta-O-galloyl-glucoside, (10) hexa-O-galloyl-glucoside, (11, 12) hepta-O-galloyl-glucoside, (13) octa-O-galloyl-glucoside, (14) nona- O-galloyl-

glucoside.

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Phenolic compounds are identified as phenolic acid and gallotannins by their mass and comparison of data in kernel

constituents, which are presented in table 5.

Table 5: Characteristic ions of phenolic acid, flavonoid and gallotannin from hulls extract of Mangifera indica L. Var. Hiesy

N° pics Retention time (mn) Identified compound [M-H]-

1 7.8 Gallic acid 169 2 13.8 Di-O-galloyl-glucoside 483 3 14.5 Catechin 289 4 14.9 Tri-O-galloyl-glucoside 635 5 16.1 Tri-O-galloyl-glucoside 635 16.5 Nd 321

6 16.9 Ethyl gallate 197 7 17.9 Tetra- O-galloyl-glucoside (four isomers) 787 8 19.2 Penta- O-galloyl-glucoside 939 9 19.6 Penta- O-galloyl-glucoside 939 10 19.9 Hexa- O-galloyl-glucoside 1091 11 20.6 Hepta- O-galloyl-glucoside 1243 12 21.3 Hepta- O-galloyl-glucoside 1243 13 21.8 Octa- O-galloyl-glucoside 1395 14 22.2 Nona- O-galloyl-glucoside 1548

Constituents of hull were identified by comparison of the chromatogram of peel and kernel. Other gallic acid (compound1) , di-O-galloylglucoside (compound 2), catechin (compound 3) and ethyl gallate (compound 6), compounds 4, 5 and 7-14 could attributed respectively to tri, tetra- (four isomers), penta-, hexa-, octa- and nona-O-galloylglucoside. The fraction ethyl acetate extract of hull containing mainly flavonoid, gallic acid and ester represented 16.55% of ethanolic extract and 4.06% of hull. Peels, kernels and hulls of “manga hiesy” contain gallotannins such the other varieties of Mangifera indica [29, 30]. Tri- to nona-O-galloylglucoside were identified using LC/MS ESI and NMR spectra. 3.3. Polyphenolic content The percent of total polyphenolic content expressed as mg gallic acid equivalent per gram of sample on a dry weight are given in table 6. Table 6: Determination of total polyphenol content of crude extracts

using Folin-Ciocalteu method

Crude extract Peel (%) Kernel (%) Hull (%) Ethanolic 22.8 35.7 11.3 Hydroacetonic 37.2 42.7 13.6

The Folin-Ciocalteu reagent is used to obtain a crude estimate of the amount of phenolic compounds present in an extract. Different crude extracts of kernel, peel and hull (ethanolic, and hydroacetonic (70/30) were prepared in order to compare their total phenolic content. As results, total phenolic content of the extracts decreased in the following order, as shown in Table 6: hydroacetonic extract of kernel > hydroacetonic extract of peel > crude ethanolic extract of kernel > crude ethanolic extract of peel > hydroacetonic extract of hull > crude ethanolic extract of hull. The total phenolic content of hydroacetonic extracts (kernel, hull, peel) was higher than those of crude ethanolic extracts. The lower polyphenol value was found in crude ethanolic extract of hull.

3.4. Content tannin The amount in hydrolysable tannin of aqueous acetone in peel, kernel and hull in “manga hiesy” are shown in table 7.

Table 7: Tannin content of hydroacetonic extract in peel, hull and kernel of “manga hiesy”

Hydroacetonic extract Peel Kernel Hull

Tannin content (%) 15,7 29,6 12,0

The higher tannin value was found in crude hydroacetonic extract of kernel. Hydroacetonic extract of kernel seems to be rich in tannin. Its result is in agreement with the literature data [31]. 4. Conclusion Results obtained showed that “manga hiesy” kernel powder contained 0.5 % moisture, 6,1% crude oil (hexane), 10.44% gallotannins, 4.63% other polyphenol compounds, and 64.25 % residual containing protein and starch. Based on the above reviews, it could be concluded that the “manga hiesy” kernel, hull and peel could be used as a potential source for bimolecular: polyphenolic (natural antioxidants), gallotannin (antimicrobial compounds), sterol (cosmetic), in addition, it could be further processed into therapeutic functional food products. Saponification of gallotannins furnished an amount quantity of gallic acid after acidification. This suggests that the kernel, hull and peel should be further utilized rather than just discarded as waste. From an economic point of view, it seems that this valorization can be envisaged only if all the valuable compounds are separated and used. An abstract of valorization method is given in Figure 7. The total percent of gallotannins and other polyphenol compounds of by-products of “manga hiesy” fruits processing is given.

~ 232 ~


Fig 7: Valorization method of kernel, hull and peel of Mangifera indica L. Var. Hiesy fruits.

Therefore, the utilization of by-products of “manga hiesy” fruits processing especially kernel, hull and peel, may be an economical way to reduce the problem of waste disposal from “manga” production in Madagascar. We can obtain 61 g oil rich in phytosterol (3%) with 1 kg of kernel. The extracts of peel, hull and defatted kernel exhibited a high phenolic content due to the occurrence of important amounts of gallotannin. This is probably the main interest of such waste. In 1kg of by-product of “manga hiesy” fruit processing, 169.4 g of gallotannins can transformed into gallic acid using in organic synthesis and 103.4 g other phenolic compounds will be employed such natural antioxidant. Moreover only very few aromatic derivatives are available from natural renewable sources gallic acid had aromatic with four oxygenated functional groups. This molecule may be transform into numerous useful reagents and intermediates using relatively simple chemistry and we work now on the transformation of the obtained gallic acid. As far as we knew, composition of “manga hiesy” peel, hull and kernel and related products have not been previously characterized in detailed knowledge as we do. This composition is of interest as far as renewable starting material is sought. 5. Acknowledgements We would like also thank all members of the laboratory “Catalyse et Synthèse Environnement” ICBMS - UMR5246 - Université Claude Bernard Lyon 1, France. The authors gratefully thank the “Agence Universitaire de la Francophonie” (AUF) and ICBMS - UMR5246 - Université Claude Bernard Lyon 1, France for financial support on this research. 6. References 1. Nathalie W, Aliou B, Elhadj SB, Marc VD, Pierre D.

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International Journal of Indigenous Medicinal Plants, ISSN: 2051- 4263, Vol.47, Issue.1 1594

© RECENT SCIENCE PUBLICATIONS ARCHIVES |May 2014|$25.00 | 27703554|

*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*

GC/MS Analysis of Volatile Compounds of Leaves

and Stem Barks of

Artabotrys Hildebrandtii O.Hffm Julio Hervé Andriamadio

Université Nord Madagascar, Department of Chemistry, Antsiranana, Madagascar.

Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique,

Department of Organic Chemistry, Université d‟Antananarivo, Madagascar.

Email:[email protected]

Léa Herilala Rasoanaivo Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique,



Radu Oprean

University of Medicine and Pharmacy 'Iuliu Hatieganu',

Department of Analytical Chemistry, Cluj-Napoca, Romania. Email:[email protected]

Ede Bodoki

University of Medicine and Pharmacy 'Iuliu Hatieganu',

Department of Analytical Chemistry, Cluj-Napoca, Romania. Email:[email protected]

Ilioara Oniga University of Medicine and Pharmacy 'Iuliu Hatieganu',

Department of Pharmacognosy, Cluj-Napoca, Romania.


Amélie Raharisololalao

Université Nord Madagascar, Department of Chemistry, Antsiranana, Madagascar. Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique,



ABSTRACT

Artabotrys hildebrandtii O. Hffm is an endemic plant of

Madagascar belonging to Annonaceae family. The

essential oils of leaves and stem barks of this plant were

extracted by hydrodistillation and analysed by gas

chromatography coupled with mass spectrometry

(GC/MS), to determine the chemical composition of the

volatile fraction and identify the chemotypes of the species.

A total of twenty-eight compounds are identified in the

two samples. The major compounds of the stem barks oil

were tau-muurolol (14.03%), α-cadinol (12.52%), δ-

cadinene (8.237%) and τ-himachalene (6.736%). The

leaves oil was found to contain δ-cadinene (15.051%),

caryophyllene (11.407%) and benzylbenzoate (11.156%)

as the major constituents. The essential oil gives yellowish

colour and presents a strong odour aromatic fragrance;

yields were 0.16% for leaves and 0.12% for stem barks.

Keywords- Artabotrys hildebrandtii, Annonaceae,

Essential oil, Medicinal plants, GC/MS

1. INTRODUCTION

The Annonaceae family are plants comprising only trees,

shrubs or rarely vines pantropical regions or they most

often develop at low altitude. The family is concentrated

in the tropics and some species are found in temperate

regions [1], [2]. About 900 species are neotropical, 450 are

afrotropical, and other species Indomalayannes [3].

In Madagascar, there are 11 genera of vegetal species:

Isolona, Uvaria, Xylopia, Hexalobus, Desmos, Artabotrys,

Polyalthia, Popowia, Cananga, Annona and Miliusa [1].

The genus Artabotrys R. Br. is strictly paleotropical [4]

and comprises over 100 species [5],[6] including seven

species are endemic of Madagascar: Artabotrys

scytophyllus Diels., Artabotrys luxurians Ghesq.,

Artabotrys mabifolius Diels., Artabotrys uncinatus Lamk.,

Artabotrys madagascariensis Miq., Artabotrys

hildebrandtii O.Hffm and Artabotrys darainensis Deroin

et L. Gaut [4]. Artabotrys hildebrandtii O. Hffm is a vine

plant, cylindrical branches, reddish-brown, glabrous. Leaf

petiole to 3 mm long, blade oblong, acuminate, obtuse at

apex, obtuse at base, 5-12 cm long and 2-5 cm wide,

subcoriace, glabrous, midrib prominent, lateral veins

prominent, well cured; 7-9 pairs of secondary veins.

Peduncle hook bearing 1-2 flowers with pedicel 2-4.5 cm

long, triangular sepals 3mm long and 5mm wide at the

base, glabrous, petals ovate, attenuated towards the apex,

thick, subequal, internal somewhat narrower 17mm long




and 10mm wide at the base, densely tomentose hairs

reddish, many stamens connective dilated apex; carpels

12-20 to 2mm long, pubescent [1].

In our traditional medicine, leaves and stems of Artabotrys

hildebrandtii O. Hffm (vernacular names fotsiavadika,

rangomanitra, manitranala, vahimbavy) is used to treat

„‟Ambo‟‟ what it means; to cure many diseases related to

liver problems, fatigue, fever with flu-like symptoms and

neurological signs. For this, decoction and steam bath of

the stem with leaves has been used by the population.

The aim of this paper is to isolate and identify these

compounds.

Herein, we report for the first time the chemical

composition of the essential oil obtained from the stem

barks and leaves of Artabotrys hildebrandtii O. Hffm.

To our knowledge no chemical and biological study has

been conducted on this plant. This is the first extraction of

volatile oil from the leaves and stem barks of Artabotrys

genus in Madagascar.

2. EXPERIMENTAL

2.1 Plant material

Artabotrys hildebrandtii O.Hffm was harvested at Joffre

Ville, located 20km from the city of Diego Suarez

Madagascar. Harvests were made in december 2013.

Leaves and stem barks were cut into pieces and spread on

screens stretched over wooden frames. Drying was done in

the shade in a well-ventilated, dry room for 30 days. The

plant was identified by botanists at the botanical and

zoological park of Tsimbazaza Antananarivo, Madagascar.

A voucher specimen has been deposited in the

“Laboratoire de Chimie des Substances Naturelles et

Chimie Organique Biologique” (DSM 423).

2.2 Essential oil extraction

The classical method with Clevenger type apparatus is

used. The essential oil was isolated by hydrodistillation for

4 h. Samples of the obtained essential oils were dissolved

in cyclohexane and stored at 4ºC until their analysis by

GC/MS.

2.3 Thin-layer chromatography (TLC)

Thin layer chromatography was performed on 0.2 mm

precoated silica plates (Kiesegel 60, Merck) with

toluen/ethyl acetate (90:10, v/v) as mobile phase. The

volatile compounds were detected using the vanillin

sulphuric reagent, and then dried at 110°C. Appearance of

pink, brown colour spots shows the presence of essential

oils [7].

1.4 Gas chromatography- mass spectrometry

(GC/MS)

The chemical analysis of essential oils was also made by

GC/MS (Agilent Technologies 5975C MS, 7890A GC

system, series 7683B) equipped with a HP-5 (5% phenyl,

95% methyl-polysiloxane) capillary column (30 m ×

25µm i.d., 0.25 µm film thickness). The injector and

interface were operated at 150˚C and 250˚C, respectively.

The oven temperature was programmed to rise from 60˚C

to 240˚C (3˚C/min). Helium was the carrier gas, 1μl

injection volume and a split ratio of 1:15. In mass

spectrometry electron-impact ionization was performed at

electron energy of 70 eV. The constituents of essential oils

were identified based on their Kovats Index, calculated in

relation to the retention time of a series of n-alkanes as

standard. The compounds were identified by comparison

of retention indices with those reported in the literature

and by comparison of their mass spectra with the NIST

and WILEY databases [8], [9].

3. RESULTS AND DISCUSSION

Artabotrys hildebrandtii O.Hffm leaf and stem bark were

submitted to hydrodistillation. The yields were 0.16% and

0.12% w/w.

The most effective and convenient method for quick

qualitative analysis is the thin layer chromatography. We

have employed this method for preliminary analysis of the

essential oil. When the TLC plate was treated with vanillin

sulfuric reagent and heated to 110 °C for a few minutes,

there is a faint pink, blue, yellow and brown colour spots

that shows the presence of essential oils (terpene

hydrocarbons).

The constituents of the essential oil were established by

comparing their MS spectra with Nist and Wiley mass

spectra database. The retention time and chemical

composition of leaves and stem barks of Artabotrys

hildebrandtii essential oil are presented in Table1.

Table 1 lists the compounds identified in the essential oils

of leaf and stem bark of Artabotrys hildebrandtii, in which

the percentage and Kovats retention indice of components

are given. The presented results indicate that seventy

compounds were identified in the stem barks of A.

hildebrandtii, which represent 92.102% of the total

essential oil components.




Table1. Composition of the essential oil from leaves and stem barks

of Artabotrys hildebrandtii O. Hffm

Constituents RI Percentage (%)

Stem barks Leaves

Benzaldehyde 965 - 0.43

Octanal 991 0.036 -

o-Cymene 1028 0.033 0.097

Benzaldehyde,4-methyl 1047 1.215 -

Limonene 1048 - 0.198

Dihydro-myrcenol 1074 0.046 -

Nonanal 1095 0.093 -

β-Linalool 1109 0.533 2.254

Benzenemethanol,2- methyl 1150 2.686 -

Trifluoroacetyl-α-terpineol 1167 0.040 -

Decanal 1188 0.041 -

cis-Geraniol 1210 0.048 -

Benzene, (1-nitroethyl)- 1211 0.212 -

α-Cumene hydroperoxide 1268 0.041 -

Diepicedrene-1-oxide 1281 0.759 -

Ledene oxide II 1293 0.158 0.427

Bicyclohexane 1304 0.731 1.002

α-Cubebene 1345 0.207 2.148

Farnesane 1365 - 0.312

1,9-Dichlorononane 1366 0.050 -

β-Elemenea 1373 - 0.257

Copaene 1375 0.474 2.911

Diphenylether 1378 0.097 -

Isolongifolene,4,5,9,10-dehydro 1380 0.513 -

7- tetramethyl[6.2.1.0(3.8)0(3.9)]undecanol,4,4,11,11-tetramethyl- 1385 0.761 -

β-Bourbonene 1386 - 0.223

Ylangene 1396 0.076 -

β-Elemenea 1398 0.048 -

β-Cubebene 1400 0.070 2.312

Tetradecane 1400 - 0.146

τ-Patchoulene 1423 0.033 -

τ-Elemene 1425 0.791 1.161

α-Guaiene 1426 - 0.263

Caryophyllene 1442 0.349 11.407

α-Caryophyllene 1448 0.361 3.437

α-Amorphene 1451 0.511 -

Alloaromadendrene 1460 1.077 1.305

epi-Bicyclosesquiphelandrene 1470 0.106 1.675

τ-Cadinene 1471 2.643 4.540

β-Guaiene 1482 0.453 -

Aristolene 1484 0.041 -

τ-Muurolene 1486 1.260 2.771

Tetracyclo(6.3.2.0(2,5).0(1,8)tridecan-9-ol,4,4-dimethyl 1490 0.613 0.188

α-Muurolenea 1495 1.597 2.052

δ-Cadinene 1497 8.237 15.051

α-Muurolenea 1498 0.459 -

τ-Himachalene 1499 6.736 -

1-Isopropyl-4,7-dimethyl-1,2,4a,5,8a-hexahydronaphthalene 1516 - 0.352

β-Cadinene 1529 0.377 -

Elemicin 1530 0.084 -

Elemol 1533 0.078 -

a Correct isomer (E or Z) is not identified. Identification is performed by MS database comparison.

RI: Retention indices relative to n-alkanes on a HP-5 column.




Table1. Composition of the essential oil from leaves and stem barks

of Artabotrys hildebrandtii O. Hffm (continued)

Constituents

RI

Percentage (%)

Stem barks Leaves

Cadinadiene-1,4 1546 0.379 0.832

4-(2-acetyl-5,5-dimethylcyclopent-2-enyldene)butan-2-one 1560 0.218 -

Caryophyllene oxide 1561 0.483 4.499

Globulol 1571 2.642 0.997

Spathulenol 1576 1.550 3.558

Isoaromadendrene epoxide 1579 0.643 -

Neoisolongifolene,8-oxo- 1587 0.291 -

Viridiflorol 1588 0.116 -

α-Calacorene 1590 2.741 0.217

tricyclo [5.2.2.0(1,6)]undecan-3-ol, 2-methylene-6,8,8-trimethyl- 1599 1.289 -

Guaiol 1600 - 1.904

5-azulene methanol,1,2,3,4,5,6,7,8-octahydro-α,α,3,8-tetramethyl 1614 0.244 -

α-Cadinol 1632 12.528 3.039

tau-Muurolol 1635 14.569 4.671

Cubenol 1645 1.705 3.727

δ-Cadinol 1646 4.371 1.713

Bulnesol 1651 - 0.407

spiro(2,7)dec-4-ene,1,1,5,6,6,9,9-heptamethyl-10-methylene 1656 0.122 -

Eudesm-7-en-4-ol 1680 1.209 -

6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydro-naphthalen-2-ol 1690 0.098 -

Heptadecane 1700 - 0.402

murolan-3,9-diene-10-peroxy 1729 2.602 -

Cadalene 1735 2.149 -

Benzylbenzoate 1738 2.139 11.156

7-Methyl-4(1-methylethyldene) bicyclo|5.3.1|undec-1-en-8-ol 1754 - 0.173

Benzylsalicylate 1824 3.201 0.299

Longifolenaldehyde 1876 0.111 -

1-butyl 2-isobutyl phthalate 1900 0.132 -

benzyl 2-methoxybenzoate 1922 1.133 -

2,9- heptadecadiene- 4,6-diyn-8-ol,(Z,E) 1924 0.118 -

Cyclodecasiloxane eicosamethyl- 2029 - 4.899

phthalic acid butyloctylester 2317 0.495 -

Total 92.102% 99.527%

a Correct isomer (E or Z) is not identified. Identification is performed by MS database comparison.

RI: Retention indices relative to n-alkanes on a HP-5 column.

These compounds can be classified in terpenic

hydrocarbons, oxygenated terpenic, alcohols, aldehydes,

esters and alkanes.

Oxygenated sesquiterpenes (46.820%), sesquiterpenes

hydrocarbons (31.688%) and esters (7.1%) were the

predominant class, of which the major components were

tau-muurolol (14.569%), α-cadinol (12.528%), δ-cadinene

(8.237%) and τ -himachalene (6.736%).

Furthermore, the essential oil obtained from the leaves of

A. hildebrandtii was found to contain forty one

compounds including oxygenated monoterpenes (2.254%),

oxygenated sesquiterpenes (25.303%), sesquiterpenes

hydrocarbons (53.226%) and esters (11.455%),

representing 99.527% of the total oil composition. These

results are shown in Table 2.

Table 2. Classification of constituents of essential oils

from leaves and stem barks of Artabotrys hildebrandtii

Class Stem

barks %

Leaves %

Monoterpene

hydrocarbons

0.033 0.410

Oxygenated

monoterpenes

0.627 2.254

Sesquiterpene

hydrocarbons

31.688 53.226

Oxygenated

sesquiterpenes

46.820 25.303

Aldehydes 1.385 0.430

Esters 7.100 11.455

Alkanes 0.731 1.550

Alcohols 2.686 -




The main constituents were δ-cadinene (15.051%),

caryophyllene (11.407%) and benzylbenzoate (11.156%)

as the major constituents. As indicated above, the stem

bark oil was rich in sesquiterpene hydrocarbones

(53.226 %), oxygenated sesquiterpenes (25.303%).

Furthermore, both oils obtained from the leaf and stem

bark are rich in compounds sesquiterpenoids. The

percentages were 78.529% and 78.508% respectively.

benzylbenzoate (11.156%) as the major constituents. As

indicated above, the stem bark oil was rich in oxygenated

sesquiterpenes. The main constituents were δ-cadinene

(15.051%), caryophyllene (11.407%) and (46.820%)

followed by sesquiterpene hydrocarbones (31.688%) while

oil of the leaves.

Artabotrys hildebrandtii contain essential oil rich in

sesquiterpenes compounds comparing with the other

species such as A. odoratissimus, A. hexapetalus and A.

lastoursvillensis from Gabon [10].

The variation of the compounds of the essential oils from

leaves and stem barks of Artabotrys hildebrandtii are

given in figure 1.

Figure 1: Variation of the compounds of the essential oils

from leaves and stem barks of Artabotrys hildebrandtii.

4. CONCLUSIONS

The aim of this study was to describe the chemical

composition of essential oil of leaves and stem barks

Artabotrys hildebrandtii O.Hffm from Madagascar. The

essential oil obtained from leaves and stem barks by

hydrodistillation were analysed by Gas Chromatography-

Mass Spectrometry (GC/MS). Seventy and forty one

compounds were identified in both oils of the leaf and

stem bark. The yields were 0.16% and 0.12%, respectively.

The identified total compounds in the stem bark accounted

for about 92.102% of the oil, and were characterized as

tau-muurolol (14.569%), α-cadinol (12.528%), δ-cadinene

(8.237%), γ-himachalene (6.736%), torreyol (4.371%), α-

calacorene (2.741%),

τ-cadinene (2.643%), globulol (2.642%), murolan-3,9-

diene-10-peroxy (2.602%), cadalene (2.149%), α-

muurolene (1.597%), spathulenol (1.55%),

tricyclo[5.2.2.0(1,6)]undecan-3-ol,2-methylene-6,8,8-

trimethyl- (1.289%), τ-muurolene (1.260%) and eudesm-

7-en-4-ol (1.209%). The essential oil obtained from the

leaves representing 99.527% of the total oil composition

and were characterized as δ-cadinene (15.051%),

caryophyllene (11.407%), tau-muurolol (4.671%), τ-

cadinene (4.54%), caryophyllene oxide (4.499%), cubenol

(3.727%), spathulenol (3.558%), α-caryophyllene

(3.437%), α-cadinol (3.039%), copaene (2.911%), τ-

muurolene (2.771%), β-cubebene (2.312%), α-cubebene

(2.148%), α-muurolene (2.052%), guaiol (1.904%) and δ-

cadinol (1.713%) .

The constituentss of leaves and stem barks were consisting

to sesquiterpenoids. It is evident that there is a relationship

between the strong antimalarial activity and the high

sesquiterpenes content of Artabotrys hildebrandtii

essential oil. Otherwise there are synergetic phenomenon

between several components of the oil from this plant.

Such efficiency may be promising for utilisation of this oil

alone or in combination to antifever drugs.

ACKNOWLEDGEMENT

We are grateful to the “Agence Universitaire de la

Francophonie (AUF)” for providing the Doctoral

Fellowship „EUGEN IONESCU‟ that facilitated the

GC/MS analysis in the Pharmacy Department, University

of Medicine and Pharmacy „Iuliu Hatieganu‟.

REFERENCES

[1] H. Perrier de la Bathier, 78ème Famille,

Annonacées, Flore de Madagascar et des Comores,

Ed. H. Humbert Muséum National d‟Histoire

Naturelle, Paris, 1958, pp. 2 ; 3 ;49 ;56.

[2] Hutchinson, J. (1964). The genera of flowering

plants (Angiospermae), Dicotyledones (Vol.1),

Oxford, Clarendon Press.

[3] Chatrou, Dr. L.W. (2005). Systématique

moléculaire d‟Annonaceae, Projets de recherche

d‟Annonaceae. Herbier Nederland de National.

[4] Deroin, T. and Gautier, L. (2008). Artabotrys

darainensis Deroin and L. Gaut. (Annonaceae), a

new species from Madagascar. In French, English

and French abstracts. Candollea 63: 93-99.

[5] Kok, K.T. and Christophe, W. (2014), Botanical

Descriptions, Ethnomedicinal and Non-Medicinal

Uses of the genus Artabotrys R.Br, International

Journal of Current Pharmaceutical Research. Vol 6,

Issue 1, pp 34-40.

[6] Brophy J., Goldsack R., Forster P. (2004), Essential

oils from the leaves of some Queensland

Annonaceae, Journal Essent Oil Res. Vol. 16, Issue.

2, pp 95-100.

●Essential oil of leaves

●Essential oil of stem barks

Are

a(%

)

Compounds




[7] Chakraborthy, G.S. (2011), Phytochemical

screening of Mirabilus jalap Linn leaf extract by

TLC, Research Journal of Pharmaceutical,

Biological and Chemical Sciences., RJPBCS Vol. 2,

Issue 1, 521.

[8] Ebru, Ç., Gökçen, Y.Ç., and I, A.M. (2010),

Essential Oil Composition and Antibacterial

Activity of Some Plant Species, Journal of Applied

Biological Sciences, Vol. 4, Issue. 1, pp 45-48.

[9] Shahramamiri and Shahramsharafzadeh. (2014) Essential Oil Components of German chamomile

Cultivated in Firoozabad, Iran. Oriental Journal of

Chemistry, Vol. 30, Issue. 1, pp 365-367.

[10] Tran D.T., Do N.D., Tran M.H., and Isiaka A.O.

(2013), Chemical compositions of the leaf essential

oils of some Annonaceae from Vietnam, Journal of

Essential Oil Research, Vol. 25, Issue. 2, pp 85-91.

HPLC/MS analysis of polyphenols, antioxidant and antimicrobial activitiesof Artabotrys hildebrandtii O. Hffm. extracts

Julio Herve Andriamadioa, Lea Herilala Rasoanaivob, Daniela Benedecc, Laurian Vlased*,

Ana-Maria Gheldiud, Mihaela Dumae, Anca Toiuc, Amelie Raharisololalaob and Ilioara Onigac

aDepartment of Chemistry, Faculty of Science, University of Antsiranana, Antsiranana 201, Madagascar;bLaboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique, Department ofOrganic Chemistry, University of Antananarivo, P.O. Box 906, Antananarivo 101, Madagascar;cDepartment of Pharmacognosy, University of Medicine and Pharmacy ‘Iuliu Hatieganu’, 12 I. CreangăStreet, Cluj-Napoca 400010, Romania; dDepartment of Pharmaceutical Technology andBiopharmaceutics, University of Medicine and Pharmacy ‘Iuliu Hatieganu’, 12 I. Creangă Street, Cluj-Napoca 400010, Romania; eState Veterinary Laboratory for Animal Health and Safety, 1 Piata MarastiStreet, Cluj-Napoca 400609, Romania

The purpose of this work was to evaluate chemical constituents, antioxidant andantimicrobial activities of Artabotrys hildebrandtii, an endemic medicinal plant fromMadagascar. Ethanol extracts from the leaves and stem bark were tested to evaluateDPPH free radical scavenging, using butylated hydroxytoluene and quercetin asstandard antioxidants. An high-performance liquid chromatography/mass spectrometrymethod was developed to investigate the presence of phenolic compounds in thestudied samples; gentisic acid, chlorogenic acid, hyperoside, isoquercitrin, rutin,quercitrin, quercetol, apigenin and luteolin were identified. Total polyphenolic contentwas determined by a spectrophotometric method using Folin–Ciocalteu reagent.Results showed the efficiency of A. hildebrandtii leaves extract against strains ofStaphylococcus aureus and Listeria monocytogenes, as the inhibitory activity is morepowerful compared to Gentamicin, used as the standard drug. The leaves of A.hildebrandtii can be considered an important source of polyphenols, especially of rutin,with good antioxidant and antimicrobial activities.

Keywords: polyphenols; antioxidant; antimicrobial; Artabotrys hildebrandtii;Madagascar

1. Introduction

Health situation is critical in developing countries such as Madagascar. However, Madagascar is

famous worldwide for its rich flora and fauna. About 13,000 species grow here, of which 80%

q 2015 Taylor & Francis

*Corresponding author. Email: [email protected]

Natural Product Research, 2015

http://dx.doi.org/10.1080/14786419.2015.1007458

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mailto:[email protected]


http://dx.doi.org/10.1080/14786419.2015.1007458

are endemic, and most of them are used in many fields, such as pharmaceuticals, cosmetics and

food, which are the same ones used in the ancient past (Cirad- Cite- Gref 1996).

The use of plants in phytotherapy is very old and evermore arouses great interest among the

public. According to World Health Organisation, nearly 6377 plants are used in Africa, there are

400 medicinal plants and they represent 90% of traditional medicines (Diallo 2005). Many

plants from Madagascar have therapeutic properties.

In the present study, the crude ethanolic extracts of Artabotrys hildebrandtii O. Hffm.,

species used in traditional medicine in Madagascar, were evaluated for phenolic compounds

content by using the Folin–Ciocalteu reagent and high-performance liquid chromatography/

mass spectrometry (HPLC/MS) analysis. Antioxidant activity was evaluated using DPPH free

radical-scavenging and antimicrobial activity was tested by disk diffusion method.

The genus Artabotrys (Annonaceae) comprises over 100 species of woody climbers and

scandent shrubs distributed mainly in tropical and subtropical regions of the world, especially

tropical Africa and Eastern Asia. Moreover, Artabotrys species have a long history of traditional

use for a wide range of medical conditions, particularly malaria, scrofula and cholera (Kok et al.

2013).

In Madagascar, seven species are endemic (Deroin & Gautier 2008); A. hildebrandtii is a

vine plant (Perrier de la Bathier 1958) and the leaves and stems are used in traditional medicine

to treat liver diseases, fatigue, fever with flu-like symptoms and neurological signs.

In this paper, we report for the first time the identification and quantification of polyphenols

by HPLC/MS in extracts obtained from leaves and stems bark of this plant species, together with

antioxidant capacity by total phenolic content (TPC), DPPH and antimicrobial activity by disk

diffusion method.

2. Results and discussions

2.1. High-performance liquid chromatography/mass spectrometry

A method of coupling HPLC with MS was optimised for the separation and identification of

phenolic acids, flavonoid glycosides and flavonoid aglycones (phenolic compounds) in the

ethanolic 50% (v/v) extracts of A. hilbebrandtii (Moldovan et al. 2014). In this study, 18

standard phenolic compounds (Table 2) have been investigated in ethanolic extracts. The

simultaneous analysis of different classes of polyphenols was performed by a single column

pass, and the separation of all examined compounds was carried out in 35min. After analysis, 16

of them were identified, eight in A. hildebrandtii leaves (one cinnamic acid derivative:

chlorogenic acid; four flavonoid glycosides: hyperoside, rutin, isoquercitrin and quercitrin; one

flavonol: quercetin; two flavones: apigenin and luteolin) and eight also in A. hildebrandtii stem

bark (one benzoic acid derivative: gentisic acid; one cinnamic acid derivative: chlorogenic acid

four flavonoid glycosides: hyperoside, rutin, isoquercitrin and quercitrin; two flavones: apigenin

and luteolin) by comparing retention times (RTs), UV and MS data with those of the reference

standards (Benedec, Vlase, Oniga, Mot, Silaghi-Dumitrescu, et al. 2013; Vlase et al. 2013;

Moldovan et al. 2014). The chromatograms of phenolic acids and flavonoids for two ethanolic

extracts are presented in Figures 1 and 2. The amounts of identified polyphenolic compounds in

the analysed samples are reported in Table 1.

In A. hildebrandtii ethanol leaves extract, three types of phenolic compounds which are

cinnamic acid derivatives, flavonoid glycosides and flavonoid aglycones were identified.

Quantitative results showed that rutin (37821 ^ 8.50mg/100 g) has the highest concentration

value over other glycosides: isoquercitrin ¼ 5282 ^ 7.50mg/100 g, hyperoside ¼ 1025 ^ 5.50 -

mg/100 g, quercitrin ¼ 926 ^ 4.72mg/100 g; three aglycones, namely quercetin (229 ^ 3.51mg/100 g), apigenin (775 ^ 3.60mg/100 g) and luteolin (537 ^ 5.13mg/100 g) were identified and

2 J.H. Andriamadio et al.

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quantified in the extract; cinnamic acid derivative (chlorogenic acid) was identified but the

concentration was too low to be quantified (Table 2).

In A. hildebrandtii, ethanol stem bark extract, chlorogenic acid, benzoic acid, hyperoside and

apigenin were identified, but the concentrations were too low to be quantified. In these samples,

three flavonoid glycosides were identified and quantified: isoquercitrin (quercetin 3-O-

Figure 1. UV and TIC chromatograms of leaves extract. ID, identified compounds: chlorogenic acid; 1,hyperoside; 2, isoquercitrin; 3, rutin; 4, quercitrin; 5, quercetol; 6, luteolin; 7, apigenin.

Figure 2. UV and TIC chromatograms of stem bark extract. ID: gentisic acid, chlorogenic acid, hyperoside;1, isoquercitrin; 2, rutin; 3, quercitrin; 4, luteolin; apigenin.

Natural Product Research 3

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glycoside) was the compound found in the highest amount (1891 ^ 6.11mg/100 g), followed byquercitrin (739 ^ 5.68mg/100 g) and rutin (412 ^ 13.01mg/100 g). One flavonoid aglycone

namely luteolin was also determined in quantities of 882 ^ 11.59mg/100 g dry matter.

Ethanolic extracts of A. hildebrandtii leaves are rich in flavonoid glycosides (hyperoside,

rutin, isoquercitrin and quercitrin), whereas those values were lower in stem bark extract.

2.2. Total polyphenolic content by Folin–Ciocalteu reagent

The TPC of the plant extracts was expressed in milligrams of gallic acid equivalent per gram of

dry material (mgGAE/g dry material), as shown in Table 3. TPC was higher in leaves than in

stem bark ethanolic extract.

2.3. Free radical-scavenging activity on DPPHz

The DPPH is a stable radical with a maximum absorption at 517 nm that can readily undergo

scavenging by antioxidant. It has been widely used to test the ability of compounds as free-

radical scavengers or hydrogen donors and to evaluate the antioxidant activity of plant extracts

and foods (Sowndhararajan & Sun 2013). The DPPH radical-scavenging activity of

A. hildebrandtii leaves, stem bark and standards synthetic antioxidant [butylated hydroxytoluene

(BHT) and quercetin] are presented in Figure 3. The scavenging activities of ethanolic extracts

Table 1. The polyphenolic compounds content in A. hildebrandtii leaves and stem bark extracts (mg/100 gdry matter).

Polyphenolic compounds m/z value RT ^ SD (min) Leaves Stem bark

Gentisic acid 153 3.69^ 0.03 – ,0.2Chlorogenic acid 353 6.43^ 0.05 (0.2 ,0.2Hyperoside 463 19.32^ 0.12 1025 ^ 5.50 ,0.2Isoquercitrin 463 20.29^ 0.10 5282 ^ 7.50 1891 ^ 6.11Rutin 609 20.76^ 0.15 37821 ^ 8.50 412 ^ 13.01Quercitrin 447 23.64^ 0.13 926 ^ 4.72 739 ^ 5.68Quercetin 301 27.55^ 0.15 229 ^ 3.51 –Luteolin 285 29.64^ 0.19 537 ^ 5.13 882 ^ 11.59Apigenin 279 33.10^ 0.15 775 ^ 3.60 ,0.2

Note: –, not found.

Table 2. RTs (min) and values of regression (r 2) for standard polyphenolic compounds.

Peakno.

Phenoliccompound m/z RT ^ SD

Peakno. r 2

Phenoliccompound m/z RT ^ SD r 2

1 Caftaric acid 311 3.54^ 0.05 10 .0.9997 Rutin 609 20.76^ 0.15 .0.99992 Gentisic acid 153 3.69^ 0.03 11 .0.9984 Myricetin 317 21.13^ 0.12 .0.99813 Caffeic acid 179 6.52^ 0.04 12 .0.9948 Fisetin 285 22.91^ 0.15 .0.99834 Chlorogenic

acid353 6.43^ 0.05 13 .0.9985 Quercitrin 447 23.64^ 0.13 .0.9948

5 p-Coumaricacid

163 9.48^ 0.08 14 .0.9996 Quercetin 301 27.55^ 0.15 .0.9989

6 Ferulic acid 193 12.8^ 0.10 15 .0.9991 Patuletin 331 29.41^ 0.12 .0.99787 Sinapic acid 223 15.00^ 0.10 16 .0.9982 Luteolin 285 29.64^ 0.19 .0.99928 Hyperoside 463 19.32^ 0.12 17 .0.9974 Kaempferol 285 32.48^ 0.17 .0.99589 Isoquercitrin 463 20.29^ 0.10 18 .0.9997 Apigenin 279 33.10^ 0.15 .0.9988

Note: SD, standard deviation.


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were concentration dependent, usually expressed as IC50 values, the amount of antioxidant

necessary to decrease the initial concentration of DPPH by 50%. Lower IC50 value indicates a

higher antioxidant activity.

The highest radical-scavenging activity was shown by the leaves extract (IC50 ¼ 38.56mg/mL), followed by stem bark extract (IC50 ¼ 57.62mg/mL). IC50 of BHT and quercetin were

15.99 and 5.59mg/mL, respectively. In this study, DPPH radical-scavenging activity of the

tested samples decreased in order quercetin . BHT . leaves extract . stem bark extract.

According to this method, ethanolic extract of A. hildebrandtii leaves exhibited a high

antioxidant capacity (IC50 #50mg/mL) and stem bark ethanol extract has a moderate

antioxidant activity (50mg/mL , IC50 # 100mg/mL) (Benedec, Vlase, Oniga, Mot, Damian,

et al. 2013).

2.4. Assay of antimicrobial activity

The antibacterial and antifungal activities of the ethanol extracts of A. hildebrandtii leaves and

stem bark are shown in the Table 4. The extracts were investigated for their in vitro antimicrobial

properties using a disk diffusion method against Staphylococcus aureus, Listeria

monocytogenes, Escherichia coli and Salmonella typhimurium. The antibacterial activity is

ranked from no activity (–: inhibition diameter , 10mm), low (þ : inhibition diameter 10–

15mm), moderate (þþ : inhibition diameter 15–20mm) and high activity (þþþ : diameter

inhibition$20mm) (Reeves &White 1983; Zbakh et al. 2012). After incubation, all plates were

examined for any zones of growth inhibition and the diameters of these zones were measured in

millimetres.

The ethanolic extract of A. hildebrandtii leaves showed a stronger antibacterial activity

against the tested Gram-positive bacterial strains: S. aureus and L. monocytogenes (inhibition

Table 3. TPC in plant extracts.

Samples TPC (mg GAE/g)

A. hildebrandtii, leaves 77.123^ 1.194A. hildebrandtii, stem bark 55.882^ 2.075

Note: GAE, gallic acid equivalents; each value represents the mean ^ SD (n ¼ 3).

Samples

0

10

20

30

40

50

60

70

IC50

(µg

/ml)

Figure 3. DPPH radical-scavenging activity of leaves and stem bark ethanol extracts.


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diameter $ 20mm), higher than Gentamicin used as reference antibiotic. This sample showed a

limited activity against the other tested bacteria: E. coli and S. typhimurium (inhibition

diameter ¼ 10mm) and a low activity on Candida albicans (inhibition diameter ¼ 10mm).

The ethanolic extract of A. hildebrandtii stem bark showed a low activity towards S. aureus

and E. coli (þ : inhibition diameter ¼ 10mm) and it was inactive against L. monocytogenes and

S. typhimurium (inhibition diameter , 10mm). The antifungal activity towards C. albicans was

high (inhibition diameter ¼ 20mm).

The results of the present investigation suggest that A. hildebrandtii leaves exhibited an

important antibacterial activity against Gram-positive bacteria, with high activity against

S. aureus and L. monocytogenes, and low activity against Gram-negative bacterial and fungal

strains. The stem bark extract had high antimicrobial activity only against C. albicans.

3. Experimental

3.1. Chemicals

Solvent 508 ethanol and HPLC grade methanol, analytical grade orthophosphoric acid,

hydrochloric acid and Folin–Ciocalteu reagent were purchased from Merck (Darmstadt,

Germany). Standard compounds: ferulic acid, sinapic acid, gentisic acid, gallic acid, patuletin

and luteolin from Roth (Karlsruhe, Germany), cichoric acid and caftaric acid from Dalton

(Toronto, ON, Canada), chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin,

isoquercitrin, quercitrin, hyperoside, kaempferol, myricetol and fisetin from Sigma (St. Louis,

MO, USA), DPPH (2,2-diphenyl-1-picrylhydrazyl), quercetin and BHT from Alfa-Aesar

(Karlruhe, Germany). The materials used for antimicrobial activity were obtained fromMueller-

Hinton (MH) Agar and MH Dextroxe Agar. The micro-organisms S. aureus ATCC 49444, L.

monocytogenes ATCC 13076, E. coli ATCC 25922, Salmonella typhymurium ATCC 14028 and

C. albicans ATCC10231 were obtained from MicroBioLogicsw (St. Cloud, MN, USA).

3.2. Plant collection, identification and extract preparation

Leaves and stems bark of A. hildebrandtii were collected at Joffre Ville, located 20 km from the

city of Diego Suarez Madagascar in December 2013 (Voucher specimen N8 DSM 423). The

species was identified by botanists at the botanical and zoological park of Tsimbazaza

Antananarivo, Madagascar and have been deposited in the ‘Laboratoire de Chimie des

Substances Naturelles et Chimie Organique Biologique (LCSN/COB)’ Madagascar. The plant

material was reduced to a powder by mechanical grinding; 1 g of dry plant material was

extracted with 10mL of 508 ethanol, for 30min by reflux apparatus, at 608C; the powder was

filtered using Whatman No. 1 filter paper. After filtration, the samples were cooled down and

Table 4. Antimicrobial activity (inhibition zone expressed in mm) of two investigated extracts ofA. hildebrandtii.

Zone of inhibition (mm)

Samples S. aureus L. monocytogenes E. coli S. typhimurium C. albicans

A. hildebrandtii stem bark 14 ^ 1.00 8 ^ 0.00 12 ^ 0.00 8 ^ 0.00 20 ^ 2.00A. hildebrandtii leaves 20 ^ 2.00 22 ^ 3.00 10 ^ 1.00 10 ^ 0.00 10 ^ 0.00Gentamicin 19 ^ 0.00 18 ^ 0.00 22 ^ 2.00 18 ^ 1.00 –Fluconazole – – – – 25 ^ 2.00

Note: The values represent the average of three determinations ^ SD. Gentamicin (10mg/well) and Fluconazole (25mg/well) were used as positive controls.


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centrifuged at 4500 rpm for 10min; the obtained solutions were collected and stored at 48C until

analysis (Benedec et al. 2012).

3.3. HPLC/MS analysis

Apparatus and chromatographic conditions: an Agilent 1100 HPLC Series system (Agilent,

Santa Clara, CA, USA) equipped with degasser G1322A, quaternary gradient pump G1311A

and auto sampler G1313A was used. The separation was performed using a Zorbax SB-C18

reverse-phase column (100 £ 3.0mm i.d., 3.5mm particle). The working temperature was 488Cand the detection of the compounds was performed at 330 nm (first 17min from chromatogram)

and 370 nm (from 17 to 38min) using a G1311A diode array detector system. The

chromatographic data were processed using the ChemStation software from Agilent. The mass

spectrometer was an Agilent 1100 SL Series ion trap equipped with turbo-ionspray (electrospray

ionisation) interface and was operated in negative ion mode. The parameters of the source were

temperature, 3608C; dry gas, nitrogen; and nebuliser, nitrogen at 65 psi.

The mobile phase was a binary gradient prepared from methanol and acetic acid solution

0.1% (v/v) in water. The gradient elution was 0–35 min, from 5% to 42% methanol; isocratic

elution followed, for the next 3min, with 42% methanol. The flow rate was 1mL/min, the

injection volume was 5mL and data were collected at 330 nm.

The identification of the polyphenols in the samples was made by comparison of their RTs,

UV and MS spectra obtained with those of pure standards in the same chromatographic

conditions and confirmed by HPLC/MS. Quantitative determinations were made using an

external standard method. Quantification was performed on the basis of linear calibration plots

of peak area against concentration. Calibration lines were constructed based on five

concentration levels of standard solutions with in 0.5–50mg/L range. The method proves good

linearity (values of regression r 2 . 0.9948, Table 2), and accuracy between 95% and 105% for

all compounds. Accuracy was checked by spiking samples with a solution containing each

phenolic compound in a concentration of 1mg/mL. All compounds were identified by both

standard addition and comparison of their RTs and MS spectra with those of standards (Meda

et al. 2011; Benedec et al. 2012).

3.4. Total polyphenol content by Folin–Ciocalteu reagent

The concentration of polyphenols in plant extracts was determined using spectrophotometric

method. Then, 1.000 g vegetal product was extracted with 10mL ethanol 50% (v/v) for 30min

on a water bath at 708C. After cooling, the solution was filtered and completed at 10mL in a

volumetric flask with the same solvent. Then, 0.5mL of this solution is diluted in a 25-mL

volumetric flask with the same solvent. At 2mL of this solution was added 1mL Folin–

Ciocalteu reagent, 10mL distilled water and completed in a volumetric flask to 25mL with

290 g/L sodium carbonate solution.

The absorbance was determined at 760 nm, using a UV-VIS JASCO V-530 (Tokyo, Japan)

spectrophotometer and distilled water as compensation liquid, after 30min storing in the

darkness (Tamas et al. 2009).

3.5 Free radical-scavenging activity on DPPHz

The free radical-scavenging assay has been efficiently used as an adequate parameter for

selecting medicinal plants with antioxidant properties. A solution of 0.25mM or 0.1 g/L 1,1-

diphenyl-2-picrylhydrazyl (DPPH) radical in methanol was prepared and 4mL of 50% ethanolic

extract was added, at an equal volume, to 50% ethanol solution of DPPH. After 308min

incubation at 408C in a thermostatic bath, the absorbance was recorded at 517 nm with a UV–


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VIS spectrophotometer Jasco V530 (Benedec et al. 2012; Benedec, Vlase, Oniga, Mot, Silaghi-

Dumitrescu, et al. 2013). Synthetic antioxidant, BHT and quercetin were used as standards.

The capability of samples to scavenge DPPHz was obtained by comparison of the sample

colour reduction effect with the control (mixture without working solution) using the

following equation and expressed as percentage values: DPPH radical-scavenging activity

(%) ¼ [(Ac–As)/(Ac)] £ 100 (Ac is the absorbance of the control; As is the the absorbance of the

sample).

3.6. Antimicrobial activity test

Here, 50% ethanolic extracts of A. hilbebrandtii leaves and stem bark were tested for

antimicrobial activity against two Gram-positive bacterial strains: S. aureus (ATCC 49444) and

L. monocytogenes (ATCC 13076), against two Gram-negative bacterial strains: E. coli (ATCC

25922) and S. typhymurium (ATCC 14028) and one fungal strain: C. albicans (ATCC10231), by

a previously described disk diffusion method, in Petri dishes. Each micro-organism was

suspended in MH broth and diluted approximately to 10E6 colony forming unit (cfu)/mL. They

were ‘flood-inoculated’ on to the surface of MH agar and MH Dextroxe Agar and then dried.

Six-millimetre diameter wells were cut from the agar using a sterile cork-borer, and 60mL of

each extract was delivered into the wells. The plates were incubated at 378C and the diameters of

the growth inhibition zones were measured after 24 h. Gentamicin (10mg/well) and Fluconazole(25mg/well) were used as standard drugs. The controls were performed with only sterile broth

and with only overnight culture and 10mL of 70% ethanol. All tests were performed in triplicate,

and clear halos greater than 10mm were considered as positive results (Reeves & White 1983;

Rota et al. 2008; Zbakh et al. 2012).

4. Conclusions

To our knowledge, this is the first report that shows the polyphenols analysis, antioxidant and

antimicrobial activities of A. hildebrandtii leaves and stem bark ethanolic extracts. Some

phenolic compounds were identified and quantified by HPLC/MS, such as rutin, isoquercitrin,

hyperoside, quercitrin, quercetin, apigenin and luteolin.

A. hildebrandtii leaves extract showed a high DPPH scavenging activity related to the total

polyphenol content. The antimicrobial activity of A. hildebrandtii was high against Gram-

positive bacteria (S. aureus and L. monocytogenes) for leaves extract and against C. albicans for

stem bark extract.

These findings justify the traditional uses of this plant. Further research is necessary in order

to know all the active principles and their pharmacological properties.

Acknowledgements

We are grateful to the Francophone University Agency (AUF) for providing the Doctoral grant ‘EugenIonescu’ that facilitated the analysis in the Pharmacy Department, University of Medicine and Pharmacy‘Iuliu Hatieganu’, Cluj-Napoca, Romania.

Funding

Laurian Vlase, Ph.D., acknowledges financial support from a POSDRU grant, no. 159/1.5/S/136893 withtitle: ‘Strategic partnership for increasing scientific research quality from medical universities throughdoctoral and postdoctoral fellowships – DocMed.Net_2.0’.


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Deroin T, Gautier L. 2008. Artabotrys darainensis Deroin and L. Gaut. (Annonaceae), a new species from Madagascar.

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(Annonaceae Juss.). Innovare J Ayurvedic Sci. 1:14–17.

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and quantification of phenolic compounds from Balanites aegyptiaca (l) Del (Balanitaceae) galls and leaves by

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antimicrobial activities for five species ofMentha cultivated in Romania. Dig J Nanomater Biostruct. 9:559–566.

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http://dx.doi.org/10.3390/molecules18088725

http://dx.doi.org/10.1080/14786419.2010.482933

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Journal of Pharmacognosy and Phytochemistry 2015; 3(6): 47-57 E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2015; 3(6): 47-57 Received: 02-12-2015 Accepted: 04-01-2015 Rivoarison Randrianasolo (a)Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101,Madagascar (b)Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Bischofsholer Damm 15/123, D-30173 Hannover, Germany

Armandine Raharinirina Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101, Madagascar

Herilala Léa Rasoanaivo Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101,Madagascar

Hans Christoph Krebs Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Bischofsholer Damm 15/123, D-30173 Hannover, Germany

Amélie Raharisolololao Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101, Madagascar

Andrianambinina Andriamarolahy Razakarivony (a)Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101,Madagascar (b)Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, D-33501 Bielefeld, Germany. Correspondence: Rivoarison Randrianasolo (a)Department of Organic Chemistry, Faculty of Science, University of Antananarivo, Madagascar, P.O. Box 906, Antananarivo 101,Madagascar (b)Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Bischofsholer Damm 15/123, D-30173 Hannover, Germany.

A New Dihydronaphtaquinone from Dianella ensifolia L.Redout

Rivoarison Randrianasolo, Armandine Raharinirina, Herilala Léa Rasoanaivo, Hans Christoph Krebs, Amélie Raharisolololao, Andrianambinina Andriamarolahy Razakarivony, Maonja Finaritra Rakotondramanga Abstract A new dihydronaphtaquinone 2-hexyl-3-(2-hydroxyethyl)-2,3-dihydronaphtaquinone 1-4, which was named armandinol, was isolated from the leaves of Dianella ensifolia L.Redouté, together with two known quinones. The structure of the new compound was elucidated using spectroscopic methods, mainly 1D and 2D NMR. Keywords: Dianella ensifolia; dihydronaphtaquinone; armandinol, NMR

1. Introduction The genus Dianella belongs to the family Liliaceae, comprises about 21 species, distributed generally in Tropic Asia, Australia and the Pacific. Dianella ensifolia L.Redouté, species recorded in Madagascar was collected in a village which name is Betafo, not far from the capital. This plant is not really used in traditional Malagasy medicine. However, according to the local people, the leaves are used an infusion as an exciting brain cells and a remedy against constipation. Although previous phytochemical research on Dianella ensifolia L.Redouté revealed phenolic and quinone compounds isolated from the root of the plant [1]. In our chemical investigation, a new dihydronaphtaquinone, 2 - hexyl -3 - (2-hydroxyethyl) -2, 3-dihydronaphtoquinone 1-4 (1) with two known quinones, chrysophanol (2) and isoeugenitol (3) were isolated from the leaves of Dianella ensifolia L. Redouté by column chromatograph. The structure of the new compound was elucidated using 1D and 2D NMR spectroscopic data. To the best of our knowledge, this is the first report on the existence of this compound from natural source.

Fig 1: Structure of compound 1, 2 and 3 2. Materials and Methods 2.1. General Melting points was measured with the melting point apparatus of Electrothermal and was not corrected. NMR spectra were recorded with a Brüker AV-400 and AV-500 with a cryoprobe for 1H, BBD, APT, HSQC, and HMBC. Chemical shift values are in δ(ppm) using the peak signals of the solvent CDCl3 (δH 7.28; and δC 70.00) as reference, and coupling constants are reported in Hz. ESIMS data were measured with a Finnigan MAT95 spectrometer (70eV) with perfluorokerosene as a reference substance for HR-EI-MS. Column chromatography was performed on silica gel 60 (6.3-20µm) (Merck, Darmstadt, Germany). Normal-phase silica gel 60 TLC plates (w/UV 254) were used for fraction detection. The spot were visualized using UV light at 254 nm and spraying with vanillin-sulfuric acid reagent.

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2.2. Plant Material The leaves of Dianella ensifolia were collected in august 2011 in one village next to Antananarivo, the capital of Madagascar. The plant was authenticated at the botanical and zoological park of Tsimbazaza Antananarivo Madagascar where a voucher specimen is deposited under the reference number DIA 011-215. 2.3 Extraction and Isolation Powdered dried of leaves from Dianella ensifolia (400 g) were extracted with 96% EtOH at room temperature. Evaporation of EtOH under reduced pressure gave a brown residue (20, 67 g). The residue was later suspended in H2O, and partitioned with Hexane, CH2Cl2 and n-BuOH. The Dichloromethane extract (3, 48g) was applied to a silica gel column with Hexane and Acetone as binary mixtures of increasing polarity afforded 12 fractions (Fr. F1-F12). Fr. F1 (Hexane – Acetone 95:05) was isolated on silica gel eluted with Hexane - Acetone step gradients (90:10→70:30) to give 4 subfractions (Fr. F5-1- F5-4). Further purification of Fr. F5-1 was applied to column chromatography on silica gel with CH2Cl2- Acetone (95:5→80:20) to obtain compound 1 (8.0 mg). Fr. F10 (Hexane – Acetone 5:5) was isolated on silica gel eluted with CH2Cl2 – Acetone (90:10→70:30) to obtain compound (2) (15 mg) and 3 (11 mg). 3. Results and Discussion 3.1. Structure elucidation Compound (1) was yellow oil with melting point of 124-125°C. Its molecular formula was determined as C18H24O3, on the basis of the HR-MS (positive) at m/z 288.1766 (calc. 288.1745). Observation of symmetrically oriented four-spin AA’BB’ type signals at δ= 7.57 ppm for H-6 and H-7 (ddd, J= 7.88, 7.37, 1.38Hz) and δ=7.69 ppm for H-5 and H-8 (ddd, J= 7.86, 1.38, 1 Hz) in the aromatic proton region of the 1H-NMR spectrum including a signal at 1,68 ppm for H-2 and H-3 (dddd, J= 9.12, 8.37, 4.11, 1.20 Hz) . The signal at δ=167,78 ppm attributed to two carbonyls carbon, two signals of an aromatic ring at δ=130.67 ppm and δ=128.62 ppm corresponding to four olefinics carbons in the 13C-NMR spectrum, suggested the presence of a dihydro-1,4-naphthaquinone skeleton having no substituent in the A-ring [2]. HSQC NMR showed that protons at δ = 7.69 ppm are attached by carbons at δ = 128.62 ppm (C-5, C-8) and protons at δ = 7.56 ppm are linked to the carbons at δ = 130.67 ppm (C-6, C-7). The signal at δ = 132.41 ppm, corresponding to quaternary carbons is attributed to the two carbons at positions C-9 and C-10. The signal at δ = 38.97 ppm is attributed to the carbon at positions C-2 and C-3. The presence of a long chain moiety in the molecule was indicated by the peaks in the region of weak field. The signal at δ = 4.21 ppm (d, J= 6.31Hz) in 1H NMR and δ=68.17 ppm in 13C NMR spectra suggested the presence of an alcohol function (-CH2OH). The correlations observed in HSQC and HMBC spectra (Figure 2) suggested that a linear chain –(CH2)6-CH3 is fused to the 2,3-dihydro-1,4-naphthaquinone skeleton and a –CH2CH2OH group at C-3 and C-2 respectively. The signal at δ = 4.21 ppm (-CH2OH) is correlated with the carbon at δ = 167.78 ppm (C-1, C-4), δ = 38.91 ppm (C-2) and δ = 30.91 ppm (C-1’). Additionally, the proton at δ = 1.68 ppm corresponding to the carbon at δ = 38.91 ppm (HSQC), are correlated with carbons at δ = 28.68 ppm (C-1’’) and δ = 22.75 ppm (C-2’’).

Thus, the structure of 1 was elucidated as an 2-heptyl-3-(2-hydroxyethyl)-2, 3-dihydronaphtaquinone 1-4 and named armandinol. Additionally, chrysophanol (2) [3] and isoeugenitol (3) [1] were identified by comparing its 1H NMR data with literature.

O

O

CHOH

CH3

H

H

H

H

H

H

A

Fig 2: The key HMBC correlations of compound 1. 3.2 Antioxidant activity Qualitative antioxidant assay was performed by the standard TLC- DPPH method [4]. The compounds 1, 2 and 3 were spotted on a TLC plate and air dried, then plates were sprayed with 0.002% ethanolic DPPH (2, 2-Diphenyl-1- picrylhydrazyl) solution using an atomizer. Positive activity was detected with the three compounds by the pale yellow spots on a reddish purple background due to the decolourization of DPPH by the antioxidant. Ascorbic acid and gallic acid were used as the positive control [5, 6, 7].

Table 1: 1H (500 Mhz) and 13C (100 Mhz) NMR chemical shifts for compound 1 in CDCl3, a,b

Carbon 1H 13C 1 2 3 4 5 6 7 8 9

10 1’ 2’ 1’’ 2’’ 3’’ 4’’ 5’’ 6’’

1,68 m (9.12, 8.41, 4.11, 1.2) 1,68 m (9.12, 8.41, 4.11, 1.2)

7,69 d (7.86, 1.38)

7,56 t (7.88, 7.37, 1.38) 7,56 t (7.88, 7.37, 1.38)

7,69 d (7.86, 1.38)

4,21 2,27 2,12 2,05 1,68 1,60 1,60

0,86 t

167,78 38,97 38,97 167,78 128,62 130,67 130,67 128,62 132,41 132,41 68,17 30,91 28,69 22,75 29,36 29,36 21,34 13,59

a Assignments were confirmed by 2D experiments. b δ ppm, 500MHz for 1H and 100 Mhz for 13C; multiplicities values (Hz) in parenthese.

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S1: 1H-NMR spectrum of compound 1

S2: 13C-NMR spectrum of compound 1

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S3: DEPT spectra of compound 1

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S4: HSQC spectrum of compound 1

S5: HMBC spectrum of compound 1 Acknowledgments The authors are grateful to the financial support of D.A.A.D (Deutscher Akademischer Austauschdienst) from its re-invitation program to Dr Rivoarison Randrianasolo and to the Organic Chemistry Institute of the University of Hanover for EIMS and NMR spectra. References 1. Vitchu Lojanapiwata, Kovit Chancharoen, Kanchana

Sakarin and Pichaet Wiriyachitra (1982), Chemical Constituants of Dianella Ensifolia Redoute, J.Sci.Soc.Thailand 8, 95-102.

2. Chihiro Ito,Yuichi Kondo,K. Sundar Rao, Harukuni Tokuda, Hoyoku Nishino, and Hiroshi Furukawa (1999), Chemical Constituents of Glycosmis pentaphylla. Isolation of A Novel Naphthoquinone and A New Acridone Alkaloid, Chem. Pharm. Bull. 47(11), 1579 - 1581.

3. Karlina García-Sosa, Ninibe Villarreal-Alvarez, Petra

Lübben and Luis M. Peña-Rodríguez (2006), Chrysophanol, an Antimicrobial Anthraquinone from the Root Extract of Colubrina greggii, J. Mex. Chem. Soc.,50(2), 76-78.

4. Sadhu, S. K., Okuyama, E., Fujimoto, H. and Ishibashi, M. (2003). SBangladesh, guided by prostaglandin inhibitory and antioxidant activitie598.

5. Atsumi T, Iwakura I, Kashiwagi Y, Fujisawa S, Ueha T. Free radical scavenging activity in the non- enzymatic fraction of human saliva: a simple DPPH assay showing the effect of physical exercise. Antioxidants and Redox Signaling 1999; 1:537–546.

6. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958; 26:1199–1200.

7. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin Journal of Science and Technology 2004; 26(2):211-219.

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Journal of Pharmacognosy and Phytochemistry 2015; 4(2): 161-171 E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2015; 4(2): 161-171 Received: 25-05-2015 Accepted: 26-06-2015

Blanche Graziella Ranisaharivony (a) Laboratoire International Associé Antananarivo-Lyon 1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. (b) Laboratoire de Chimie et de Valorisation des Produits Naturels, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. Voahangy Ramanandraibe Laboratoire International Associé Antananarivo-Lyon 1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. Léa Herilala Rasoanaivo (a) Laboratoire International Associé Antananarivo-Lyon 1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. (b) Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. Marcelle Rakotovao Laboratoire de Chimie et de Valorisation des Produits Naturels, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. Marc Lemaire (a) Laboratoire International Associé Antananarivo-Lyon 1, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar. (b) Laboratoire CASYEN-ICBMS-UMR5246- Université Claude Bernard Lyon 1, France. Correspondence: Marcelle Rakotovao Laboratoire de Chimie et de Valorisation des Produits Naturels, Faculté des Sciences BP 906, Université d’Antananarivo, Madagascar.

Separation and potential valorization of chemical

constituents of soursop seeds

Blanche Graziella Ranisaharivony, Voahangy Ramanandraibe, Léa Herilala Rasoanaivo, Marcelle Rakotovao, Marc Lemaire Abstract Seeds of Annona muricata, by-products from the edible fruit, were assessed chemically and biologically. Using larvicidal bioassay, activity-directed fractionation of ethanolic extract led to the isolation of three known acetogenins: annonacin, murisolin and annonacinone. Their structures were established by spectroscopy experiments. Synergistic activity of these acetogenins was observed. The low water solubility of annonacin was demonstrated to be one of the limiting factors of its larvicidal activity. The catalytic hydrogenation of annonacin afforded a mixture of diastereoisomers which is more active against Culex quinquefasciatus larvae than annonacin. Micro-Kjeldahl analysis showed that residues of extraction (insoluble matter) contained 25.6% crude proteins. Solvent partitioning of the kernel ethanolic extract yielded: oil (30.4% of kernels), mixture of acetogenins (4.2% of kernels) and mixture of carbohydrates (5.2% of kernels). The oil was weakly toxic to Artemia salina nauplii. Eight fatty acids were identified. Sucrose was the major carbohydrate. Potential valorization of these components is discussed. Keywords: Annona muricata, seed, oil, acetogenin, valorization, Madagascar 1. Introduction Dealing with waste issues is one of the greatest challenges to mankind of this century. Even if agriculture wastes are already valorized (for example rice or wheat straws are used for animal feeding) their collection and transport are often difficult and expensive. On the opposite, industrial wastes obtained during food processes are already gathered and well defined physically and chemically. Their transformation could be performed if an efficient industrial ecology was set up [1, 2]. The main drawback is the relatively smaller amount of these agro-industrial wastes compared to that of other agricultural wastes. Nevertheless, this disadvantage is limited as far as the high value materials are obtained from the conversion of these wastes. As part of our research for industrial ecology, we have already described the tentative valorization of waste from mango processing [3]. In the present article we describe an attempt to evaluate the potential application of soursop seeds. Soursop, Annona muricata (Annonaceae) is a tropical tree [4] that grows in different localities in Madagascar. Pulp of the edible fruit is largely used for juice or jam preparation [5]. Seeds are discarded due to their toxicity. They are used in folkloric medicine for treating skin diseases [6] as cited in Le Ven [7]. In India, they are used as emetic, astringent or fish poison [8]. In Brazil, they are destinated to soil fertilization [9]. Since the isolation of uvaricin [10], the first annonaceous acetogenin, they have been mainly studied [11, 12, 13] for the research of a new class of cytotoxic drug candidates. Some authors reported pesticidal or larvicidal activity from soursop seed extract [7, 14, 15]. Soursop seeds contain fibers and proteins [16, 17] which could be useful for feeding animals. They also contain large amount of oil [18, 19, 20, 21] which is toxic due to lipophilic acetogenins [22]. These molecules exhibit a broad range of biological activities: mitochondrial inhibitor or cytotoxic [11, 12, 13, 22], pesticidal and larvicidal [8, 9]. Therefore, they may be proposed to control mosquito populations which constitute vectors of various tropical diseases in Madagascar. However, oil which contains these acetogenins, has restricted potential applications although it is used to kill head lice in India and Mexico [23]. To expand these potential applications, separation of toxic molecules from oil is useful. For this purpose, the methodology consisted of extraction with Soxhlet apparatus, followed by liquid/liquid partition, using low toxicity, more affordable and available solvents. The derived components from the treatment of soursop seeds can be valorized depending on their potential uses.

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2. Materais and Methods 2.1 Plant material Fruits of Annona muricata were bought at the market of Antananarivo. Seeds were provided by restaurants of Antananarivo from August 2012 to August 2013. Pulp, peels, core and seeds of the fruits (2261 g) were separated and weighted. Seeds were dried in a well-ventilated area. Husks (37.4%) were separated from kernels (62.2%). 2.2 Solvents Solvents (hexane, dichloromethane, ethyl acetate, ethanol and methanol) were distilled before use. Toluene (Merck), diethyl ether (Panreac Quimica) and dimethylsulfoxyde (Fisher Scientific) were analytical grade. 2.3 General analysis Silica gel 60 Å, 230-400 mesh (Merck) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. The spots were detected by spraying with various reagents: Kedde’s reagent (acetogenins), thymol (carbohydrates), Rhodamin B (fatty acids), ninhydrin (amino acids), vanillin sulfuric acid (universal reagent) followed by heating. 1D (1H, 13C, DEPT) and 2D (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC) NMR spectra were recorded on a Bruker Varian 400 NMR operating at 400.15/125.15 MHz using CDCl3 as solvent and TMS as an internal standard. Fragments were obtained by a Micro TOF-Q Bruker Daltonics (Bremen, Germany) mass spectrometer fitted with ESI source. Positive ions mass spectra of the column eluate were recorded in the range m/z 50-2000. Nitrogen was used as the drying gas at a flow rate 4 L/min. The nebulizer temperature was set at 200 °C and the pressure was 0.6 bar. GC/MS experiments were performed on a Focus GC chromatograph linked to a mass spectrometer (70 eV) with an electron ionization system. The carrier gas was helium with a flow rate of 50 mL/min. A DB-5MS apolar capillary column (length 30 m, inner diameter 0.25 mm, film thickness 0.25 µm) was used with the following temperature program: 70 °C (2 min), 70-310 °C at 15 °C/min, and 310 °C (10 min). The injector temperature was 220 °C. 2.4 Extraction and isolation of the constituents 2.4.1 Ethanolic extracts Maceration: The powdered seeds (600 g) were repeatedly macerated with ethanol 95° (3 L) during (24 hours x 3) at room temperature. The combined ethanol extracts were evaporated and yielded (65.9 g, 11%) EtOH extract (EM). Soxhlet: The ground materials were extracted with EtOH 95° (700 ml) by Soxhlet apparatus (200 mL) with flow rate 5 mL/min: husks (50 g) yielded (5.4%) ethanolic extract (EH) after 16 hours; kernels (50 g) provided (40.5%) ethanolic extract (EK) and (53.8%) insoluble matter (IM) after 13 hours. Partition of ethanolic extract Method 1: 64.2 g of EtOH extract (EM) were partitioned successively between H2O and C6H14 to yield (58.6%) C6H14 extract (A) and between H2O and CH2Cl2 to afford (22.3%) CH2Cl2 extract (B) and (10.6%) aqueous extract(C). Fractionation was monitored by the larvicidal bioassay [24]. Method 2: The EtOH extract (EK) (50 g) was partitioned between Hexane/MeOH/Water: 50/25/25 to yield 75.2% hexane extract (A’), the MeOH/Water phase was extracted with CH2Cl2 and yielded 10.3% CH2Cl2 extract (B’) and 12.7% aqueous extract (C’).

Method 3: The method 2 was applied but CH2Cl2 was superseded by ethyl acetate. 75.5% hexane extract (A*), 13.8% ethyl acetate extract (E*) and 8.8% aqueous extract (C*) were obtained. 2.4.2 Isolation of acetogenins The bioactive CH2Cl2 extract (B) (10 g) was subjected to column chromatography (elution with CH2Cl2/MeOH: 95/5, 90/10 and 80/20) and yielded three fractions F1 (32.8%), F2 (59.9%) and F3 (4.5%). The bioactive fraction F2 (3 g) was subjected to silica gel column chromatography and eluted with CH2Cl2 containing increasing amounts of MeOH (CH2Cl2/MeOH: 99/1 to 90/10). Three bioactive fractions were obtained: B1 which contained mainly murisolin, B2 which was a mixture of three acetogenins (murisolin, annonacinone and annonacin) and B3 which contained a bioactive mono-tetrahydrofuran γ-lactone acetogenin: annonacin (1) (1.33 g, 44.3% of F2 and 26.6% of B). The CH2Cl2 extract (B’) (3 g) was separated by column chromatography on silica gel. Elution with Hexane/EtOAc: 90/10 to 80/20 led to the isolation of three mono-tetrahydrofuran α, β-unsaturated γ-lactone acetogenins: annonacin (1) (282.3 mg), annonacinone (2) (82.8 mg) and murisolin (3) (40 mg). These acetogenins reacted positively with Kedde’s reagent and were displayed as orange spot after the treatment of the chromatoplate with vanillin sulfuric acid. Annonacin (1) White amorphous powder. Mp=60 °C. Identified by MS and 1H, 13C NMR. Rf=0.23 in CH2Cl2/MeOH: 95/5 (v/v). C35H64O7; MS: m/z 619.6 [M+Na]+, 597.4 [M+H]+, 579.4 [MH-H2O]+, 561.5 [MH-2H2O]+, 543.5[MH-3H2O]+, 525.5 [MH-4H2O]+. NMR chemical shifts are consistent with the literature [25]. Annonacinone (2) Needle-like crystals. Identified by MS and 1H, 13C NMR. Rf=0.27 in CH2Cl2/MeOH: 95/5. C35H62O7 ; MS: m/z 617.4 [M+Na]+, 595.4 [M+H]+, 577.4 [MH-H2O]+, 559.4 [MH-2H2O]+, 541.4 [MH-3H2O]+, 523.4 [MH-4H2O]+. NMR chemical shifts were compared with the literature [26]. Murisolin (3) Needle-like crystals. Identified by MS and 1H, 13C NMR and by comparison of data with the literature [27]. Rf=0.34 in CH2Cl2/MeOH: 95/5. C35H64O6 ; SM: m/z 603.4 [M+Na]+, 581.4 [M+H]+, 563.4 [MH-H2O]+, 545.4 [MH-2H2O]+, 527.4 [MH-3H2O]+. Catalytic hydrogenation of annonacin Catalytic hydrogenation of annonacin was performed in an ethyl acetate solution with 5% Pd/C at room temperature under an atmospheric pressure of H2 and moderate stirring for 18 hours. The mixture was filtered through a column of silica gel and eluted with methanol. A mixture of diastereoisomers (94% of annonacin by weight) was obtained. 2.4.3 Isolation of sucrose The aqueous extract (C’) (523 mg) was separated by column chromatography on silica gel. Elution with CH2Cl2/MeOH /Water: 10/3.5/0.5 to 1/3.5/0.5 afforded sucrose (50.1 mg, 9.6%). White crystals identified by MS and 1H, 13C NMR. Rf=0.26 in CH2Cl2/MeOH/H2O: 5.5/3.5/0.5 (v/v/v). C12H22O11; MS: m/z

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365.1 [M+Na]+, 203.1 (cleavage of the glycosidic bond between the two sugars); 13C chemical shifts are consistent with the literature [28]. 2.4.4 Seed oils The powdered materials: husks (10 g), kernels (10 g) were separately extracted with hexane by soxhlet (10 hours, flow rate: 5 mL/min) until materials were completely defatted and yielded respectively (1.6%) (HO) and (36.8%) (KO) oil extract. Kernel oil was a pale yellow liquid. Physical and chemical characteristics of kernel oil Specific gravity of kernel oil was determined in triplicate and the average result reported. Chemical characteristics of kernel oil (acid value, saponification value and content of unsaponifiable matter) were determined by standardized methods [29]. Transesterification of fatty esters Methylation of kernel oil and fatty acids of reference was performed according to Christie [30] with slight adjustments. The lipid sample (250 mg) was dissolved in toluene (3 mL) and 2% sulfuric acid in methanol (5 mL). The mixture was left overnight in a stoppered tube at 50 °C. Then, aqueous sodium chloride solution (5%, 10 mL) was added. The required methyl esters were extracted with hexane (3 x 10 mL). The hexane layer was dried over anhydrous sodium sulfate. The solution was filtered and the solvent was removed under reduced pressure in rotary evaporator. Diluted solutions were prepared before injection for GC analysis. Analysis of fatty acid composition The obtained fatty acid methyl esters were separated using a GC-14A chromatograph (Shimadzu) equipped with a flame ionization detector (FID). The carrier gas was nitrogen with a flow rate of 5 mL/min. A DB-5MS-UI apolar capillary column (length 30 m, inner diameter 0.25 mm, film thickness 0.25 µm) was used with the following temperature program: 170 °C (1 min), 170-230 °C at 3 °C/min, 230 °C (1 min), 230-310 °C at 15 °C/min, and 300 °C (1 min). Fatty acids were identified by equivalent length chain method [31, 32] prior to the confirmation by GC/MS analysis. Data were compared with those in MS library. 2.5 Crude protein content of kernels After the extraction of kernel with ethanol, insoluble matter (IM) remained. Crude protein content of the latter was determined according to Kjeldahl method [33]. 2.6 Biological assays 2.6.1 Feeding experiments Fifteen swiss mice were divided into three batches. Each batch was fed with, respectively, 10 g, 5 g and 2.5 g insoluble matter (IM) mixed in their diet. The mice ate this for two days and were deprived of food during the third day. The behavior of mice was observed during the five days following their ingestion of the residues. 2.6.2 Larvicidal bioassay Larvae of Culex quinquefasciatus were collected from their breeding habitat (the runoff channels in downtown Antananarivo). The larvicidal bioassay was performed according to World Health Organization protocol [24]. Solutions were prepared by dissolving extracts in ethanol. 1 mL of ethanol was used as control test. Stock solution 3.2%

of CH2Cl2 extract (B) was prepared. 1 mL of this solution added into the vial containing 250 mL of spring water and larvae corresponded to 127.5 ppm. Different levels of concentration were prepared by diluting the stock solution with ethanol. Tested concentrations : (for CH2Cl2 extract (B): 1 ppm, 2 ppm, 4 ppm, 8 ppm, 16 ppm, 31.9 ppm, 63.8 ppm, and 127.5 ppm), (for annonacin: 5 ppm, 10 ppm, 20 ppm, and 40 ppm), (for the mixture of dihydro-annonacin: 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm, and 60 ppm), (for ethyl acetate extract (E*): 15 ppm, 30 ppm, 45 ppm, 60 ppm, 75 ppm, and 90 ppm). 2.6.3 Toxicity bioassay The toxicity bioassay was performed according to Nando et al. [34] with slight adjustments. Stock solutions were prepared by dissolving the extracts in DMSO. Different levels of concentration were prepared by diluting the stock solution with DMSO. Each level of concentration was tested in quadruplicate. One negative control was used. Ten brine shrimp larvae of 24 hours old were transferred into each vial containing 3 mL of artificial seawater (38 g of sea salt / 1 L of spring water, pH adjusted to 8). 100 µL of solution extract were added. The volume was then adjusted to 5 mL with artificial seawater. 100 µL of DMSO were used as control test. The vials were maintained under illumination. The number of dead Artemia salina nauplii was recorded after 24 hours. Tested concentrations: (for annonacin: 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 10 ppm, 20 ppm, 50 ppm, and 100 ppm), (for the mixture of dihydro-annonacin: 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, and 6 ppm), (for the ethyl acetate extract (E*): 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, and 60 ppm), (for oils: 10 ppm, 20 ppm, 50 ppm, and 100 ppm). 2.7 Statistical analysis Mortality data were corrected using Abbot’s formula. LC50 and LC90 values were determined by log-probit regression using XLSTAT 2014 software. 3. Results and discussion 3.1 Separation of plant material The compositions of the fruit are reported in Figure 1.

Fig 1: Fruit composition Results slightly differed from data: 67.5% pulp, 20% peels and 4% core reported by the literature [35] as cited in Badrie and Schauss [36]. Seed content is similar to that of soursop from Nigeria [19]. In general, the average is ranging from 2.4% to 8.5% [12, 20, 35, 36]. During the drying process, the seeds had lost about 30% of their weight. Kernels and husks accounted respectively for 62.2% and 37.4% of dry seeds by weight. Literature reported that kernels represented 67% [18] to 68.55% [20]. Since the water content was evaluated at 8.9%, no drying process prior to any extraction of kernels was necessary [29].

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3.2 Ethanolic extracts Compared to maceration, the treatment with Soxhlet apparatus provided a better yield of ethanolic extract. Moreover, it required neither filtration nor renewal of solvent. It also reduced the time of extraction. Ethanolic extract obtained from the treatment of kernels (40.5%) was more important than that obtained from husks (5.4%). This finding showed little interest to proceed to extraction of the latter. 3.3 Solvent partitioning of the ethanolic extracts Three methods, according to solvent system, were used for the partition of kernel ethanolic extract. The binary system, hexane/water (Method 1), produced a stable emulsion that took many hours to resolve. Several extractions with hexane were necessary to obtain the apolar products. Compared with hexane/water, the ternary system: hexane/methanol/water: 50/25/25 (Method 2) reduced the formation of emulsion. It improved the separation of products. Nevertheless, the use of dichloromethane, a toxic solvent, would not be suitable in large scale. In this regard, the method 3 (Figure 2) was the best one. Ethyl acetate is less toxic than dichloromethane. Chromatographic profile of ethyl acetate extract differed from that of dichloromethane extract by the occurrence of polar products in the latter. This fact was supported by the increase of yield from 10.3% to 13.8%.

Fig 2: Solvent partitioning of the ethanolic extract 3.4 Acetogenins of soursop seeds Three known acetogenins were isolated as main products (13.5%) from CH2Cl2 extract (B’): annonacin (1), annonacinone (2) and murisolin (3) (Figure 3). Annonacin was obtained with good yield (26.6% of CH2Cl2 extract (B)) when elution was undergone with CH2Cl2/MeOH. Indeed, these solvents are preferably used to extract acetogenins [37]. These acetogenins bear a tetrahydrofuran ring flanked with two hydroxyl groups with a threo-trans-threo relative configuration. This relative configuration is also supported by the melting point of annonacin (60 °C) which is far below 77 °C (the melting point of cis-annonacin) [12]. Based upon the investigations of Curran and co-workers [38], relative configuration of C-4 and C-34 in the hydroxybutenolide subunit was assigned. Since the difference between resonances of C-33 and C-4 is 82 ppm for annonacin (1) and 81.8 ppm for murisolin (3), the relative configuration of C-4 and C-34 is syn (4S, 34S) for the former compound whereas anti (4R, 34S) for the latter. As the difference between resonances of C-33 and C-4 is 82.1 ppm for annonacinone, the relative configuration of C-4 and C-34 cannot be assigned. However, considering the results of Rieser and co-workers [39] which assigned 4R and 34S as the absolute configuration of annonacin and annonacinone the preparation of Mosher ester derivatives of the currently isolated acetogenins would be useful.

Fig 3: Acetogenins of soursop seeds: annonacin (1), annonacinone (2), murisolin (3) Isolated annonacin was

hydrogenated as shown in Figure 4.

Fig 4: Hydrogenation of annonacin The mixture obtained after hydrogenation of the isolated annonacin was analysed by TLC. After spraying the plate with vanillin sulfuric acid, followed by heating, three orange spots were displayed at Rf 0.36, 0.32 and a very intense one at 0.23 in CH2Cl2/MeOH: 95/5. These results suggested the existence of two isomers of the isolated annonacin since catalytic hydrogenation of a 4-hydroxylated α, β-unsaturated γ-lactone acetogenin performed with Pd/C affords a 1:1 mixture of diastereoisomers (the cis and the trans products) [40, 41]. Therefore, we hypothesized the existence of four diastereomers of dihydro-annonacin and two of them were unresolvable in the solvent system used. Main differences between the chemical shifts of annonacin and those of the mixture of dihydro-annonacin are reported in Table 1. Table 1: Chemical shifts of hydroxybutenolide subunit of annonacin

and those of the mixture of dihydro-annonacin

N° δ 13C (δ 1H) ppm

Annonacin Dihydro-annonacin 1 174,8 180.4 2 131.1 39.5 (2.89 ; m) / 41.7 (2.35 ; m) 3 33.2 (2.41 dd; 2.53 ; dt) 37.4 (2.47 ; 2.45 ; m) 4 69.9 (3.82 ; m) 66.7 (3.82m) / 70.5 (3.61 ; m) 33 151.9 (7.21 ; d) 25.7- 29.9 (1.19-2.6 ; m)34 79.1 (5.08 ; q) 76.0 (4.53 ; m) / 79.7 (4.39 ; m) 35 19.2 (1.45 ; d) 21.1 (1.36 ; m) / 24.5 (1.19 ; m)

The occurrence of murisolin, annonacin and annonacinone in soursop seeds has already been reported in the literature [11, 12]. The employed method permitted the isolation of almost all molecules with such structure using solvent extraction and liquid/liquid partition. Relatively cheap and low-toxicity

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solvents were used. Acetogenins could be used as starting material for chemical modification and biological evaluation. The following results concerning larvicidal properties are only one of the examples of what could be performed. 3.5 Larvicidal activity and toxicity of crude extracts and that of acetogenins 3.5.1 Larvicidal activity and toxicity of crude extracts Dichloromethane extract (B) showed larvicidal activity with LC50=74.4 ppm (95% confidence intervals: 60 ppm – 97 ppm). Three fractions were obtained from the dichloromethane

extract (B). Fraction (B1) containing the murisolin, showed weak larvicidal activity. Fraction (B2) consisting of a mixture of three acetogenins (1, 2 and 3) was more active than fraction (B3) which contained annonacin. These results may indicate the synergistic action of acetogenins. The ethyl acetate extract (E*) exhibited larvicidal activity with LC50=14.5 ppm (95% confidence intervals: 12 ppm – 17 ppm) and LC90=60.1 ppm (95% confidence intervals: 53 ppm – 71 ppm) as shown in Figure 5. It was about five times more effective than the dichloromethane extract (B).

Fig 5: (a) Dose-larvicidal activity of the dichloromethane extract (B), (b) Dose-larvicidal activity of the ethyl acetate extract (E*)

Nevertheless, the ethyl acetate extract (E*) was also proved to be toxic to Artemia salina nauplii with LC50=2.7 ppm (95% confidence intervals: 2.3 ppm – 3.2 ppm) which limits the application in this field. Its toxicity was five times higher than its larvicidal effect. 3.5.2. Larvicidal activity of annonacin and that of dihydro-annonacin derivatives Annonacin was active at low concentrations. However at higher concentrations (10 to 40 ppm), the mortality rate induced by the molecule remained constant and did not exceed

50%. LC50 was estimated at 246 ppm. Annonacin is lipophilic [22]. From 10 ppm, it formed a colloidal aqueous solution. Considering this fact, it is obvious that the median lethal concentration of annonacin would not be achieved. So, the low water solubility of annonacin constitutes a limiting factor of its larvicidal activity. The mixture of dihydro-annonacin diastereoisomers exhibited larvicidal activity with LC50=64.6 ppm (95% confidence intervals: 52.2 ppm – 87.5 ppm). Compared to the larvicidal activity of annonacin, that of the hydrogenated compound has increased (Figure 6).

Fig 6: (a) Dose-larvicidal effect of annonacin, (b) dose-larvicidal effect of the mixture of dihydro-annonacin

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This increase of activity can be explained by two phenomena: the solubility and the synergistic action. Firstly, the mixture of dihydro-annonacin diastereomers was more soluble in water than the starting molecule. Secondly, molecules acted synergistically. This fact was already noted with the fraction (B2) which contained the three acetogenins (annonacin, annonacinone and murisolin) and with the crude extract which contained mainly acetogenins. 3.5.3. Toxicity of annonacin and that of dihydro-annonacin derivatives Annonacin exhibited potent toxicity toward Artemia salina nauplii with LC50=1.5 ppm (95% confidence intervals: 1.2 ppm – 1.9 ppm). Previous work reported the toxicity of isomers of annonacin toward brine shrimp with LC50 values ranging from 2.3 ppm for cis-annonacin [12] to 3.3 ppm for trans-annonacin [42]. Therefore, the isolated annonacin, bearing trans relative configuration, was more toxic compared to the literature data. The isomers of dihydro-annonacin were toxic to brine shrimp larvae with LC50=0.8 ppm (95% confidence intervals: 0.5 ppm - 1.1 ppm). At low concentrations, they were slightly more toxic than annonacin. At higher concentrations, their toxicities did not show significant difference (Table 2). Table 2: LC50 and LC90 values of annonacin and dihydro-annonacin

against Artemia salina nauplii

Lethal dose Annonacin Dihydro-

annonacin LC50 (95% confidence

intervals) 1.5 ppm (1.2 -

1.9) 0.8 ppm (0.5 -

1.1) LC90 (95% confidence

intervals) 11.9 ppm (9.3 -

16.5) 10.4 ppm (7.6 -

17.2) The catalytic hydrogenation slightly increased the larvicidal activity of the molecule, but did not reduce its toxicity. The toxicity of crude extract containing acetogenins and that of annonacin against Artemia salina nauplii confirms the use of Annona muricata seeds as fish poisons [6] and should be considered as a serious drawback. Based upon median lethal concentrations, crude extract is more efficient than pure compound. The use of crude extract offers two main advantages: (1) it does not require isolation of pure molecule which involves high costs and (2) insect resistance is much less likely to occur. Thus, ethyl acetate crude extract may be employed as effective larvicide but its toxicity should be reduced by chemical modification. 3.6 Carbohydrates and aqueous extract of soursop seeds Aqueous extract (C’) represented 5.2% of kernels. TLC analysis of the extract revealed the occurrence of amino acids and carbohydrates. The latter was mainly composed by sucrose (9,6%) and induced the caramel texture of the aqueous extract. Sucrose was already isolated from seeds of Annona reticulata [43]. Carbohydrates can be used as raw materials for fermentation process or for the synthesis of nonionic surfactants such as sugar esters [44, 45] or alkylpolyglycoside. 3.7 Seed oils Oil extracted from husks (HO) accounted for 1.6% by weight. Literature reported that husks of soursop seeds from Nigeria contained no trace of oil [16]. However, this content was much lower compared to that obtained from kernels. Since husks contain low level of lipid and acetogenins, their extraction is

not worthy. Separation of kernels from husks is essential. With an ash content of 2.58% [16], husks can serve as alternative of wood-fuel. Extraction of kernels with hexane yielded 36.5% oils (KO). Taking into account the water content of the kernels (8.9%), the oil content represents 40.4% of dry kernels by weight. This content is very high compared to the literature data: 22.1% (extracted with petroleum ether) [20], 22.57% (extracted with ether) [16]. This difference is probably due to several factors: origin, climate, variety, cultivar, the ripening stage, the harvesting period or the solvent used. The oil content of kernels of soursop seeds is lower than those of peanuts (46.57 to 53.05%) [46] but higher compared to those of some varieties of olives (18 to 23.6%) [47]. The kernel oil (A’) (30.4% of kernel) obtained by liquid/liquid partition of the ethanol extract and the one extracted with hexane (KO) were compared. 3.7.1 TLC analysis of kernel oils TLC analysis revealed the existence of acetogenins in the kernel oil (KO) extracted with hexane contrary to the other one (A’) obtained by liquid/liquid partition. These acetogenins were displayed as orange spots after spraying the plate with vanillin sulfuric acid followed by heating (Figure 7).

Fig 7: Chromatographic profile of the kernel oil (A’) obtained by liquid/liquid partition of the ethanolic extract and that of the kernel oil

(KO) extracted with hexane Acetogenins are lipohilic. Diluted in oils, they can be extracted by hexane during the treatment of kernels. However, due to the hydroxyl groups (in annonacin, annonacinone and murisolin), they are soluble in methanol. Thus, most of them were removed from the hexane layer during the partition process using the solvent system hexane/methanol/water. The kernel oil extracted directly with hexane contained acetogenins. It confirmed the toxicity of the oil and its folkloric use as treatment to kill head lice. Thus, this oil is not well-fitted for consumption. 3.7.2 Physical and chemical characteristics of kernel oils The physico-chemical characteristics of the two oils obtained by the two methods are summarized in Table 3.

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Table 3: Chemical and physical characteristics of the kernel oils obtained by the two methods

Physico-chemical characteristics

Kernel oil (KO) extracted with

hexane

Kernel oil (A’) obtained by

solvent partition Specific gravity at 25 °C 0.9136 0.9093 Acid value (mg KOH/g) 2.02 2.24

Saponification value (mg KOH/g)

215.3 221.6

Ester value (mg KOH/g) 213.28 219.36 Content of unsaponifiable

matter (%) 0.4 0.39

The physico-chemical characteristics of the two oils showed no major difference. The specific gravity of the oil extracted with hexane (KO) (0.9136) was close to 0.9178 [18]. The oil obtained by liquid/liquid partition was lighter compared to that extracted with hexane. This may be explained by the absence of the acetogenins. The acid values of both oils showed no significant difference and were close to 2.29 mg KOH/g [18]. This index ranges from 0.93 [20] to 10.02 [19]. Codex alimentarius has set the maximum value of 0.6 mg KOH/g for refined oils [48]. If the kernel oil obtained by liquid/liquid partition is considered as refined one, its high level of acid value makes it unsuitable for consumption. The saponification value of both oils was superior to the data mentioned by some literatures: 117 mg KOH/g [21], 157 mg KOH/g [19], 197 mg KOH/g [18] but close to 227.48 mg KOH/g [20]. The high level of saponification value makes the kernel oil usable for specialty chemical (green gasoil, soap…). 3.7.3. Fatty acid composition of kernel oils Fatty acid composition of the kernel oil extracted with hexane and that of obtained by liquid/liquid partition did not differ. The GC/MS spectrum of the kernel oil (KO) extracted with hexane is reported in Figure 8.

Fig 8: Separation of fatty acids in kernel oil of Annona muricata seeds by GC/MS. Peak assignment: tr=12.54: palmitoleic acid; tr=12.68: palmitic acid; tr=13.81: linoleic acid; tr=13.87: oleic acid; tr=13.98: stearic acid, tr=15.02: gondoic acid; tr=15.18: arachidic acid

The GC/MS analysis did not reveal the occurrence of myristic acid. This saturated fatty acid is weakly ionized by electron impact. Ravaomanarivo and co-workers [15] reported the presence of this fatty acid in soursop seeds. The monounsaturated fatty acid with 20 carbons can be gondoic or paullinic acid depending on the location of the double bond (C-11 or C-13). The previously mentioned authors designated this fatty acid as paullinic. Due to the migration of the double bond in the FAMEs, the preparation of pyrrolidide would be useful to determine this fatty acid without ambiguity. The fatty acid composition of the kernel oils extracted with hexane and that obtained by solvent partition is reported in Table 4.

Table 4: Fatty acid composition of the kernel oils

Fatty acid Relative percentage of fatty acid

Noun Symbol Kernel oil (KO) extracted with

hexane

Kernel oil (A’) obtained by solvent

partition

Myristic n-14:0 0.05 0.05

Palmitoleic 9-16:1 1.30 1.29

Palmitic n-16:0 19.65 19.77

Linoleic 9,12-18:2 35.86 35.68

Oleic 9-18:1 38.32 38.11

Stearic n-18:0 4.05 4.19

Gondoic 11-20:1 0.23 0.33

Arachidic n-20:0 0.55 0.58

% of saturated fatty acids 24.30 24.59

% of unsaturated fatty acids

75.70 75.41

Relative percentage of fatty acids in both oil samples did not differ. Unsaturated fatty acids predominated up to 75%. The literature reported different values: 75.45% [21], 71.93% [20] and 70.02% [18]. Oleic (38.1%) and linoleic (35.7%) were the major fatty acids. These results are consistent with the literature since the oil was classified among the oleic-linoleic acid group [21]. The main saturated fatty acid was palmitic (up to 19.7%). The value was close to 20.3% [21]. Palmitoleic acid was present in small amount and linolenic acid was not detected. These findings are consistent with the literatures which mentionned their existence in small or trace amount [15, 21]. In general, the relative percentage of each fatty acid slightly differed from that reported by literature [15, 21]. 3.7.4 Toxicity of the kernel oils against Artemia salina nauplii The kernel oil (A’) obtained by liquid/liquid partition of ethanolic extract was less toxic than that extracted with hexane (KO). From 10 ppm to 50 ppm, mortality rate varied from 9% to 11%. At 100 ppm, it remained far below 50%. By contrast, the kernel oil (KO) extracted with hexane showed strong toxicity with LC50=22.4 ppm (95% confidence intervals: 18.8 ppm – 26.4 ppm) and LC90=84.4 ppm (95% confidence intervals: 66.1 ppm – 118.7 ppm). Logistic regression which allowed the obtention of those lethal doses is depicted in Figure 9.

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0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 0,5 1 1,5 2 2,5

Mortality

Log(Dose (ppm))

Logistic regression of Mortality by Log(Dose (ppm))

Active Model

Natural mortality Lower bound (95%)

Upper bound (95%)

Fig 9: Dose-toxicity of the kernel oil (KO) extracted with hexane

These results confirmed the occurrence of acetogenins in the kernel oil extracted with hexane as it was noted in TLC analysis. The weak toxicity of the oil obtained by liquid/liquid partition could be induced by acetogenins in trace amount so that they were not detected in TLC analysis. For this reason, kernel oils obtained by the two methods are not suitable for consumption and their contact with living cells should be avoided as much as possible. However, they may be used in diverse areas of lipid chemistry for instance as intermediate for synthesis of surfactants [44, 45]. With high oleic content and saponification value, the kernel oil is appropriate for soap manufacturing. Methylated oil can serve as gasoil or lubricant for drilling machines. Oil has a myriad of potential applications: for treatment of wood, as industrial degreasing products, as fluxing for bitumen, as concrete form release agents or in the formulation of printing inks [49]. 3.8 Residues of extraction, the insoluble matter Insoluble matter (53.8% of kernels) remained after 13 hours of extraction of kernels with ethanol. Micro-Kjeldahl analysis of these residues showed a crude protein content of 25.6%. The average is ranging from 21.43% [20] to 27.34% [16]. The major part of these residues could be fibers. In fact, crude fiber content was estimated at 43.4% of kernels [16]. Due to these components, the residues may be considered as potential material for animal feeding.

During the feeding experiments, swiss mice tolerated residues of extraction 2.5 g, 5 g and 10 g mixed in their diet. No death was recorded. No metabolic changes or adverse effects were observed during the five days following the ingestion of the residues. Although these doses were administered once, the mice ate this for two days. Considering the period of exposure to residues and the frequency of administration, the results showed no subacute toxicity. Thus, the use of residues of extraction as animal feed is safe. However subchronic and chronic toxicity tests are necessary to assess the effects of a long-term ingestion of the residues. 4. Conclusion Husks of soursop seeds are not worthy of extraction but they can supersede wood-fuel. The treatment of the kernels led to four components: oils, a mixture of acetogenins, a mixture of carbohydrates and residues containing fiber and crude protein up to 25%. These components are more valuable than the by-products themselves. These results showed potential valorization of soursop seeds. The methodology used was relatively simple, feasible in large scale and reproducible. Optimal yield was reached at each step of the treatment. The solvents used were weakly toxic, affordable and available in large amount. A summary of the potential valorization of soursop seeds is depicted in Figure 10.

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Fig 10: Method of soursop seed valorization

One kilogram of kernels can provide 304 g of oils, 42 g of mixture of acetogenins and 52 g of mixture of carbohydrates. The resultant oil was less toxic than the one extracted with hexane. It is not appropriate for dietary purpose but may be used in lipid chemistry. The mixture of acetogenins may be applied as biolarvicides but the toxicity should be reduced. Catalytic hydrogenation of annonacin, the major acetogenin of Annona muricata, can increase the larvicidal activity of the molecule. The quantity of seeds depends closely on fruit biomass. Therefore, cultivation of the fruit should be promoted. Nevertheless, since atypical parkinsonism has been associated with the consumption of plants of the Annonaceae family [50], studies permitting a safe consumption is crucial. The valorization of soursop seeds is worthy and this current methodology can be applied to seeds of other species or genus of the Annonaceae family and even of other plants in general. Implementation of a pilot project is in progress. Treatment of by-products is a promising sector and is helpful to deal with waste issues.

5. Acknowledgments We thank all members of the laboratory « Catalyse et Synthèse Environnement » ICBMS – UMR5246 – Université Claude Bernard Lyon 1, France for their help. We are also grateful to the whole members of “Centre Commun de Spectrométrie de Masse – Lyon 1” and “Centre Commun de RMN – Lyon 1” for the analysis. We express our gratitude to Mr David Ramanitrahasimbola (Institut Malgache de Recherches Appliquées) who performed the toxicity bioassay of the residues of extraction. 6. References 1. Jelinski LW, Graedel TE, Laudise RA, McCall DW, Patel

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Colloque BioMad III, 12 et 13 décembre 2013, Hôtel Les Roches Rouges, Mahajanga Page 31

Evaluation de la sensibilité des moustiques de Madagascar aux répulsifs et attractants chimiques vers un développement de pièges in natura.

F. Y. Rasoloharijaonaa,b, G. Ranisaharivonya, V. Ramanandraibea,c, L. Rasoanaivoa, M. Rakotovaoa,c, P. Mavinguia,d, M. Lemairea,e, V. Jeannodab aLaboratoire International Associé (LIA), Antananarivo bLaboratoire de Biochimie fondamentale et Appliquée, Université Antananarivo cLaboratoire de Chimie et de Valorisation des Produits Naturels (LCVPN), Université d’Ankatso dLaboratoire d’Ecologie microbienne, Université Claude Bernard Lyon1 eLaboratoire de Catalyse Synthèse et Environnement (CASYEN), Université Claude Bernard Lyon1 [email protected] Bien que plusieurs méthodes prometteuses ont été mises au point pour évaluer l’efficacité de kairomones vis-à-vis des moustiques1, elles ne sont pas adaptées pour les pays en développement comme Madagascar. Un exemple est l’olfactomètre qui peut atteindre un coût très élevé et utilisé sous des conditions particulières2. Le LIA « Etude et Valorisation de la biodiversité Malgache » mène des recherches sur de nouvelles molécules actives contre les moustiques par une approche d’écologie chimique. Dans cet objectif, il est nécessaire de disposer de biotests efficaces, faciles à mettre en œuvre et à faibles coûts. Nous avons développé un dispositif expérimental original qui répond aux critères précités. Il a été utilisé pour évaluer la sensibilité des moustiques de Madagascar vis-à-vis de 2 répulsifs connus : l’acide 1-piperidinecarboxylique, 2-(2-hydroxyéthyl)-1-méthylpropylester (Picaridine) et le N,N-diéthyl-3-méthylbenzamide (DEET)3 et 2 produits supposés attractants :le 1 -octèn-3-ol et l’acide isovalérique4. Les produits dilués dans l’éthanol sont déposés dans le dispositif confectionné à Antananarivo, lequel est placé dans l’insectarium du LIA. Les tests sont réalisés en présence de CO2 pour potentialiser l’attraction longue distance. Les résultats obtenus sur Culex quinquefasciatus montrent un effet répulsif avec ED50 et ED90 respectivement 1,2 mg et 1,6 mg pour le DEET et 1,8 mg et 2,1 mg pour la Picaridine. A faible dose, l’octénol et l’acide isovalérique attirent faiblement C. quinquefasciatus mais semblent être répulsifs à forte dose. La validation du procédé mis au point ouvre des perspectives d’utilisation sur les moustiques vecteurs du paludisme (Anophèles) et d’arbovirose (Aèdes), avec des molécules Malgaches obtenues au LIA.

Mots clés : tests biologiques, kairomones, moustiques vecteurs, Culex quinquefasciatus



Valorisation des graines de corossol (Annona muricata) comme source de biopesticides pour le contrôle des moustiques hématophages vecteurs

Graziella Ranisaharivonya, Franck Rasoloharijaonaa, Voahangy Ramanandraibea,b, Léa Rasoanaivoa, Marcelle Rakotovaoa,b, Patrick Mavinguia,c, Marc Lemairea,d aLaboratoire International Associé (LIA), Antananarivo bLaboratoire de Chimie et de Valorisation des Produits Naturels (LCVPN), Université d’Antananarivo cLaboratoire d’Ecologie microbienne, Université Claude Bernard Lyon1 dLaboratoire de Catalyse Synthèse et Environnement (CASYEN), Université Claude Bernard Lyon1 [email protected] Les moustiques hématophages occupent le premier rang de vecteurs de maladies infectieuses. Par exemple le paludisme, qui est la première parasitose mondiale avec un million de décès par an, est transmis par les moustiques Anophèles1. Le paludisme demeure une des principales causes de mortalité à Madagascar. Une des voies pour combattre ce fléau consiste à éradiquer les moustiques vecteurs par le biais de la chimie verte en valorisant les biomasses et les produits tout en préservant l’environnement2. Parmi les objectifs de la présente étude figurent l’identification de molécules actives à partir des graines de corossol, réputées pour leur propriété larvicide3, l’optimisation de leurs activités en modifiant leurs structures et en réduisant leur cytotoxicité si nécessaire. Des extraits éthanoliques ont été obtenus à partir de graines de corossol,puis soumis à un partage liquide-liquide avec des mélanges aqueux de solvants organiques de polarité croissante (hexane, dichlorométhane). Les différentes phases obtenues sont évaporées puis testées selon la méthode de l’OMS (WHO/VBC/81.807). L’extrait dichlorométhanique présentant une activité larvicide avec une DL50 = 16,33ppm a été fractionné sur colonne de silice, dans le système CH2Cl2/MeOH en gradient de polarité croissante afin d’isoler les principes actifs. Les analyses structurales des extraits sont effectuées au Laboratoire de Catalyse Synthèse et Environnement, Université Lyon 1. Comparés aux données de la littérature, l’extrait hexanique n’a présenté aucune activité larvicide3. Par contre l’extrait dichlorométhanique s’est avéré actif contre les larves de Culex quinquefasciatus4. Les extraits biolarvicides et la richesse en dérivés d’acides gras valorisent les graines de corossol. Mots clés : corossol, Annona muricata, biolarvicide, acide gras, Culex

quinquefasciatus



Valorisation des ressources naturelles Malgaches :Etude de l’activité anti-inflammatoire de l’extrait de la plante FRD (Euphorbiacées)

ANDRIAMAMPIANINA Tianarilalaina Tantely, RASOANAIVO Léa, RANDIMBIVOLOLONA Fanantenanirainy, Assistant d’ESR – Doctorante en Pharmacologie Université d’Antananarivo, Faculté des sciences, Département de physiologie animale et de pharmacologie, Laboratoire de pharmacologie générale, de pharmacocinétique et de cosmétologie [email protected]

Les plantes sont une des principales sources médicamenteuses. A Madagascar, de nombreuses plantes sont utilisées empiriquement pour traiter les inflammations. A titre d’exemples, citons Harungana madagascariensis (Harongana), Nymphea stellata (voahirana), Strychnos madagascariensis (vakakoana). Les objectifs de ce travail d’une part est de valoriser l’utilisation des ressources naturelles malgaches et d’autre part d’étudier l’activité anti-inflammatoire de l’extrait de la plante FRD appartenant à la famille des Euphorbiacées et est utilisée en médecine traditionnelle malgache pour atténuer la fièvre, et traiter les affections cutanées et les symptômes grippaux. Pour vérifier que la plante a une activité anti-inflammatoire, des modèles expérimentaux d’inflammation aiguë, subchronique et chronique ont été utilisés. Les résultats expérimentaux démontrent que FRD est actif pour inhiber significativement l’inflammation expérimentale aiguë, subchronique et chronique. Ces effets observés varient avec la dose. L’activité de FRD sur l’inflammation aiguë est plus marquée que sur les inflammations chroniques et subchroniques. Ce travail nous a permis de démontrer que l’utilisation empirique de cette plante est prouvée expérimentalement, et des travaux de recherche sur la détermination de la molécule active et l’élucidation des mécanismes d’action possibles sont en cours d’exécution. Mots clés : Plantes – FRD – Anti-inflammatoire



Molécules isolées de Pervillaea phillipsonii klack. plante toxique et biologiquement active endémique de Madagascar †* Maonja F. Rakotondramanga, *Amélie Raharisololalao ,*Pr. Jeannot V. Rakotoarimanga, †Hans C. Krebs, *Léa Rasoanaivo, *Rivoarison Randrianasolo * Laboratoire de Chimie des Substances Naturelles et de Chimie Organique Biologique,

Université d’Antananarivo, Q211 Antananarivo 101, Madagascar † Laboratoire de Chimie des Substances Naturelles. Institut des aliments toxiques et Chimie

Analytique, Ecole Supérieure de Médicine Vétérinaire Hanovre Fondation, Allemagne

[email protected] La valorisation des ressources naturelles, notamment végétale, dans le système de soins de notre pays est une priorité importante pour minimiser notre dépendance vis-à-vis des médicaments importés. La plante codée TSK est de la famille des Asclepadaceae, elle est toxique et endémique à Madagascar. Basée sur la forte toxicité des racines, l’extrait CH2Cl2 a été sélectionné pour isoler les constituants actifs. La majorité des constituants de TSK sont des cardénolides glycosidiques, des produits naturels de grande importance, utilisés pour le traitement de la défaillance cardiaqueet la première génération de cette famille chimique est en phase d’essai clinique pour le traitement du cancer. Les objectifs de ce travail sont l’étude phytochimique des plantes toxiques endémiques malagasy afin d’isoler des molécules pures responsable de la toxicité, d’élaborer leurs structures et de faire des tests de toxicité des molécules obtenus. Ainsi que de découvrir des nouveaux molécules actives. Des méthodes chromatographiques ont été utilisées pour isoler les constituants de l’extrait CH2Cl2. Une partie de cette étude est l’analyse phytochimique de la plante, c'est-à-dire l’isolement et la détermination de structure des produits isolés. Pour élucider la structure des produits isolés, des méthodes spectroscopiques ont été établies tel que UV, IR, MS ainsi que les études des spectres RMN monodimensionelle 1H et 13C ; mais pour les composés dont les structures ne sont pas bien connues, une étude des spectres RMN bidimensionnelle heteronucléaire 1H-13C HSQC, HMBC, et homonucleaire 1H-1H COSY et ROESY a été faite pour voir la structure concrète et la stéréochimie relative du composé. Cinq molécules connues et deux nouvelles molécules TSK40, TSK45, ont été isolées de cette plante. Les tests sur les oreillettes isolées de cobaye des produits TSK 40 et TSK 45 possèdent une propriété tonicardiaque. Les résultats obtenus in vitro, reflètent l’activité toxique de TSK brut chez les souris avec une valeur de DL50 égale à 239.8 mg/Kg. Cette étude nous donne une idée sur la perspective de continuer les travaux déjà effectués sur TSK, comme l’étude biologique et pharmacologique des produits isolés et des extraits bruts. La réduction de la double liaison C=C du cycle lactonique des hétérosides cardiotoniques dans le but de diminuer la toxicité du produit et de savoir s’il est encore actif ou non. Des tests sur des cellules cancéreuses feront aussi l’objet d’un projet de recherche. Mots clés : Asclepiadaceae, hétérosides cadiotoniques, RMN, tonicardiaque



Contribution à l’étude chimique - Activité biologique des tiges feuillées de Cryptocarya sp Lauraceae Volasoa Herilalaina Victorine Rambeloson , Amélie Raharisololalao, Léa Herilala Rasoanaivo Laboratoire de Chimie des Substances Naturelles et Chimie Organique Biologique (LCSN/COB), Université d’Antananarivo [email protected] Une déficience ou une absence d’antioxydants entraîne un stress oxydatif pouvant endommager ou détruire les cellules. Il a été mis en cause dans la pathogénèse de nombreuses maladies humaines telles les accidents vasculaires cérébraux et les maladies neurodégénératives. Les antioxydants sont aussi des ingrédients importants des compléments alimentaires. Dans le but d'entretenir la santé et de prévenir certaines maladies une recherche d’antioxydants naturels est appropriée. Le présent travail sera axé sur l’étude chimique de Cryptocarya sp (Lauraceae), une plante endémique de Madagascar .Le décoction des tiges et des feuilles de cette plante sont très utilisées en médecine traditionnelle pour soigner les irritations de la peau ; l’infusion des feuilles est prise pour traiter les maux de dent ; les maux de tête. Le but est de valoriser Cryptocarya sp à cause de ses propriétés thérapeutiques. Le criblage phytochimique a montré la présence des composés phénoliques dans les tiges feuillées de Cryptocarya sp. La présence abondante de flavonoïdes nous a conduit à procéder à une extraction par partage de l’’extrait hydroalcoolique des tiges feuillées de Cryptocarya sp successivement l’hexane, le diclorométhane, le n-butanol. Le test antioxydant est effectué utilisant le 2,2-diphénylpicrylhydrazyle ont montré que l’extrait butanolique contient des composés antioxydants. Le fractionnement bioguidé de l’’extrait butanolique sur chromatographie sur colonne a conduit à l’isolement des flavonoïdes glycosides. Leurs structures ont été élucidées et déterminées par des méthodes spectroscopiques notamment la RMN. Mots clés : Antioxydant, Cryptocarya sp, Flavonoides glycosides, Lauraceae.


http://fr.wikipedia.org/wiki/Stress_oxydant

http://fr.wikipedia.org/wiki/Cellule_%28biologie%29

http://fr.wikipedia.org/wiki/Pathog%C3%A9n%C3%A8se

http://fr.wikipedia.org/wiki/Accident_vasculaire_c%C3%A9r%C3%A9bral

http://fr.wikipedia.org/wiki/Maladie_neurod%C3%A9g%C3%A9n%C3%A9rative

http://fr.wikipedia.org/wiki/Maladie_neurod%C3%A9g%C3%A9n%C3%A9rative

http://fr.wikipedia.org/wiki/Compl%C3%A9ment_alimentaire

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