Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 3(1), 27-31, January (2013) Res.J.Chem. Sci. International Science Congress Association 27 Volatile oil composition and antiproliferative activity of Hyptis spicigera Lamagainsthuman breast adenocarcinoma cells MCF-7 Bogninou-Agbidinoukoun G.S.1,3,4, Yedomonhan H.Avlessi F.1*, Sohounhloué D., Chalard P.3,4, Chalchat J-C.3,4, Delort L., Billard H., Caldefie-Chézet F. and Figueredo G.Laboratoire d’Etude et de Recherche en Chimie Appliquée (LERCA) EPAC/UAC, 01 BP 2009 Cotonou, BENIN Herbier National, Département de Botanique, Faculté des Sciences et Techniques, Université d’Abomey-Calavi, BENIN Clermont Université, ENSCCF, Institut de Chimie de Clermont-Ferrand, BP10448, F-63000 Clermont-Ferrand, FRANCE CNRS, UMR 6296, ICCF, F-63177 Aubière, FRANCE Université d’Auvergne-Faculté de Pharmacie, Laboratoire des Sciences Végétales et Fongiques pharmaceutiques, Equipe EA4233 «Nutrition, Cancérogenèse et Thérapie antitumorale», FRANCE LEXVA Analytique, 460 rue du Montant, 63110 Beaumont, FRANCEAvailable online at: www.isca.in Received 22nd August 2012, revised 30th September 2012, accepted 30th October 2012Abstract This study reported the chemical composition and the evaluation of the in vitro antiproliferative activity of volatile extracts of Hyptis spicigera (HS) on breast cancer cells MCF-7. Essential oils (EO) HS1 and HS2 extracted from leafy stems of H. spicigera by hydrodistillation after different harvesting periods were characterized by gas chromatography/flame ionization detector (GC/FID) and gas chromatography/mass spectrometry (GC/MS). The cytotoxicity of these volatile extracts was tested in order to assess their antiproliferative activity on cancer cells MCF-7 by the resazurin test. Chromatographic analysis revealed several major compounds depending on the harvest period of vegetable. Sample HS1 was characterized by -pinene (16.9%), sabinene (13.8%), -pinene (9.6%) and 1,8-cineole (9.6%), while HS2 was potentially rich in caryophyllene (53.2%), caryophyllene oxide (5.4%) and -humulene (3.8%). Biological investigations performed have shown that HS1 and HS2 were cytotoxic against MCF-7 cells. Inhibitory concentrations values (IC50) determined for this purpose were 170 and 84 µg/mL respectively for HS1 and HS2. Major compounds of the EO should be responsible for this inhibitory activity. Keywords: Hyptis spicigera, volatile extract, MCF-7, -caryophyllene, -pinene, Benin. Introduction Hyptis spicigera (Lamiaceae) is an aromatic plant annual, hairy, smartweed, woody at the base. Leaves are opposite and lanceolate. Inflorescences are terminal, dense, cylindral spike consisting to very small flowers with white corolla and mauve mark on the lips. H. spicigera is a ruderal herborous. It grows in savannas periodically flooded and fields. Although endemic to Brazil, it is widespread in tropical Africa and in Asia (figure-1). The decoction is used as bath water or tea, as eupneic, or expectorant to treat bronchial secretions. The powder obtained from the aerial organs is used as an antimigraine drug. Fresh inflorescences of this plant are used to treat headaches and coryza. In most Southern-Africa countries, this species is exploited by farmers to control grain infestations in storage. H. spicigera is also used by farmers against insect pests and to fight mosquitoes2-5. Phytochemical investigations lead to the isolation of seven (7) new labdane diterpenes. Due to its aromatic character, essential oil produced from this plant has been subjected to numerous studies. Indeed, a high proportion of -caryophyllene (57-66%) was revealed in the volatile extract of H. spicigera from Burkina Faso. On the other hand, the proportion of -caryophyllene (23-28%) of H. spicigerareported from the sample of Mali was down compared to those of this sesquiterpene compound noted from variety harvested in Nigeria (68%) 8,9. Otherwise, the presence of -caryophyllene (33.8%), -bergamotene (11.3%) and -caryophyllene (7.4%) had been reveled in majority in the sample from Togo10. The sample of H. spicigera from Cameroon contained a large proportion of terpenes with a relatively low level of caryophyllene (19.1%)11. Further investigations on the insecticidal, anticonvulsant, sedative and antimicrobial properties of the plant have been highlighted in the literature12-15. Spicigerolide (6-tetraacetyl oxyhetenyl-5,6-dihydro-pyrone), isolated from H. spicigera of Mexico was found to be active on cancer cells line KB16. Nevertheless, the antiproliferative character of the essential oil ofH. spicigera has not been studied in Benin. The aims of the present work is to determine the chemical composition by GC/FID and GC/MS of volatile oils extracted from leafy stems of H. spicigeraharvested in Benin and to evaluate their cytotoxic activity on cancer cells line MCF-7. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 3(1), 27-31, January (2013) Res. J. Chem. Sci. International Science Congress Association 28 Figure-1 Hyptis spicigera Lam Material and Methods Plant Material: The aerial parts of H. spicigera were harvested at Tele (Benin) in January (HS1) and October (HS2) 2010. In the laboratory, plant material was stored at 20°C and protected from sunlight during the extractions. The identification and authentication were made at the National Herbarium of Abomey-Calavi University (Benin). Voucher number was assigned to the specimen. Extraction of essential oils: Essential oils were obtained by hydrodistillation using a Clevenger-type apparatus for a period of three hours. The volatile oils collected after decantation was dried over anhydrous sodium sulfate and then stored at 4°C in amber glass vials until analysis. Gas chromatography-mass spectrometry analysis of the volatile oils: The essential oils were analysed on a AGILENT gas chromatograph Model 7890, coupled to a AGILENT mass spectrometer model 5975 equipped with a DB5 MS column (20m X 0.20 mm. 0.20µm) programming from 50°C (5 min) to 300°C at 8°C/min with a 5 min hold. Helium was used as the carrier gas (1.0 mL/min); injection in split mode (1: 250); injector and detector temperature 250 and 280°C respectively. The mass spectrometer worked in electron impact mode at 70 eV; electron-multiplier: 1500 eV; ion source temperature: 230°C; mass spectra data were acquired in the scan mode in m/zrange 33-450. The essential oil was diluted in hexane: 1/30. Cancer cells: The cell line under investigation was human breast adenocarcinoma (MCF-7). MCF-7 stands for Michigan Cancer Foundation-7, in reference to the Detroit Institute where the line was established17,18. Cytotoxicity assays: Biological investigations were performed on cancer cells of human breast adenocarcinoma MCF-7. According to the cells growth profile, MCF-7 cells were seeded into the wells of microplates at a concentration of 50.000 cells/mL. The microplates were kept in an incubator at 37°C, in an humidified atmosphere containing 5% carbon dioxide. After 24 hours, the cells were treated with volatile oils initially dissolved in dimethylsulfoxide (DMSO) and sonicatedIn parallel, for each tested essential oil, a control of dimethylsulfoxide DMSO (solvent of dilution of the EO) is produced. Plates were returned into the incubator for 72 hours under the same conditions. After this final period of incubation, the culture medium was replaced with a solution of resazurin (25 mg/mL) which in the presence of metabolically active cells is oxidized into resofurin, fluorescent at 590 nm. The intensity of fluorescence was proportional to the number of viable cancer cells. Fluorescence was then measured using a plate reader (Fluoroskan Ascent ® FL. Thermo Electron Corporation France) at 590 nm. The antiproliferative activity was evaluated using the following concentration ranges: 0.04% to 0.01% for HS1 and 0.04% to 0.002% for HS2. The intensity of fluorescence obtained after reading with Fluoroskan Ascent (expressed in arbitrary units) was converted into percentage of inhibition of proliferation relative to cells proliferation in the control (DMSO). It was established that DMSO got no influence on cell proliferation, in comparison with a control performed without DMSO under the same conditions. Results and Discussion Yield and chemical composition of essential oils: The yield of essential oil extracted from leafy stems of H. spicigera varied from 0.23% (HS1) to 0.2% (HS2) depending on harvest periods. These values were different from those obtained respectively from varieties of H. spicigera from Mali (0.1-0.3%), Cameroon (0.12%), Nigeria (0.1%) and Togo (1.2%)8-11. These differences could be attributed to the geographical characteristics of the ecological zone and the vegetative state of the plant species. The chemical composition of the essential oils is given in table-1. According to table-1, 58 to 60 compounds were identified representing 98.9 to 92.8% of the total identified in investigated essential oils. Depending on the period of collection of vegetable, chemical compositions of the essential oils, analyzed by GC/MS, varied remarkably in hydrogenated and oxygenated terpenes. Indeed, a large proportion of hydrogenated monoterpenes (62.5%) and oxygenated monoterpenes (21.5%) were noted in HS1. Contrary, sample HS2 was dominated by hydrogenated sesquiterpenes (68.1%) and oxygenated sesquiterpenes (8.5%). The results of the chromatographic analysis showed that essential oils HS1 and HS2 were characterized by -thujene (6.5%), -pinene (2.5-16.9%), sabinene (13.8%), -pinene (9.6%), 1,8-cineole (9.6%), terpinene (6.0%); terpinolene (5.3%), -caryophyllene (7.0- 53.2%), -humulene (3.8%), caryophyllene oxide (2.1-5.4%) and cembrene A (2.6%). These main components, apart from their concentrations in each of the two samples, had already been revealed in earlier works 8-11. On the other hand, the rate of -thujene (6.5%) in HS1 is higher than those obtained in HS2 and essential oils extracted from H. spicigera collected in Mali, Togo, Cameroon and Nigeria8-11. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 3(1), 27-31, January (2013) Res. J. Chem. Sci. International Science Congress Association 29 Table-1 Chemical composition of essential oils of HS1 and HS2N° Compounds KI HS1 HS2 1 -thujene 923 6.5 0.6 2 -pinene 931 16.9 2.5 3 sabinene 970 13.8 1.1 4 -pinene 974 9.6 1.4 5 octen-3-ol 978 0.1 0.2 6 myrcene 987 1.0 0.1 7 octan-3-ol 995 - 0.1 8 -phellandrene 1003 1.7 0.4 9 -terpinene 1014 1.1 0.1 10 p-cymene 1023 3.6 1.4 11 limonene 1027 2 0.5 12 -phallandrene 1029 - 0.3 13 1,8 cineole 1033 9.6 - 14 (E)--ocimene 1046 0.2 0.1 15 -terpinene 1058 6.0 1.2 16 sabinene cis hydrate 1071 0.4 - 17 terpinolene 1085 5.3 1.6 18 p-cymenene 1089 0.2 0.1 19 linalool 1099 - 0.1 20 sabinene trans hydrate 1101 0.5 - 21 2-methyl-1-butyle 2-methylbutanoate 1103 0.3 - 22 2-methyl-1-buthyle isovalerate 1109 0.7 - 23 (E)-thujone 1117 0.1 - 24 cis-p-menth-2-en-1-ol 1125 0.1 - 25 campholenal 1127 0.1 - 26 nopinone 1138 0.1 - 27 (E)-pinocarveol 1141 0.4 - 28 (E)-p-menth-2-en-1-ol 1143 0.5 - 29 (E)-verbenol 1147 0.5 - 30 sabinacetone 1158 0.1 - 31 pinocarvone 1162 0.1 - 32 -terpineol 1172 0.2 - 33 borneol 1174 0.2 - 34 terpinen-4-ol 1181 1.9 - 35 p-cymen-8-ol 1187 0.5 0.1 36 myrtenal 1196 0.6 0.1 37 myrtenol 1197 0.6 - 38 verbenone 1207 0.1 - 39 cuminic aldehyde 1243 0.1 - 40 carvacrol 1303 0.1 - 41 -elemene 1336 - 0.1 42 eugenol 1351 - 0.1 43 -ylangene 1370 0.1 0.3 44 -copaene 1376 - 0.2 45 -bourbonnene 1384 0.1 0.3 46 -elemene 1389 0.1 0.3 47 isocaryophyllene 1405 0.1 0.4 48 -cedrene 1410 - 0.1 49 -caryophyllene 1426 7.0 53.2 50 -elemene 1430 - 0.7 51 -guaiene 1436 - 0.3 52 guaia-6,9-diene 1443 0.1 0.5 53 guaia-1(5),6-diene 1448 0.2 - 54 selina-4(15),6-diene 1451 - 0.9 55 -humulene 1459 0.4 3.8 56 -himachalene 1478 - 0.2 57 (E)--ionone 1479 - 0.1 58 germacrene-D 1484 0.2 1.3 59 -amorphene 1505 - 0.3 60 -cadinene 1516 0.3 1.3 61 -cadinene 1520 - 0.3 62 -cadinene 1540 - 0.1 63 germacrene-B 1562 0.1 0.7 64 caryophyllene oxide 1586 2.1 5.4 65 -gurjunene 1592 - 0.3 66 -muurolene 1596 - 0.1 67 curzerenone 1601 - 0.2 68 humulene-epoxyde II 1613 0.1 0.3 69 1,10-di-epi-cubenol 1618 0.1 0.4 70 isospathulenol 1630 - 0.2 71 caryophylla-4(12).8(13)-dien-5--ol 1641 0.2 0.9 72 -cadinol 1658 0.2 0.8 73 6,10,14-trimethyl pentadecan-2-one. 1842 - 0.2 74 thumbergene 1926 - 0.2 75 cembrene A 1957 0.1 2.6 76 isophyllocladene 1966 - 0.1 77 palmitic acid 1964 0.5 - 78 bicyclogermacrene 2022 - 1.0 79 valenrena-4,7(11)-diene 2045 - 0.9 80 phytol 2106 - 0.3 81 oleic acid 2144 0.4 0.5 82 octadecanoïc acid 2167 1.0 - Hydrogenated monoterpenes 62.5 9.9 Oxygenated monoterpenes 21.5 1.9 Hydrogenated sesquiterpenes 7.9 68.1 Oxygenated sesquiterpenes 2.6 8.5 Others 4.4 3.8 Total identified 98.9 92.2 Anti-proliferative activities against MCF-7 cells: The cytotoxicity activities of test samples (HS1 and HS2) were performed against MCF-7. Analysis of the results revealed a difference of behavior of MCF-7 cells in the presence of various volatile extracts of H. spicigera. IC50 values of volatile extracts HS1 and HS2 were respectively 170 and 84 µg/mL. Based on these values, it appeared that the two essential oils had inhibited the growth of MCF-7 cells. However, HS2 had a higher efficiency than HS1. This activity was justified by the presence of -pinene, sabinene, -pinene, 1,8-cineole, -caryophyllene, caryophyllene oxide, -humulene which were identified as having antiproliferative properties on cancer MCF-719-22. Indeed, essential oil of Eugeniazuchowskiae from Costa Rica, rich in -pinene (23.3%), -caryophyllene (13.2%), -humulene (13.1%) was cytotoxic to MCF-7 cells19. Also, 1,8-cineole, the main component of essential oils extracted from of Laurus nobilis and Salvia tribola had demonstrated significant Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 3(1), 27-31, January (2013) Res. J. Chem. Sci. International Science Congress Association 30 inhibitory activity on cells20. The antiproliferative ability of essential oils of Heteropyxis dehniae and Pentadesma butyraceae, respectively rich in caryophyllene oxide and caryophyllene, was also demonstrated on the mammary adenocarcinoma cells MCF-721,22. Volatile extracts of current work were less effective than that of Origanumvulgare (IC50 = 30.1 g/mL) and the methanolic extract of Vitex trifolia, but HS2 possessed higher antiproliferative activity on MCF-7 cells than that of essential oil of Laurus nobilis (IC50 = 101.7 g/mL)20, 23. Conclusion This work was devoted to the determination of the chemical composition by GC/FID and GC/MS and has evaluated the anti- proliferative activity of two volatile extracts (HS1 and HS2) of the leafy stems of H. spicigera harvested in Benin. HS1 is potentially rich in monoterpenoic compounds (-pinene: 16.9% sabinene: 13.8% -pinene: 9.6%, 1,8-cineole: 9.6% -thujene: 6.5%, -terpinene: 6.0% and terpinolene: 5.3%) while that of HS2 is mainly composed by sesquiterpenoic compounds ( caryophyllene: 53.2%, caryophyllene oxide: 5.4%, -humulene: 3.8%). The significant antiproliferative activity of HS2 essential oil could be due to its high content in sesquiterpenoic compounds. Acknowledgement The authors like to address the greatest consideration to Doctors Noudogbessi J-P.A. and Yehouenou B. for reading this manuscript. 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