Journal of Ethnopharmacology 151 (2014) 652–659 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep Chemical composition, cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin$ Salomé Kpoviessi a,b,c, Joanne Bero b, Pierre Agbani d, Fernand Gbaguidi a,c, Bénédicta Kpadonou-Kpoviessi a, Brice Sinsin d, Georges Accrombessi a, Michel Frédérich e, Mansourou Moudachirou c, Joëlle Quetin-Leclercq b,n a Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), University of Abomey-Calavi (UAC), Faculty of Sciences and Technics (FAST), BP: 4521 Cotonou, Benin b Pharmacognosy Research Group, Louvain Drug Research Institute, Université catholique de Louvain, B1 7203 Av. E. Mounier 72, B-1200 Bruxelles, Belgium c Laboratory of Pharmacognosy and Essential oils (LAPHE), University of Abomey-Calavi (UAC), Faculty of health Sciences (FSS), Faculty of Sciences et Technics (FAST), 01 BP: 188 Cotonou, Benin d Laboratory of Applied Ecology (LEA), University of Abomey-Calavi (UAC), Faculty of Agronomic Sciences (FSA), 03 BP: 1974 Cotonou, Benin e Université de Liège, Drug Research Center, Laboratoire de Pharmacognosie, Av. de l'Hôpital 1, B36, B-4000 Liège, Belgium art ic l e i nf o a b s t r a c t Article history: Received 25 April 2013 Received in revised form 8 November 2013 Accepted 9 November 2013 Available online 21 November 2013 Ethnopharmacological relevance: Cymbopogon species are largely used in folk medicine for the treatment of many diseases some of which related to parasitical diseases as fevers and headaches. As part of our research on antiparasitic essential oils from Beninese plants, we decided to evaluate the in vitro antiplasmodial and antitrypanosomal activities of essential oils of four Cymbopogon species used in traditional medicine as well as their cytotoxicity. Materials and methods: The essential oils of four Cymbopogon species Cymbopogon citratus (I), Cymbopogon giganteus (II), Cymbopogon nardus (III) and Cymbopogon schoenantus (IV) from Benin obtained by hydrodistillation were analysed by GC/MS and GC/FID and were tested in vitro against Trypanosoma brucei brucei and Plasmodium falciparum respectively for antitrypanosomal and antiplasmodial activities. Cytotoxicity was evaluated in vitro against Chinese Hamster Ovary (CHO) cells and the human non cancer fibroblast cell line (WI38) through MTT assay to evaluate the selectivity. Results: All tested oils showed a strong antitrypanosomal activity with a good selectivity. Sample II was the most active against Trypanosoma brucei brucei and could be considered as a good candidate. It was less active against Plasmodium falciparum. Samples II, III and IV had low or no cytotoxicity, but the essential oil of Cymbopogon citratus (I), was toxic against CHO cells and moderately toxic against WI38 cells and needs further toxicological studies. Sample I (29 compounds) was characterised by the presence as main constituents of geranial, neral, β-pinene and cis-geraniol; sample II (53 compounds) by trans-pmentha-1(7),8-dien-2-ol, trans-carveol, trans-p-mentha-2,8-dienol, cis-p-mentha-2,8-dienol, cis-pmentha-1(7),8-dien-2-ol, limonene, cis-carveol and cis-carvone; sample III (28 compounds) by βcitronellal, nerol, β-citronellol, elemol and limonene and sample IV (41 compounds) by piperitone, ( þ)-2-carene, limonene, elemol and β-eudesmol. Conclusions: Our study shows that essential oils of Cymbopogon genus can be a good source of antitrypanosomal agents. This is the first report on the activity of these essential oils against Trypanosoma brucei brucei, Plasmodium falciparum and analysis of their cytotoxicity. & 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: Cymbopogon species Essential oils Chemical composition Antitrypanosomal activity Antiplasmodial activity Cytotoxicity ☆ Chemical compounds studied in this article. 1. Introduction β-Myrcene (PubChem CID: 31253); Limonene (PubChem CID: 22311); β-Citronellal (PubChem CID: 7794); Geranial (PubChem CID: 638011); Neral (PubChem CID: 643779); β-Pinene (PubChem CID: 14896); Piperitone (PubChem CID: 6987); ( þ)-2-Carene (PubChem CID: 78249); cis-p-Mentha-1(7),8-dien-2-ol (PubChem CID: 6429040); Nerol (PubChem CID: 643820). n Corresponding author. Tel.: þ 32 2 764 72 54; fax: þ 32 2 764 72 93. E-mail address: joelle.leclercq@uclouvain.be (J. Quetin-Leclercq). 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.11.027 Aromatic plants are used since ancient times for their medicinal properties (Bakkali et al., 2008). Essential oils may be used as alternatives or adjuvants to current antiparasitic therapies and the emergence of parasites resistant to current chemotherapies highlights the importance of plant essential oils as potential novel S. Kpoviessi et al. / Journal of Ethnopharmacology 151 (2014) 652–659 antiparasitic agents (Anthony et al., 2005; Moon et al., 2006; Nibret and Wink, 2010; Cheikh-Ali et al., 2011; Monzote et al., 2011; Palmeira-de-Oliveira et al., 2012). Cymbopogon species are commonly used in folk medicine for the treatment of many diseases. Cymbopogon citratus is used for the treatment of nervous and gastrointestinal disturbances, and as an antispasmodic, analgesic, anti-inflammatory, anti-pyretic, diuretic and sedative (Santin et al., 2009). Decoctions of the leaves and flowers of Cymbopogon giganteus are used as an effective treatment against skin disorders, conjunctiva, headaches and hepatitis (Adjanohoun et al., 1979; Adjanohoun and Aké Assi, 1979, 1985). Cymbopogon nardus is used for cooking, perfumery, rheumatism and in the treatment of fevers, intestinal parasites and of digestive and menstrual problems (Konwar and Gohain, 1999; Abena et al., 2007). Cymbopogon schoenathus is used as an embrocation, a diuretic, an insecticide, an aphrodisiac, for fever, snake-bite and for the treatment of rheumatism. The smoke from the burning grass is said to dispel temporary maniacal symptoms (IUCN, 2005, Khadri et al., 2010). Essential oils of these species are known for antimicrobial, antifungal, antioxidant, analgesic, antinociceptive, neurobehavioral and insecticidal properties (Bassolé et al., 2011; Innsan et al., 2011, Khadri et al., 2010; Abena et al., 2007; Jirovetz et al. 2007; de Billerbeck et al., 2001; Viana et al., 2000; Onawunmia et al., 1984) and as repellent against mosquitos, the major vector of malaria (Nonviho et al., 2010; Samarasekera et al., 2006; Tyagi et al., 1998). Their direct activity against Trypanosoma brucei and Plasmodium falciparum was not very documented excepted for essential oil from Cymbopogon nardus of Malaysia whose in vitro antitrypanosomal activity was recently reported by Muhd Haffiz et al. (2013). Furthermore, they are used in traditional medicine for the treatment of symptoms given by malaria or sleeping sickness (as fevers, headaches,…). So it seemed interesting to study the antiplasmodial and antitrypanosomal activities of these essential oils and its components. Trypanosoma brucei is the parasite responsible for human African trypanosomiasis or sleeping sickness, an illness affecting 300,000–500,000 people, while up to 60 million people in 36 countries are at risk of contracting the disease (World Health Organisation (WHO), 2002). This parasite is transmitted by the bite of infected tsetse flies of the genus Glossina. Malaria is also a disease caused by a protozoan parasite of Plasmodium species and still remains a major public health problem in the world. Five hundred million people are exposed to this disease, with an annual death rate that the World Health Organisation (WHO/World Health Statistic, 2011) estimates to more than 800,000 people in 2009. These two parasitic diseases are the cause of considerable mortality and morbidity throughout the world (WHO/World Health Statistic, 2011) and parasites develop resistance to most of the drugs used. Some of these drugs need a long course parenteral administration, show toxicity and a variable efficacy between strains or species. These reasons led to the search for new antitrypanosomal and antiplasmodial compounds and it is known that plants used in traditional medicine are a source of new leads with a new mechanism of action (Hoet et al., 2004, Bero et al., 2011). The present study investigates the in vitro antitrypanosomal and antiplasmodial activity of essential oils from four plants of Cymbopogon genus used in traditional medicine in Benin. Oils from fresh leaves of each plant were prepared and analysed by GC/FID and GC/MS. They were evaluated for their antitrypanosomal and antiplasmodial activities and their selectivity was assessed by analysing their cytotoxicity against Chinese Hamster Ovary cells (CHO) and a human non cancer fibroblast cell line (WI38). 653 2. Materials and methods 2.1. Plant material Fresh leaves of Cymbopogon citratus (DC) Stapf, Cymbopogon giganteus (Hochst.) Chiov., Cymbopogon nardius (L.) Rendle and Cymbopogon schoenantus (L.) Spreng. (Poaceae) were collected in March 2011, from the Botanical Garden of the Abomey-Calavi University. Voucher specimens (nos. AA6387, AA6388, AA6389 and 6390/HNB respectively) of these leaves were conserved at the University of Abomey-Calavi Herbarium. 2.2. Chemicals and drugs DMEM and Ham's-F12 culture media were purchased from Life technologies corporation (Grand Island, NY 14072, USA); Dulbecco's Phosphate Buffered Saline (DPBS 1  ) from Invitrogen (Grand Island, NY 14072, USA); tetrazolium salt (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium-bromide) (MTT), (S)-( þ)-camptothecin, suramine, chloroquine, artemisinin, dimethyl sulfoxide (DMSO), α-pinene, β-pinene, camphene, p-cymene, myrcene, α-terpinene, γ-terpinene,1,8-cineol, terpinolene, borneol, citronellyl acetate, terpine-4-ol, α-terpineol, geraniol, verbenone, carvacrol, thymol, bornyl acetate, α-copaene, β-caryophyllene, fenchone, thujone, trans-pinocarveol, trans-verbenol, lavandulol, myrtenal, transcarveol, carvone, aromadendrene, allo-aromadendrene, γ-gurjunene, cis-ocimene, camphor and n-alkanes “C7–C28” were obtained from Sigma-Aldrich (Steinhein, Germany), Acros Organics (New jersey, USA), and Fluka Chemie (Buchs, Switzerland); α-thujene, sabinene, γ-3-carene, limonene, linalool, αhumulene, cis-pinane, α-phellandrene, p-cymenene, myrtenyl acetate and valencene were purchased from extrasynthese (Genay, France). All compounds were of analytical standard grade. TerButyl methyl ether (TBME) was an analytical grade solvent purchased from Fluka Chemie, and anhydrous Na2SO4 was of analytical reagent grade from UCB (Bruxelles, Belgium). 2.3. Isolation of essential oils Five hundred grams (500 g) of fresh leaves were steam distillated for 3 h in a modified Clevenger-type apparatus (Bruneton, 1993). The extraction was carried out in triplicate. The oils were preserved in a sealed vial at 4 1C. The essential oil yields were calculated based on the fresh plant material. 2.4. Chemical analysis of essential oils 2.4.1. GC/FID and GC/MS analysis The GC/FID analysis was carried out on a FOCUS GC (Thermo Finigan; Milan, Italy) using the following operating conditions: HP 5MS column (30 m  0.25 mm, film thickness: 0.25 μm) (J&W Scientific Column of Agilent Technologies, No. US167072Ã, USA); injection mode: splitless; injection volume: 1 mL (TBME solution); split flow: 10 mL/min; splitless time: 0.80 min; injector temperature: 260 1C; oven temperature was programmed as following: 50–250 1C at 6 1C/min and held at 250 1C for 5 min; the carrier gas was helium with a constant flow of 1.2 mL/min; FID detector temperature was: 260 1C. The data were recorded and treated with the ChromCard software. The GC/MS analysis was carried out using a TRACE GC 2000 series (Thermo-Quest, Rodano, Italy), equipped with an autosampler AS2000 Thermo-Quest. The GC system was interfaced to a Trace MS mass spectrometer (ThermoQuest) operating in the electronic impact mode at 70 eV. HP 5 MS column (30 m  0.25 mm, film thickness: 0.25 μm) was used in the same operating conditions as above. The coupling temperature of the GC was 654 S. Kpoviessi et al. / Journal of Ethnopharmacology 151 (2014) 652–659 260 1C and the temperature of the source of the electrons was 260 1C. The data were recorded and analysed with the Xcalibur 1.1 software (ThermoQuest). in 96-well microtiter plates. The parasitaemia and the haematocrit were 2% and 1%, respectively. All tests were performed in triplicate. 2.7. In vitro test for antitrypanosomal activity 2.4.2. Identification of oil components Individual components of the volatile oils were identified by computer matching against commercial EI-MS spectra library (NIST, 1998; Adams, 1995), home-made mass spectra library obtained from pure substances and components of known oils (Kpoviessi et al., 2011). These identifications were supported by comparison of the GC retention times of a series of n-alkanes “C7– C28” mixture on a non-polar column (Kovats indices (KI)) (VanDenDool and Kratz, 1963). These indices calculated were in agreement with those reported by Adams (1995). For several compounds, comparison of data and retention times with those of authentic reference standards further confirmed the identifications. Quantification (expressed as percentages) was carried out by the normalisation procedure using peak areas obtained by FID. Values are expressed as mean 7standard deviation (n ¼3). 2.5. Parasites, cell lines and media Trypanosoma brucei brucei strain 427 (Molteno Institute in Cambridge, UK) bloodstream forms were cultured in vitro in HMI9 medium containing 10% heat-inactivated foetal bovine serum (Hirumi and Hirumi, 1994). Plasmodium falciparum chloroquine-sensitive strain 3D7 (from Prof. Grellier of Museum d'Histoire Naturelle, Paris, France) asexual erythrocytic stages were cultivated continuously in vitro according to the procedure described by Trager and Jensen (1976) at 37 1C and under an atmosphere of 5% CO2, 5% O2 and 90% N2. The host cells were human red blood cells (A or O Rhþ). The culture medium was RPMI 1640 (Gibco) containing 32 mM NaHCO3, 25 mM HEPES and 2.05 mM L-glutamine. The medium was supplemented with 1.76 g/L glucose (Sigma-Aldrich), 44 mg/ mL hypoxanthin (Sigma-Aldrich), 100 mg/L gentamycin (Gibco) and 10% human pooled serum (A or O Rhþ ). Parasites were subcultured every 3–4 days with initial conditions of 0.5% parasitaemia and 1% haematocrit. The macrophage-like cell line, CHO Chinese Hamster Ovary cells (ATCC no. CCL-61, batch 4765275), were cultivated in vitro in Ham's-F12 Nutrient Mixture 21765 medium (Gibco) containing 2 mM L-glutamine supplemented with 10% heat-inactivated foetal bovine serum (Gibco), penicillin–streptomycin (100 UI/mL–100 μg/ mL) and fungizone (ampotericine D 250 UG/mL). The human non cancer fibroblast cell line, WI38 (ATCC no. CCL-75 from LGC Standards) was cultivated in vitro in DMEM medium (Gibco) containing 4 mM L-glutamine, 1 mM sodium pyruvate supplemented with 10% heat-inactivated foetal bovine serum (Gibco), penicillin–streptomycin (100 UI/mL–100 μg/mL) and fungizone (amphotericin D 250 UG/mL). 2.6. In vitro test for antiplasmodial activity Parasite viability was measured using parasite lactate dehydrogenase (pLDH) activity according to the method described by Makler et al. (1993). The in vitro test was performed as described by Murebwayire et al. (2008). Chloroquine (Sigma) or artemisinin (Sigma) were used as positive controls in all experiments with an initial concentration of 100 ng/mL. First stock solutions of essential oils and pure compounds were prepared in DMSO at 20 mg/mL. The solutions were further diluted in medium to give 2 mg/mL stock solutions. The highest concentration of solvent to which the parasites were exposed was 1%, which was shown to have no measurable effect on parasite viability. Essential oils were tested in eight serial threefold dilutions (final concentration rang: 200–0.09 μg/mL, two wells/concentration) The in vitro test was performed as described by Hoet et al. (2004). Suramine (a commercial antitrypanosomal drug, MP Biomedicals, Eschwege, Germany) was used as positive control in all experiments with an initial concentration of 1 μg/mL. First stock solutions of essential oils and compounds were prepared in DMSO at 20 mg/mL. The solutions were further diluted in medium to give 0.2 mg/mL stock solutions. Essential oils and compounds were tested in eight serial threefold dilutions (final concentration range: 100–0.05 μg/mL, two wells/concentration) in 96-well microtiter plates. All tests were performed in triplicate. 2.8. Cytotoxicity assay The cytotoxicity of the oils against CHO and WI38 cells was evaluated as described by Stevigny et al. (2002), using the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (Sigma)) colorimetric method based on the cleavage of the reagent by dehydrogenases in viable cells (Mosmann, 1983). Camptothecin (Sigma) was used as positive cytotoxic reference compound. Stock solutions of compounds and essential oils were prepared in DMSO at 10 mg/mL. The solutions were further diluted in medium with final concentrations of 50-1.56 μg/mL (four wells/concentration). The highest concentration of solvent to which the cells were exposed was 1%, which was shown to be non-toxic. Each oil was tested in six serial twofold dilutions in 96-well microtitre plates. All experiments were made at least in duplicate. 2.9. Statistical analysis Student's t-test was used to test the significance of differences between results obtained for different samples, and between results for samples and controls (GraphPad Prism 4.0; GraphPad Software Inc., San Diego, USA). Statistical significance was set at Po 0.05. 3. Results and discussion 3.1. Chemical composition of the essential oils The oils extracted from fresh leaves of Cymbopogon citratus (I), Cymbopogon giganteus (II), Cymbopogon nardus (III) and Cymbopogon schoenantus (IV) collected in the same place and in the same time, were obtained with different yields (w/w) (Table 1). This may be explained by the difference between species. Moreover, these yields were less than those described by Nonviho et al. (2010) on dried leaves of three of these plants (I, II and IV), but they were calculated on the dry material (Bourkhiss et al., 2009; Nebie et al., 2011). A total of 29 (I), 53 (II), 28 (III) and 41 (IV) compounds, representing respectively 98.1% (I), 98.6% (II), 98.5% (III) and 98.6% (IV) of the hydrodistillate, were identified (Table 1). The oils were characterised by two major chemical groups: monoterpenes and sesquiterpenes, with a high amount of oxygenated monoterpenes in all studied oils (Table 2) followed by hydrocarbon monoterpenes in IV, I and II; and by oxygenated sesquiterpenes in III (Table 2). Almost all constituents of I and II were monoterpenes while III and IV contained monoterpenes with sesquiterpenes (Table 2). Non terpenic compounds were mainly identified in II, where 4,4-dimethyl androst-5en-3-one was the major component of this class (Table 1). The sample I (29 compounds, 98.1%) obtained from Cymbopogon citratus 655 S. Kpoviessi et al. / Journal of Ethnopharmacology 151 (2014) 652–659 Table 1 Chemical composition and yield of essential oils from Cymbopogon species (mean 7standard deviation, n¼ 3). No. Compoundsa KIb I II III IV 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 α-Pinenec,f,i Camphenec,f,i β-Myrcenec,f,i β-Pinenec,f,i ( þ )-2-Carenec,f,i α-Phellandrenec,f,i p-Cymenec,f,i Limonenec,f,i (Z)-β-ocimenec,f,i (E)-β-ocimenec,f,i Melonalc,g α-Terpinolenec,f,i Thujolc,g,i Fenchone-Dc,g Myrcenolc,g,i β-Linaloolc,g,i trans-3(10)-caren-2-olc,g p-Mentha-1,3,8-trienolc,g trans-p-mentha-2,8-dienolc,g cis-p-mentha-2,8-dienolc,g Isopulegolc,g 4-Isopropylidene-cyclohexanole,g α-Phellandren-8-olc,g,i trans-2-caren-4-olc,g,i cis-α-terpineolc,g,i trans-p-mentha-1(7),8-dien-2-olc,g β-Citronellalc,g,i cis-verbenolc,g trans-carane, 4,5-epoxy-c,g cis-p-mentha-1(7),8-dien-2-olc,g trans-piperitolc,g,i p-Menth-1-en-9-alc,g cis-carveolc,g,i 7-Methyl-3-methylene-6-octen-1-ole,g β-Citronellolc,g,i trans-carveolc,g cis-carvonec,g,i Neralc,g,i Isoamyl hexanoatee,g trans-3-caren-2-olc,g cis-geraniolc,g,i Perillalc,g Nerolc,g,i Piperitonec,g,i p-Mentha-1(7),8(10)-dien-9-olc,g 1-Methyl-2-decalonec,g Limonene dioxidec,g 2-Caren-10-alc,g Geranialc,g,i Piperitone oxidec,g Exo-2-hydroxycineole acetatec,g Nopolc,g β-Bourbonened,f Geranyl acetatec,g β-Elemened,f,i 2-Undecanoned,g 3-Oxo-α-ionold,g β-Caryophyllened,f,i Neric acidc,g Isoamyl caprylated,g α-Humulened,f,i β-Cubebened,f Geranic acidc,g α-Himachalened,f Germacrene-Dd,f,i β-Eudesmened,f,i τ-Gurjunened,f α-Muurolened,f Seychellened,f τ-Muurolened,f α-Bergamotened,f δ-Cadinened,f,i Elemold,g,i Geranyl butyratec,g Cubenold,g,i 949 963 993 996 1005 1017 1023 1028 1032 1042 1045 1055 1070 1090 1092 1101 1110 1111 1120 1133 1142 1146 1161 1171 1173 1181 1192 1199 1201 1206 1211 1215 1227 1230 1244 1246 1267 1268 1278 1288 1291 1292 1294 1296 1298 1304 1315 1320 1328 1331 1335 1338 1340 1344 1353 1368 1392 1394 1423 1427 1443 1454 1467 1470 1477 1483 1493 1499 1505 1514 1521 1523 1556 1568 1579 t – – 10.1 70.04 – – 0.5 70.00 – 0.4 70.00 0.2 70.00 – 0.2 70.00 – – 0.4 70.00 0.9 70.00 0.17 0.00 – – 0.17 0.00 – – 0.5 70.00 – – – 0.4 70.00 1.77 0.01 – – – – – – 0.4 70.00 – – 35.57 0.15 – – 4.3 70.02 – – – 0.17 0.00 – – – 39.5 70.00 – – 0.4 70.00 0.5 70.00 1.07 0.00 – 0.17 0.00 – 0.2 70.00 0.17 0.00 – – – 0.17 0.00 – – – 0.17 0.00 – – – 0.17 0.00 – – – – – – t – 0.47 0.00 – – 8.37 0.08 – – t 0.1 70.00 0.1 70.00 0.37 0.00 – 0.1 70.00 – 0.27 0.00 15.5 7 0.15 11.3 7 0.03 – 0.1 70.00 0.87 0.01 – t 18.3 70.17 t – 1.57 0.01 8.97 0.08 – 0.27 0.00 7.37 0.07 – – 17.47 0.16 3.47 0.03 – 0.37 0.00 0.27 0.00 – 0.57 0.01 – 0.1 70.00 0.1 70.01 0.1 70.00 0.1 70.00 – – 0.1 70.00 0.1 70.00 t – 0.1 70.00 t – 0.1 70.03 t – 0.27 0.00 – – – t – t – t – t – – – – – – – 0.27 0.00 – – – – 2.27 0.02 – – 0.27 0.00 – – – – 0.47 0.00 – – – – 0.27 0.00 – – – – – 35.9 7 0.34 – – – – – – – 11.6 7 0.11 – – 0.47 0.00 – – – – 24.3 7 0.23 – – – – – 0.67 0.01 – – – – 1.37 0.01 1.97 0.02 – – 0.1 70.00 – – 0.1 70.00 0.1 70.00 – – 1.57 0.01 – 0.27 0.00 0.27 0.00 0.27 0.00 0.47 0.00 – 1.17 0.01 9.07 0.08 – 1.87 0.02 0.1 70.00 0.1 70.00 0.27 0.00 – 13.0 7 0.20 0.27 0.00 – 6.47 0.10 – 0.77 0.01 – 0.27 0.00 – 0.37 0.01 – – – – 1.8 7 0.03 1.3 7 0.02 – – 0.47 0.01 0.1 70.00 – 0.37 0.00 – – – – 0.57 0.01 – – 0.27 0.00 – – – – – – – – – 60.37 0.92 – – – 0.1 70.00 – – – – 0.1 70.00 – 0.47 0.01 – – 0.87 0.01 – – 0.1 70.00 – – 0.1 70.00 0.1 70.00 0.1 70.00 0.1 70.00 t 0.1 70.00 0.1 70.00 – 0.27 0.00 4.97 0.08 0.47 0.01 t 656 S. Kpoviessi et al. / Journal of Ethnopharmacology 151 (2014) 652–659 Table 1 (continued ) No. Compoundsa KIb I II III IV 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 β-Caryophyllene oxided,g Hedycaryold,g Ledold,g,i Eudesm-7(11)-en-4-old,g Phenylethyl caproatee,g Guaiold,g τ-Eudesmold,g τ-Cadinold,g,i β-Eudesmold,g,i α-Cadinold,g,i Isoaromadendrene epoxided,g (Z,E)-farnesold,g Geranyl caproatec,g ( )-Spathulenold,g Ledene alcohold,g Phenylethyl octanoatee,g 4,4-Dimethylandrost-5-en-3-onee,g Bolasteronee,g Norethindronee,g 1585 1610 1615 1617 1619 1620 1630 1639 1648 1650 1661 1699 1748 1779 1809 1888 2184 2209 2336 0.17 0.00 – – 0.17 0.00 – – – – – – – – – – – – – – – 0.1 70.00 – – – 0.1 70.00 – – – t – t t 0.1 70.03 t t 0.1 70.01 1.7 7 0.02 0.27 0.00 0.1 70.04 – – 0.6 7 0.01 – – – 0.6 7 0.01 1.17 0.01 – 2.0 7 0.02 – 0.2 7 0.00 – – – – – – – 0.4 7 0.01 t – – – 0.17 0.00 1.17 0.02 0.3 7 0.00 3.17 0.05 – 0.17 0.00 0.17 0.00 – – – – – – – Total identified 98.17 0.41 98.67 0.97 98.5 7 0.92 98.6 7 0.52 h 0.717 0.02 0.657 0.02 1.067 0.10 1.88 7 0.12 Yield (%) a Compounds listed in order of elution from HP-5 MS column. Kovats indices (KI) on HP-5 MS column; I ¼Essential oil from Cymbopogon citratus; II ¼essential oil from Cymbopogon giganteus; III¼ essential oil from Cymbopogon nardus; IV ¼essential oil from Cymbopogon schoenantus; t ¼traces (inferior or equal to 0.05%); and ( ) ¼ absence or not detected. c Monoterpenes. d Sesquiterpenes. e Non-terpenes. f Hydrocarbons. g Oxygenetad. h Yield calculated based on the fresh plant material. i Comparison of data with reference standard. b Table 2 Chemical groups in essential oils from Cymbopogon species (mean 7standard deviation, n¼3). No. Chemical groups I II III IV 1 2 3 4 5 6 7 Hydrocarbon monoterpenes Oxygenated monoterpenes Monoterpenes Hydrocarbon sesquiterpenes Oxygenated sesquiterpenes Sesquiterpenes Others 11.4 7 0.04 85.5 7 0.35 96.97 0.39 0.4 7 0.00 0.2 7 0.00 0.67 0.00 0.67 0.04 8.8 70.08 86.7 70.73 95.57 0.81 t 0.3 70.06 0.37 0.06 2.87 0.07 2.4 70.02 74.9 7 0.7 77.37 0.09 5.8 70.04 15.3 7 0.15 21.1 70.19 – 20.9 7 0.31 65.5 7 1.01 86.47 1.32 2.2 7 0.02 10.0 7 0.16 12.27 0.18 0.37 0.00 I¼ Essential oil of Cymbopogon citratus, II¼ essential oil of Cymbopogon giganteus, III¼essential oil of Cymbopogon nardus, IV¼ essential oil of Cymbopogon schoenantus, ( absence or not detected, and t¼ traces (inferior or equal to 0.05%). was characterised by the presence as main constituents of geranial, neral, β-pinene, cis-geraniol, cis-verbenol and geranyl acetate (Table 1). The mixture of geranial and neral, two geometrical isomers constituting of citral, accounts for about 75% of the total of oil I (Table 1). Sakirigui et al. (2011) obtained 70.13% of citral, Nonviho et al. (2010) 74% and Blanco et al. (2009) 77% in Cymbopogon citratus oil. The sample II (53 compounds, 98.6%) obtained from Cymbopogon giganteus was characterised by the presence as major constituents of trans-p-mentha-1(7),8-dien-2-ol, trans-carveol, trans-p-mentha-2,8dienol, cis-p-mentha-2,8-dienol, cis-p-mentha-1(7),8-dien-2-ol, limonene, cis-carveol, cis-carvone, 4,4-dimethylandrost-5-en-3-one and trans-carane-4,5-epoxy (Table 1). Its composition, except for limonene, approached that found in leaves from Togo (Nyamador et al., 2010). The sample III (28 compounds, 98.5%) obtained from Cymbopogon nadus was characterised by a high concentration of βcitronellal, nerol, β-citronellol, elemol, limonene, α-cadinol, β-elemene, cubenol, germacrene-D, geranyl acetate, δ-cadinene and τcadinol (Table 1). These results seem similar to those of Oliveira et al. )¼ (2011) that identified in the Brazilian species 34.61% of citronellal followed by 23.18% of geraniol and 12.10% of citronellol. The sample IV (41 compounds, 98.6%) obtained from Cymbopogon schoenantus showed the presence as main constituents of piperitone, (þ)-2carene, limonene, elemol, β-eudesmol, trans-p-mentha-2,8-dienol, cis-p-mentha-2,8-dienol and τ-eudesmol (Table 1). This composition was different from that described by Nonviho et al. (2010) which found 68% of piperitone in a sample from Akogbato (Benin) but was close to the results reported by Ayedoun et al. (1997) in a sample from Bassila (Benin). The major components represented over 90% of the studied oils. The concentrations of all the other constituents were less than 1.2%. Each oil was thus characterised by known compounds with percentages sometimes different from those described in the literature (Nonviho et al., 2010; Abena et al., 2007, Alitonou et al., 2006; Sidibé et al., 2001; Shahi and Tava, 1993). This variation can be due to the influence of the moment or the place of harvest in the chemical composition of these oils (Singh et al., 1994; Boruah et al., 1995; Kpoviessi et al., 2011; Kpadonou Kpoviessi et al., 2012). 657 S. Kpoviessi et al. / Journal of Ethnopharmacology 151 (2014) 652–659 Table 3 In vitro antitrypanosomal and antiplasmodial activity, cytotoxicity and selectivity index of essential oils from Cymbopogon species and some of their major components. Samples I II III IV Myrcene R( þ )-limonene Citral Citronellal Nerol β-Citronellol β-Pinene 6-Acetoxy-pmentha-1,8diene p-Cymene Campthotecin Suramine Chloroquine Artemisinin Cytotoxicity (IC50, μg/ mL) average7 standard deviation Antitrypanosomal activity Tbb (IC50, μg/mL) average7 standard deviation Antiplasmodial activity Pf (IC50, μg/mL) average7 standard deviation CHO WI38 10.63 7 0.72 450 450 450 450 450 20.62 7 1.59 450 450 450 450 450 39.777 3.31 4 50 4 50 4 50 4 50 4 50 39.487 1.59 4 50 4 50 4 50 4 50 4 50 1.83 7 0.13b 0.25 70.11a 5.717 1.40c 2.107 0.89b 2.247 0.27b 4.247 2.27c 5.98 70.54c 2.76 7 1.55b 4100 6.45 74..86c 47.377 15.65e 28.82 72.91d 47.977 13.09c 11.22 7 5.35b 52.61 74.79c 43.15 713.19c nd nd nd nd nd nd nd nd 450 0.747 0.09 nd nd nd 4 50 0.44 70.12 nd nd nd 76.32 7 13.27f nd 0.117 0.02a nd nd nd nd 0.02 7 0.01a 0.01 70.001a τ Selectivity indices WI38/ Tbb WI38/ 3D7 3D7/ Tbb 21.73 4200 48.76 423.81 411.76 48.94 4.01 47.41 o0.5 41.53 41.06 44.64 0.93 44.46 40.95 41.16 26.21 3.14 9.21 20.55 I ¼Essential oil from Cymbopogon citratus, II ¼ essential oil from Cymbopogon giganteus, III¼ essential oil from Cymbopogon nardus, IV¼ essential oil from Cymbopogon schoenantus, WI38 ¼human normal fibroblast cells, CHO ¼ Chinese Hamster Ovary cells, nd¼ not determined, Tbb ¼ Trypanosoma brucei brucei, 3D7 ¼chloroquine-sensitive strain of Plasmodium falciparum, and IC50 ¼ sample concentration providing 50% death of cells or parasites. τSelectivity index ¼IC50 (WI38)/IC50 (Tbb or 3D7). Data in the same column followed by different letters (a,b,c,…) are statistically different by Student's t-test (Po 0.05). Values are means7 standard deviation of three separate experiments. 3.2. Antitrypanosomal, antiplasmodial activities and cytotoxicity All the studied oils were tested in vitro for their antitrypanosomal and antiplasmodial activities respectively on Trypanosoma brucei brucei and Plasmodium falciparum 3D7 and their cytotoxicity against WI38 and CHO cells. Results are summarised in Table 3. All oils showed a stronger effect against Trypanosoma brucei brucei with IC50 values r6 μg/mL. These oils were in increased order of activity. Cymbopogon nardus (III, IC50 ¼ 5.71 71.40 μg/mL), Cymbopogon schoenantus (IV, IC50 ¼2.10 70.89 μg/mL), Cymbopogon citratus (I, IC50 ¼1.83 7 0.13 μg/mL) and Cymbopogon giganteus (II, IC50 ¼0.25 7 0.11 μg/mL). The student's t-test showed a highly significant difference between the activity of II and the other tested oils. However the activity of the essential oil from Cymbopogon giganteus (II) was not significantly different (P value 4 0.1) to that of suramine (IC50 ¼0.11 70.02 μg/mL) the standard compound used against this parasite. The selectivity index of the four tested oils (I ¼21.73; II 4200; III48.75 and IV4 23.81) showed that II was also the most selective. In vivo studies should be performed to assess its efficacy on sleeping sickness and determine if the essential oil from Cymbopogon giganteus (II), already used extensively in traditional medicine, can be recommended for the treatment of this illness. It will necessary to search for adequate formulation as LBDDS (lipid based drug delivery systems) (Mu et al., 2013) and to verify the absence of toxicity. Recently, Muhd Haffiz et al. (2013) reported a different IC50 value (0.31 70.03 μg/mL) for the essential oil of Cymbopogon nardus from Malaysia, tested against the BS221 strain of Trypanosoma brucei brucei. This difference on IC50 value may be due to differences in the origin and the composition of the essential oil and in the strain of Trypanosoma brucei brucei. The antitrypanosomal activity of available major compounds of these studied oils was also evaluated. β-Myrcene (IC50 ¼2.24 μg/mL), citronellal (IC50 ¼2.76 μg/mL), R(þ)-limonene (IC50 ¼ 4.24 μg/mL), citral (IC50 ¼5.98 μg/mL) and β-citronellol (IC50 ¼6.45 μg/mL) showed antitrypanosomal IC50 values near to those of studied oils and can in part explain their activities. The antitrypanosomal IC50 values of the other tested major components: 6-acetoxy-p-mentha-1,8diene (IC50 ¼ 28.82 μg/mL), β-pinene (IC50 ¼47.37 μg/mL) and pcymene (IC50 ¼ 76.32 μg/mL); and those of (7)-linalool (39.32 μg/ mL), β-caryophylene (13.78 μg/mL), 1,8-cineole (83.15 μg/mL), ( )-carvone (12.94 μg/mL), piperitone (41.12 μg/mL), ( )-verbenone (30.24 μg/mL), limonene epoxide (22.58 μg/mL) and caryophyllene oxide (17.70 μg/mL) (Hoet et al., 2006; Nibret and Wink, 2010), were higher than 10 μg/mL and could not explain the strong activity of these oils but synergic effect is possible. Furthermore, most of the major constituents of II were not available and could not be tested. Even though sesquiterpenes as artemisinin are good antiparasitic agents, we note here that the two most active antitrypanosomal oils only contain trace amounts of sesquiterpenes and are particularly rich in oxygenated monoterpenes (Table 2). Two of them: trans-p-mentha-1(7),8-dien-2-ol and trans-carveol are major compounds of II, possessing the best activities and should be tested to elucidate their contribution to the antitrypanosomal activity of II. Concerning the antiplasmodial activity against the chloroquinosensitive strain of Plasmodium falciparum (3D7), we observed that Cymbopogon giganteus essential oil (II) was the only one which could be considered as having an interesting activity with an IC50 value r20 μg/mL, the other oils had a moderate activity (IC50 values between 21 and 60 μg/mL). With a selectivity index 44.46, the essential oil Cymbopogon giganteus (II) can also be a good candidate for bio-guided fractionation to yield a more active fraction against Plasmodium falciparum. It would also be interesting to test trans-pmentha-1(7),8-dien-2-ol and trans-carveol, the major compounds of this oil, on Plasmodium. Moreover these results showed the selectivity of the activity of the studied oils on Trypanosoma brucei brucei as compared to Plasmodium falciparum (SI43 for all studied oils). The cytotoxicity tests against the Chinese Hamster Ovary (CHO) cells and the human non cancer fibroblast cell line (WI38) showed that all tested oils and components had a low cytotoxicity (IC50 450 μg/mL) (Table 2). The only exception was Cymbopogon citratus essential oil (I) which was toxic against CHO cells (IC50 ¼10.63 μg/mL) and moderately toxic against WI38 cells 658 S. Kpoviessi et al. / Journal of Ethnopharmacology 151 (2014) 652–659 (IC50 ¼39.77 μg/mL). The major component of this oil (citral¼neralþgeranial ¼74%) was also toxic against CHO cells (IC50 ¼20.62 μg/mL) and moderately toxic against WI38 cells (IC50 ¼39.48 μg/mL). The second major component (β-pinene¼ 10.10%) was not toxic against these cells (IC50 450 μg/mL). 4. Conclusions Our study shows that the essential oils of Cymbopogon citratus (I), Cymbopogon giganteus (II), Cymbopogon nardus (III) and Cymbopogon schoenantus (IV) from Benin were more active on Trypanosoma brucei brucei than on Plasmodium falciparum (3D7). The essential oil of Cymbopogon giganteus (II) already used extensively in traditional medicine is the most active and could be interesting for the treatment of sleeping sickness but may also have some interest on Plasmodium. These oils had a low cytotoxicity except Cymbopogon citratus essential oil (I) which was toxic against CHO cells and moderately toxic against WI38 cells. Its major component (citral¼neralþgeranial) was also toxic against CHO cells and moderately toxic against WI38 cells. Cymbopogon citratus already largely used in folk medicine and cooking should need further research on its toxicity and the population sensitised about it. 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