In vitro antitrypanosomal and antiplasmodial activities of crude extracts and essential oils of Ocimum gratissimum Linn from Benin and influence of vegetative stage.

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Ocimum gratissimum Linn are largely used in folk medicine for the treatment of many diseases, some of which related to parasitical infections as fevers and headaches. In order to validate their use and to clarify the plant part which possesses the best antiparasitic properties, we decided to evaluate the in vitro antiplasmodial and antitrypanosomal activities of essential oils and crude extracts from leaves, stems and seeds of Ocimum gratissimum as well as their cytotoxicity. MATERIALS AND METHODS: The essential oils and ethanol crude extracts of leaves and stems of Ocimum gratissimum from Benin, were obtained in pre and full flowering stages. Seeds obtained only in full flowering stage, were also extracted. The oils were isolated by hydrodistillation and analyzed by GC/MS and GC/FID. Extracts and essential oils were tested in vitro against Trypanosoma brucei brucei and Plasmodium falciparum. 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 and toxicity was assessed against Artemia salina Leach. RESULTS: The essential oils and non-volatile crude extracts of Ocimum gratissimum were more active on Trypanosoma brucei brucei than on Plasmodium falciparum (3D7). This activity varies according to the vegetative stage (pre and full flowering) and the plant part (seeds, stems and leaves) extracted. The best growth inhibition of Trypanosoma brucei brucei was observed with ethanol crude extracts of leaves (IC50=1.66 ± 0.48 µg/mL) and seeds (IC50=1.29 ± 0.42 µg/mL) in full flowering stage with good selectivity (SI>10). The chemical composition of the essential oil from aerial parts (47 compounds), characterized by the presence as main constituents of p-cymene, thymol, γ-terpinene, ß-myrcene and α-thujene, depends on the vegetative stage. The oil contained some minor compounds such as myrcene (IC50=2.24 ± 0.27µg/mL), citronellal (IC50=2.76 ± 1.55µg/mL), limonene (IC50=4.24 ± 2.27µg/mL), with good antitrypanosomal activities. These oils and crude extracts were not toxic against Artemia salina Leach and had a low cytotoxicity except leaves and seeds ethanol extracts obtained in full flowering which showed toxicity against CHO and WI38 cells. CONCLUSIONS: Our study shows that ethanol crude extracts of leaves and seeds of Ocimum gratissimum in full flowering stage can be a good source of antitrypanosomal agents. This is the first report about the relation between the plant part extracted, the vegetative stage of the plant, the antitrypanosomal and antiplasmodial activities and the cytotoxicity of essential oils and non-volatile extracts of Ocimum gratissimum from Benin.

Chemical composition, cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin.

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, ß-pinene and cis-geraniol; sample II (53 compounds) by trans-p-mentha-1(7),8-dien-2-ol, trans-carveol, trans-p-mentha-2,8-dienol, cis-p-mentha-2,8-dienol, cis-p-mentha-1(7),8-dien-2-ol, limonene, cis-carveol and cis-carvone; sample III (28 compounds) by ß-citronellal, nerol, ß-citronellol, elemol and limonene and sample IV (41 compounds) by piperitone, (+)-2-carene, limonene, elemol and ß-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.

Chemical variation of essential oil constituents of Ocimum gratissimum L. from Benin, and impact on antimicrobial properties and toxicity against Artemia salina leach.

To determine the period of harvest that optimizes the antimicrobial activities of the essential oil of Ocimum gratissimum L. from Benin, aerial plant parts were collected at two vegetative stages (pre- and full-flowering) and three sampling times (7 am, 1 pm, and 7 pm). Extraction by hydrodistillation yielded between 0.65 and 0.78% of essential oils. Characterization of the oils by GC-FID and GC/MS analysis revealed the presence of monoterpenes (87.26-93.81%), sesquiterpenes (5.57-11.34%), and aliphatic compounds (0.15-0.18%), with p-cymene (1; 28.08-53.82%), thymol (2; 3.32-29.13%), γ-terpinene (3; 1.11-10.91%), α-thujene (4; 3.37-10.77%), and ß-myrcene (5; 4.24-8.28%) as major components. Two chemotypes were observed, i.e., a p-cymene/thymol and a p-cymene chemotype, for plants harvested at 7 am for the former and at 1 pm or 7 pm for the latter, respectively. The oils were fungicidal against Candida albicans, with the sample from full-flowering plants collected at 7 am being the most active (MIC = 0.06±0.00 mg/ml). The chemical variation of the oils also influenced the antimicrobial effect against Staphylococcus aureus; the most active oil was obtained from plants at the pre-flowering stage collected at 7 am (MIC=0.24±0.01 mg/ml). Escherichia coli was insensitive to the chemical variation of the oils (MICs of ca. 0.48±0.02 mg/ml for all oils). Moreover, the essential oils showed low toxicity against Artemia salina Leach larvae, with LC(50) values in the range of 43-146 µg/ml. This is the first study of the interaction between the daytime of collection and vegetative stage of the plants and the antimicrobial properties and toxicity of the essential oil of O. gratissimum from Benin.