Chemical composition and antimicrobial properties of different basil essential oils chemotypes from Togo

Koffi Koba1, P.W. Poutouli2, Christine Raynaud3, Jean-Pierre Chaumont4 and Komla Sanda1 Unité de Recherche sur les Agroressources et la Santé Environnementale, Ecole Supérieure d’Agronomie, Université de Lomé, BP. 1515, Lomé Togo; Département de Biologie Animale et de zoologie, Faculté des Sciences, Université de Lomé, B.P. 1515 Lomé Togo; Laboratoire de Chimie Agro-Industrielle, UMR 1010, INRA/INPENSIACET, 118, route de Narbonne, 31077 Toulouse cedex, France; Equipe des Sciences Séparatives et Biopharmaceutiques EA 3924, Université de Franche-Comté, Faculté de Médecine et de Pharmacie, Place SaintJacques, 25030, Besançon, Cedex France.


Introduction
The genus Ocimum collectively called basil (in English), includes around 30 plant species from tropical and subtropical areas (Paton, 1992).Ocimum is widely cultivated and extensively used for food, perfumery, cosmetics, pesticides, medicine, and traditional rituals because of their natural aroma and flavor and other properties (Alburquerque, 1996;Darrah, 1974).Literature reports that O. basilicum leaf essential oils or leaf powder have effective insecticidal and pesticidal actvities against Vigna unguiculata pests Callosobruchus maculatus (F) (Coleoptera: Bruchidae) (Keita et al., 2001;Ketoh et al., 2002).O. basilicum commonly grows semiwild and is cultivated in Togo at small scale in vegetable gardens.Current domestic uses are only for food and folk medicine (Adjanohoun et al., 1986).
Nevertheless, a large scale production of the basil essential for well-known value-added applications (Berrada et al., 1987;Archtander, 1994) is quite viable under local circumstances.
The chemical composition of O. basilicum essential oils has been intensively investigated throughout the world (Ekundayo et al., 1987;Sanda et al., 1998;Yayi et al., 2001), indicating that the estragole chemotype and the linalool -estragole one are the most widely distributed.
At the same time, only very little work has been done on the chemical composition and the antimicrobial activities of basil oils from plants growing in Togo (Sanda et al., 1998;Koumaglo et al., 1996).In addition, there are very few data available either for practical use or for basic research needs about antimicrobial properties of the essential oils of O. basilicum growing in Togo (Baba-Moussa et al., 1997;Chaumont et al., 2001).
In order to help fill this deficiency, partly at least, the aim of the present work was to investigate basil oils extract of plants sampled in different localities belonging to different ecological areas of Togo.Specifically, oils percentage composition was determined along with their bacteriostatic and fungistatic activity using 14 bacterial and 14 fungal strains.
The principal finding expected from this applied research is the candidate basil oil(s) that could be produced in Togo for use as source(s) of active ingredients in natural antibiotics or cosmetics.

Plant material sampling and volatile oils isolation
Leaves and inflorescences of O. basilicum used in this work were harvested from plants at full flowering stage in vegetable gardens in five various locations of Togo from May to October 2007.
Plant specimen was identified by Prof. Akpagana, Departement de Botanique, Faculté des Sciences at the Université de Lomé (Togo), where voucher specimens were deposited in the herbarium.
A sample (50 g) of air-dried plant material was extracted by the hydrodistillation technique during 2 hours in a modified Clevenger-type glass apparatus (Craveiro et al., 1976).The extracted crude essential oils were stored in hermetically sealed dark glass flasks with rubber lids, covered with aluminium foil to protect the contents from light and kept under refrigeration at 4°C until use without any prior purification.

Essential oil analyses by gas chromatography
Gas chromatographic analysis was carried out on a Varian 3300 type gas chromatograph equipped with FID detector.An apolar capillary column DB-5 (30 m x 0.25 mm i.d.; film thickness 0.25 µm) and on a polar column supelcowax 10 with the same characteristics as above mentioned were used.DB-5 column operating conditions were as follows: From 50°C (5 min), 50 to 250°C at the rate of 2°C/min and supelcowax 10 from 50°C (5 min), 50 to 200°C at 2°C/min.The injector and detector temperatures were respectively 250 and 300°C.The carrier gas was helium at a flow rate of 1.5 mL/ min.Samples (0.2 µL) of undiluted essential oil were injected manually.

Gas chromatography-mass spectrometry analysis
The GC/MS analysis was carried out on a Hewlett Packard 5890 series II chromatograph, coupled with a mass spectrometer of the Hewlett Packard 5971 series type operating in the EI mode at 70 eV.The capillary column type was DB5-MS (30 m x 0.25 mm i.d.; film thickness 0.25 µm).The amount of sample injected and GC/MS parameters were the same as above.

Identification of components
The components of oils samples were identified by their retention time, retention indices relative to C8-C24 n-alkanes, computer matching with Wiley 275L library and as well as by comparison of their mass spectra with the authentic samples or with data already available in the literature (Kondjoyan and Berdagué, 1996;Adams, 2001).
The percentage of composition of the identified compounds was computed from the GC peak area without any correction factor and was calculated relatively.

In vitro antifungal testing
Fungal strains used are listed in Table I.They were supplied by Institut Pasteur de Paris and Hôpital Saint Jacques de Besançon, France.The fungi were cultivated on a sabouraud agar medium in which was added chloramphenicol 1%, all purchased from BioMerieux Co. (Paris, France).Pure estragole, linalool and methyleugenol commercial standards were also purchased from BioMerieux Co. (Paris, France).
The antimicrobial activities of the essential oils were assessed according to agar dilution method (Benjilali et al., 1986;Griffin et al., 2000).The tested essential oil and its pure major components from commercial origin were diluted in a minimal quantity of ethanol 95% 1/10 v/v to which was added an aqueous solution of Tween 80 (final concentration of 1% v/v) in order to obtain a homogeneous mixture.The later was incorporated as appropriate to the microbiological culture medium under solidification to obtain final concentrations of the active ingredient that ranged from 10 to 500 µL/L.The mixture was then poured into 3 cm diameter petri dishes.
After solidification fungal strains were respectively seeded as described below: (i) dermatophytes were seeded with a disc of approximately 2 mm, from a mycelia carpet of preculture, laid in the middle area of a new petri dishes, upper side against the new culture medium; (ii) 1 mL of a suspension of 10 5 conidia per mL of Aspergillus fumigatus or 10 5 blastospores per mL of yeast was poured on the surface of the culture medium.Incubation time and temperatures depended on the fungal strains: 24 hours at 37°C for Candida albicans and Aspergillus, 48 hours at 37°C for Cryptococcus, 14 days at 24°C for the dermatophytes and Scopulariopsis brevicaulis.

Antibacterial testing
Bacterial strains were supplied by Institut Pasteur Collection de Paris and Leeds University Microbiology Laboratory, United Kingdom.They were cultivated on 1.3% (m/v) nutrient broth-agar-granulated (NB-AG) culture medium (AES Labortories), in 9 cm diameter petri dishes.After cooling and solidification, the petri dishes were automatically inoculated with the bacterial suspensions 10 5 CFU/mL with STEERS apparatus.The petri dishes were incubated at 37°C for 48 hours under aerobic conditions.
To our knowledge, the last three basil oil chemotypes have not yet been reported.O. basilicum has been thoroughly investigated with regards to volatile oil composition but some of our findings are supportive of the idea that continuing chemical inventory on this species is still of scientific interest to support action aimed at plant biodiversity knowledge, conservation and sustainable exploitation.
The findings on the chemical composition of basil oils in Togo were very instructive with regard to the possible applications.Hence, the estragole chemotype found here resembled that described by Ekundayo et al. (1987) and Baritaux et al. (1992), which is well-known for its valuable usage in perfumery and food (Kayibou, 1992).In addition, this chemotype has been recently indicated as a possible pesticide against larva and adult of Callosobruchus maculatus known as a major postharvest pest of Vigna unguiculata in storage (Keita et al., 2001;Ketoh et al., 2002).
The pesticidal activity of the volatile oil of Clausena anisata, containing mainly estragole and t-anethole (Moudachirou et al., 1997;Okunde and Olaifa, 1987).Such indications on the pesticidal properties of this essential oil containing estragole and t-anethole is certainly indicative of the biocide potential of these natural molecules which might be endowed with antimicrobial properties (Friedman et al., 2002), our focus in this investigation.(Alvarez et al., 2000).The African Trichophyton soudanense, a parasite frequent in school environment (Ouaffak et al., 2001;Vandemeulebroucke et al., 1999) was interestingly also sensitive to the tested essential oils (MICs 100 and 200 µL/L).It was also the case for Cryptococcus neoformans, a hazardous opportunist yeast, which is a resistant germ usually infecting humans affected by HIV/AIDS, which group of patients is known to be generally at high-risk with regard to mycosis opportune affections.
The high antifungal activity of this basil oil chemotype on those pathogenic fungi like dermatophytes, filamentous fungi and yeasts confirmed the excellent fungal growth inhibition properties previously reported as a characteristic of essential oils rich in methyleugenol and/or other phenol derivatives (Chaumont and Leger, 1989).In this work there is no doubt that the antifungal activity of O. basilicum chemotype methyleugenol against tested fungi is predictable consequence of its high content in methyleugenol known as one of the phenolic volatile molecules endowed with antimicrobial properties (Chaumont and Leger, 1989;Viollon and Chaumont, 1994).In comparison, commercial linalool and estragole tested as standards in this study were found noneffective unlike pure methyleugenol standard.
The methyleugenol/t-anethole chemotype showed a moderate antifungal activity only on dermatophytes with MICs ranged from 250-300 µL/L but not filamentous fungi and yeasts were not affected.The estragole type, the linalool/estragole type, and the tanethole type were ineffective on all fungal strain tested in this work.A lesson learned from the findings mentioned above was that estragole and t-anethole, which are reported as possible natural pesticides, could not serve as natural fungicidal ingredients unlike methyleugenol against the fungal strains tested in this work.This simply indicates the broader understanding of what is generically termed to as the biological activity of essential oils.
Table III shows that all feet microflora bacterial strains tested were only sensitive to methyleugenol and methyleugenol/t-anethole oil chemotypes.
The MICs, which were in the range of 200-500 µL/L were slightly higher than those recorded on fungal strains in this study, indicating a lesser activity.The results recorded against common armpit microflora bacterial strains, were especially promising for a possible use against Corybacterium xerosis, the principal one of the bacteria responsible for bad odors.Its growth was interestingly inhibited with at a dose of 100 µL/L antibacterial test suspension.Note that this bacteria metabolizes human steroids produced via transpiration to yield small and bad-smelling volatile molecules (Rennie et al., 1991).
The findings in this study indicated bacteriostatic effects against feet microflora germs like Staphylococcus epidermidis and Staphylococcus hominis (MIC: 200 and 300 µL/L) partly responsible for feet bad odors (Marshall et al., 1987;Marshall et al., 1988).
The proven antibacterial activities of the methyleugenol and metyleugenol/t-anethole types of basil oil found in this study are quite indicative of their potential as possible active ingredients for use in the formulation of deodorants for armpits or feet.Developing sustainable large scale consumer goods like natural drugs and cosmetics based on basil oil as well as on other essential oils bearing aromatic plants growing in Togo is of an obvious economic interest while helping preserve plant biodiversity.
Culture media with or without ethanol 95% and Tween 80 were used as controls.
The antimicrobial activities were evaluated by the determination of the minimum inhibitory concentration (MIC).The MIC of a tested active ingredient was determined as the lowest concentration of the test antimicrobial ingredient sample that resulted in a complete inhibition of visible growth of the microorganisms.All tests were carried out in triplicate.

Results and Discussion
Basil oil extraction yields were in the range of 1.4% to 2.2%, based on plant material dry weight.Identified oil constituents and percentage and their relative The experimental data in Table II show that all fungal strains tested were sensitive to the metyleugenol type of basil oil, but dermatophytes and pathogenic yeasts were more particularly affected.Hence, markedly low MICs (80 µL/L) were recorded with T. mentagrophytes var mentagrophytes.Along with dermatophytes, percentages are listed in Table II.
Antibacterial activities are shown in Table III.

Conclusion
The prospective investigation of the percentage composition of O. basilicum essential oils along with the evaluation of their antimicrobial properties on human normal cutaneous microflora bacterial and fungal strains is quite a typical applied research.The ultimate goal of this type of investigation is to help protect plant biodiversity and validate on a scientific basis the potential of some valuable species whether for food or non-food applications.The present study has shown that it is feasible to use some kinds of the leaf essential oil of O. basilicum growing in Togo as a natural powerful antimicrobial ingredient whether in traditional or in modern medicines as well as in cosmetics.Such oils are the methyleugenol and the methyleugenol/t-anethole types.Conversely, the estragole type, the linalool/estragole type, and the tanethole type, which were also included in the investigated samples, did show any useful antimicrobial properties.