The family Lamiaceae of which the genus Ocimum belongs is composed of diverse and rich source of essential oil containing plants. Ocimum contains between fifty to one hundred and fifty species of herbs and shrubs from the tropical regions of Asia, Africa, and Central and South America [1]. O. gratissimum L. is referred to as holy basil. In Sierra Leone, it is known as “Tea bush” (Krio) and “orgbethor” (Themne) while it is known as “efirin nla” in the Yoruba speaking culture of Nigeria. O. basilicum L. is also referred to as sweet basil. It is known as “Patmenji” (Krio) and “Sorow” (Themne) of Sierra Leone and “efinrin wewe” by the Yorubas. O. americanum L. (synonym of O. canum Sims) [2,3] is known as American basil or hoary basil. In Sierra Leone both the available varieties of O. americanum L and O. basilicum L. are used in culinary as “Patmengy” indiscriminately. The different varieties of O. gratissimum L. [implying different chemotypes] have been used extensively in the traditional system of medicine in many countries as had been reviewed [4-10]. The main issues of concern with the use of herbal drugs remain safety, validation of claims and standardisation of product. There exist the problems of significant variation in the content of Ocimum plants across and within species, with implication of varied biological activities. In spite of the popular use of the genus Ocimum in food and confectionery as spices and application in the treatment of gastrointestinal infections and conjunctivitis in Sierra Leone, the varieties of the plant growing in Sierra Leone have not been, hitherto, investigated. The present study therefore determined the essential oil constituents of the Ocimum species collected in Freetown, Sierra Leone compared with varieties growing in Ile-Ife, Nigeria in order to establish the specific chemotypes in the specific regions.

Plant materials

Aerial parts of cultivated O. basilicum (OBS), O. gratissimum (OGS) and (OAS) at full flowering stage were collected from Krootown Road, Central Freetown, Sierra Leone while the Nigerian varieties O. basilicum (OBN), O. gratissimum (OGN) and O. americanum (OAN) were collected in Ile-Ife, Osun- State, Nigeria. The plants were identified at the Department of Botany, Faculty of Sciences, OAU, Ile-Ife, Nigeria where Voucher specimens were deposited.

Extraction of essential oils

Fresh or air-dried leaves of the plants were subjected to hydrodistillation using a Clevenger-type apparatus for 4 hours. The volatile oils were dried over anhydrous magnesium sulphate and stored in amber sealed vials at 4°C until analysis.

Analysis of the essential oils

The GC-MS analyses of the essential oils were carried out on Agilent 6890N coupled to a quadrupole MSD 5973. Column: DB-5MS (5% phenyl 95% dimethylarylene siloxane) 30 m × 0.25 mm × 1 mm film thickness (Agilent). Temperature program: from 50˚C (5 min) to 200˚C (10 min) at 2˚C/min. Injection temperature: 25˚C. Injection volume: 1.0 μL. Inlet pressure: 10.3 kPa PSI. Carrier gas: He, Linear velocity: 40 cm/sec. Injection mode: split (10:1). MS interface temp.: 230˚C; MS mode: EI at 70 eV; mass range: 30-400. The oil sample was dissolved in dichloromethane before injection.

The relative compositions of the essential oils were determined on Agilent 7890B coupled to FID and an autoinjector (Agilent G45138). Column: 19091J-413 HP-5 (5% phenyl methyl siloxane) 30 m × 0.32 mm × 0.25 mm film thickness (Agilent). Temperature program: from 50˚C (5 min) to 200˚C (10 min) at 2˚C/min. Injection temperature: 250˚C. Injection volume: 1.0 μL. Inlet pressure: 66.7 kPa. Carrier gas:He, Linear velocity: 40 cm/sec. Injection mode: split (50:1). FID temp.: 230˚C; H2 flow: 40 mL/min; air flow: 400 mL/min. The oil sample was dissolved in dichloromethane before injection.

Identification of the compounds

Compounds were identified by their retention indices determined using C8-C40 alkane standards and compared with literature data [11].

The identities were confirmed by the mass spectral data observed (Table 1) supported with library (National Institute of Standards and Technology).

Table 1: Observed Mass spectral features of the prominent components of the essential oils of Ocimum plants from Sierra Leone and Nigeria.

Antimicrobial Tests

Overnight broth cultures of the test organisms were diluted to give cell suspensions of 10/6 -10/7 CFU which were used to surfaceinoculate nutrient agar (Oxoid, London) (bacteria) and Sabouraud dextrose agar (Oxoid, London) (C. albicans). The antimicrobial activity tests of the oils were performed using paper disc method with Whatman No. 1 paper of size 6 mm which were each saturated with the test neat essential oil and subsequently placed on the surface of the inoculated plates [12]. The plates were kept at 4˚C for 1 h to allow for diffusion before subsequent incubation at 37˚C for 24 h for bacteria and 25˚C for 48 h for C. albicans. The reference antibacterial agents were Streptomycin (1 mg/ml) or acriflavine (1%). The tests were carried out in duplicate and the average zones of inhibition were determined.

The yields of the essential oils from the six plants are 0.34% (OAS), 0.98% (OAN), 0.71% (OBS), 0.96% (OBN), 0.93% (OGS) and 0.84% (OGN). The results of the antimicrobial test of the oils are presented below. The compounds identified in the different oil samples are presented in Table 2.

Table 2: The composition of essential oils of Ocimum species collected from Sierra Leone and Nigeria.

The chemotyping of Ocimum plants had been defined in terms of the combination of all major components constituting more than 20% rather than a dominant single component of the essential oil of the plant [13]. In this respect, both the Sierra Leone and Nigeria O. americanum L. leaf materials were thus identified as the linalool chemotype, constituting 49.1% and 39.6% respectively, in the present study. This chemotype of O. americanum L. is consistent with those previously reported in Benin [14], Rwanda [15], Cameroon [6] and Brazil [16]. The high content of linalool in some varieties of O. basilicum characterises the varieties as the best grade of commercial sweet basil for culinary use [17,18].

The Sierra Leone O. basilicum examined in the present study was clearly identified as the methyl eugenol chemotype (89.7%). This chemotype had been reported in Togo [19] and Turkey [20]. Methyl eugenol has been known in literature as a powerful insect attractant, [7,21-23], known to attract fruit flies from a distance as far as 0.8 km [7]. In the present study, attracted dead fruit insects (Drosophilia melanogaster) were found in the waste distillate (aqueous portion) drained into a bowl and left overnight. This observation underscores the potential use of methyl eugenol, or even this chemotype of O. basilicum co-formulated with an insecticide, as a powerful insecticidal product. On the other hand, the essential oil of the Nigerian O. basilicum variety was the methyl chavicol (89.8%) chemotype.

Both the Sierra Leone and Nigerian O. gratissimum L. varieties examined in this study are identified as the thymol chemotype having 60.5% and 42.2% thymol content respectively. This chemotype is widespread in West Africa (Nigeria [24], Cameroon [25], Togo [26] and Sao Tome [27]) although the presence of a Thymol-Cymene- Terpinene chemotype has been reported from the sub-region (Benin [14]). While O. gratissimum L. is the commercial source of eugenol in India [18,28], this component was not detected in the present material. The second most abundant constituent of Sierra Leone variety of L. is linalool (10.3%) which is a minor component in the Nigerian variety [0.4%] while the second most abundant constituent of the Nigerian variety is γ-terpinene (10.8%) which is only a minor component of the Sierra Leone variety (0.8%). Caryophyllene oxide constitutes a prominent sesquiterpene in both varieties of O. gratissimum L. The predominant thymol content probably explains the superior antibacterial activities of both O. gratissimum collections in the antimicrobial test (Table 3).

Table 3: The zones of inhibition (mm) of essential oils from Ocimum species collected from Sierra Leone and Nigeria (average of duplicate) compared with reference standards. Note: R=completely resistant, nt=not tested; OAN=O. americanum (Nigeria); OBS=O. basilicum (Sierra Leone); OBN=O. basilicum (Nigeria); OGS=O. gratissimum (Sierra Leone) and OGN=O. gratissimum (Nigeria).

Both Sierra Leone and Nigeria O. americanum L. varieties were identified as linalool chemotype. The Sierra Leone O. basilicum L. variety is the methyl eugenol chemotype, while the Nigerian variety is predominantly methyl chavicol. Both varieties of O. gratissimum L collected from Sierra Leone and Nigeria are of thymol chemotype.

MIK is grateful to Obafemi Awolowo University Central Science Laboratory - Carnegie Corporation of New York Project for Visiting Fellowship. TAO is grateful to COMAHS - Ministry of Health and Sanitation World Bank Assisted Project RCHP for visiting appointment. The support of Nigeria TetFund for TETF/NRF/OAU grant is also acknowledged. The assistance of Mr. I. I. Ogunlowo of the Department of Pharmacognosy, OAU, for the collection of plants, is similarly acknowledged.