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Phytochemical Constituents and Antimicrobial and Grain Protectant Activities of Clove Basil (Ocimum gratissimum L.) Grown in Nigeria

Ocimum gratissimum Linn (Lamiaceae) is an herbaceous plant reputed for many medicinal and agronomic practices amongst Nigerian peasant farmers. O. gratissimum was investigated for antimicrobial activity against ten micro-organisms and for grain protectant activity against Callosobrochus maculatus. The phytocontituents of the aerial part of O. gratissimum were extracted with 95% ethanol using the percolation method. The crude ethanol extracted was further fractionated into hexane, chloroform and methanol fractions. The fractions obtained were screened for phytoconstiteunts, antimicrobial and grain protectant properties. Result showed that hexane fraction exhibited the highest antimicrobial activity against Vibrio cholera and Klebsiella pneumonia. Similarly hexane fraction also showed the highest grain protectant activity. The other extracts of the O. gratissimum did not significantly inhibit both bacterial growth and grain infestation. However, the methanol fraction contains phytocompounds such as phenolic compounds associated with antioxidant properties. The study shows that O. gratissimum extractants are potential sources of antimicrobial and preservative agents.


Introduction
Traditional medicine continues to provide health coverage for over 80% of the world population, especially in the developing world [1]. Plants are the major constituents of traditional medicine [2]. Many of the plant materials used in herbal medicine are readily available in rural areas and this has made it relatively cheaper than orthodox medicine [3]. The upsurge in the prevalence of side effects of many synthetic antimicrobial agents and incidence of multi-drug resistant bacteria and pests has spurred scientists onto the research for plant based antimicrobial of therapeutic and pesticidal potentials [4][5][6][7].
Several ethnobotanical surveys show that Ocimum gratissimum was among the plants reported in Nigeria communities to be used traditionally to treat bacterial infections such as enteric diseases viz: diarrhoea, dysentery and other gastrointestinal infections; upper respiratory tract infections associated with coughing pneumonia, asthma and bronchitis; urogenital infections including sexually transmitted diseases, skin infections (dermatitis, eczema, scabies), wounds and ulcers; headache, ophthalmic, insect bites, nasal bleeding, stroke, measles, paludism; and bacterial fevers such as typhoid fever and diabetes and veterinary problems . It is also used in the treatment of epilepsy, shigellosis, trypanosomiasis, convulsion, pile and anaemia in Nigeria [37]. It is also implicated in the oral hygiene and veterinary in Nigeria [38,39].
Comprehensive biological activities of O. gratissimum has been reviewed [14] and it is associated with antibacterial, antifungal, hypoglycaemic, antipyretic, anti-nociceptive, antioxidant, anti-inflammatory, anthelmintic, chemo-preventive, anti-carcinogenic, free radical scavenging, radio protective, antidermatophytic activities, and numerous others pharmacological use . Earlier reports have also shown the smooth muscle contracting and antimutagenic activity [62] as well as its anti-diarrhoeal effects in experimental animals [63], high antiviral indices against HIV-1 and HIV-2 [64]; shigellocidal properties [65,66], anti-trypanosomal effects [67], immunobiologicalactivity [68], gastroprotective properties [69], controlling agent for food spoilage and mycotoxin producing fungi [70], disintegrant properties of its seed mucilage [71], and as a relaxant on isolated ileum from guinea pig [72]. Its essential oil has mosquito repellant, insecticidal properties [73,74]. The essential oil of O. gratissimum and its main component eugenol were reported to be efficient in inhibiting Haemonchus contortus [75,76]. Currently, basil is mainly used as a culinary herb as well as perfumes and cosmetics [77]. It is therefore important that phytochemical composition be correlated to the antimicrobial activity in order to verify the therapeutic value proclaimed by the traditional healers.
One major factor responsible for promoting grain production in developed countries has been attributed to the usage of insecticides in grain protection and storage. Many plant components are now known to possess herbicidal, insecticidal or fungicidal properties [78][79][80][81]. Preparations made from Nigerian plants are identified with pesticidal activities [82][83][84][85][86][87][88]. The discovery and development of these products were based on the significance of the Nigerian plants in folklore medicine and agronomic practices amongst our peasant farmers. Peasant farmers in northern Nigeria indigenously use various plants to protect cereals and legumes against pest damage during storage with O. gratissimum being one of such plants [7]. It is based on this view that, bioassay screening method [87] was adopted for screening Ocimum gratissimum for grain protectant activities. Despite these scientific and medicinal values, comparative analyses of its phytochemical evaluation vis-à-vis the antimicrobial and pesticidal potentials have not been investigated. Therefore, the present study reports phytochemical, antimicrobial and grain protectant activities of O. gratissimum used for the treatment of human infections and agronomic pests.

Collection and Identification of Materials Used
The aerial parts of O. gratissimum were collected from Kuchi-gbako, a village along Bida-Doko road of Lavun Local Government, Niger State as described by the traditional healers and farmers.

Extraction Procedure
The plant material was air-dried under the laboratory room conditions for one week and then milled into coarse powder by using clean mortar and pestle. The powdered material (200g) was percolated with 95% ethanol (2.5L) for two weeks. The extract was filtered and evaporated to dryness using a rotavapor to give a dark greenish residue (43.5g) [89].

Test procedure for Antimicrobial Activity
The crude ethanol extract of the aerial parts of O. gratissimum was screened in vitro for antimicrobial activity against ten pathogenic microorganisms ( Table 2) using Agar-dilution streak technique [90] as follows: the test organisms were prepared by incubating them in freshly prepared nutrient broth at 37 o C for 8 h the cultures were serially diluted with sterile normal saline. 48 mg of the test extract was dissolved in 1ml of absolute ethanol and made up to 3ml with sterile distilled water to give a concentration of 16 mg/ml of extract. 1ml of the prepared extract was then introduced into 15 ml of molten Agar placed in water at 54 o C these were mixed well and poured into sterile petri-dish plates to give a concentration of 1000 µg/ml of Agar. Other concentrations were similarly prepared. The plates were then hardened in a refrigerator for 15 min. Thereafter the standardized test organisms (1000 ml each) were inoculated onto the nutrient Agar plate and incubated at 37℃ for 24-48 h the results of the tests done in triplicate are shown in Table 2.

Fractionation of Extracts
The crude extract (100 g) was extracted with hexane and 70% aqueous methanol (150 ml, 1:1), hexane soluble fraction was separated and concentrated in vacuo to give a hexane residue (15.56 g), and the methanol layer was then extracted with chloroform (150 ml). The resultant chloroform soluble and methanol soluble portions were separated and concentrated in vacuo to give chloroform residue (5.4 g) and methanol residue (3.2 g) respectively.

Cowpea Weevil Bioassay with the Plant Extracts
The residues obtained from the fractionation of O. gratissimum were screened for grain protectant activity against C. maculatus using cowpea weevil bioassay techniques [87] as follows: Unperforated cowpea seeds (50 g) from newly harvested dry pods were weighed out and from 10 g were transferred to each of four Erlenmeyer flasks. The cowpea seeds in each of these three flasks were separately treated with the various extracted residues (1 g) each. The untreated seeds in the fourth flask served as a control.
Freshly emerged adults of C. maculatus (age, 0-8 h) were used to infest the cowpea seeds in each flask. The flasks were covered with mesh net and left on the shelf at room temperature. The control and the three treated samples are trip-licated. After 4 months the cowpea seeds in each flask were examined for perforations. The number of cowpea seeds perforated in treated and control were counted for determination of weevil perforation index (WPI). The weevil perforation index, defined as percentage of treated cowpea seeds perforated X 100/percentage of control cowpea seeds perforated + percentage of treated cowpea seeds perforated, was calculated for each extract for comparison of grain protectant properties of O. gratissimum.

Test Organisms
Stock culture of Neisseria gonorrheae, Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumoniae, Vibrio cholerae, Streptococcus faecalis and Bacillus anthracis were obtained from Microbiology Laboratory, Federal University of Technology, Minna. These cultures were checked for viability and purity and maintained on nutrient agar slopes.

Statistical Analysis
The data was analyzed with ANOVA and the means were separated using Duncan Multiple Range Test at a probability level of 5%.

Results and Discussion
The crude 95% ethanol extract of O. gratissimum has demonstrated antimicrobial activity against N. gonorrhoeae S. typhi, P. aeruginosa, K. pneumoniae, and V. cholera at a concentration of 1000 µg/ml ( Table 1). Most of the fractions obtained had exhibited broad-spectrum antimicrobial activity for many test organisms at 1000 µg/ml which is a good inhibitory concentration. Since crude extracts with activity at concentrations of 1000 µg/ml and below are considered as promising bioactive agents for further work [94].  The antimicrobial potency of plants is associated with the secondary metabolites found in its extracts. Fractionation of the crude extracts allows the distribution of these metabolites in to petroleum ether, chloroform, ethyl acetate and methanol fractions according to their polarity [95]. By this, phytocompounds can easily be separated and the associated activity might be correlated to the presence of alkaloids, flavonoids, saponins, sterols and tannins in the individual fraction [96][97][98][99]. The result of the preliminary phytochemical screening revealed the presence of tannins, phenolic compounds, terpenoids, sterols, saponins and alkaloids in O. gratissimum. In particular the flavonoids, terpenoids and alkaloids were detected in most of the extracts of the basil grown in Nigeria. However, ketones, cardiac glycosides, and flavonols were not detected at all (Table 2).
Carbohydrates, anthraquinones, phenolics, tannins and saponins were present in alcoholic fractions only. Sterols were found in crude ethanolic extract as well as hexane chloroform soluble fractions. The antimicrobial activity of flavonoids is may be due to their ability to complex with extracellular and soluble protein and to complex with bacterial cell wall; thereby disrupting their membrane integrity [100]. Antioxidants are molecules that can delay or prevent an oxidative reaction catalysed by free radicals. It is noteworthy that phytochemicals are the most important antioxidants in dietary. Such vital metabolites include polyphenols, quinones, flavonoids, catechins, coumarins, terpenoids and in addition to the smaller molecules like ascorbic acid (Vitamin C) and alpha-tocopherol (Vitamin E) [101]. Therefore, the presence of these phytochemicals could support the herbal medicine uses of O. gratissimum as antioxidant and its edible leaves being used to prepare soup and tea. The antioxidant effect is mainly due to the presence of phenolic components such as flavonoids and phenolic acids [101]. On further fractionation, flavonoids and phenolic compounds were found to be predominantly present in the methanol fraction of O. gratissimum. Saponins which are glycosides with soapy characteristic are often reported to possess bioactive agents [102]. Tannins have been reported to hinder the development of micro-organisms by their ability to precipitate and inactivate microbial adhesions enzymes and cell envelope proteins [4]. The significant activity observed in this study could thus be attributed to the interaction of one or more of the identified metabolites against the test organisms. On further fractionation, the hexane soluble fraction obtained demonstrated the highest sensitivity towards V. cholera (18.5 mm) and K. pneumoniae (18.0 mm), followed by the methanol soluble fraction with Escherichia coli (17.0 mm) and Staphylococcus aureus (17.0 mm). The present results justify the traditional medical uses of O. gratissimum for treating diarrhoea, respiratory tract infection and fever. In addition, it could also be used for the range of organisms inhibited, for which the plants is not traditional used for. A full investigation of the toxicity of this plant is required, even though many ethnic groups of Nigeria use the edible leaves O. gratissimum to prepare soup and tea for decades. The 'Green' movement in Western society has changed attitudes of the general public who now viewed naturally derived substances and extracts as being inherently safer and more desirable than synthetic chemicals products thereby leading to the net increase in sales of herbal preparations [95]. Therefore, 80% of people in the developing world rely on natural products for primary healthcare for man [1][2][3].
Of the various screening procedures, the cowpea weevil bioassay [87] is the most convenient for general use in a small laboratory. Hence it was adopted for the activity screening of O. gratissimum. Weevil perforation index values are recorded for tests in which the damage levels of control seeds are not less than 50%. In this bioassay, a WPI value of 45 or less after 4 months of storage with plant extracts at a dosage of 10% (wt/wt) is considered to be a strong activity, a WPI value of 50 shows that the equal amounts of treated and untreated cowpea seeds were performed. Of all the extracted fractions screened, hexane fraction with 3WPI value showed the best grain protectant activity (Table 3). From the results of the phytochemical screening, some phytocompounds identified have previously been associated with repellency activity [73,74,103]. The principal constituent of the essential oil of O. gratissimum is eugenol which is responsible for the biological activities observed. The volatile oil of O. gratissimum also contain phenols, particularly thymol which was earlier reported to be responsible for the antimicrobial action [11,104]. The studies of the chemical composition of a related species O. suave showed that the major components of the oil are p-cymene, α-thujene, myrcene and thymol, in addition to phenols [105][106]. Therefore, these compounds may be responsible for the grains protectant and antimicrobial activities of the hexane fraction of O.
gratissimum and it could serve as a potential source of pharmaceutical applications and in the control of agronomic pests.

Conclusions
The preliminary biological and phytochemical screenings of O. gratissimum results are quite promising and have strongly indicated the grain protectant property as well as the antimicrobial activity spectra of aerial parts of the plant. The present result also showed the possible phytocompounds to which the biological activity may be attributable. Further work is ongoing to isolate and elucidate the structure of the bioactive compounds and to screen its pure active constituents against agronomic pests.