Phytochemical and Antibacterial Activities of Combined Leaves and Flower Extracts of English Camphor Basil ( Ocimum canum ) on Some Selected Bacteria Associated with Skin Infections

Phytochemical and antibacterial activities of combined leaves and flower extracts of English camphor basil (Ocimum canum) on some selected bacteria associated with skin infections was investigated. Plant samples were collected at Mista Ali, Bassa LGA of Plateau State. Phytochemical analysis was carried out in the Biochemistry Laboratory of National Veterinary Research Institute Vom (NVRI), located in Jos South LGA. The test organisms, S. aureus, S. pyogenes, P. aeruginosa, S. marcescens, E. faecalis were obtained from the microbial banks of bacteriology and dermatophilosis sections of NVRI, Vom and were standardized with a Nephelometer. Quantitatively, the leaves and flower ethanolic extracts were tested individually and in combination against bacteria associated with skin infections both extracts showed the presence of steroids, cardiac glycosides and flavonoids while tannins were detected in the leaves extract only. There was significant (p<0.05) variation in the quantity of inherent phytochemical components of the extracts. Cardiac glycoside had the highest concentrations (0.64±0.02 and 0.54±0.02) in the leaves and flower extracts respectively. Leaves extract showed activity against test bacteria except E. faecalis. Strains of S. pyogenes and S. aureus were only susceptible to flower extract. The MBC ranged from 50-100mg/ml for the sensitive bacterial isolates. Fractional Inhibitory Concentration Index (FICI) from the combined extracts varied from 1.89 to 3.97 on the test bacteria that showed lack of interaction (FICI<4). Thus, combination of leaves and flower of O. canum may not exhibit any advantage. However, the plant contain bioactive constituents of pharmacological significance and should be explore for more potentials.

Synergistic interactions allows the use of lower doses of the combination constituents, a situation that reduces adverse reactions (Tallarida, 2006).Modern science has been relatively slow in its efforts to understand the herb-herb interactions, a number of studies validate the concept of synergisms between components of herbal concoctions.Some bacteria and fungi are extremely pathogenic causing serious human infections.The discovery of antibiotics to combats these pathogens marked a resolution in the 20 th century (Evans, 1992).

Materials and Methods: Sample Collection:
The plant materials (leaves and flowers) were collected at Mista-Ali, Bassa Local Government Area of Plateau State.Authentication was carried out at Federal College of Forestry located at Bauchi Road, Jos North, Plateau States with the aid of botanical keys (Arber, 1972), to obtain specimen(plant) voucher.
Test organisms (Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Pseudomonas aeruginosa and Serratia marcescens were obtained from the microbial banks of Mycological and Bacteriological sections of National Vertinary Research Institute, Vom in Jos South Local Government Area of Plateau State. Biochemical tests were performed for authentication of test organisms.Catalase, oxidase, indole and coagulase production were employed for identification of bacteria (Cheesbrough, 2006,).

Preparation of Plant Materials:
The plant leaves and flowers were separately cleaned with tap water and dried at room temperature in a shade for a period of 2-4 weeks.The dried leaves and flowers were then pulverized into fine powder using mortar and pestle and stored in an airtight plastic containers.

Ethanolic Extraction:
The method of Fatope et al. (1993) was used for the ethanolic extraction.Two hundred and fifty grams of the powdered flower material was successively extracted separately using cold maceration with 450ml of ethanol (95%) for 24 hours, and one hundred and eighty grams of powdered leave material was also successively extracted with 180ml of the same solvent.Extracts were filtered using Whatman No.1 filter paper and allowed to evaporate in a rotary evaporator at 45 o C. The dried extracts were preserved at refrigeration temperature.

Phytochemical Screening:
Quantitative phytochemical analysis of the extracts of the plant was determined by the methods used by ( Edeoga et al., 2005).
The extracts were screened for the presence of alkaloids, saponins, flavonoids, tannins, cardiac glycosides, carbohydrate, steroids and terpenes.

Standardization of Microbial Isolates:
Standardization of test organisms was done using a Nephelometer based on McFarland standard.The agar well diffusion method was performed to exploit anti-bacterial potential of the extracts as described by (Bibi et al., 2010).The standardized cultures of each test bacteria was spread on Nutrient agar plates so as to achieve even growth on the plates.The plates were allowed to dry in an incubator for 30 min at 37 0 C and a sterile cork borer (5.0 diameters) was used to bore 5 holes at equidistance in each agar plate.The bottoms of each well was sealed with sterile agar to prevent seepages of extracts under the agar.Exactly 0.5ml of each prepared concentration (25mg, 50mg and 100mg) was introduced aseptically into wells 1-3.The 4 th and 5 th wells served as positive and negative controls.Ciprofloxacin (10ug/ml) was used as positive control and distilled water as negative control.The plates were allowed on the bench for 40 minutes for prediffusion followed by incubation at 37 0 C for 24 hours.The resulting zones of inhibition were measured to the nearest mm.

Combination Assay:
Well Diffusion Method: Varying concentrations of the leaves and flowers extracts were added into wells on prepared plates aseptically.

Determination of Minimum Inhibitory Concentration (MIC):
This was determined using broth dilution method as described by Junaid (2006).Exactly 4 extract concentrations each of 100, 50, 25 and 12.5, mg/ml of plant materials was used.The 5 th and 6 th tubes served as positive and negative controls.One (1) ml the extract was added in each test tube containing sterile Mueller Hinton medium.The tubes were inoculated with 0.1ml of bacterial suspension except for the positive and negative control.The MIC was examined for turbidity (cloudiness) after 24 hours of incubation.
The MIC was read as the least concentration of extract that showed no growth (clear) after incubation period.

Determination of Minimum Bacteriocidal Concentration (MBC):
The MBC and MFC were determined by sub-culturing the test dilutions which showed no growth onto fresh drug-free solid media (Mueller Hinton agar) and were incubated for 24 hours at 37 0 C. The lowest concentration that yielded no single bacterial colony on the medium was taken as the MBC.

Discussion:
The increase in bacteria resistance to common available antibacterial agents has also increased the need for the invention of new drugs.In this study, both the leaves and flower extracts of Ocimum canum showed the presence of pharmacologically active components.The predominance of steroids, flavonoids and tannins detected in the extracts of Ocimum canum agrees with earlier reports (Aluko et al.,2012).
Quantitatively, the predominance of cardiac glycosides in both the leaves and flower extracts of Ocimum canum is contrary to the findings of A high MIC value indicates low activity and vice versa.In this study, no significant different was observed between the MICs and MBCs of both Gram-negative and Gram-positive organisms.This result suggests their susceptibility and in vitro broad spectrum antibacterial activity of the leaves and flower extracts of Ocimum canum as reported by coworkers (Biswas, 2013).The high MICs of 25 to 50mg/ml do not conforms with the findings of Biswas et al. (2013) who reported lower MIC (0.156 to 0.625) similarly on species of Pseudomonas aeruginosa, and Staphylococcus aureus.Preliminary result on the effect of combination of leaves with flower extracts showed decrease interaction against the test bacteria evaluated.These suggest that combination of the plant extracts affected the potency of either phytochemical agent against the organisms (Ofokansi et al., 2013).Thus, combination of the leaves and flower extracts resulted in indifference and may not exhibit any advantage when used.

Conclusion:
English camphor basil (Ocimum canum) leaves and flower extracts showed antibacterial activity on bacterial isolates used.The plant contain chemical constituent of pharmacological significance and can be used to treat infections of bacterial origin.

Gberikon, G.M et
al. Phytochemical and Antibacterial Activities of Combined Leaves and Flower Extracts of English Camphor Basil (Ocimum canum) on Some Selected Bacteria Associated with Skin Infections

Table 2 : Antibacterial Activity of Leaves and Flower Extracts of Ocimum canum
Values are Mean± Standard Error of Mean for duplicate determinations.Values with different superscripts within column or row vary significantly at p< 0.05.
Key: DMSO = Dimethyl sulfoxide Cipro = Ciprofloxacin.Values are Mean ± Standard Error of duplicate determinations and values with different letters as superscript across the row are significantly different at P≤ 0.05.

Table 3 : Minimum Inhibitory Concentration of Leaves and Flower Extracts of Ocimum canum
bValues are mean ± standard error of duplicate determinations; values with different letters as superscript across the row are significantly different at P≤ 0.05 -No activity

Table 5 : Antibacterial Activity of Combined Leaves and Flower Extracts of Ocimum canum
Values are mean ± standard error of duplicate determinations; values with different letters as superscript across the row and column are significantly different at P≤ 0.05.-not determined

Table 6 : Effect of Combined Extracts on Strains of Bacteria
(Bassole et al., 2005)1)man et al., 2017))high level of flavonoids (10.00 %) in the leaves were reported.Comparatively, the flavonoids content (0.47±0.02 to 0.31±0.02mg/ml)reported in this study is significantly lower than values (10.00 %) obtained byAluko et al. (2012)in the species of O. canum obtained in southern Nigeria.Lower tannin composition was found in leaves extracts of Ocimum canum compare to 4.73 ± 0.01 mg/ml reported in the study by coworkers(Aluko et al., 2012).In this study, leaves and flower extracts of the novel Ocimum canum exhibited antibacterial activity.This finding is not against the backdrop that susceptibility of a wide range of microorganisms to extracts of Ocimum canum had been established(Bassole et al., 2005).Studies by coworkers similarly reported inhibitory properties of the extracts of Ocimum canum to both Gram positive and Gram negative bacteria(Bassole et al., 2005;Usman et al., 2017).Conforming to the result of this study however, the extracts showed better antibacterial activity and higher efficacy on Gram-positive bacteria to Gram-negative bacteria(Usman et al., 2017).Of particular importance, is the susceptibility of Staphylococcus aureus and Streptococcus pyogenes to both the leaves and flower extracts of Ocimum canum.Although some earlier reports had shown that Staphylococcus aureus exhibited strong resistance against a host of plant extracts(Nwachukwu et al., 2001).Antibacterial activity of the leaves and flower extracts of Ocimum canum on the bacteria species evaluated exhibited by diameter of inhibition range of 12.00±0.12to24.30±0.15mm.In this study, susceptibility of Staphylococcus aureus to the leaves extract (21.00±0.58-24.30±0.15mm)andflower(19.10±0.06-21.5±0.06 mm) extract is higher than values obtained by Biswas, (2013) for the ethanolic and aqueous extracts(7.8 -15.5mm).The diameter of inhibition of Streptococcus pyogenes ranged between 12.00±0.29 to 20.0±0.20 mm for both leaves and flower extracts of Ocimum canum is lower than ethanolic extract at 5 mg/ml concentration for most bacteria evaluated(Bassole et al., 2005).