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Antibacterial Investigation using Spectrophotometric Assay of the Polar Leaf Extracts of Ficus capensis (Moraceae)

This study was designed to explore new antibacterial agent(s) from the defatted methanol and water extracts of Ficus capensis. The extracts were obtained using various solvents, namely, cyclohexane, dichloromethane, methanol, and water by serial exhaustive extraction, after which both extracts were challenged with pure clinical isolates of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter baumannii, and Burkolderia cepacia viz-a-viz standard antibiotics of chloramphenicol, gentamicin, and amoxicillin at 25 μg/ml, 50 μg/ml, and 100 μg/ml concentrations using spectrophotometric method. Using the zone of inhibition as an inhibitory parameter, both extracts of F. capensis showed promising antimicrobial activity in a concentration-dependent manner comparable to the standard antibiotics for all tested microorganisms. This research holds promise for the exploration of various potentially active secondary metabolites which would help in developing pharmaceuticals, especially antibacterial drugs, for combating common microbial infections. This validates the use of the plant leaves in the treatment of broad-spectrum microbial infections in ethnomedicine.


IntroductIon
Ficus capensis, a native of tropical Africa and the Cape Islands, belongs to the family Moraceae. The plant is a deciduous tree with spreading roots and branches and broad green leaves. It is known as "uwaryara" in Hausa, "opoto" in Yoruba, "rima bichehi" in Fulani, "akokoro" in Igbo, and "obada" in Edo. [1,2] Conventionally, preparations from the plant parts are used in the treatment of dysentery, edema, epilepsy, chest ailments, leprosy, tuberculosis, anemia, and rickets in infants among others. [2][3][4][5] Aside its tradomedicinal applications, F. capensis has been reported based on modern scientific investigations to possess anti-sickling, [6,7] antibacterial, [4] antiabortifacient, [8] immune-stimulatory, [9] antidiarrheal, [10] antioxidant, [11] pro-fertility in treating azoospermia, [12] as well as its leaf, stem, and roots recently established to manage and treat typhoid disease-causing Salmonella typhi organism. [13] The world over relies one way or the other on medicinal plants as alternative sources of drugs for the treatment of several disease conditions [14] as a result of the fact that majority of the populations, especially in developing countries like Nigeria experience inadequate contacts with orthodox health-care facilities, the nonavailability and unbearable side effects associated with synthetic drugs. [2] Phytochemicals are naturally occurring compounds in medicinal plants which offer defense mechanism and protection from various diseases. [15] They are active secondary metabolites playing an important role in the prevention of various diseases conditions. [14] This study attempts to give scientific credence or otherwise to the claim that F. capensis leaf as used in Igala traditional medicine have positive effect for the treatment of febrile ailments and infective disorders, hence as part of a wider study, the broadspectrum antibacterial activities of the polar extracts of the plant were investigated.

Collection of Microorganisms
Clinical microbial strains used in this study (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Acinectobacter baumanii, and Burkolderia cepacia) from different specimens of patients referred to the Medical Microbiology Department, University of Lagos Teaching Hospital, Lagos State Nigeria. The bacteria were characterized to species level using different laboratory procedures, including gram's stain, cultural characterization, and biochemical tests, notably Indole, Methyl red, Vougues Proskeaur, Catalase, Citrate utilization, and coagulase tests. [16,17] The isolates were maintained on nutrient agar slants at 4°C until ready for use.

Standard Antibiotics Employed for the Study
Chloramphenicol (Kuka Consumer Healthcare, Nigeria), Gentamicin (Jinhing Pharmaceuticals, China), and Amoxicillin (Clarion Medicals, Nigeria) were the employed reference drugs employed for the study purchased from reputable drug outlets.

Extracts Preparation
The defatted methanol extract of F. capensis (FC_dMeOH) was obtained by cold macerating 300 g of the pulverized plant sample within 3 L of cyclohexane for 24 h with the resulting residue after filtration subjected to further cold maceration sequentially using 3 L of dichloromethane and methanol for 24 h each. The residue of the methanol extraction was cold macerated in distilled water, allowed to stand for 24 h and filtered with Whatmann No. 1 filter paper. The filtrate was concentrated to dryness using a freeze dryer. The yield of the crude extract (FC_dHOH) was determined relative to the starting material. The extracts were kept in an airtight glass container and refrigerated at −4°C until use.

Preparation and Reconstitution of the Plant Extracts for Antibacterial Study
The inocula were reconstituted and standardized according to the procedure described by Rajarkaruna et al. [18] The FC_dMeOH and FC_dHOH were individually reconstituted by dissolving 1 mg of the extract in 2 ml of dimethyl sulfoxide (DMSO) to obtain a stock solution concentration of 0.5 mg/ml. One milliliter of the stock solution was added to 4 ml nutrient broth give a concentration of 100 μg/ml. By double serial dilution, the concentrations of 50 μg/ml and 25 μg/ml were obtained. Twelve test tubes were sorted into two groups (FC_dMeOH and FC_dHOH) with each group having six test tubes, three for the test concentrations of 25 μg/ml, 50 μg/ml, and 100 μg/ml, respectively, while the other three test tubes were each for the standard antibiotics (positive control) prepared using the same concentration. The negative control was prepared by adding 1 ml DMSO to 4 ml nutrient broth.

Antibacterial Screening
The spectrophotometric method described by Banjara et al. [19], as modified by Musa et al. [20] was used to determine the antibacterial activity of the extracts.

Statistical Analysis
Data obtained from the study were subjected to a one-way analysis of variance, and variant means were separated post hoc using the least significant difference method. Statistical Package for the Social Sciences software, version 16.0, was used for the analysis. Significance was accepted at P < 0.05.

results
The kinetics of the optical densities of the microbial growth of the six test bacteria is as shown in Figure 1.
The results of the percentage inhibitions of the FC_dMeOH and FC_dHOH in comparison to the standard antibiotics when challenged with the test organisms at the different investigated concentrations are represented in Figures 2a-f.

dIscussIon
Percentage Yield of the Extracts 12.30 g and 35.40 g of the methanol and aqueous extracts corresponding to a percentage yield of 4.1% and 11.8%, respectively, were obtained for the extraction of the pulverized leaves of F. capensis. These results are comparable to the findings of Dickson et al. [13] but higher than the 8.78% reported by Ayinde and Owolabi [2] and Ayinde et al. [10]

Antimicrobial Study
The microbial strains growth and inhibitions when monitored spectrophotometrically were seen to increase on an hourly basis before peaking after 12 h, the decline between 12 and 15 h before increasing during the 18 h monitoring period. The negative control which contained DMSO without the plant extract or standard antibiotics produced no inhibition of any of the test organisms at 12 h post-inoculation.
Both extracts exhibit an increase in the inhibition of all the microbial isolates in a dose-dependent manner. All the standard antibiotics exhibit a higher microbial inhibitions greater than both extracts when challenged with E. coli, P. aeruginosa and K. pneumoniae except for E. coli which exhibits a 100% inhibition at the highest concentration of 100 μg/mL. This, however differs, from that of S. aureus, A. baumanii, and B. cepacia, where the standard antibiotics exhibit a higher inhibition than both extracts at the highest investigated concentration. The disparity between the activities of the extracts and the standard antimicrobial drugs may be due to the mixtures of bioactive compounds present in the extracts compared to the pure compound contained in the standard antibiotics. [14] The antimicrobial activities of the extracts are in agreement with the report of Cooper et al. [21]   that the presence of more group of phytochemical diversity gives synergic effects in many biological applications. The phytoconstituents present in the leaves of the plant, as reported by Dickson et al. [13,19] as well as Uzoekwe and Mohammed, [22] who confirms the presence of flavonoid, alkaloid, saponins, phenols, cardiac glycoside, terpenoid, steroids, and tannins. The presence of these phytochemicals in F. capensis leaf extracts has also been reported by many researchers [23][24][25] to confer the therapeutic potentials of all medicinal plants. Alkaloids, saponins, and tannins specifically have been reported to inhibit bacterial growth as well as offer protection to plants from microbial infections. [26] The leaves of the plant have been reported to exhibit remarkable inhibitory activities against the growth of enteric bacteria notably E. coli, B. subtilis, S. aureus, and Shigella species as reported by Oyeleke et al., [4] François et al., [27] and Solomon-Wisdom et al. [28]  The standard antibiotics showed a better antibacterial activity than both extracts thou not statistically significant. All tested concentrations of FC_dMeOH have a better antibacterial agent than FC_dHOH except when challenged with P. aeruginosa and S. aureus at 25 μg/mL and 25 μg/mL, respectively. These reports in combination with the results of the present work may justify the use of the F. capensis leaves in the treatment of broadspectrum microorganisms in ethnomedicine as well as encourages researchers to do further in vitro and in vivo research that will explore the role of the bioactive constituents responsible for these activities as well as carry out studies at molecular level.