Raw materials
Oranges were purchased from New Benin Market in Benin City, Nigeria. Oranges were obtained same day they were plucked from local farm trees not under any pesticide treatment. The fruits were washed with distilled water and the peels removed with the aid of a sharp knife. Outer peel removal was carried out to ensure that the flavedo was not harvested alongside the albedo. The flavedo were divided into two equal groups. One group was air-dried in a shade at 30–33 °C for seven days and then pulverized. The other group was pulverized immediately after peeling the oranges and then subjected to extraction. Pulverization was carried out using a sterile mortar and pestle till fine granular or powdery consistency was obtained.
Preparation of plant extract
The pulverized samples were subjected to Soxhlet extraction for a period of 12 h with 500 mL of ethanol and then concentrated using a rotary evaporator at reduced pressure. The extracts were stored in the refrigerator till required for use.
Determination of Total phenolic content
Total phenolic content was determined according to Folin-Ciocalteau reagent method of Cicco [14]. Concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg/mL of gallic acid were prepared in methanol. Extracts were also prepared in methanol to obtain concentrations of 1 mg/mL. Then 4.5 mL of distilled water was added to 0.5 mL of the extract and mixed with 0.5 mL of a ten-fold diluted Folin- Ciocalteau reagent. Subsequently, 5 mL of 7% sodium carbonate and 2 mL of distilled water were added. The mixture was allowed to stand for 90 min at room temperature before the absorbance was read at 760 nm. All determinations were performed in triplicates with gallic acid utilized as the positive control. The total phenolic content was expressed as gallic acid equivalent (GAE).
Determination of Total flavonoid content
Total flavonoid contents were estimated using the method described by Ebrahimzadeh et al, [15]. Extracts (0.5 mL of 1 mg/mL) were mixed with 1.5 mL of methanol. To this mixture, 0.1 mL of 10% aluminium chloride was added, followed by 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water. The mixture was incubated at room temperature for 30 min. The absorbance was measured by a spectrophotometer at 420 nm. The results were expressed as milligrams quercetin equivalents (QE) per gram of extract (mg QE/g extract).
Determination of Total tannin content
The total tannin content was determined by modified method of Polsheltiwar et al., [16]. To 0.1 mL of 1 mg/mL sample extracts was added 0.5 mL of Folin-Denis reagent followed by 1 mL of Na2CO3 (0.5% W/V) solution and distilled water up to 5 mL. The absorbance was measured at 755 nm within 30 min of reaction against blank. The total tannin in the extract was expressed as the equivalent to tannin acid.
Antimicrobial assay
Test microorganisms
Eight (8) microorganisms were used in this study - Five bacterial strains and three fungal strains. Two were gram positive (Staphylococcus aureus and Enterococcus faecalis) while the other three were gram negative (Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium). The three fungal strains used are Candida albicans, Aspergillus niger and Penicillium notatum. All microorganisms were clinical isolates obtained from Lahor Research and Diagnostic Laboratories, Benin City, Nigeria. The identities of the test organisms were confirmed to the specie levels using standard biochemical and morphological procedures.
Antimicrobial susceptibility assay
Test organisms and 2 control strains (S. aureus ATCC 25923 and E. coli ATCC 25922) were sub-cultured onto fresh suitable broth medium. Broth cultures were then incubated at 37 °C till the turbidity of 0.5 McFarland’s standard (1.5 × 108 CFU/mL). Mueller-Hinton agar was used as bacterial medium and Sabouraud agar as fungal medium. All were incubated appropriately as specified for each test organism. The turbidity of the actively growing broth culture was adjusted with sterile saline to obtain 0.5 McFarland’s standard turbidity. One milliliter of the suspension was then used to flood the surface of solid Mueller-Hinton agar plates and drained dry. Wells of 5 mm in diameter and about 2 cm apart were punched in the culture media with sterile cork borer. The extracts (0.2 mL) were thereafter used to fill the boreholes. Each plate was kept in the refrigerator at 4 °C for 1 h before incubating at 37 °C for 24 h (bacteria) and 72 h (fungi). Zones of inhibition around the wells, measured in millimeters, were used as positive bioactivity. All experiments were carried out in triplicates.
Minimum inhibitory concentration (MIC)
The organisms that showed susceptibility to the different solvent extracts were introduced into the broths containing different concentrations of each extract (Serial dilutions of the extracts corresponding to 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL and 12.5 μg/mL). The tubes were thereafter incubated for 24 h at 37 °C. The MIC was taken as the lowest concentration of the extracts that did not permit any visible growth.
Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC)
The tubes that showed no turbidity in the MIC test were taken and a loop-full from each tube was streaked on Mueller Hinton agar. The plates were incubated for 24 h at 37 °C and the absence of growth was observed. The concentration of the extracts that showed no growth was recorded as the MBC / MFC.
Statistical analysis
The data were expressed as mean ± SEM of three replicates. The data were subjected to one-way analysis of variance (ANOVA), and differences between means were determined by Duncan’s multiple range test using the Statistical Analysis System (SPSS Statistics 17.0) where applicable. P values ≤0.05 were regarded as significant.
