Chemicals and Reagents The standards catechin, gallic acid, 1,1-diphenyl-2- picrylhydrazyl radical (DPPH) and 2,4,6-tris (2-pyridyl)- 1,3,5-triazine (TPTZ) were purchased from Sigma-Aldrich (St. Louis, MO). Tannic acid, L-ascorbic acid, Folin– Ciocalteu’s phenol reagent were purchased from Merck Co. (Darmstadt, Germany). The Mueller Hinton agar was purchased from HIMEDIA (Mumbai, India). All of the chemicals and reagents used in this study were of analytical grade.
Fruit Sample Collection The matured green BAU Kul was manually harvested at the commercial maturity stage in the month of February 2012 from the northern region of Bangladesh. After storage, the fruits were packed in cardboard boxes and transported by road (with an approximate transportation time of 4–6 h). The fruits were sent to the microbiology laboratory in the Department of Biochemistry and Molecular Biology, Jahangirnagar University. In the laboratory, the fruits were cleaned with sterile water in a laminar blower to remove any possible contamination and were subsequently stored in the refrigerator (4C) for a day before further processing.
Preparation of Extracts The fruit extract was prepared according to method described by Saradha and Rao (2011) and Walter et al. (2011). The fresh matured fruits were randomly selected and thoroughly rinsed with cold sterile water before being cut into small pieces with a stainless steel blade. The fruit pieces were then ground with a blender. For the preparation of a methanol extract, the ground fruit (400 g) was extracted in methanol using a Soxhlet extractor for 7 h. The crude extract was further concentrated to dryness in a rotary evaporator (Buchi, Tokyo, Japan) at reduced pressure (100 psi) and controlled temperature (40C). Semisolid extract was kept in the incubator at 30C for 2 days to make sure of complete evaporation of residual methanol. The dried extract (37 g) was preserved for up to 2 months at −20C for subsequent use.
Phytochemical Analysis Estimation of Total Polyphenol Content. The total polyphenol content in BAU Kul extracts was estimated by spectrophotometric determination following Amin et al. (2006). Briefly, the solution (0.4 mL) containing 1 mg of extract was mixed with 1.6 mL of 7.5% Na2CO3 solution. Then, 2 mL of 10-fold diluted Folin–Ciocalteu’s reagent was added and the final reaction mixture was incubated for 1 h in the dark. The intensity of the blue colored complex was measured at 765 nm using a PD-303S spectrophotometer (APEL, Angyouryou Negishi, Kawaguchi Saitama, Japan). The total polyphenol content present was determined as gallic acid equivalents (GAEs) (5–80 μg/mL) and expressed as milligram of GAEs per 100 g of BAU Kul.
Estimation of Total Flavonoid Content. The total flavonoid content in BAU Kul extracts was estimated using an aluminum chloride colorimetric assay (Chang et al. 2002). Briefly, 1 mL of the solution containing 1 mg of extract was mixed with 4 mL of distilled water and then 0.3 mL of 5% NaNO2 was added. After 5 min, 0.3 mL of 10% AlCl3 was added. Six minutes later, 2 mL of 1 M NaOH was added, followed by the immediate addition of 2.4 mL distilled water to reach a total volume of 10 mL. The solution was mixed well and the intensity of the developed flavonoid-aluminum colored complex was measured at 510 nm. The total flavonoid concentration was determined as catechin equivalents (CE; milligram of CEs per 100 g of BAU Kul).
Estimation of Total Tannin Content. The total tannin content in BAU Kul extracts was estimated using Folin– Ciocalteu’s method (Folin and Ciocalteu 1927) with tannic acid as standard. Briefly, 0.1 mL of the solution containing 1 mg of the extract was mixed with 7.5 mL of distilled water and 0.5 mL of Folin–Ciocalteu’s reagent was added. One milliliter of 35% Na2CO3 and 0.9 mL of distilled water were added to the aforementioned mixture. The solution was mixed and then incubated for 30 min. The intensity of the developed blue colored complex was measured at 725 nm. The results were expressed as milligram of tannic acid equivalents (TEs) per 100 g of BAU Kul.
Estimation of Ascorbic Acid Content. The ascorbic acid content in BAU Kul extracts was estimated by the method established by Omaye et al. (1979) with slight modifications. Briefly, 1 mg of extract was mixed with 1 mL of 5% trichloroacetic acid solution and centrifuged for 15 min at 3,500 rpm. Then, 0.5 mL of supernatant was mixed with 0.1 mL of 2,4-Dinitrophenylhydrazine/ thiourea/copper solution and incubated for 3 h at 37C. The reaction was followed by addition of 0.75 mL of icecold 65% H2SO4. The solution was allowed to stand for an additional 30 min at room temperature. The developed colored derivatives were monitored at 520 nm. The ascorbic acid concentration was determined as ascorbate equivalents (AEs; 1.25–20.00 μg/mL) and ascorbic acid content was expressed as milligram of AEs per 100 g of BAU Kul.
Sterility of Extracts. The BAU Kul extracts were filtered using Millipore nylon membranes (0.45 μm) and tested for sterility by introducing 2 mL of the extract into 10 mL of sterile nutrient broth and incubated at 37C for 24 h. The sterile extract was indicated by the absence of turbidity (i.e., broth clarity) after the incubation period (Atlas 1995).
Bacterial Strains. Five pathogenic bacterial strains (Salmonella paratyphi, Escherichia coli, Chromobacterium violaceum, Staphylococcus aureus and Pseudomonas aeruginosa) were used in the antibacterial activity tests. The strains were obtained from the Bangladesh Institute for Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders. The bacterial strains were reidentified as an additional confirmatory step on the basis of their morphological, culture and biochemical characteristics according to the method established by Cheesbrough (2000).
Statistical Analysis All analyses were performed in triplicate and the data are reported as the mean ± standard deviations (SD). Data were analyzed using SPSS (Statistical Packages for Social Science, version 20.0, IBM Corporation, New York, NY) and Microsoft Excel 2007 (Redmond, WA).