2.1. Preparation of tea extract four different varieties of tea such as FBOP, BOP, red dust and green tea were collected from the local market as granular powder of a particular local brand. Tea extract was prepared according to the method described previously. Briefly, tea powder (50 g) was mixed with 300 mL distilled water and boiled for 45 min. All the granular materials were removed by filtration with Whatman No. 1filter paper. Filtered tea extract was then lyophilized in order to obtain dry fine powder. The yield of boiling water extracts for BOP, FBOP, red dust and green tea were 13.94%, 13.32%, 13.56% and 11.87%, respectively. Dryfine powder was stored at 4C and when necessary it was dissolved in phosphate-buffered saline (PBS)to make 400 mg/mL working solution.
2.2. Determination of total polyphenol content (TPC) of tea extract THC was determined by spectrophotometry against gallic acid (GA) as standard by the Folin–Ciocalteu method. An aqueous solution of tea extract at a concentration of 500mg/mL was used in this analysis. Briefly, 0.5 mL of the diluted aqueous extract was mixed with 2.5 mL of 10% Folin–Ciocalteu reagent dissolved in water and then vortexed. Afterward, 2.5 mL of 7.5% NaHCO3was added. The tubes were then allowed to stand at room temperature for 60 min. Absorbance was measured at 765 nm against water. Each sample was prepared in triplicate. The same procedures were repeated for the standard solution. The TPC was expressed as gallic acid equivalents (GAE) in mg of GA/g of tea extract.
2.3. Determination of total flavonoid content (TFC) of tea extract THC content of tea extract was determined by spectrophotometry against catechin as standard using aluminum chloridecolorimetric assay[20]. Briefly, 1 mL of aqueous tea extract at concentration of 30 mg/mL was dissolved in 10% 0.3 mL AlCl3solution and 5% 0.3 mL NaNO2followed by addition of 200mLNaOH. The sample was incubated for 1 h at room temperature. Absorbance was measured at 510 nm against water. Each sample was prepared in triplicate. The same procedure was repeated for the standard solution. The TFC was expressed as catechin equivalents in mg of catechin/g of tea extract.
2.4. Reducing power assay (iron-reducing activity)The total reducing power of four varieties of C. Sinensis-tracts was determined according to the previously described method. Briefly, 2.5 mL of various concentrations of four varieties of tea extracts were mixed with 2.5 mL of 0.2 mol/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50C for 20 min. Next, 2.5 mL of 10% trichloroacetic acid was added and centrifuged at 650 r/min for 10 min. The upper layer (5 mL) of the solution was gently mixed with 5 mL deionized water and1 mL of 0.1% of ferric chloride. After that, the absorbance was measured at 700 nm. Each assay was performed in triplicate and the results are expressed as mean ± SD.
2.5. Determination of total antioxidant capacity using phosphomolybdenum methodThe antioxidant activity of four varieties of tea extracts was determined by the phosphomolybdenum method according to the method described previously. Briefly, 0.3 mL of the extract (200mg/mL, 600mg/mL, and 1000mg/mL) was mixed with3 mL of reagent solution (0.6 mol/L sulfuric acid, 28.0 mmol/L sodium phosphate, 4.0 mmol/L ammonium molybdate). For blank, 0.3 mL of methanol was used instead of tea extract. The mixture was then incubated in water bath at 95C for 90 min. After the reaction became cooled, the absorbance was taken at695 nm. Each sample was prepared in triplicate. The antioxidant capacity of each sample was expressed as ascorbic acid equivalent (AAE) using the following linear equation established using ascorbic acid as standard.
A = 0.0037C + 0.0343;R2= 0.991
where A is the absorbance at 695 nm and C is the concentrationas AAE (mg/mL).
2.6. Bacterial strains and growth conditions bacterial strains were collected from the Department of GeneticEngineering and Biotechnology, University of Dhaka.Shigelladysenteriae(S. dysenteriae), Shigella boydii(S. boydii), Vibriocholerae(V. cholerae), Salmonella typhi(S. typhi), Salmonellaparatyphi(S. paratyphi), Klebsiella pneumonia (K. pneumonia), Escherichia Coli (E.coli) were routinely cultivated in either nutrient agar or broth medium (HiMedia, India) at 37Cwithshaking. Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were cultivated in trypticase soy medium (HiMedia,India) at 37C. Streptococcus sp. were cultivated in brain heart infusion medium (Scharlau Co., EU), if necessary supplemented with 5% sheep blood at 37C in microaerophilic conditions.
2.7. Determination of the antibacterial activity of tea extractAntibacterialactivitywas measured using the standard agar well diffusion method described in earlier studies. Briefly, overnight grown bacteria were adjusted to McFarland turbidity standard (~1.0 × 108CFU/mL) and then 0.1 mL of each culture of bacteria was spread on agar plate surfaces. Wells were made on the agar medium using a sterile borer and the test extracts or control(PBS) were then added into the wells at 100 mg/ml concentration and incubated for 24 h. The zones of inhibition around the wells were observed and measured as millimeters (mm) in diameter after incubation at 37C in microaerophilic conditions. For the antibacterial assay, all bacterial strains were grown in Mueller Hinton agar medium (Difco, USA). ForStreptococcusspp., 5% sheep blood was added for antibacterial assay.