Chemicals and Reagents Gallic acid, catechin, 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2, 4, 6-tris (2-pyridyl)-1, 3, 5-triazine (TPTZ) standards were purchased from Sigma-Aldrich (St. Louis, MO). Tannic acid, L-ascorbic acid, trichloroacetic acid, ammonium molybdate, sodium carbonate (Na2CO3), aluminum chloride (AlCl3), sodium nitrite (NaNO2), ferrous sulfate heptahydrate (FeSO4.7H2O), sodium hydroxide (NaOH) and methanol were purchased from Merck Co. (Darrmstadt, Germany). Folin-Ciocalteu’s phenol reagent was purchased from LOBA Chemie (Mumbai, India), while Mueller Hinton Agar was purchased from HIMEDIA (Mumbai, India). All of the chemicals and reagents used in this study were of analytic grade.
Sample Collection Mature Apple kul fruits were purchased from the local markets in Savar, Dhaka, Bangladesh during the late winter of February 2012 and were authenticated by Professor Nuhu Alam from the Botany Department, Jahangirnagar University.
Preparation of the Extract The fresh matured Apple kul fruits were thoroughly rinsed with cold water and then cut into small pieces using a sterile stainless steel knife. Then, small pieces of the pulp were mashed using a household blender. To prepare the methanolic extract, the mashed fruit (200 g) was extracted with methanol by a soxhlet extractor for 6 h. The crude extract was concentrated in a rotary evaporator (Buchi, Tokyo, Japan) under reduced pressure (100 psi) and at a controlled temperature (40C). Following this procedure, 22.50 g of the extract was collected and finally preserved at −20C for the subsequent studies.
Phytochemical Analysis Estimation of the Total Polyphenol Contents. The total polyphenol content in the Apple kul extract was determined using a modified Folin-Ciocalteu’s method (Amin et al. 2006). Briefly, the solution (0.4 mL) containing 1 mg of the extract was mixed with 1.6 mL of 7.5% of Na2CO, solution. After mixing, 2 mL of the 10-fold diluted Folin-Ciocalteu’s reagent was added. The final reaction mixture was incubated in the dark for 1 h. In an alkaline solution, any phosphotungustomolybdic acid present in Folin-Ciocalteu’s reagent is reduced by the polyphenols present in the extract to produce a mixture with a very strong blue color. The intensity of the blue-colored complex was measured at 765 nm using a PD-303S Spectrophotometer (APEL, Angyouryou Negishi, Kawaguchi Saitama, Japan). The concentration of the total polyphenol content was determined as gallic acid equivalents (GAEs) at several concentrations (5, 10, 20, 40, 80 μg/mL) and is expressed as mg of GAEs per 100 g of Apple kul.
Estimation of the Total Flavonoid Content. In an alkaline solution, any flavonoid molecules present in the extract will react with sodium nitrite and aluminum chloride to form a colored flavonoid–aluminum complex. The flavonoid content in the Apple kul extract was estimated according to the aluminum chloride colorimetric assay method (Shiv 2011). Briefly, 1 mL of the extract solution containing 1 mg of Apple kul extract was mixed with 4 mL of distilled water. Then, 0.3 mL of 5% NaNO2 was added to the reaction mixture and after approximately 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 of distilled water to make a total volume of 10 mL. Then, the reaction mixture was properly mixed and the intensity of the colored flavonoid–aluminum complex was measured at 510 nm. The concentration of the total flavonoid was determined as catechin equivalents (CEQs; 5, 10, 20, 40, 80 0μg/mL) and the results are expressed as mg of CEQs per 100 g of Apple kul.
Estimation of the Total Tannin Content. The total tannin content in the Apple kul extract was estimated according to Folin-Ciocalteu’s method (Folin and Ciocalteu 1927). Briefly, 0.1 mL of solution containing 1 mg of the extract was mixed with 7.5 mL of distilled water and then 0.5 mL of Folin-Ciocalteu’s reagent was added to the solution. Then, 1.0 mL of 35% Na2CO3 and 0.9 mL of distilled water were added to the solution. The solution was properly mixed and incubated for 30 min. In an alkaline solution, the phosphotungustomolybdic acid present in the FolinCiocalteu’s reagent is reduced by the tannin molecules present in the extract to produce a very strong blue color. The intensity of this developed blue-colored complex was measured at 725 nm. The concentration of the total tannins was estimated as tannic acid equivalents (TEs; 12.5, 25.0, 50.0, 100.0, 200.0 μg/mL) and the results are expressed as mg of TEs per 100 g of Apple kul.
Estimation of the Total Protein Content. The total protein content in the Apple kul extract was estimated using Lowry’s method (Lowry et al. 1951). This method is based on the formation of a copper–protein complex because of the reduction of phosphomolybdate and phosphotungstate (present in Folin-Ciocalteu’s reagent) to heteropolymolybdenum blue and tungsten blue, respectively. Bovine serum albumin (BSA) (0.05–1.00 mg/mL) was used as a standard to prepare a calibration curve and the final results are expressed as g of BSA equivalents per 100 g.
Estimation of Reducing Sugar Content. The content of reducing sugars in the Apple kul extract was estimated according to the Nelson-Somgi method. Briefly, 2 mL of the extract (0.25 mg/mL) and standards were transferred into different test tubes followed by the addition of 2 mL of copper reagent to each tube. The tubes were heated for 15 min in a water bath at 100C before a cooling step. Finally, an arsenomolybdate color reagent (1 mL) was added and the solution was mixed. The absorbance was read at 520 nm. Dextrose was used as a standard for the preparation of the calibration curve (6.25, 12.50, 25.00, 50.00, 100.00 μg/mL) and the reducing sugar content is expressed as g of D-glucose per 100 g of Apple kul.