Evaluating Antioxidant Profiles of Pomegranate Cultivars

ChemicalsAll reagents and solvents were purchased from Merck (Darmstadt, Germany) and Sigma (St. Louis, MO) unless otherwise mentioned. All chemicals used in the experiments were of analytical grade.
Sample preparationNine cultivars of pomegranate were donated from Saveh Agricultural Investigation Center during September 2007. A total of 27 pomegranate fruits (three numbers of each cultivar) were collected and washed three times with distilled water. To prepare pomegranate extract, fresh fruits were peeled and their edible portions (seed coats and juice) were separated. 30 g of pulps and peels were weighted and extracted separately for 4 h by Soxhlet apparatus with acetone, followed by ethyl acetate, methanol and water solvent respectively (four hours for each solvent) to extract different kinds of antioxidant components. The four different extracts of every cultivar were mixed and dried on a water bath. 1 g of pulp and peel extracts were dissolved and diluted with methanol 80% (v/v) to 25 mL. To assay total antioxidant and phenolic content, pulp extracts were diluted 1 : 10 (v/v) where it was 1: 100 (v/v) for peel extracts by 80% methanol.
Total flavonoid assay Total flavonoid content was measured by the aluminum chloride colorimetric assay (21 ). An aliquot (1 mL) of extracts or standard solution of catechin (50, 100, 150, 200, 250 and 300 mg/L) was added to 10 mL volumetric flask containing 4 mL of double distilled water. Then 0.3 mL 5% NaNO₂ was added to the flask and after 5 min, 0.3 mL AlCl₃ (10%) was also added. At 6^th min, 2 mL NaOH (1 M) was added and the total volume was made up to 10 mL with double distilled water. The solution was mixed completely and the absorbance level was measured versus prepared reagent blank at 510 nm. Total flavonoid content was expressed as mg catechin equivalents (CE) per one gram dry extract. The total flavonoid assay was measured three times for each pomegranate extract.
Total phenolic assayTotal phenolics were determined using Folin-Ciocalteu reagent as described by Velioglu et al. (22 ) with slight modifications. The extract (200 μL) was mixed with 1.5 mL of Folin-Ciocalteu reagent (previously diluted 10 times with distilled water) and allowed to stand at room temperature for 5 min. 1.5 mL sodium bicarbonate solution (60 g/L) was added to the mixture and after incubation for 90 min at room temperature, the absorbance level was measured at 750 nm using a UV-Visible spectrophotometer (GBC, Cintra 40). Total phenolics were quantified by calibration curve obtained from measuring the absorbance of the known concentrations of gallic acid standard solutions (25-150 μg/mL in 80% methanol). The results were calculated as gallic acid equivalent (GAE) per one gram dry extract and reported as mean value ± SD.
Total antioxidant activityThe FRAP (ferric reducing antioxidant power) assay was described initially by Benzie and Strain (23 ). It is based on the reduction of a ferric tripyridyl triazine complex to its ferrous blue colored form in the presence of antioxidants. It is a relatively simple method frequently used in the assessment of antioxidant activity of various fruits, vegetables and some biological samples (24 ).
Briefly, the FRAP reagent contained 5 mL of TPTZ (2, 4, 6-tripyridyl-s-triazine) solution (10 mmol/L) in HCl (40 mmol/L) plus 5 mL of FeCl₃ (20 mmol/L) and 50 mL of acetate buffer (0.3 mol/L, pH 3.6). It was freshly prepared and warmed up to 37°C. A volume of 100 μL pulp or peel extract was mixed with 3 mL of FRAP reagent and the absorbance of the reaction mixture was measured at 593 nm after incubation at 37°C for 10 min. To construct calibration curve, five concentrations of FeSO₄ 7H₂O (1000, 750, 500, 250, 125 μmol ∕ L) were used and the absorbance were measured as sample solution. The values were expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of 1 mmol/L FeSO₄ and also as vitamin E and C equivalent. All the measurements were taken in triplicate and expressed as mean value ± standard deviation (SD).
Statistical analysisThree replicates of each sample were used for statistical analysis and the values were reported as mean ± SD. Pearson’s correlation and regression analysis were carried out using SPSS statistical program to study the relationship between antioxidant activity and total phenolic and flavonoid content. Data were also subjected to the analysis of variance and mean values were compared by Dunnett T3 post-hoc multi-comparison tests. Differences at p < 0.05 were considered to be significant.