2.1. 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) were purchased from SigmaAldrich (St. Louis, MO, USA). L-ascorbic acid, tannic acid, Folin-Ciocalteu’s phenol reagent, and ferrous sulfate heptahydrate (FeSO4 7H2O) were purchased from Merck Co. (Darmstadt, Germany). All of the chemicals and reagents used in this study were of analytical grade.
2.2. Turmeric Collection. Two different turmeric varieties (“mura” and “chora”) were collected from the Khulna district of Khulna division and the Khagrachari district of Chittagong division in Bangladesh in July 2013. Following collection, the turmeric samples were packed into sterile polybags before transportation to the Laboratory of Preventive and Integrative Biomedicine in the Biochemistry and Molecular Biology Department, Jahangirnagar University, Savar, Dhaka, Bangladesh.
2.3. Extract Preparation and Yield Determination. Turmeric samples were cleaned and air-dried in the shade for two days before being ground to a fine powder in a blender (CM/L7360065, Jaipan, Mumbai, India). The fine powder was used to prepare both ethanolic and aqueous extracts based on Kang’s method [13] with slight modification. Briefly, 20% ethanolic extract was prepared by adding turmeric powder (20 g) in 70% ethanol solution to make a 100 mL solution. Similarly, for 20% aqueous extract preparation, 20 g of turmeric powder was dissolved in water to make a 100 mL solution. Both ethanol and aqueous extract solutions were placed in the dark to avoid reactions that may occur in the presence of light and were shaken in a shaker for 72 h at room temperature. Then, the solutions were filtered through Whatman No. 1 filter paper and concentrated in a rotary evaporator (Buchi, Tokyo, Japan) under reduced pressure (100 psi) at 400C (for ethanol) and 550C (for water). The dried extracts were collected and preserved at −200C for subsequent analysis. The percentage of yield of the extracts was determined according to the following formula: % yield = [weight of sample extract/initial weight of sample] × 100. Eight different turmeric extracts were prepared for antioxidant analysis.
2.4. Phytochemical Analysis 2.4.1. Estimation of Total Polyphenol Content. The total polyphenol content (TPC) of the turmeric extracts was estimated spectrometrically according to the Folin-Ciocalteu method and adopted by Afroz et al. Briefly, 0.4 mL of the extract (0.25 mg/mL) was mixed with 1.6 mL of 7.5% sodium carbonate solution. Then, 2 mL of 10-fold diluted Folin-Ciocalteu 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 PD303S spectrophotometer (APEL, Japan). The total polyphenol content present was determined as gallic acid equivalent (GAE) (6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 g/mL, 2 = 0.9970) and was expressed as g of GAE/100 g of turmeric.
2.4.2. Estimation of the Total Flavonoid Content. The total flavonoid content (TFC) was estimated using an aluminum chloride colorimetric assay [16]. First, 1 mL of the extract (1 mg/mL) was mixed with 0.3 mL of 5% sodium nitrite and added to the reaction mixture. After approximately 5 min, 0.3 mL of 10% aluminum chloride was added. Subsequently, after 6 min, another 2 mL of 1 M sodium hydroxide (NaOH) was added, followed by the immediate addition of 2.4 mL of distilled water to produce a total volume of 10 mL. The color intensity of the flavonoid-aluminum complex was measured at 510 nm. The total flavonoid content was determined as catechin equivalent (CE) (6.25–200.00 ?g/mL) and was expressed as g of CE/100 g of turmeric. 2.4.3. Estimation of the Total Tannin Content. The total tannin content (TTC) in the turmeric extracts was estimated using the Folin-Ciocalteu method with tannic acid as a 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 reagent was added. To the above mixture, 1 mL of 35% sodium carbonate and 0.9 mL of distilled water were added. 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 g of tannic acid equivalent (TE) per 100 g of turmeric. 2.4.4. Determination of the Ascorbic Acid Content. The ascorbic acid content (AAC) in the turmeric samples was estimated as described by Omaye et al. [18] with slight modifications. Briefly, 1 mL of extract (500 mg/mL) was mixed with 1 mL of a 5% trichloroacetic acid (TCA) solution, followed by centrifugation for 15 min at 3500 rpm. Then, 0.5 mL of the supernatant was mixed with 0.1 mL of DTC (2,4- dinitrophenylhydrazine/thiourea/copper) solution and incubated for 3 h at 37? C. To the mixture, 0.75 mL of ice-cold 65% sulfuric acid (H2SO4) was added. The solution was allowed to stand for an additional 30 min at room temperature. The developed colored complex was monitored at 520 nm. The ascorbic acid concentration was determined as ascorbate equivalent (AE) and expressed as mg of AE per 100 g of turmeric.
2.5. Antioxidant Activity. The antioxidant activity of the turmeric samples was determined using the DPPH radicalscavenging activity and FRAP values. 2.5.1. DPPH Free Radical-Scavenging Activity. The antioxidant activities of all turmeric extracts were evaluated according to the DPPH radical-scavenging activity as described by Braca et al. [19]. Briefly, 1 mL of the extract was mixed with 1.2 mL of 0.003% DPPH in methanol at varying concentrations (2.5–80.0 ?g/mL). The percentage of DPPH inhibition was calculated using the following equation:
2.5.2. Ferric Reducing Antioxidant Power (FRAP) Assay. The FRAP assay was performed as described by Benzie and Strain. The reduction of a ferric tripyridyltriazine complex into its ferrous form produces an intense blue color at low pH that can be monitored by measuring the absorbance at 593 nm. Briefly, 200 ?L of the extract solution at different concentrations (62.5–1000.0 ?g/mL) was mixed with 1.5 mL of the FRAP reagent, and the reaction mixture was incubated at 37? C for 4 min. The FRAP reagent was prepared by mixing 10 volumes of 300 mM acetate buffer (pH 3.6) with 1 volume of 10 mM TPTZ solution in 40 mM hydrochloric acid and 1 volume of 20 mM ferric chloride (FeCl3⋅6H2O). The FRAP reagent was prewarmed to 370 C and was always freshly prepared. A standard curve was plotted using an aqueous solution of ferrous sulfate (FeSO4⋅7H2O) (100–1000 0?mol), with FRAP values expressed as micromoles of ferrous equivalent (0?M Fe [II] per 100 g of sample).
2.6. Statistical Analysis. All analyses were performed in triplicate, and the data are reported as the mean ± standard deviation (SD). Data were analyzed using SPSS (Statistical Packages for Social Science, version 16.0, IBM Corporation, NY, USA) and Microsoft Excel 2007 (Redmond, WA, USA). Statistical analyses of the biochemical data were conducted using Tukey’s test. ? < 0.05 was considered statistically significant.