Procedures .1Sample treatment
2.2 Procedures 2.2.1Sample treatment
The vegetables were immediately chopped into small pieces. Then samples were placed into round-bottomed flasks and stored in a freezer (-20 °
C) overnight prior to freeze drying (Virtis, Freeze mobile 25 EL, Gardiner, New York). Each freeze-dried sample was subsequently milled using a food processor (Philips) and passed through
30 mm mesh size sieve. The samples were stored in dark brown bottles and kept in a refrigerator (4 °
C) until used for analysis.
2.2.2 Methanolic extract of sample
Briefly, 500 mg of sample was placed in 15 ml falcon tubes and 5 ml of methanol-HCl (1%) solution was added. The tubes were vortexed and subsequently placed in a roller extractor for 15 min. After that, the tubes were centrifuged at 2790 x g for 20 min at 4 °
C. The mixture was then filtered using Whatman No.1 filter paper and the supernatant were collected. The extraction was repeated three times and the supernatants were combined, dried using rotary evaporator, and stored until further used.
2.2.3 Phenolic analysis
Folin Ciocalteau method by Singleton and Rossi (1965) was applied to determine total phenolic content. Briefly, 0.1 ml extract was placed in a tube, and 0.5 ml Folin Ciocalteu reagent mixed with ultrapure water (1:1) was added, mixed, and allowed to
stand for 8 min. Then, 4.5 ml of 2% sodium carbonate (Na 2 CO 3 ) solution were added, mixed and stored in a dark room for 1 h at room temperature. Absorbance of the resulting blue complex was then measured at 765 nm using a spectrophotometer. Methanol was used as the blank and gallic acid was used as standard. The results were expressed as mg Gallic acid equivalents/100 g dry weight of sample. Data were reported as means ± SD for three replications.
2.2.4 DPPH ( 2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity
The DPPH radical scavenging activity was measured according to method described by Astadi et al ., [18]. In brief, 0.5 ml of freshly prepared 0.5 mM DPPH solution was vigorously mixed with 0.1 ml of extract. Then, 4 ml of methanol was added to the mixture and vortexed thoroughly before allowing to stands for 60 min in a dark at room temperature. The absorbance was then measured using spectrophotometry at 516 nm against a blank. DPPH radical scavenging activity was calculated using the following formula, % DPPH radical scavenging = [(control absorbance-sample absorbance)/control absorbance] x 100%. BHT was used for reference. Data were reported as means ± SD for three replications.
Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a
2.2.5 Ferric Reducing Antioxidant Power (FRAP)
The FRAP was determined following a method reported by Vadivel and Biesalski (2012). Briefly, 1.8 ml freshly prepared FRAP reagent was mixed with 180 μl distilled water and 60 ° μl extract. Then the mixture was incubated at 37 C for 30 min. The
absorbance readings were taken immediately at 593 nm using a spectrophotometer. The reducing power was calculated from the calibration curve prepared with different
concentrations of Fe [II] (FeSO 4 . 7 H 2 O, 100–2000 mM). Methanol and BHT were used for the reagent blank and positive control, respectively.
2.2.6 Superoxide radical scavenging capacity
The superoxide radical scavenging activity of extracts was measured based on method reported by Zhishen et al ., (1999). Briefly, 4.9 ml of reagent (riboflavin, methionine and NBT in 0.05 M phosphate buffer pH 7.8 with final concentration of 3
X 10 -4 , 1 X 10 and 1 X 10 mol/l, respectively) was mixed with 100 μl of extract. The mixture was then illuminated at 25 ° C for 25 min using a 20 W fluorescent lamp. The
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un-illuminated reaction mixture was used as a blank and the absorbance was measured at 560 nm.
2.2.7 Statistical Analysis
All of the experiments were performed in three replications. SPPS version 13 was used for statistical analysis, and significant test was performed with Least Significant Difference (LSD) test. Data were reported as means ± SD for three replications.