Antioxidant assay (Free radical scavenging effect): About 100 μL of sample with various concentration (1, 0.5, 0.25 and 0.125%) was diluted with 1800 μL methanol p.a then was mixed with 200 μL of DPPH (1000 ppm). Incubation of mixed reagent and samples were performed for 30 min, at room temperature. Then the absorbance was measured using Hitachi spectrophotometer U 2000 at 517 nm.
Total phenolic content: Total phenolic contents of extract were determined by using Folin-Ciocalteu reagent and external calibration with pyrogallol. Briefly, 100 μL of extract (1 % w/v) was mixed with 4.5 mL of aquadest then added with 100 μL of Folin-Ciocalteau reagent and the reagents were mixed throughly. After three minutes 300 μL of Na2CO3 was added and then the mixtures were allowed to stand in room temperature for 2 h. The absorbance was measured at 760 nm using a HITACHI, U-2000 spectrophotometer. The estimation of total phenolic in the extract was determined as equivalent mg g-1 of pyrogallol through an equation obtained from pyrogallol calibration curve (y = 2.5957x +0.0784, R2 = 0.9857).