Assay of ABTS.

+ scavenging activity

This method measures the antioxidant activity of both water-soluble and lipid-

soluble antioxidants, as well as extracts from natural sources (Erkan et al., 2008). ABTS
.
radical cation (ABTS +) scavenging activity was determined according to the method

.
decribed by Re et al. (1999) with some modify. ABTS + was produced by reacting 38.43

mg ABTS (final concentration 7 mM ) and 6.90 mg potassium persulfate (final

concentration 2.55 mM) and 10 mL demineralised H2O in an Erlenmeyer flask and keeping
.
the mixture in the dark at 26 ± 3 ºC for 12 h. An aliquot the blue–green ABTS + solution

thus obtained was diluted with 95 % ethanol to give an absorbance of 0.70 ± 0.02 at 734

nm. ABTS.+ adjusted solution (1 mL) and ethanol (0.02 mL) was mixing in a semi-

microcuvette and absorbance at 734 nm corresponding to blank (E1) was recorded

(Spectronic Genesys 5, Germany). Then test sample (0.02 mL) was added into semi-

microcuvette and mixed with a disposable spatula. For each extract, concentrated extract as

well seven dilutions extract (1:5, 1:10, 1:20, 1:150, 1:80, 1:100 and 1:125 extract:H2Od)

were analysing. The reaction mixture was allowed to stand for exactly 6 min (E2). The

ABTS.+ scavenge % was calculated as:

 E1  E 2 
ABTS. scavenge %    *100
 E1 

The ABTS.+ scavenge % was plotted against the antioxidant extract concentration.

Only values concentrtion between 20-80 ABTS.+ scavenge % were considered to make the

curve.
 The radical-scavenging activity was expressed in terms of IC50 value (the

concentration of the antioxidant sample required to scavenge 50% of ABTS.+) calculated by

a linear regression analysis.