2.6. Antioxidant activity determined using the
thiocyanate method
The antioxidant activity of extract and purified compound
on inhibition of linoleic acid peroxidation was
assayed using the thiocyanate method (Mitsuda, Yasumodo,
& Iwami, 1966). A 0.5 ml methanol solution of
the isolated compounds was mixed with linoleic acid
emulsion (2.5 ml, 0.02 M, pH 7.0) and phosphate
buffer (2 ml, 0.2 M, pH 7.0). The linoleic acid emulsion
was prepared by mixing 0.2804 g of linoleic
acid, 0.2804 g of Tween 20 as emulsifier and 50 ml
phosphate buffer, and then the mixture was homogenized.
The reaction mixture was incubated at 37 C
to accelerate the oxidation process. The levels of oxidation
were determined by measuring the absorbance at
500 nm on a Hitachi U-2000 spectrophotometer following
reaction with ferrous chloride and ammonium
thiocyanate. The antioxidant activity was expressed as a
percentage of inhibition of peroxidation (IP%):
IP%=[1(absorbance of sample at 500 nm)/(absorbance
of control at 500 nm)]100. The antioxidant
activities of BHA and a-tocopherol were also assayed at
the same concentration for comparison purposes. All
the tests were performed in triplicate and the results
averaged.