The contents of superoxide anion radical and hydrogen peroxide
were extracted and determined according to the method of
Able et al. [31] and Patterson et al. [32].
0.5 g leaves was extracted with 5 ml 5% (w/v) trichloroacetic
acid, and then centrifuged at 16,000g for 10 min. The supernatant
For hydrogen peroxide assay, the extract was divided into two
aliquots of 100 ll. A 20-unit of catalase (CAT, EC 1.11.1.6) was
added to one aliquot (blank). Both the blank and the other aliquot
without CAT were added to 0.5 ml with 100 mM Tris–HCl (pH 8.5),
and then kept at room temperature for 10 min, following the addition
of 0.5 ml colorimetric reagent. The colorimetric reagent was
made daily by mixing 1:1 (v/v) 0.3 mM potassium titanium oxalate
and 0.3 mM 4-(2-pyridylazo) resorcinol monosodium salt. The assay
mixture was incubated at room temperature for 15 min and
then the absorbance at 508 nm was monitored