**2.5 Determination of antioxidant enzymes**

One hundred milligrams of leaves were homogenized in 2 ml of 50 mM phosphate buffer (pH 7.8) which contained 0.4 % polyvinyl pyrrolidone (PVP), an inhibitor of phenolic compounds in a pre-chilled mortar and pestle on ice. The homogenate was centrifuged at 10 000×g for 20 min at 4 °C and the supernatant was collected as crude enzyme extraction (Tan et al., 2008).

Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured by monitoring the inhibition of nitro blue tetrazolium (NBT) reduction at 560 nm. The reaction mixture (3 ml) contained 195 mM methionine, 1.125 mM NBT, 3 μM EDTA, and 100 μl of enzyme extract in 50 mM PBS (pH 7.8). After addition of 20 μM riboflavin, the cuvettes were exposed to a 15- W circular "white light" tube for photoreaction 10 min. Then the reaction mixture was measured as absorbance of 1 cm cuvette at 560 nm. One unit of SOD activity was defined as the amount of enzyme per fresh mass sample causing 50 % inhibition of the photochemical reduction of NBT (Beauchamp et al., 1971).

Ascorbate peroxidase (APX, EC 1.11.1.11) activity was assayed by measuring the oxidation of ascorbate at 290 nm according to Nakano and Asada (1981). Total 3 ml of reaction solution contained 50 mM PBS (pH 7.0), 1 mM H2O2, and 1mM ascorbate. The reaction was started by adding 100 μl of enzyme extraction. Changes of absorbance at 290 nm were then recorded within 3 min after the start of the reaction at 1 min intervals.

Catalase (CAT, EC 1.11.1.6) activity was determined according to the method of Zavaleta-Mancera et al. (2007). The total reaction mixture (3 mL) contained 50 mM PBS pH 7.0 and 100 μL of enzyme extract. The reaction was initiated by the addition of 10 mM H2O2. The decomposition was followed directly by the decrease in absorbance at 240 nm every 20 s for 3 min.
