Dichromate

The CAT activity was assayed following the method of Sinha (1972) (link). In this method, dichromate (Sigma–Aldrich) in acetic acid (Sigma–Aldrich) is reduced to chromic acetate when heated in the presence of H₂O₂. The blue perchromic acid, an unstable intermediate is then formed. The reaction mixture consisted of 187.5 μl phosphate buffer (0.1 M, pH 7.5) and 12.5 μl of homogenate. The reaction was started by adding 50 μl of H₂O₂ (50 mM, Sigma–Aldrich). After 1 min, the reaction was stopped by the addition of 500 μl of dichromate acetic acid reagent. The tubes were immediately kept in a boiling water bath at 100°C for 10 min, and the green color developed during the reaction was read at 570 nm on a spectrophotometer against the blank. Blank tube, devoid of enzyme, was also processed in parallel. The amount of H₂O₂ remaining was determined using a standard curve. The enzyme activity was expressed in mmol of H₂O₂ consumed/min/mg of protein in brain tissue. The specific activity of CAT was calculated as follows: CAT activity = [(A of sample – A of blank) × f/(a × t × mi)]. Where A is the absorbance, f the dilution factor, a standard curve coefficient, t the time in minute and mi the weight of tissue processed.

Vitamin C, PTZ, scopolamine methyl nitrate, diethyl ether, pilocarpine hydrochloride, sodium valproate, Biuret reagent, acetylcholine iodide, 5′5-dithiobis-(2-nitrobenzoic acid) (DNTB), adrenaline, acetic acid, dichromate, hydrogen peroxide (H₂O₂), Tris-Hcl, trichloroacetic acid, thiobarbituric acid, sodium phosphate buffer, Griess reagent were purchased from Sigma Chemical Co., St. Louis (United States), while diazepam was purchased from Roche, Neuilly sur-Seine, France. The minimal dose of chemoconvulsant at which 99% of the animals showed a convulsion was determined based on the doses used by other researchers and by a dose-percentage effect curve (Miller and Tainter, 1944 (link); Ahmadiani et al., 2003 (link)). Vitamin C and sodium valproate were dissolved in distilled water. All solutions were prepared freshly in the day of the experiment and were administered intraperitoneally at a volume of 10 ml/kg, except for distilled water and aqueous extract of P. daemia administered per os at the same volume.