Pyrogallol solution

β-Carotene, trichloroacetic acid solution, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) reagent, phosphate buffer solution, pyrogallol solution, dipotassium hydrogen phosphate, potassium hydroxide, potassium dihydrogen phosphate, hydrogen peroxide from Sigma Aldrich, Darmstadt, Germany and diazepam from Pfizer, Karachi, Pakistan. All chemicals were purchased from the local market.

Rotenone (Catalog number (Cat.) R 8875, Sigma, St. Louis, MO, USA) solution was freshly prepared in dimethylsulfoxide (DMSO) (0.5 mg/ml) and adjusted to pH 7.4 with potassium hydroxide. The solution was immediately used because it is only stable at 25°C for 24 h. Curcumin (Cat.: 458‐37‐7), acetylcholinesterase (AChE) (Cat. C2888), and pyrogallol solution (Cat. 254002) were purchased from Sigma‐Aldrich, Inc. (St Louis, MO, USA). ELISA kits for reduced glutathione (GSH) (Cat.: MBS724319) and lipid peroxidation (malondialdehyde, MDA) (Cat.: MBS738685) were purchased from MyBioSource. Astrocyte‐specialty glial fibrillary acidic protein (GFAP) was purchased from Lab Vision Corp, Medico Co., Egypt (Cat. # MS‐280‐B0), rabbit anti‐tyrosine hydroxylase (TH) polyclonal antibody (Cat #ab117112) was purchased from Abcam, and biotinylated secondary antibody (horse anti‐mouse IgG antibodies) was obtained from Vector Laboratories, Burlingame, CA (BA‐2000‐1.5).

The method is based on spectrophotometric monitoring of the inhibition of pyrogallol autoxidation as described by Xie et al. [29 (link)] with slight modifications. Briefly, the samples were diluted with 50 mM TRIS-HCl buffer containing 1 mM EDTA (pH 8.3) to a final concentration of 5 mg/mL, and an aliquot of 0.16 mL was conveyed into a clear microplate well. After that, 0.16 mL of 1.5 mM pyrogallol solution (Sigma Aldrich) was put into each well, and the plate was incubated at 25 °C in the dark for 8 min. The absorbance was measured at 320 nm immediately after the addition of the pyrogallol over 8 min using a microplate reader (Multiscan GO, Thermo Fisher Scientific, Waltham, MA, USA). The SRS activity was calculated as follows: $$SRS (\%)=\frac{{\left(\frac{\mathsf{\Delta }\mathrm{A}}{min}\right)}_b-{\left(\frac{\mathsf{\Delta }\mathrm{A}}{min}\right)}_s}{{\left(\frac{\mathsf{\Delta }\mathrm{A}}{min}\right)}_b}·100$$
where b and s are blank and sample, respectively.