U 2000 double beam uv vis spectrophotometer

10 times the phosphate-buffered saline (PBS) was added to the homogenized kidney. It was centrifugated at 4°C with 12000 rpm for 15 minutes. The supernatant was used to measure malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT). In the present study, the contents of malondialdehyde (MDA) of renal tissue were evaluated according to the thiobarbituric acid (TBA) method, similar to the method described by Buege and Aust [32 ]. Absorption was read at 532 nm by spectrophotometer (Hitachi's U-2000 Double-Beam UV/Vis Spectrophotometer). Renal levels of GSH were determined in accordance with the Ellman method [33 (link)]. GPX activity was measured by the Rotruck et al. method. In brief, tert-butyl hydroperoxide and hydrogen peroxide (H₂O₂) were mixed with the kidney sample, and then, its absorption was read by the ELISA reader at 420 nm [34 , 35 (link)]. SOD activity was measured by the Minami et al. method [36 (link)]. For the CAT reaction, 0.7 ml of potassium phosphate buffer (pH: 7.0), 0.1 ml of hydrogen peroxide (H₂O₂), and 100 μl of a homogenized renal sample were mixed, and its absorption at 240 nm was read by a spectrophotometer (Hitachi's U-2000 Double-Beam UV/Vis Spectrophotometer). [37 (link)].

All chemicals used were purchased from Aldrich Chemical Co., Ltd. (St. Louis, MO, USA), and were of highest commercially available purity. Silica gel for column chromatography (CC) was performed on silica gel (Merck silica gel 60H, particle size 5–40 μm) and Sephadex LH-20 gel. Thin layer chromatography (TLC) was performed on Merck aluminium backed plates, precoated with silica (0.2 mm, 60F254). UV spectra were recorded on Hitachi's U-2000 Double-Beam UV/Vis Spectrophotometer. Mass spectra were carried out on a Thermo Finnigan PolarisQ Ion Trap system (Waltham, MA, USA) using a direct exposure probe. Nuclear magnetic resonance ¹H NMR and ¹³C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Varian Gemini spectrometer (Palo Alto, CA, USA). Melting points were determined using a Stuart SMP10 melting point apparatus (Stone, Staffordshire, UK).
Diazepam was kindly donated by the Department of Pharmacy, University of Peshawar. Imipramine, DMSO, Tween solution (TWEEN® 80), flumazenil (Sigma-Aldrich, USA), and pentylenetetrazol (Tokyo Chemical Industries Co., Ltd.) were purchased for the study. All chemicals and solvents used in this research were of analytical grade.