Aflatoxin b1 standard

Aflatoxin B₁ standard, paraquat, sodium ascorbate, and lyophilized powder of Cu/ZnSOD from bovine erythrocytes were purchased from Sigma-Aldrich (#S7571; St Louis, MO, USA). MitoSOX and DHE were purchased from Thermo Fisher Scientific (Waltham, MA, USA). paraquat and sodium ascorbate were dissolved in water to be 100 mM and 3 M, respectively. Bovine Cu/ZnSOD was dissolved in phosphate buffered saline (pH7.4) to be 10 U/μL. According to Sigma-Aldrich, one unit will inhibit reduction of cytochrome c by 50% in a coupled system with xanthine oxidase at pH 7.8 at 25 °C in a 3.0 mL reaction volume. Enzyme concentration is about 3000 units/mg protein.

Aflatoxin was extracted from top-agar inoculated cultures (three cores—16 mm diameter) with 5 ml chloroform. The chloroform phase was then collected and allowed to evaporate overnight. Dried residues were resuspended in 300 μl of chloroform. Twenty-five microliters of each extracts were separated by thin layer chromatography (TLC) as previously described [37 (link)] on silica plate (Si250F, J.T. Baker) using chloroform:acetone (85:15,v/v) as solvent system. Then plates were allowed to air dry, sprayed with 12.5% AlCl₃ solution in 95% ethanol and baked at 80°C for 10 minutes. Presence of aflatoxin was detected under ultraviolet light at a wavelength of 375 nm. The aflatoxin B₁ standard was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).