13 Superoxide dismutase (SOD) activity in liver homogenate was determined according to the method described by Nandi and Chatterjee.14 This method is based on the ability of SOD to inhibit the auto-oxidation of pyrogallol at an alkaline pH. One unit of SOD is described as the amount of enzyme required to cause 50% inhibition of pyrogallol auto-oxidation. The glutathione (GSH) content

in the liver homogenate was determined using the method of Van Dooran et al.15 The basis of the GSH determination method is the reaction of Ellman’s reagent (5,5′-dithiobis-[2-nitrobenzoic acid]) with thiol groups of GSH at pH 8.0 to produce the yellow 5-thiol-2-nitrobenzoate anion. Glutathione S-transferase (GST) activity was determined according to the method of Habig et al.16 In this assay, GST catalyzes the conjugation of GSH with 1-chloro-2,4-dinitrobenzene, producing Dorsomorphin molecular weight a chromophore at 340 nm. The total protein contents of liver tissues were determined according to the Lowry method as modified by Peterson.17 Absorbances were recorded using a Shimadzu recording

spectrophotometer (UV-160) in all measurements. Liver cancer cell MI-503 manufacturer line HepG2 were maintained in Roswell Park Memorial Institute-160 medium with 10% fetal bovine serum and 1% of 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C inside a humidified incubator with 5% CO2 and 95% room air. Cells were subcultured every 4-7 days with trypsin/ethylenediamine tetraacetic acid (1:250; PAA Laboratory, Germany). Cells were treated with several concentrations of saffron extract for several time points. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) proliferation assay was used in the HepG2 cell line to assess the effect on cell proliferation of a range of concentrations of saffron extract.

Cells (104) were plated and grown in 200 μL of growth medium in 96-well microtiter plates. After an overnight attachment period, cells were treated with varying concentrations of saffron extract (1.0, 2.0, 4.0, and 6.0 mg/mL) prepared from a 100 上海皓元医药股份有限公司 mg/mL stock solution dissolved in water. All studies were performed in triplicate and repeated three times independently. Cell growth was quantified by the ability of living cells to reduce the yellow dye MTT to a purple formazan product. Cells were incubated with MTT (Sigma) at 37°C in a humidified 5% CO2 atmosphere for 2 hours. The MTT formazan product was then dissolved in dimethylsulfoxide, and absorbance was measured at 570 nm in a microplate reader. One day before treatment, cells were seeded at a density of 1.2 × 106 cells per plate. After the indicated times, the cells were harvested by trypsin release, washed twice with phosphate-buffered saline, fixed with 70% ethanol, treated with 1% ribonuclease, and finally stained with propidium iodide (100 μg/mL final concentration).