Abstract
Ultrafiltration coupled with liquid chromatography-mass spectrometry (LC-MS) was established to screen xanthine oxidase (XO) inhibitors from Pueraria lobata root extract. Four compounds were screened out and identified as puerarin, daidzin, daidzein and genistein with half-maximal inhibitory concentration (IC50) values of 30.8, 5.31, 14.5 and 3.02 µg mL−1 on XO, respectively. The interactions between these compounds and XO were investigated by fluorescence spectroscopic method. The hydrogen peroxide induced oxidative stress model of human normal gastric epithelial cell lines (GES-1) was used to investigate the protections on injured cell. As a result, four XO inhibitors exhibited protective effects without cytotoxicity. With the increased concentrations of four inhibitors, cell viability was higher with decreased mortality rate, the decrease of superoxide dismutase activity, leakage of lactate dehydrogenase and increase of intracellular superoxide anion production induced by hydrogen peroxide were restrained. It showed that these four XO inhibitors could effectively enhance cell viability and protect injury of GES-1 cells from oxidative stress.
Highlights
Natural extracts with bioactive compounds play an invaluable role in drug discovery process, in the areas of cancer and infectious diseases.[1]
The interactions between four inhibitors and xanthine oxidase (XO) were investigated by fluorescence spectroscopic methods, and the protection of four inhibitors on hydrogen peroxide (H2O2) induced oxidative stress model of human normal gastric epithelial cell lines (GES-1 cells) was conducted by evaluating cell viability, intracellular reactive oxygen species (ROS), superoxide dismutase (SOD), lactate dehydrogenase (LDH) release and apoptosis assays
The results indicated that the P. lobata root extract contains XO inhibitors and the following screening and identification of XO inhibitors from extract are worthwhile
Summary
Natural extracts with bioactive compounds play an invaluable role in drug discovery process, in the areas of cancer and infectious diseases.[1]. The quantitative analysis of LDH was completed with commercially available LDH Cytotoxicity Assay Kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions.[35] The GES-1 cells were exposed to 250 μmol L−1 H2O2 in the presence of 10, 25, 50 and 100 μmol L−1 of four screened compounds for 72 h. Identification of screened XO binders daidzein (3) and genistein (4) (the structures were shown in Figure 2).[36,37,38] The chromatograms of four authentic references were shown in Figure 3 together with that of P. lobata root extract.
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