Analysis of Xanthine Oxidase Inhibitors fromPuerariae flosUsing Centrifugal Ultrafiltration Coupled with HPLC-MS

Abstract

In this study, centrifugal ultrafiltration coupled with high performance liquid chromatographymass spectrometry was utilized to screen and identify xanthine oxidase inhibitors from Puerariae flos extract. The experimental conditions of centrifugal ultrafiltration including xanthine oxidase concentration, incubation time, pH and temperature were optimized. At the optimum condition (xanthine oxidase concentration: 30.0 μg mL -1 , incubation time: 20 min, pH 7.0 and temperature: 25 °C), four compounds were successfully screened from P. flos extract and identified as tectoridin, daidzin, ononin and biochanin A. The yields of tectoridin, daidzin, ononin and biochanin A were 0.231, 0.117, 0.303 and 0.089 g from 50.0 g crude P. flos samples. The inhibitory activities of these compounds were verified by xanthine oxidase inhibition assays. The experimental half maximal inhibitory concentration (IC 50 ) values of tectoridin, daidzin, ononin and biochanin A were 88.5, 85.1, 88.8 and 87.0 μmol L -1 , and the binding degree of them were 5. 70, 8.28, 6.31 and 37.83% at the optimum condition, respectively. The proposed method provided a rapid and effective way to screen and analyze active compounds from natural products. Keywords: HPLC-MS, inhibitor, Puerariae flos, ultrafiltration, xanthine oxidase IntroductionNatural products have been used as the most consistently successful resources of new drug discovery for a long time because of their great diversity of the chemical structures and better drug-like properties compared to the synthetic compounds.1 However, natural products resources like plant extracts were very complex and usually contained various kinds of components. Separation and purification of natural compounds were time-consuming and laborious processes.2 Hence, simple and effective methods aiming at directly screening natural product extracts would be greatly helpful for drug discovery. Centrifugal ultrafiltration (CU) utilized centrifugal force and a semi-permeable membrane to retain suspended solids and high molecular weight solutes, while liquid and low molecular weight solutes were allowed to pass through depending on the nominal molecular weight cut-off of the membrane. 4 Based on these features, active compounds could be retained by membrane together with enzyme due to the binding with enzyme. Thus, CU became a useful technique for screening active compounds bound to biomacromolecules such as bovine serum albumin, 5 α-glucosidase, 6 quinone reductase-2, 7 deoxyribonucleic acid (DNA) and liposomes. 8,9 High performance liquid chromatography-mass spectrometry (HPLC-MS or LC-MS) has been widely applied for the simultaneous separation and identification of active compounds in complex mixtures. 10The combination of CU and LC-MS (CU-LC-MS) became a powerful tool in analyzing active compounds due to its simple operation, high speed and low sample consumption. Compared with immobilized enzyme screening assay, complex synthesis procedures could be avoided in CU-LC-MS as well.11 Moreover, the efficient screeni...

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