Screening Potential Xanthine Oxidase Inhibitors From Natural Products In Vitro

In recent years, the incidence of gout is rising. Gout is a metabolic disease caused by purine metabolic disorder. Hyperuricemia is the most important biochemical basis of gout. For gout patients, the accumulation of uric acid was rapid in vivo but excretion of that was slow. Too much uric acid in the body leads to the deposition of urate crystals in the joints and organs. Uricosuric and inhibiting the production of uric acid are the two basic means for the treatment of gout. Xanthine oxidase inhibitors can reduce the number of fatal complications of gout but side effects can not be ignored in clinical. Traditional Chinese medicine(TCM) has been a long history for the treatment of gout in China. The chemical compounds from TCM have various structures and targets, as well as small side effects. Therefore, screening efficiency and low toxicity xanthine oxidase inhibitor from natural products has a bright future.Currently, the screening methods for xanthine oxidase inhibitors include ultraviolet spectrophotometry, electrochemical method and ultra-high performance liquid chromatography. Due to their disadvantage, it is necessary to establish a simple, accurate and suitable method to screen efficiency and low toxicity xanthine oxidase inhibitors from natural products.Objectives: To develop a method for screening xanthine oxidase inhibitor from natural products in vitro. In order to obtain the lead compouds, bioassay-guide isolation of TCM were conducted by above method.Methods: The activity of xanthine oxidase inhibitor was evaluated by the concentration of xanthine assayed by HPLC. The chromatographic conditions were as follows: column, Agilent SB-C18(4.6 mm ? 250 mm, 5 ?m); mobile phase, 0.02 mol·L-1 KH2PO4(1% methanol); flow rate, 1.0 m L·min-1; detection wavelength, 254 nm; injection volume, 20 ?L; column temperature, room temperature. Using this method, xanthine oxidase inhibitory activity of 14 TCMs(Caesalpinia sappan, Aralia chinensis, Paeonia suffruticosa, cortex fraxint, Polygonum cuspidatum, radix clematidis, stigmata maydis, rhizoma smilacis glabrae, desmodium, Terminalia chebula, flos puerariae, radix gentianae macrophyclae, rhizome alpiniae officinarum and cortex ailantht) were evaluated.Results: Xanthine was separated perfectly from the other components with the selected chromatographic conditions. Method validation indicated a good linearity, precision, stability, repeatability and recovery of established HPLC method. The experiment of screening xanthine oxidase inhibitory activity of 14 TCMs showed that 70% ethanol extract of T. chebula, C. sappan, P. cuspidatum and P. suffruticosa had significant activity with IC50 of 10.04, 8.02, 2.06 and 1.36 mg·m L-1, respectively. A phytochemical investigation on the Et OH extract of P. suffruticosa resulted in the isolation of fiftteen compounds. Their stuctures were identified as gallic acid(1), methyl gallate(2), ethyl gallate(3), quercetin(4), catechin(5), daucusterol(6) paeoniflorin(7), mudanpioside D(8), 4-O-methyloxypaeoniflorin(9), galloyl-oxypaeoniflorin(10), paenonol(11), apiopaeonoside(12), auffruticoside B(13), paeonolide(14) and suffruticoside D(15). Componds 1-3, 10, 13, 15 showed significant activity with IC50 of 2.04, 1.91, 3.60, 0.75, 1.28, 0.97 mmol·L-1. In additon, the activity of 24 compounds isolated from C. sappan previously by our group were also evaluated. 7,3’,4’-trihydroxy-3-benzyl-2H-chromene, 3-deoxysappanone B, sappanchalcone and protosappanin A showed moderate xanthine oxidase inhibitory activity.Conclusion: This bioassay method based on HPLC is simple, accurate and suitable for screening xanthine oxidase inhibitors in vitro, which providing a new way to screen xanthine oxidase inhibitors from natural products. Eight componds with xanthine oxidase inhibitory activity from P. Suffruticosa were obtained by above method.