Fundamental Study On Inhibiting Xanthine Oxidase From Medicago Sativa And Citrus

Xanthine Oxidase(XOD) is the important enzyme in the metabolism of nucleic acids in vivo, which can catalyze the production of uric acid by intensifing oxidation of xanthine and hypoxanthine with the releasing of the free radical. High serum uric acid levels will lead to hyperuricemia and gout. The cornerstone of effective gout management is persistent reduction of s UA levels, which can be achieved by XO inhibitors.According to the related reports, Melilotus Albus belonging to Melilotus and Aurantii Fructus Immaturus, Citri Reticulatae Pericarpium Viride, Citri Reticulatae Pericarpium,Citri Exocarpium Rubrum, Fructus Citri Sarcodactylis, Citri Fructus belonging to medicinal and edible Chinese materia medica in Citrus are in rich of flavonoids.Flavonoids have strong biological activity, which can blocking the production of uric acid by inhibiting XOD activity. This comprehensive analysis combinated the establishment of the HPLC fingerprints of different components from Melilotus albus and Citrus herbs with in vitro xanthine oxidase test, and in order to separate and identify the activity compounds and clarify its material basis and mechanism of action.This study established Melilotus Albus and citrus herbs extracts HPLC analysis method by optimizing different chromatographic column, mobile phase and detection wavelength, and degree of enzyme inhibition was calculated by measuring the formation amount of uric acid in the xanthine oxidase-xanthine reaction system by HPLC.According to the results date, all extracts have inhibitory activity except pure water extract for Melilotus Albus, ethyl acetate extract has higher inhibitory activity,inhibition is 66.93 ± 1.52% with concentration of 200 μg/m L. And for citrus herbs extracts, ethyl acetate extracts of Aurantii Fructus Immaturus and Citri Reticulatae Pericarpium have higher inhibitory activity, the inhibition is 60.70 ± 0.74% and 50.15 ± 0.44% respectively,with concentration of 200 μg/m L.By using eighteen alkylbonded silica gel and preparative liquid chromatography, and other means to separate the 2 activity chemical compounds from Melilotus Albus, the structure of the compounds was identified by 1H-NMR and 13C-NMR,their identification results:apigenin(compound 1);tricin(compound 2). We separated seven compounds from the ethyl acetate extract of Aurantii Fructus Immaturus, the five flavanones were identified as naringin(compound 3), hesperidin(compound 4), nohesperidin(compound5), naringenin(compound 6), hesperetin(compound 7), respectively, and the two polymethoxyflavones were nobiletin(compound 8), targeretin(compound 9).The five activity compounds apigenin, tricin, hesperetin, nobiletin and tangeretin were found by in vitro xanthine oxidase inhibitor experiment. Apigenin, tricin, hesperetin and nobiletin have higher inhibitory activity, IC50 is 0.42μM, 4.13μM, 16.48μM and107.53μM. The inhibitory mode is determined by Lineweaver–Burk plots of different concentrations and the intersection of these lines. The inhibitory mode of apigenin, tricin,hesperetin are mixed and the nobiletin is noncompetitive. Tricin was first separated from Melilotus Albus and xanthine oxidase direct inhibiting effect of tricin and nobiletin are reported for the first time.