| Xanthine oxidase (EC1.2.3.2， XOD) is a kind of composite flavin enzyme,widely distributed in bacteria, fungi and mammalian tissue. It is a key enzyme in theprocess of nucleic acid metabolism in vivo, which catalyzes xanthine andhypoxanthine produce uric acid and peroxide radicals. Excessive uric acidconcentration would lead to hyperuricemia and gout. In recent years, the XODinhibitor has been widespread concern of research scholars at home and abroad, as avery effective anti-gout drug. So, the study of screening new naturally XOD inhibitorwould provide a theoretical basement for the development of XOD targeting anti-goutdrug and health products.This article made a deep research on the XOD inhibition of the ethanol extractsthat extracting from Laminaria japonica Aresch and Gymnogongrus flabelliformisHarv. The optimum extracting conditions of the substances from two seaweeds wereconfirmed by single factor and orthogonal experiment. The inhibitory mechanism andtypes of the extracts on XOD were studied using enzyme kinetic method. The jointinhibitor effects of the substance in water from two seaweeds with allopurinol wereexamined using a toxic equivalency method. This research determined the free radicalscavenging activity of the substance in water from two seaweeds and made a simpleanalysis of their chemical composition. The contents and results were as follows.The XOD inhibitor screening model was established based on the method ofspectrophotography., using0.6mmol/L xanthine as substrate, the wave length being294nm, the final concentration of XOD reached to4μg/mL, the pH of the bufferbeing7.5,the optimum reaction temperature and time being25℃and1min.Using ultrasonic-assisted constant temperature water extraction method to extractthe antienzyme components of L．japonica Areseh and G．flabelliformis, using single factor and orthogonal experiment method to optimize the extracting conditions. Theoptimum extracting conditions of L．japonica Areseh were extracting temperature30℃,ethanol concentration50%,ultrasonic time15min, extracting time4h; the optimumextracting conditions of G．flabelliformis were extracting temperature50℃, ethanolconcentration60%,ultrasonic time10min, extracting time4h.After solvent extracting by ethyl acetate, the ethanol extract of the seaweedswere separated into substance in water and substance in ethyl acetate. The result are asfollows: Dry powder yield of the substance in water and substance in ethyl acetatefrom L．japonica Areseh were22.51%and1.23%respectively. There was significantdifference (p<0.01). The results showed that the IC50were0.71mg/mL and2.37mg/mL.The inhibition kinetics analyzed by Lineweaver-Burk plots showed thatthe substance in water extract of L．japonica Areseh was a competitive inhibitor toXOD and the inhibition constant（ki） was determined to be0.086mg/mL. The thejoint inhibitory effects of the substance in water from L．japonica Areseh withallopurinol on XOD activity were examined using a toxic equivalency method, resultshowed that the joint inhibitory effects of the substance in water from L．japonicaAreseh with allopurinol was synergistic effect. This study also detect the free radicalscavenging activity of the substance in water from L．japonica Areseh, result showedthat the substance in water from L． japonica Areseh has scavenging activity toO_~-2·and·OH.Dry powder yield of the substance in water and substance in ethyl acetate fromG．flabelliformis were12.7%and0.30%respectively. There was significant difference(p<0.01). The results showed that the IC50were0.43mg/mL and0.51mg/mL.Theinhibition kinetics analyzed by Lineweaver-Burk plots showed that the substance inwater extract of G．flabelliformis was a competitive inhibitor to xanthine oxidase andthe inhibition constant（ki） was determined to be0.37mg/mL. The joint inhibitoryeffects of the substance in water from G．flabelliformis with allopurinol on XODactivity were examined using a toxic equivalency method, result showed that the jointinhibitory effects of the substance in water from G．flabelliformis with allopurinol was additive effect. This study also detect the free radical scavenging activity of thesubstance in water from G．flabelliformis, result showed that the substance in water fromG．flabelliformis has scavenging activity to·OH.Synthesizing the XOD inhibition of the two seaweeds, the antienzymecomponents of L．japonica Areseh and G．flabelliformis are both concentrated in thesubstance in water. |
PDF Full Text Request