Inhibition Of Xanthine Oxidase By Flavone From Rose Hip,Preparation And Characterization Of Quercetin Microcapsules

Rose hip is a kind of wild plant widely distributed in northeast China.Its fruit contains a variety of effective components,with anti-fatigue,anti-oxidation,anti-inflammatory and other active functions.Flavonoid compounds in rose hip are abundant,among which quercetin,kaempferol,rutin,hyperoside and luteolin are common ones.Xanthine oxidase（XOD）is a very key enzyme involved in purine metabolism,which can catalyze the production of uric acid.If the content of uric acid is too high,hyperuricemia will be triggered,and then lead to gout.Flavonoid compounds can be used to develop natural non-toxic XOD inhibitors to inhibit uric acid production.Due to the poor stability of flavonoid compounds,they are prone to oxidation and decomposition under the influence of p H,temperature and light during long-term storage.Therefore,in this paper,quercetin is used as the core material and sodium alginate as the wall material to prepare and characterize microcapsules,which further provide theoretical basis for the application of flavonoid compounds in the food and drug industry.The specific research work of this paper is as follows:（1）The ultrasound-assisted enzymatic hydrolysis method was used to extract flavonoids from rose hip.The effects of enzymatic hydrolysis time,ultrasonic time,enzymatic hydrolysis temperature,cellulase concentration,ethanol volume fraction and extraction methods on the extraction rate of total flavonoids were investigated.The optimal extraction parameters were determined and response surface analysis was carried out to obtain the optimal extraction conditions:The enzymatic hydrolysis time was 2.07 h,the ethanol volume fraction was 54.09%,the digestion temperature was50.82℃,and the cellulase concentration was 8.50 mg/g.Under this condition,the maximum extraction rate of the flavonoids was 44.83%.The optimal extraction condition was verified,and the resulting total flavonoids extraction rate was 46.80%,which was close to the predicted value of the response surface.（2）The effects of quercetin,rutin and hyperoside on the activity of XOD were investigated by enzyme reaction kinetics experiment with allopurinol as positive control.The results showed that the three flavonoids had good inhibitory effects on XOD activity,and their half-inhibitory concentrations(IC₅₀)were（8.33±0.36）μmol/L,（60.81±0.19）μmol/L,（35.22±0.4）μmol/L.In the linear relationship between enzyme concentration and enzymatic reaction rate,the three flavonoids with different concentrations all pass through the origin.In the Lineweaver-Burk diagram,they all intersect at a point in the second quadrant,which proves that quercetin,rutin and hyperoside all have reversible competition-non-competitive mixed inhibition on XOD.（3）Quercetin,rutin and hyperoside were used for fluorescence titration of XOD,and the quenching constant K_SVwas calculated according to Stern-Volmer equation.The results showed that the K_SVvalues of the three flavonoids decreased with the increase of temperature,and the quenching rate constant K_qof the three flavonoids was greater than the maximum quenching constant at different temperatures.It was proved that quercetin,rutin and hyperoside all quenched XOD by static fluorescence.The thermodynamic parameters were calculated according to the van’t Hoff equation,and the enthalpy change,entropy change and free energy change were all negative values.It was proved that the three flavonoids spontaneously combined with XOD through van der Waals force and hydrogen bond.The results of synchronous fluorescence experiment showed that the combination of quercetin and XOD could redshift the maximum wavelength of tyrosine and tryptophan,which proved that it reduced the hydrophobicity of the microenvironment of both,while the combination of rutin and hypericin with XOD only caused obvious redshift of tryptophan,which proved that it had little effect on tyrosine.According to calculation of Synchronous Fluorescence Quenching rate（RSFQ）between tyrosine and tryptophan,RSFQ value of tryptophan is higher than tyrosine at the same concentration,proving that in the fluorescence quenching process,The effect of tryptophan residues was greater than that of tyrosine residues,and the binding site of flavonoids was similar to tryptophan.（4）Using quercetin as core material and sodium alginate as wall material,quercetin microcapsules were prepared by the sharp-hole method.Single factor experiment and orthogonal experiment were designed to determine the optimal preparation conditions as follows:Core wall ratio was 1:1,sodium alginate concentration was 2%,Ca Cl₂solution concentration was 4%,the highest embedding rate of 96.88%.Simulated digestion and characterization of microcapsules prepared under optimal conditions in vitro:After 7 h,the cumulative release amount of intestinal fluid was 65.38%and gastric fluid was 24.77%,indicating that microcapsules had a good slow-release effect.The microcapsule microstructure（SEM）showed that the microcapsule structure was tight and the molding effect was good.By means of fourier infrared spectroscopy（FTIR）,X-ray diffraction（XRD）and Differential scanning calorimetry（DSC）analysis,most of the characteristic peaks of quercetin can be found in the characteristic peaks of the microcapsules,which proves that quercetin has been successfully embedded.Four antioxidant activities were compared between free quercetin and microcapsules.The results showed that the antioxidant activity decreased after embedding.