| Xanthine oxidase(XOD)is an important target to reduce the content of uric acid in human body.Screening new xanthine oxidase inhibitors with good effect and low side effects from natural products is of great significance in curing gout and hyperuricemia.Perilla(Perilla frutescens(L.)Britt.)is an annual herb,which is classified as a medicinal and edible plant by the Ministry of Health of China.Among them,perilla leaf can not only be used in food,cosmetics and other fields,but also can be used to treat a variety of diseases.Therefore,using perilla leaf as raw material,the inhibitory effect of perilla leaf extract on xanthine oxidase was determined,the extraction and purification technology of xanthine oxidase inhibitor from perilla leaf was studied,and its antioxidant activity and antibacterial activity were further determined.The main results are as follows:(1)A UV spectrophotometric method was established to determine the inhibitory effect of perilla leaf extract on xanthine oxidase activity.Through the analysis of enzyme inhibition and inhibition kinetics,it was determined that the inhibition type of perilla leaf extract on xanthine oxidase was competitive and reversible,which was the same as that of allopurinol,and its IC50 to xanthine oxidase was 5.80mg/m L,and the inhibition kinetic constant Ki was2.19mg/m L.Using this detection system,perilla leaves collected from 24 species of perilla in5 growth stages were screened.The results showed that the best time for leaf collection was seedling stage,and the best perilla variety was ZY7.(2)The extraction process of xanthine oxidase inhibitor from perilla leaves was optimized by response surface method.The effects of particle size,ultrasonic time,ethanol concentration,extraction temperature,ultrasonic power and ratio of material to liquid on the xanthine oxidase inhibition rate of perilla leaves extract were investigated by ultrasound-assisted extraction.Based on the single factor test,the response surface methodwas used to optimize the extraction process,and the optimum process was obtained as follows: ultrasonic time 18 min,ethanol concentration 48 %,extraction temperature 40?,material-liquid ratio 1:30.Under these conditions,the inhibition rate of perilla leaf extract on xanthine oxidase reached 68.71%.(3)The xanthine oxidase inhibitory components in perilla leaves were further isolated and purified by the combination of organic solvent extraction and macroporous resin purification.The 50% ethanol extract of perilla leaf was extracted in petroleum ether,ethyl acetate,chloroform and n-butanol,respectively.The results showed that the n-butanol extract had the best inhibitory effect on xanthine oxidase,and the inhibition rate was 69.14%.Through the static adsorption-analysis experiment,the NKA-9 macroporous resin was selected as the best purification resin type.The static adsorption rate of the resin was 83.27%,the resolution rate was 83.41%,the best adsorption equilibrium time was 9.0h,the analytical equilibrium time was 3.5h,and the p H of the sample solution was 6.0h.Through the dynamic adsorption-elution experiment,the best purification process was determined as follows: the concentration of the sample solution was 15mg/m L,the flow rate was 3BV/h,the volume fraction of ethanol was 60%,and the elution flow rate was 2BV/h.After purification,the inhibition rate of perilla leaf xanthine oxidase inhibitor on xanthine oxidase increased from68.52% to 80.86%.By HPLC-MS analysis of the purified samples,four main compounds were identified as apigenin-7mure O-diglucuronide,luteolin-7mure O-glucuronide,baicalin and 7-hydroxycoumarin.(4)Five models of scavenging ABTS+ free radical,scavenging DPPH free radical,scavenging hydroxyl radical,iron ion reduction ability(FRAP)and superoxide anion scavenging ability were used to study the antioxidation of pure and crude xanthine oxidase inhibitors in Vc and perilla leaves.The results showed that the IC50 values of the three samples for scavenging ABTS+ free radicals were 0.220,0.558 and 0.771 mg/m L,respectively,the IC50 values for scavenging DPPH free radicals were 0.449,0.945 and 3.518mg/m L,respectively,and the IC50 values for scavenging superoxide anions were 0.473,0.703 and 0.890 mg/m L,respectively.In the above three models,the scavenging abilities wereVc > pure product > crude product.In the(FRAP)model of iron ion reduction ability,the FRAP value of pure product was also significantly higher than that of crude product,while in the hydroxyl radical scavenging model,the scavenging ability of Vc > crude product > pure product,and their IC50 values were 1.559,2.231 and 1.896mg/m L,respectively.At the same time,the antibacterial activity of xanthine oxidase inhibitor in perilla leaves against Escherichia coli,Bacillus subtilis and Staphylococcus aureus showed that the minimum inhibitory concentrations of xanthine oxidase inhibitor against the three tested strains were2.5,0.3125 and 1.25mg/m L,respectively. |
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