| The incidences of hyperuricemia and gout are rising year by year.Allopurinol and febuxostat were mainly used for clinical treatment of these diseases,but they had a series of side effects.It had very important practical significance that developing healthy foods with natural and non-toxic side effects were used to prevent or improve the symptoms of hyperuricemia or gout,because prevention was better than cure.Xanthine oxidase as an important enzyme in the process of uric acid metabolism,can directly control the generation of uric acid.Renal uric acid transporter,such as URAT1,OAT1,ABCG2,UAT and GLUT9,can adjust the secretion and reabsorption of uric acid.For these reasons,xanthine oxidase and renal uric acid transporters can be regarded as targets in the process of uric acid metabolism.Polyphenol compounds had many biological activities and they get the favour of people gradually.It has been the widespread concern of scholars that searching and selecting the effective xanthine oxidase inhibitors from polyphenol compounds were used to provide new options for the hyperuricemia prevention and control.Acteoside,caffeic acid and hydroxytyrosol were polyphenol compounds,and they had many physiological activity.A variety of methods were used to study the molecular mechanisms of acteoside and its aglyconeson reducing uric acid.Hope to provide theoretical basis for the development of health food products of acteoside and its aglycones.Meanwhile,hope to provide methods for other active substances to reduce uric acid levels.The main contents and results in this article were summarized as follows:(1)Hyperuricemia model of rats were established.The kit methods were used to analyze acteoside and its aglycones effects on the related physiological indexes.Results showed that the concentrations of serum uric acid,serum creatinine,serum urea nitrogen and the activity of xanthine oxidase and adenosine deaminase in hyperuria rats,were maintained at a higher level.Compared to the normal group,they had significant differences.Compared to the model group,the five indexes in the rats with treatment of acteoside and its aglycones were reduced in a different degree.It showed that acteoside and its aglycones can adjust the concentrations of serum uric acid,serum creatinine and serum urea nitrogen,and it also can inhibit the activity of xanthine oxidase and adenosine deaminase in vivo to reduce the formation of uric acid.For a single material,a high concentration of acteoside and its aglycones more significant effected on the above five indexes,and for different materials the effects on the above indicators of rats were different.(2)Hyperuricemia model of rats were established.Fluorescence quantitative PCR method was used to analyze acteoside and its aglycones effects on the renal uric acid transporter gene expression.In the hyperuria rats,the relative expressions of URAT1 m RNA and GLUT9 m RNA,which associated with uric acid reabsorption,were maintained at a higher level.The relative expressions of OAT1 m RNA,UAT m RNA and ABCG2 m RNA,which associated with uric acid secretion,were maintained at a lower level.While,in the rats with treatment of acteoside and its aglycones,the relative expressions of URAT1 m RNA and GLUT9 m RNA were decreased,and the relative expressions of OAT1 m RNA,UAT m RNA and ABCG2 m RNA were increased.It indicated that acteoside and its aglycones can adjust the expression of the renal uric acid transporter URAT1,GLUT9,OAT1,UAT and ABCG2,and the ability of kidney to absorb uric acid was decreased and its secretion capacity increased,which promotes the excretion of uric acid in the body.Hence the uric acid levels was reduced.(3)In vitro,the methods of high performance liquid chromatography(HPLC),spectrophotometry and circular dichroism spectroscopy was used to study the inhibitory mechanism of acteoside and its aglycones on xanthine oxidase.The contents of the free acteoside and its aglycones after the combination with xanthine oxidase were tested by HPLC.Then,the binding rates of acteoside,caffeic acid and hydroxytyrosol with xanthine oxidase within 15 minutes were calculated for 5.87%,2.05% and 4.72%,respectively.The half inhibitory concentrations of acteoside,caffeic acid and hydroxytyrosol inhibiting xanthine oxidase detected by color-developing method with NBT were 13.05 ?M,72.10 ?M and 33.95 ?M,respectively.The results of Lineweaver-Burk showed that the inhibition types of acteoside and hydroxytyrosol on xanthine oxidase were mixed inhibition,while caffeic acid was competitive inhibition.The results of circular dichroism spectroscopy method showed that the main secondary structure of xanthine oxidase was the ?-helix,up to 59.3%,while the contents of ?-sheet and ?-ture were less.As it combined with acteoside and its aglycones,the secondary structures of xanthine oxidase were changed.With the increase of the concentration of acteoside and its aglycones,the ?-helix was reduced,while ?-sheet,?-ture and random coil were increased gradually.According to the results of in vitro inhibitory activity,acteoside and its aglycones combined with xanthine oxidase,and then changed the secondary structures of xanthine oxidase,so that the order of molecular structure was decreased.Then,the combination of xanthine and xanthine oxidase was blocked and the enzyme activity was inhibited.(4)The molecular docking technology was used to simulation acteoside and its aglycones docking with the Mo-pt and FAD of xanthine oxidase.The results showed that acteoside and hydroxytyrosol can dock with the Mo-pt and FAD of xanthine oxidase.The binding energy of acteoside docked with FAD(-6.95 kcal/mol)was lower than that of the acteoside docked with Mo-pt(-6.82 kcal/mol).In contrast,the binding energy of hydroxytyrosol docked with Mo-pt(-6.14 kcal/mol)was lower than that of the hydroxytyrosol docked with FAD(-5.07 kcal/mol).While caffeic acid was only docked with Mo-pt,and the binding energy was-5.72 kcal/mol.According to the results of docking,acteoside and its aglycones can be used as xanthine oxidase inhibitors,but the inhibition mechanisms were different.Acteoside and hydroxytyrosol can occupy the channel of the substrate into the active center,and also can block the electron transfer;whereas caffeic acid can competitively combined with the substrate xanthine in the activity area of xanthine oxidase. |
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