| This paper is mainly divided into five parts.The first part is the extraction,separation,purification,and identification of the active components of robustaside B and robustaside A from Vaccinium dunalianum Wight.In the second part,the anti xanthine oxidase（XOD）activity of robustaside B and caffeic acid,the metabolite of robustaside B,was tested in vitro.In the third part,the effects of robustaside B,robustaside A,and caffeic acid on deceaciing uric acid level in acute hyperuricemia（HUA）mice were investigated.The crude extract of Vaccinium dunalianum Wight and caffeic acid on relieving pain,and anti-inflammatory activity in acute pain,and acute inflammation model mice were investigated.In the fourth part,a rat model of chronic hyperuricemic renal injury was constructed and applied to the pharmacodynamic study in vivo.In the fifth part,the crude extract of Vaccinium dunalianum Wight and caffeic acid were applied to rats with chronic hyperuricemic renal injury to investigate their effects on decreasing uric acid and blood lipids in rats with hyperuricemic renal injury.Objective（s）:To prepare high purity chemical constituents,robustaside B and robustaside A from Vaccinium dunalianum Wight.Then the pharmacodynamics,including uric acid decrease,kidney protection,liver protection,anti-inflammatory and analgesic effects,and lowering blood lipids of two active compounds and metabolite of robustaside B,and the crude extract of Vaccinium dunalianum Wight were investigated in vitro and in vivo,in order to providing reference for the in-depth development of Vaccinium dunalianum Wight resources and the development of new drugs for reducing uric acid.Methods:（1）The ethanol ultrasonic extraction method was used to obtain the crude extract of Vaccinium dunalianum Wight,and then the crude extract was separated by MPLC to obtain robustaside B and robustaside A.（2）The inhibitory effects of robustaside B and caffeic acid on xanthine oxidase（XOD）activity in vitro were tested using nitrotetrazolium blue chloride（NBT）colorimetry.（3）（1）A mouse model of acute hyperuricemia was established by intraperitoneal injection of 500mg·kg-1potassium oxyzinate.The levels of serum uric acid（UA）were measured using a fully automated biochemical analyzer to evaluate the effect of robustaside B and robustaside A,and caffeic acid on reducing uric acid in mice with acute hyperuricemia.（2）An inflammatory mouse model was induced by applying xylene to the mouse auricle;a mouse model of pain was induced by intraperitoneal injection of0.2%glacial acetic acid.The anti-inflammatory effects of the crude extract of Vaccinium dunalianum Wight and caffeic acid on mice were evaluated by calculating the degree of ear swelling in mice.The analgesic effects of the crude extract of Vaccinium dunalianum Wight and caffeic acid on mice were evaluated by observing the number of body twists in mice within 20 minutes.（4）A stable rat model of chronic hyperuricemic renal injury were screened by comparing four modeling methods,300mg·kg-1adenine,2500mg·kg-1potassium oxazinate,100mg·kg-1adenine+1500mg·kg-1potassium oxazinate,100mg·kg-1adenine+250mg·kg-1ethambutol..（5）Using the rat model of chronic hyperuric acid kidney injury,the crude extract of Vaccinium dunalianum Wight and caffeic acid were administered by gavage respectively.（1）Weigh and record the weight of rats at different time periods to evaluate the effects of the crude extract of Vaccinium dunalianum Wight and caffeic acid on the weight of rats;detection of serum uric acid（UA）level using a fully automated biochemical analyzer;detection of liver xanthine oxidase（XOD）activity using a kit,to evaluate the effect of the crude extract of Vaccinium dunalianum Wight and caffeic acid on reducing uric acid in rats with chronic hyperuricemic renal injury and its mechanism in vivo.（2）Detect serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）levels,to evaluate the protective effects of the crude extract of Vaccinium dunalianum Wight and caffeic acid on the liver of rats with chronic hyperuricemia.（3）Detection of serum creatinine（Cr）and urea nitrogen（BUN）levels,the renal weight to body ratio,the renal tissue morphology,and the pathological changes in renal tissue using hematoxylin eosin staining（HE）,to evaluate the protective effects of the crude extract of Vaccinium dunalianum Wight and caffeic acid on chronic hyperuricemic kidney injury in rats.（4）Detect the levels of serum total cholesterol（TC）,low density lipoprotein（LDL-C）,and high density lipoprotein（HDL-C）,to evaluate the effect of Vaccinium dunalianum Wight and caffeic acid on lowering blood lipids in rats with chronic hyperuric acid kidney injury.Results:（1）Robustaside B and robustaside A were prepared with the purity95.1%and 91.5%respectively.（2）Robustaside B and caffeic acid have a certain inhibitory effect on XOD activity,with inhibition rates of 67.2%and 77.2%at a concentration of 1 mg·ml-1,respectively.（3）Compared with the blank group,the serum UA level of mice in the acute hyperuricemia model group was significantly increased（P<0.05）.（1）Compared with the model group,robustaside B,robustaside A and the metabolite of robustaside B which named caffeic acid all significantly reduced the serum UA levels in mice（P<0.05）.（2）Compared with the xylene induced inflammatory mouse model group,both the crude extract of Vaccinium dunalianum Wight and caffeic acid significantly reduced the degree of ear swelling in mice（P<0.05）.Compared with the mice model group induced by glacial acetic acid,each dose group of the crude extract of Vaccinium dunalianum Wight and the high dose group of caffeic acid have significant inhibitory effects on the writhing reaction in mice（P<0.05）.（4）Compared with the blank group,the serum UA,Cr and BUN levels were significantly increased in the 100mg·kg-1adenine+1500mg·kg-1potassium oxazinate group（P<0.05）,and further screening of 100mg·kg-1+1000mg·kg-1,60mg·kg-1+1500mg·kg-1,100mg·kg-1+800mg·kg-1of adenine combined with potassium oxazinate also significantly increased serum UA,Cr and BUN levels（P<0.05）.（5）Compared with the blank group,the serum UA,Cr,BUN levels in the model group were significantly increased（P<0.05）.（1）Compared with the blank group,there was no difference in the weight of rats in each treatment group（P>0.05）.Compared with the model group,the crude extract of Vaccinium dunalianum Wight and caffeic acid in each dose group could significantly reduce the serum UA level and inhibit the liver XOD activity in rats（P<0.05）.（2）Compared with the blank group,the serum ALT level in the model group was significantly increased（P<0.05）.Compared with the model group,except for the benzbromarone group,the serum ALT level of rats in each treatment group was significantly decreased（P<0.05）.Compared with the blank group,the serum AST level in the model group increased,but there was no statistical difference.Compared with the model group,only the high dose group of the crude extract of Vaccinium dunalianum Wight,the high dose group of caffeic acid,and the extremely low dose group of caffeic acid significantly decreased the serum AST level of rats in the three groups（P<0.05）.（3）Compared with the model group,the crude extract of Vaccinium dunalianum Wight and caffeic acid in each dose group can significantly reduce the levels of serum Cr and BUN in rats,and improve the renal tissue morphology and pathological changes in rats by comparing the weight of both kidneys to the renal body（P<0.05）.（4）Compared with the model group,each dose of the crude extract of Vaccinium dunalianum Wight and caffeic acid significantly reduced the levels of TC and HDL-C in serum groups of rats in each group,except for the low dose group of caffeic acid,the serum LDL-C levels of rats were significantly reduced in all treatment groups（P<0.05）.Conclusion（s）:（1）The selected method can be used to prepare high purity robustaside B and robustaside A.（2）Robustaside B and caffeic acid have a certain inhibitory effect on XOD activity in vitro.（3）Robustaside B,robustaside A and caffeic acid have a certain effect on reducing uric acid in acute hyperuricemia mouse models.The crude extract of Vaccinium dunalianum Wight and caffeic acid have a certain anti-inflammatory and analgesic effect on acute inflammation mouse models and acute pain mouse models.（4）100mg·kg-1+800mg·kg-1of adenine combined with potassium oxazinate can be used as a model for constructing chronic hyperuric acid kidney injury in rats.（5）The crude extract of Vaccinium dunalianum Wight and caffeic acid have certain effects on reducing uric acid,renal protection,liver protection,lowering blood lipids,and inhibiting liver XOD activity in a rat model of chronic hyperuric acid kidney injury. |
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