Research On The Effects Of Ganoderma Applanatum And Poria Cocos Extracts Against Hyperuricemia

Objective:Based on the hyperuricemia mice model,this study explored the urate-lowering effects,toxicology and mechanism of Ganoderma applanatum and Poria cocos.It provides new ideas and methods for the study of the mechanism of traditional diuretic medicinal fungi provide ideas and methods for reducing uric acid.Method:1.Extracting the main components of Ganoderma applanatum and Poria cocos.(1)Extracting the liposolubilic components in Ganoderma applanatum and Poria cocos by heating ethanol extraction.(2)Extracting the hydrophilic substances in Ganoderma applanatum and Poria cocos by heating water extraction.(3)Filtering and evaporating under reduced pressure and freeze dring the ethanol extract and the water extract,then calculating the yield.2.Based on the hyperuricemia mice model to explore the lowering-urate action of Ganoderma applanatum and Poria cocos.(1)Establishing hyperuricemia mice model using administration combined with potassium oxonate and hypoxanthine.(2)Administering hyperuricemia mice with Ganoderma applanatum and Poria cocos extracts,in contrast with allopurinol and benzbromarone as positive control.(3)The whole blood samples were collected for separating to the serum for determining serum uric acid.The urine samples were collected for detecting urine uric acid.3.Exploring toxicology and potential targets of Ganoderma applanatum and Poria cocos based on hyperuricemic mice(1)Determination of liver and kidney function parameters in serum of hyperuricemia mice.(2)Liver,kideney,spleen and thymus were excised,weighted and calculated for organ coefficients.(3)Taking renal and intestinal tissues for reverse transcription polymerase chain reaction and Western blotting to determine the gene and protein expression of proteins related urate transport and metabolism.(4)Fixing,embedding and slicing liver and kidney tissues for staining with haematoxylin and eosin to analyze histological examination for liver and kidney.4.Exploring the molecular mechanism of the improvement of hyperuricemia by Ganoderma applanatum and Poria cocos.(1)Determining the activity level of xanthine oxidase(XOD).(2)Retrieving the known compounds in Ganoderma applanatum and Poria cocos from the existing free and open source Traditional Chinese medicine database and literature,and integrating them into the small molecular library of Ganoderma applanatum and Poria cocos,respectively.(3)According to results of gene and protein expression of urate transport metabolism related proteins and XOD activities,selecting protein targets to dock with the small molecular library of Ganoderma applanatum and Poria cocos,respectively.(4)Verification of urate-lowering effect,toxicology and mechanism of candidate small molecule,DHAP.Results:1.Extraction yeilds of Ganoderma applanatum and Poria cocos extracts.Ethanol extracts yield of Ganoderma applanatum(GAE)was 3.78%,and water extracts yield of Ganoderma applanatum(GAW)was 8.53%.Ethanol extracts yield of Poria cocos(PCE)was 1.22%,and water extracts yield of Poria cocos(PCW)was 1.29%.2.The hypouricemic effects of Ganoderma applanatum and Poria cocos based on hyperuricemia mice model.GAE,GAW,PCE and PCW effectively reduced the serum uric acid in hyperuricemia mice depended on dose.The serum uric acid levels of GAE,GAW and PCW even similar to the normal control group.Simultaneously,the extracts of Ganoderma applanatum and Poria cocos promote the discharge of uric acid from the urine.3.Toxicological effects of Ganoderma applanatum and Poria cocos on hyperuricemia miceHepatic and renal function parametres,organ eoefficients and HE analysis showed all extracts have nephronprotective and hepatoprotective effects.4.Impacts of Ganoderma applanatum and Poria cocos on potential targets for hyporuricemia.The extracts of Ganoderma applanatum showed slightly inhibitory effect on XOD,and the extracts of Poria cocos inhibit hepatic XOD activity.GAE and GAW are able to down-regulate renal GLUT9 and intestinal CNT2 but up-regulate renal OAT1.Specially,GAE inhibit renal URAT1.PCE and PCW presented up-regulation of mRNA and protein of renal ABCG2,OAT1,OAT3 and OCT2.5.Screening results of hypouricemic compounds in Ganoderma applanatum and Poria cocos based on hyperuricemia mice model.A total of 84 compounds of Ganoderma applanatum were obtained for docking to XOD and URAT1,and 54 compounds Poria cocos were collected for docking to XOD and ABCG2.Through molecular docking,it was found compounds in Ganoderma applanatum and Poria eocos both impacted on XOD,and Poria cocos have stronger XOD inhibition.Among them,the compound 2,5-Dihydroxyacetophenone(DHAP)was selected as the further research object.6.Verification of hypouricemic effect,toxicology and underlying mechanism of candidate compound.DHAP effectively reduced serum uric acid in hyperuricemia mice.By molecular docking in slico to simulate the binding mode of DHAP and XOD,determining the XOD inhibition of DHAP in vitro and the XOD activities of hyperuricmic mice in vivo,DHAP was found to inhibitory effect on XOD.Renal indicators and kidney coefficient indicated that DHAP relieved renal injury.The liver coefficient indicated a possibility of hepatic side-effects of DHAP.The results of WB and RT-PCR demonstrated that anti-hyperuricemia mechanism of DHAP maybe though inhibiting URAT1,GLUT9 and CNT2 and increasing OAT1 mRNA.Conclusions:1.The Ganoderma applanatum and Poria cocos extracts both effectively reducing the serum uric acid level and promoting urate excretion in hyperuricemia mice,in line with the definition of diuretics for traditional Chinese medicine.Morever,the Ganoderma applanatum and Poria cocos extracts relived the injury attributed to hyperuricemia modeling.2.The Ganoderma applanatum and Poria cocos extracts may both inhibit XOD.The Ganoderma applanatum extracts could promote uric acid excretion by regulating the uric acid transport metabolism protein in the kidney and intestine;the Poria cocos may promotes uric acid excretion by regulating renal urate transporter.3.Molecular docking results shown that both Ganoderma applanatum and Poria cocos have compounds targeted at least two proteins.What's more,Ganoderma applanatum and Poria cocos both produce inhibition of XOD.4.A compound in Ganoderma applanatum,DHAP,has hypouricemia activities in hyperuricemia mice.DHAP mainly play a role in inhibiting XOD activity.However,there is a possibility of impairing hepatic function.