Study On The Effect And Mechanism Of Clerodendranthus Spicatus Polyphenols On Hyperuricemia

Objective:Hyperuricemia(HUA)has been an important cause for gout,hypertension,atherosclerosis and cardiovascular disease.At present,although the treatment with allopurinol and benzbromarone can control the content of blood uric acid effectively,there are serious side effects and unsatisfactory long-term effect.Therefore,finding safe and effective uric acid lowering drugs has become a hot spot for the prevention and treatment of hyperuricemia and gout.Clerodendranthus spicatus(Thunb.)C.Y.Wu generally used in folk as tea or herbal medicine to treat urinary-related diseases with potent effect.In recent years,relevant studies have reported the anti-hyperuricemic effect of C.spicatus extract,but its mechanism has not been fully elucidated,and the substance basis of the extract with its in vitro and in vivo activities have not been clarified yet.Therefore,the aim of this study was to investigate the anti-hyperuricemic effect of C.spicatus and its underlying mechanism.Methods:The solvent extraction process of C.spicatus polyphenols(CSP)was optimized through single factor investigation of solvent dosage,material-liquid ratio,extraction duration and times of extraction.Antioxidant activity of CSP was detected by in vitro DPPH,ABTS,hydroxyl radical and superoxide anion radical scavenging experiments.The cytotoxicity of CSP was determined by cell viability test.In vitro enzyme catalytic systems were established to explore the inhibitory effect of CSP on XOD,ADA and PNP.Potassium oxazidate was used as an inducer,KunMing mice were selected as experimental animals to establish the animal model of acute hyperuricemia.Anti-hyperuricemic effect of CSP on hyperuricemia mice was investigated and its mechanism was explored at the molecular level.Results:(1)Optimized solvent extraction process of CSP:50%ethanol ratio,material-liquid ratio(1:20 g/mL),extraction time 40 min,and extraction 3 times.Under this condition,the extraction rate of CSP reached its highest 3.61%.(2)UPLC/ESI-MS analysis indentified 8 chemical components in CSP,namely Protocatechualdehyde,Caffeic acid,Rosmarinic acid,Caffeic acid tetramer,Lithospermic acid,Salvianolic acid B,Isopropyl Rhamnin-3-oxohexoside and Caffeic acid derivatives.(3)Antioxidant experiment showed that CSP had great abilities to scavenge DPPH,ABTS and hydroxyl radicals.(4)MTT result showed that CSP had a tiny cytotoxity,the dose of CSP less than 1 mg/mL had little effect on the viability of Beas-2B cells.(5)In vitro enzyme activity experiments showed that CSP had potent inhibitory activities on xanthine oxidase(XOD),adenosine deaminase(ADA)and purine nucleoside phosphorylase(PNP).(6)Animal experiment showed that 1 g/kg and 2 g/kg doses of CSP could significantly reduce serum uric acid content in hyperuricemic mice.While 0.5,1,and 2 g/kg doses of CSP were capable of clearing serum creatinine and urea nitrogen in hyperuricemic mice effectively;Hepatic enzyme experiment displayed that CSP potently inhibited hepatic activities of XOD and ADA in hyperuricemic mice;Renal histopathology analysis exhibited that CSP had nephroprotective effect and could significantly alleviate kidney injury caused by hyperuricemia;Q-PCR and Western Blot experiments presented that CSP significantly down-regulated the expressions of urate anion transporter 1(URAT1)and glucose transporter 9(GLUT9)genes,and up-regulated the expressions of organic anion transporter 1(OAT1)and organic anion transporter 3(OAT3)genes.Conclusions:This thesis investigated the anti-hyperuricemic activity of CSP,and preliminarily revealed that CSP could alleviate hyperuricemia by inhibiting the enzyme activities participating in uric acid synthesis and regulating the expression of uric acid transporters,which provides more evidence for further developing the medical value of C.spicatus and anti-hyperuricemic drugs.