Discovery,Synthesis And Anti-hyperuricemia Of Oxyberberrubine,a Novel Metabolite Of Berberrubine

ObjectiveBerberine(BBR)exerts multiple pharmacological activities including anti-oxidation,anti-inflammation and anti-hyperuricemia.The contradiction between various pharmacological activities and poor bioavailability of BBR attracts interest to researchers.Berberrubine(BRB),one of the major metabolites of BBR,exerts an similar pharmacological effects even superior to BBR.Therefore,some studies suggest that BRB is one of the metabolites of berberine which plays a pharmacological role.Nevertheless,there are few studies on the bioactivities and metabolites of BRB.Liver is an important location for BRB transformation.Liver microsomes were used to incubate BRB for studying its biotransformation and oxyberberrubine(OBR)was found in the liver microsomes incubation system.Hyperuricemia(HUA)is a metabolic disease characterized by elevated serum uric acid(SUA)levels over a prolonged period,resulting from the metabolism disorder of purine and decreased SUA excretion in the human body.The clinical drugs currently used for the treatment of hyperuricemia are associated with various side effects,which limit their clinical applications.Recently,more and more studies have found that traditional Chinese medicine and natural products have significant therapeutic effects on hyperuricemia,and no visible toxic effects as well as side effects were observed.According to our previous study,BBR can effectively reduce the UA level and the renal damage.Many metabolites exhibit similar pharmacological activities to their prodrugs.Therefore,we reasonably speculate that OBR and BRB have similar uric acid lowering effects.The effect of oxyberberrubine on hyperuricemia mice was studied.Methods1.The biotransformation of BRB by mouse liver microsomes and Synthesis and identification of OBRThe liver microsomes incubation system was prepared to incubate berberrubine,and the metabolites were preliminarily speculated and identified by LC-MS.The hypothesized metabolites were synthesized by chemical synthesis method.UV,NMR and LC-MS were used to verify the structure of the synthesized products and compare whether the mass spectrum peak of the metabolites was consistent with that of the synthesized products.Then,the synthesized product was compared with biotransformation mixtures of BRB by HPLC analysis to determine whether it was a metabolite of BRB.Finally,the reaction was optimized from the solvent,oxide,reactant ingredient ratio,reaction time and reaction temperature to get enough drugs for subsequent pharmacological experiments.2.Acute Toxicity of OBR in Miceoral administration of berberine oxide in SPF grade Kunming mice was conducted,and the median lethal dose of berberine oxide was determined by the maximum dose experiment.The differences in the behavior,body weight,organ index and tissue section between the administration group and the control group were observed.3.Anti-hyperuricemia effect and mechanism of OBR in vivoIn this work,the hyperuricemic mice model was established by receiving potassium oxonate(PO)and hypoxanthine(HX)for 7 consecutive days.1 h after modeling,different dosages of OBR(5,10 and 20 mg/kg),BRB(20 mg/kg)or febuxostat(Fex,5 mg/kg)were given mice by gavage.Animals were sacrificed after fasting overnight and 1 h later the drug treatment.Serum,liver and kidney tissues were harvested and stored.Histopathological changes in kidney tissues were observed by hematoxylin and eosin staining.The content changes of UA,CRE,BUN,ALT,AST,XOD and ADA were detected by corresponding kits.Kidney inflammatory factors(TNF-α,IL-1β,IL-6,IL-18)were determined by Elisa kits,and gene expressions of XOD,GLUT9,URAT1,NLRP3,ASC,Caspase-1,IL-1β,SIRT1 were determined by q RT-PCR.The expressions of XOD,GLUT9,URAT1,NLRP3,ASC,Caspase-1,IL-1β,IL-18,AMPK and PAMPK were detected by Western-blot.Results1.NMR,LC-MS and HPLC results showed that BRB could be converted to OBR in the incubation system of mouse liver microsomes.The chemical synthesis method was successfully used to obtain OBR and optimize the reaction conditions: in DBN as a solvent,chloro-peroxybenzoic acid and berberine material ratio =2:1,reaction time is 8 h,reaction temperature 45℃ conditions,OBR yield is the highest.2.After oral administration of OBR,no mice died within 14 days and no significant differences in body weight between the control group and OBR treatment group were observed.There was no morphological difference in HE slices of liver,kidney and heart between the control group and the OBR treatment group.3.Serum uric acid(UA)level was lowered,and the activities of xanthine oxidase(XOD)as well as adenosine deaminase(ADA)in the liver were suppressed after treatment with OBR.Hepatic expressions of XOD were remarkably decreased at m RNA and protein levels by OBR treatment.In addition,OBR prominently alleviated renal injury,embodied in markedly reduced serum creatinine and blood urea nitrogen(BUN)levels,decreased inflammatory mediators(TNF-α,IL-1β,IL-6 and IL-18)levels and repairment of renal tissues damage.OBR could up-regulated renal m RNA expression of SIRT1 and renal protein expression of PAMPK/AMPK.Besides,OBR down-regulated renal expression of urate transporter 1(URAT1),glucose transporter 9(GLUT9),NOD-like receptor 3(NLRP3),apoptosisassociated speck-like protein containing CARD(ASC),and caspase-1 at m RNA and protein levels.Conclusion1.BRB could be transformed into OBR in the incubation system of mouse liver microsomes.2.The optimum chemical synthesis conditions of OBR were DBN as solvent,chloroperoxybenzoic acid and berberine material ratio = 2:1,reaction time is 8h,reaction temperature 45 ℃.Moreover,the acute toxicity test results of OBR showed that its LD50 was > 5000 mg/kg,which had good safety.3.OBR prominently alleviated PO/HX-induced hyperuricemia.And the decreasing UA effect may be partially associated with inhibition of XOD in vivo to reduce production of UA,as well as down-regulation of urate reabsorption transporters(URAT1 and GLUT9)to promote UA excretion.Furthermore,OBR exerted reno-protective effect,promoting the activation of AMPK/SIRT1 pathway and inhibiting NLRP3-ASC-Caspase-1 pathway activation to reduce renal inflammation and lesion.Therefore,it is reasonable to speculate that OBR is a candidate drug for the treatment of hyperuricemia.