Isolation Of Hypouricemic Fraction Of Sonneratia Apetala Leaves And Branches And Its Mechanisms Investigation

Objective:Recently,the hypouricemic effect of Sonneratia apetala leaves and branches(SAL)has been reported,but the active compound and its mechanism are unclear.Thus,this study aims to explore the effective fraction of SAL and the mechanism of its active compound on uric acid formation and excretion.Methods:1.Isolation of the uric acid lowering fraction in SALSAL was extracted with ethyl acetate and concentrated to obtain solvent-free extracts(SAL-EA).The remains fraction(SAL-E)and the supernatant fraction(SALS)of SAL resulting from water extraction and alcohol precipitation were collected and dried.The effects of different fractions were explored on hyperuricemic mice(dose equivalent to 200 mg/kg SAL).2.Analysis and identification of the main active compoundThe SAL-S fraction was diluted with 60% methanol and was analyzed by HPLC(LC-20AT)for it’s active compound.The mobile phase composition was 0.05%phosphoric acid : methanol(93:7)at an isocratic mode throughout the analysis.The separation was conducted on a C18 column(4.6 × 250 mm,5 μm)at a wavelength of271 nm and a flow rate of 1.0 m L/min for 30 min.3.Study on hypouricemic mechanisms of GAThe experimental mice were random Ly divided into six groups(8 mice per group):N group,MO group,BZM group(20 mg/kg)and GA groups(25,50 and 100 mg/kg).Potassium oxonate(PO),hypoxanthine(HX),BZM and GA were administered as a suspension formulated by 0.5% CMC-Na.Hyperuricemic mice were induced by 300mg/kg HX by gavage and 300 mg/kg PO by subcutaneous injection for 7 constitutive days.On the 8th day,serum samples were obtained from the orbital and uric acid(UA),creatinine(CRE),blood urea nitrogen(BUN),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were assayed to estimate renal and hepatic functions.The liver,kidney and intestine tissues were harvested,and activities of adenosine deaminase(ADA)and xanthine oxidase(XOD)in the liver were also determined.Kidney indexes were carefully measured and histopathological examination was also performed to evaluate the nephroprotective activity of GA.To estimate the anti‐oxidant capacity of the kidney,activities of SOD,GSH-Px,CAT in the kidneys were tested.For assessment of inflammation,renal levels of MDA、IL-1β、IL-6、TNF-α、TGF-β1 and COX-2 were detected.For hypouricemic mechanistic investigation,the protein expression levels of URAT1 GLUT9 and ABCG2 analyzed by western blot and the m RNA expression levels of NPT1,CNT2,OAT1,OAT3,TGF-β1 and COX-2 were also quantified by q PCR analysis.Results:1.Isolation of the uric acid lowering fraction in SALOf all the isolated fractions,only SAL-S showed excellent activities in decreasing the levels of uric acid(UA),blood urea nitrogen(BUN),and creatinine(CRE)in serum and in attenuating kidney damage.Thus,we deduced that SAL-S was the major fraction with hypouricemic activity in SAL and subsequently determined it’s main active compound.2.Analysis and identification of the main active compoundThe HPLC chromatogram result showed that gallic acid(GA)has the highest intensity in SAL-S.Thus,we deduced that GA was the main active component in SALS and subsequently explore its hypouricemic mechanism3.Study on hypouricemic mechanisms of GAThe results show that the hypouricemic effect of GA was probably associated with the downregulation of URAT1 and GLUT9,upregulation of NPT1,CNT2,ABCG2 and decreased activities of adenosine deaminase(ADA)and xanthine oxidase(XOD).Moreover,GA suppressed the level of MDA、IL-1β、IL-6、TNF-α、TGF-β1 and COX-2,and enhanced the activities of SOD,GSH-Px and CAT in the kidneys.These results indicated that GA protects against hyperuricemia-induced kidney injury via suppressing oxidative stress and inflammation as well as decreasing the serum levels of UA by regulating urate transporters.Conclusions:Our findings elaborated that GA treatment in PO/HX-induced HUA mice exhibited remarkable dual hypouricemic and nephroprotective effects.GA exerted hyperuricemic effects mainly byprohibiting the production of urate by suppressing hepatic XOD and ADA activity,and simultaneously accelerating the excretion of urate by modulating the expression of URAT1,GLUT9,and ABCG2.GA also possesses a renoprotective property by reducing renal inflammation and oxidative stress.These findings for the first time suggested that GA may be possibly developed as a complementary treatment for HUA in the future.