Study On Chemical Constituents Of Dipterocarpus Alatus And The Mechanism By Which Its Main Compounds Alleviate Hyperuricemia

Hyperuricemia is one of the most common and extensive metabolic diseases,which is caused by over-production and/or under-excretion of unic acid.Natural products have been attracted extensive attention because of their potential effects on hyperuricemia.Dipterocarpaceae is well-known as rich in various resveratrol oligomers,which exhibite a wide spectrum of bioactivities,such as antioxidantion,anti-inflammation,hepatic protection and immunoregulation.In order to find out favorable natural products with large quanity and alleviation of hyperuricemia,the chemical constituents of Dipterocarpus alatus(Dipterocarpaceae)branches and twigs were investigated systematically.Using silica gel column chromatography,reversed phase column chromatography,Sephadex-H20 column chromatography and high performance liquid chromatography(HPLC)methods,21 compounds were isolated from 85%ethanol extracts and identified through spectrum analysis methods combined with reference comparison.Among them,18 compounds(No.4-21)belong to resveratrol oligomers and the derivates,including one sesqui-resveratrol(Diptoindonesin D),seven resveratrol dimmers(Hopeahainol C,Hopeafuran,Isoampelopsin F,Ampelopsin A,Heimiol A,Balanocarpol and Hopeahainol A),three resveratrol tripolymers(Vaticanol A,Vaticanol E and Caraphenol A),five resveratrol tetramers(Pauciflorol C,Neoisohopeaphenol A,Hopeaphenol A,Vaticaffinol and Hemsleyanol C),one resveratrol glucoside(Piceid),one resveratrol tetramer glucoside(Vatalbinoside A),respectively.Other three compounds were Bergenin,11-O-Acetylbergenin and Quercetrin(not resveratrol oligomers).Among them,two compounds with high yield were vaticaffinol(No.19,1.3 g,yield 1.79%)and vatalbinoside A(No.21,8.83g,yield 6.41%),respectively.To evaluate the anti-hyperuricemic and anti-inflammatory effects and the mechanisms of vaticaffinol and vatalbinoside A,uricase inhibitor potassium oxonate was used to induce hyperuricemia in mice.In this animal model of hyperuricemia,firstly,effective dosages of vaticaffinol and vatalbinoside A were screened to have anti-hyperuricemic action.10,20,40,60,80 and 100 mg/kg vaticaffinol and vatalbinoside A were orally administrated to hyperuricemic animals by gavage for 1 week,respectively.Groups of control and positive drug allopurinol(5 mg/kg)were designed simultaneously.The effective dosages were 20,40,60,80 and 100 mg/kg for vaticaffinol,and 40,60,80 and 100 mg/kg for vatalbinoside A,respectively.Accordingly,the present thesis further investigated the effects of vaticaffinol and vatalbinoside A on hepatic activity of xanthine dehydrogenase(XDH)and xanthine oxidase(XOD),renal expression of organic ion transporters,and NOD-like receptor(NLR)superfamily,pyrin domain containing 3(NLRP3)inflammasome in hyperuricemic mice.Vaticaffinol(20,40 and 60 mg/kg),vatalbinoside A(40,60,80 mg/kg),as well as allopurinol(5 mg/kg)were orally administrated to hyperuricemic animals by gavage for 1 week,respectively.Uric acid and creatinine levels in serum and urine,blood urea nitrogen(BUN)levels in serum,pro-inflammatory cytokines,interleukin(IL)-1?,IL-6,IL-18 and tumor necrosis factor(TNF)-? in serum and/or kidney,as well as XDH and XOD activity in liver were determined by standard diagnostic kits,respectively.Renal mRNA and/or protein levels of mouse urate transporter 1(mURAT1),glucose transporter 9(mGLUT9),organic anion transporter 1(mOAT1),organic cation transporter 1(mOCT1),mOCT2,organic cation/carnitine transporterl(mOCTN1),mOCTN2,renal protein levels of NLRP3,Apoptosis-associated speck-like protein(ASC)and Caspase-1,as well as IL-1? were analysed by semi-quantitative RT-PCR and/or Western blot methods,respectively.The present study found that vaticaffinol and vatalbinoside A significantly decreased serum urate,creatinine and BUN levels,and increased fractional excretion of uric acid(FEUA)in hyperuricemic mice.Additionally,vaticaffinol at 20,40,60 and 80 mg/kg remarkedly inhibited hepatic XDH and XOD activity in this animal model.Stimulateuously,vaticaffinol was found to down-regulate renal mRNA and protein levels of mURAT1 and mGLUT9,and up-regulate renal mRNA and protein levels of mOATl,mOCT1,mOCT2,mOCTN1 and mOCTN2 in hyperuricemic mice.Vatalbinoside A also remarkably down-regulated mURAT1 and mGLUT9,and up-regulated mOAT1,mOCT1,mOCT2,mOCTN1 and mOCTN2 at protein levels in the kidney of hyperuricemic mice,but failed to inhibit hepatic enzymes activity.Interestingly,vaticaffinol and vatalbinoside A were found to notablely down-regulate renal mNLRP3,mASC and mCaspase-1 protein levels,reduce IL-1?,IL-18,IL-6 and TNF-? levels,and alleviate kidney inflammatory injury in hyperuricemic mice.These results showed that vaticaffinol and vatalbinoside A had anti-hyperuricemic effects in animal model of hyperuricemia,which may be associated with the reduction of urate reabsorption by down-regulating renal mURAT1 and mGLUT9 expression,and the increase of urate secretion by up-regulating renal mOAT1 expression,resulting in the enhancement of kidney urate excretion.The inhibition of vaticaffinol on hepatic XDH and XOD activity to reduce urate production may be other effective way to exhihit its anti-hyperuricemic action.Furthermore,vaticaffinol and vatalbinoside A could improve kidney function and reduce inflammatory injury by up-regulating renal mOCT1,mOCT2,mOCTN1 and mOCTN2,as well as suppressing renal NLRP3 inflammation activation in hyperuricemic mice.Brief summary,this study importantly analysed chemical constituents of Dipterocarpus Alatus branches and twigs,obtained a series of resveratrol oligomers and the derivatives.Amoung those constituents,vaticaffinol and vatalbinoside A were found to possess anti-hyperuricemic,anti-inflammatory effects with kidney function improvement in hyperuricemia,and the mechanisms underlying were revealed preliminarily.These results may provide experimental basis for D.alatus in the prevention and treatment of hyperuricemia and kidney injury.