Study On Anti-gout Constituent From Vernonia Amygdalina Del.

With the improvement of living standards,the incidence of gout has increased year by year and has progressed toward younger age on account of the increasedly intake of purines coupled with irregular life.Currently mainstream drugs for the treatment of gout,such as colchicine,allopurinol,etc.exit in toxic effects,drugs allergies and other potential issues.Natural products are an important sources of new active anti-gout ingredients.Vernonia amygdalina Del.,also named Nanfiye,has been widely used for the treatment of gout,hypertension,hyperlipidemia,and other diseases in Taiwan and Southeast Asia.Recent studies have shown that the extract of V.amygdalina could reduce uric acid and decreas the activity of xanthine oxidase.V amygdalina shows powerful aid in the prevention and treatment of gouty disease,while its substantial basis of effect is still unclear.Thus,the study was conducted on V.amygdalina for the purpose of searching for highly effective anti-gout small-molecule lead compound candidates with low-toxicity.In this study,the 60%ethanol extracts of tender twigs of V.amygdalina and their extracts in petroleum ether,methylene chloride,ethyl acetate and n-butanol layers were screened for xanthine oxidase inhibition activities.The results showed that the 60%ethanol extract fraction,the dichloromethane and the ethyl acetate fractions showed potent inhibitory activities.Various chromatographic techniques like silica gel column chromatography,Sephadex LH-20 column chromatography,reverse column chromatography,medium and low pressure column chromatography,recrystallization,thin layer preparative chromatography,preparation and semi-preparative HPLC,etc were used for the purification and thirty-four compounds were isolated from active dichloromethane part of V.amygdalina.Their structures and absoluted configurations were elucidated by high-resolution mass spectrometry,nuclear magnetic resonance spectroscopy,CD spectroscopy,X-ray single crystal diffraction,molybdenum reaction,modified Mosher Method and calculation of ECD.All 34 compounds were assigned to stigmastane-type steroid saponins and aglycones,among them,1-12.15-20.23-30?32-34 were new compounds.The compound 25 has a relatively novel cyclization mode with a six-membered oxygen ring formed by a 16,23-epoxy group and cis-conjugated a five-membered oxygen ring formed by a 22,28-epoxy group.This type of cyclization was firstly found in this type of compoundsThe anti-gout activities of isolated compounds were investigated by the xanthine oxidase inhibition and the NF-?B Dual-luciferase reporter assays.The results showed that compound 21 had significant xanthine oxidase inhibition activity,compounds 6?17?18?19?21?25?27 exhibited potent NF-?B inhibitory activities.Therefore,these steroid compounds might have anti-gout activities,and their mechanism of action need further investigation.Preliminarily structure-activity relationship study showed that 21,23 and 22,28-epoxy stigmastane-type steroids exhibited more potent effects than other stigmastane-type steroids.However,further research on the structure-activity relationship is needed.Our work has laid a chemical foundation for revealing the anti-gout ingredients of V.amygdalina and provide scientific basis for further rational utilization of V.amygdalina.