巴西人参皂苷的纯化富集及其体外促雄激素活性研究

Purification and Enrichment of Pfaffia Glomerata Saponin and Its Promoting Effect on Androgen in vitro

目的:探讨巴西人参提取物中齐墩果酸型皂苷类化合物对小鼠睾丸间质细胞(TM3细胞)雄激素合成与分泌的促进作用,并根据细胞试验筛选出活性较强的组分。方法:以AB-8与XAD1600两种大孔吸附树脂分离富集得到的不同组分为试材,分别采用四甲基偶氮唑蓝(MTT)法、酶联免疫吸附法、硝酸还原法、羟胺法、硫代巴比妥酸法检测不同组分在不同浓度下对TM3细胞增殖活率、睾酮(T)含量、二氢睾酮(DHT)含量、环磷酸鸟苷(cGMP)含量、一氧化氮(NO)含量、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量的影响。结果:经AB-8大孔吸附树脂分离得到的70%乙醇组分较其他组分具有更强的促进TM3细胞雄激素分泌的活性,再以XAD1600大孔吸附树脂对70%乙醇组分进一步纯化富集,获得的GP-1与GP-2组分均可显著促进TM3细胞T、DHT、cGMP的分泌量(P<0.05,P<0.01),并提高SOD酶活性(P<0.01),降低MDA含量(P<0.05,P<0.01),且GP-2组分效果优于GP-1组分。结论:巴西人参各分离纯化组分可以显著促进TM3细胞雄激素的合成与分泌,并提高TM3细胞的抗氧化能力,其中GP-2组分活性最强,其主要活性成分为齐墩果酸型皂苷。

Objective:To investigate the effect of oleanolic acid saponins from Pfaffia glomerata extract on the synthesis and secretion of androgen in TM3 mouse Leydig cells,and screen out the components with strong activity according to the cell test.Methods:Two kinds of macroporous adsorption resins(AB-8 and XAD1600)were used to separate and enrich different components as test materials.Methyl thiazolyl tetrazolium(MTT)method and enzyme-linked immunosorbent assay were used to determine the effects of different components and concentrations on TM3 cell activity,testosterone(T)content,dihydrotestosterone(DHT)content and cyclic guanosine monophosphate(cGMP)content.Nitric oxide(NO)content,superoxide dismutase(SOD)activity and malondialdehyde(MDA)content of TM3 cells were detected by nitrate reduction method,hydroxylamine method and thiobarbituric acid method,respectively.Results:The 70%ethanol fraction isolated by AB-8 macroporous adsorption resin had a stronger activity in promoting the secretion of androgen in TM3 cells than other fractions.The 70%ethanol fraction was further purified and enriched by XAD1600 macroporous adsorption resin.The obtained GP-1 and GP-2 fractions significantly promoted the secretion of T,DHT and cGMP in TM3 cells(P<0.05,P<0.01),increased the activity of SOD enzyme(P<0.01)and reduced the content of MDA(P<0.05,P<0.01),and the effect of GP-2 fraction was better than that of GP-1 fraction.Conclusion:The synthesis and secretion of androgen and antioxidant capacity of TM3 cells were significantly improved by separation and purification components of P.glomerat.Among them,the activity of GP-2 component was stronger,and its main active component was oleanolic acid saponin.