波棱酮纳米混悬剂的制备及其体外抗肝纤活性评价

Preparation and in vitro anti-hepatic fibrosis evaluation of herpetone nanosuspensions

波棱酮(herpetone,HPT)是从波棱瓜子总木脂素中分离得到的单体化合物,具有保肝降酶及抗乙肝病毒活性。然而,波棱酮存在水溶性差、生物利用度低等问题,其是否对肝纤细胞有抑制作用也未见报道。为了提高HPT溶出度并研究其抗肝纤活性的作用及机制,课题组采用微型化介质研磨法制备波棱酮纳米混悬液(HPT-NS),并通过单因素实验优化处方及工艺,扫描电镜(SEM)观察HPT-NS的形态;透析法考察体外溶出情况; CCK8法测定HPT-NS对大鼠肝星状细胞(HSC-T6)增殖的影响;流式细胞术检测HPT-NS对HSC-T6细胞周期及凋亡的影响。结果显示,优化所得的HPT-NS平均粒径为(196±7) nm,多分散指数为0. 279±0. 009; SEM显示HPT-NS呈规则的棒状;体外溶出实验显示HPT-NS在18 h内累积溶出度达93%,显著高于其物理混合物组(HPT-CS,28%); CCK8实验显示HPT-NS对HSC-T6细胞抑制率高于HPT-CS;流式细胞检测结果显示HPT-NS可将HSC-T6细胞阻止在G2/M期,诱导HSC-T6细胞的晚期凋亡,且作用显著强于HPT-CS。研究表明,HPT-NS显著提高了HPT的体外溶出度,增强了对HSC-T6细胞增殖的抑制作用,其作用机制为阻滞细胞于G2/M期,并诱导其晚期凋亡。

Herpetone( HPT) is a bioactive lignan extracted from Herpetospermum pedunculosum,which can protect liver,lower aminotransferase and inhibit hepatitis B virus. However,HPT has a poor oral bioavailability due to its poor water solubility. And there is no report about whether HPT has an anti-hepatic fibrosis activity. To improve the dissolution of HPT and study its anti-hepatic fibrosis activity and mechanism,the study group prepared herpetone nanosuspensions( HPT-NS) by the miniaturized media milling method. The formulation and process of HPT-NS were optimized by the single factor experiment. Scanning electron microscopy was used to observe morphology of HPT-NS. Dialysis method was used to study dissolution of HPT-NS in vitro. CCK8 method was used to assess the effect of HPT-NS on proliferation of the rat hepatic stellate cells( HSC-T6). Flow cytometry was used to assess the effect of HPT-NS on apoptosis and cell cycle of HSC-T6. The mean particle size of optimized HPT-NS was( 196±7) nm with a polydispersity index of 0.279±0.009.SEM showed that HPT-NS was in a regular rod shape. The cumulative dissolution rate of HPT-NS reached 93% in 18 h,and was higher than that of herpetone coarse suspensions( HPT-CS,28%). CCK8 experiment showed that the inhibition rate of HPT-NS on HSC-T6 was higher than that of HPT-CS. Flow cytometry showed that HPT-NS could block HSC-T6 cells in G2/M phase and induce apoptosis of HSC-T6 cells,with a significantly stronger effect than HPT-CS. The results revealed that HPT-NS significantly increased the in vitro dissolution of HPT,and enhanced the inhibitive effect on HSC-T6 cell proliferation by blocking cells in the G2/M phase and inducing late apoptosis.