补骨脂酚通过MAPK途径诱导人肝癌HepG2细胞凋亡的研究

目的:研究补骨脂酚对人肝癌细胞凋亡的影响及其作用机制。方法:应用噻唑蓝法（MTT法）和倒置显微镜技术考察补骨脂酚对HepG2细胞生长的影响;采用荧光显微镜观察补骨脂酚处理后细胞凋亡;利用蛋白免疫印迹法检测补骨脂酚对HepG2细胞中凋亡相关蛋白及MAPK家族蛋白表达的影响;引入MAPK家族蛋白抑制剂考察生长抑制率和凋亡相关蛋白表达的变化。结果:补骨脂酚可以剂量依赖性地抑制人肝癌HepG2细胞增殖,其生长抑制作用明显强于临床上常用的抗肿瘤药5-氟尿嘧啶,并且补骨脂酚对人正常肝L02细胞具有较低的毒性。进一步研究发现,补骨脂酚可以诱导HepG2细胞发生凋亡。此外,MAPK家族参与到补骨脂酚诱导的HepG2细胞凋亡过程中,补骨脂酚可以剂量依赖性激活JNK的表达发挥促凋亡的作用,同时抑制了ERK促存活通路,然而对p38无明显影响。结论:本研究首次阐明了补骨脂酚诱导人肝癌HepG2细胞生长抑制作用机制,为进一步的临床应用提供了理论依据。     

Evaluation Of The Pharmacological Properties Of Nutmeg Oil In Rats And Mice

The pharmacological properties of nutmeg oil in animal models were investigated. The oil exhibited an anti-inflammatory effect on acute inflammation. An analgesic effect was produced by the oil in the acetic acid-induced writhing model and in the late phase of the formalin-induced licking. The oil showed an antipyretic activity, and antithrombotic and anti-diarrhoeal properties. Sub-acute administration of the oil produced gastric ulcerations. These data indicate that nutmeg oil showed pharmacological activities similar to those of non-steroidal anti-inflammatory drugs, in addition to anti-diarrhoeal activity.     

补骨脂酚的提取纯化工艺优选及其对骨质疏松症的治疗作用分析

目的:优选补骨脂酚的提取纯化工艺,并考察其对骨质疏松症的治疗作用。方法:使用乙醇回流法提取,经D101型大孔树脂纯化,通过单因素试验优选工艺条件,冷冻干燥后得到补骨脂酚;采用斑马鱼体内试验评价补骨脂酚对骨密度和骨骼发育钙化的影响。结果:优选的工艺条件为加8倍量85%乙醇浸泡过夜,回流提取3次,每次2 h,边搅拌边往补骨脂酚粗提物中加入0.2%氢氧化钠水溶液至完全溶解,过预处理好的D101型大孔树脂柱处理,上样流速1 BV·h~(-1),静置4 h,加水6 BV洗脱,用60%乙醇2 BV洗脱,加95%乙醇4 BV洗脱,收集95%乙醇的洗脱液;制备的补骨脂酚质量分数80%,收率10%。斑马鱼体内活性研究表明补骨脂酚能提高斑马鱼骨质疏松症的骨密度,且能促进正常斑马鱼骨骼发育钙化。结论:此工艺具有绿色环保、操作简便等优点,所得补骨脂酚的纯度和收率均比较理想,可开发成预防或治疗骨质疏松症的药品和食品。     

补骨脂酚通过线粒体自噬抑制人肝癌Hep3B细胞生长的机制研究

[目的] 探讨中药补骨脂活性成分补骨脂酚（BAK）诱导人肝癌Hep3B细胞生长抑制的机制。[方法] 采用噻唑蓝法（MTT法）检测BAK对人肝癌Hep3B细胞生长抑制作用;相差显微镜观察BAK处理后Hep3B细胞形态变化;Hoechst/Mitotracker/Rhodamine荧光探针染色结合高内涵细胞成像系统分析BAK对线粒体功能的影响;免疫印迹法（Western blot法）考察BAK处理后自噬标志性蛋白溶酶体相关蛋白1（LAMP1）、微管相关蛋白1轻链3（LC3）、SQSTM1蛋白（p62）以及线粒体自噬标志性蛋白PTEN诱导假定激酶1（PINK1）、帕金蛋白（Parkin）、线粒体融合蛋白2（MFN2）、线粒体膜蛋白（Tom20）的表达情况;MTT法考察线粒体自噬抑制剂氯喹（CQ）、巴弗洛霉素A1（Baf-A1）对BAK诱导的肝癌细胞生长抑制的影响。[结果] BAK时间剂量依赖性抑制Hep3B细胞生长;高内涵实验结果表明BAK能够升高线粒体质量,降低线粒体膜电位;BAK可以上调LAMP1、LC3、PINK1、Parkin表达量,下调p62、 MFN2及Tom20蛋白表达;加入线粒体自噬抑制剂可显著逆转BAK诱导的Hep3B细胞生长抑制。[结论] 补骨脂酚通过线粒体自噬抑制人肝癌Hep3B细胞生长。     

Antioxidative components of Psoralea corylifolia (Leguminosae)

A meroterpene and four flavonoids were isolated from the seeds of Psoralea corylifolia as antioxidative components. Their structures were elucidated by spectral data and identified as bakuchiol (1), bavachinin (2), bavachin (3), isobavachin (4) and isobavachalcone (5). In particular, meroterpene 1 and flavonoids 4 and 5 showed broad antioxidative activities in rat liver microsomes and mitochondria. They inhibited NADPH-, ascorbate-, t-BuOOH- and CCl(4)-induced lipid peroxidation in microsomes. They also prevented NADH-dependent and ascorbate-induced mitochondrial lipid peroxidation. Bakuchiol (1) was the most potent antioxidant in microsomes and the inhibition of oxygen consumption induced by lipid peroxidation was time-dependent. Furthermore, bakuchiol (1) protected human red blood cells against oxidative haemolysis. These phenolic compounds in P. corylifolia were shown to be effective in protecting biological membranes against various oxidative stresses.     

In vitro evidence for bakuchiol's influence towards drug metabolism through inhibition of UDP-glucuronosyltransferase (UGT) 2B7

Background

Inhibition of drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reason for clinical drug-drug interaction.

Aim

The aim of the present study is to evaluate the inhibition of bakuchiol towards UDP-glucuronosyltransferase (UGT) 2B isoforms.

Methods

In vitro recombinant UGT2B-catalyzed 4-methylumbelliferone glucuronidation was used as the probe reaction. Dixon plot and Lineweaver-Burk plot were employed to determine the inhibition kinetic type, and nonlinear regression of data was utilized to calculate the inhibition kinetic parameter (Ki). In vitro-in vivo extrapolation (IVIVE) was carried out to predict in vivo inhibition magnitude.

Results

Among the tested UGT2B isoforms, UGT2B7 was inhibited by the strongest intensity. The noncompetitive inhibition was demonstrated by the results obtained from Dixon plot and Lineweaver-Burk plot. The K_i value was calculated to be 10.7 µM. In combination with the reported concentration after an intravenous administration of bakuchiol (15 mg/kg) in rats, the high risk of in vivo inhibition of bakuchiol towards UGT2B7-catalyzed metabolism of drugs was indicated.

Conclusion

All these results provide an important information for the risk evaluation of the clinical utilization of bakuchiol.     

酚-黄复方对痤疮致病菌的体外抗菌作用研究

[目的]通过体外抑菌实验,考察补骨脂酚与黄芩苷组合用药的体外抗痤疮菌活性。[方法]采用菌落计数法,以不同浓度补骨脂酚及加入黄芩苷后对表皮葡萄球菌和痤疮丙酸杆菌生长的抑菌率为指标考察其抑菌效果。[结果]当补骨脂酚浓度达到2560mg/L时对表皮葡萄球菌及痤疮丙酸杆菌的抑菌率分别为100.0%、61.1%。在补骨脂酚溶液中加入黄芩苷后两药浓度分别为2 560 mg/L和800 mg/L,对两种痤疮菌的抑菌率均为100.0%,在此浓度下两药合用对痤疮丙酸杆菌的抑制作用更为明显,抑菌率是单用补骨脂酚的1.64倍。[结论]酚-黄复方对痤疮菌的抑菌率增加显著,表明酚-黄复方共同发挥抗痤疮菌作用,揭示酚-黄合用的必要性和合理性。     

D-最优设计响应面法结合UHPLC优选补骨脂药材炮制工艺

目的采用D-最优设计响应面法结合UHPLC技术优选补骨脂药材炮制工艺。方法以补骨脂药材炮制工艺的关键参数闷润时间、炒制温度及加盐量为自变量,进行D-最优设计;以UHPLC检测获得的补骨脂药材中9种主要指标成分补骨脂素、异补骨脂素、新补骨脂异黄酮、补骨脂甲素、补骨脂定、补骨脂乙素、补骨脂宁、补骨脂查耳酮和补骨脂酚炮制前后的质量分数变化为因变量,进行响应面分析;并结合单因素试验优化炒制时间,优选出补骨脂药材炮制工艺。结果补骨脂药材的最佳炮制工艺为在100 g补骨脂药材中加入2.10 g盐,闷润12 h,在80℃炒制30 min。结论利用D-最优设计响应面法结合UHPLC能够优选补骨脂药材炮制工艺参数,提高炮制工艺的稳定性和重复性,为提高补骨脂盐炙品的质量均一性和临床用药安全性提供技术支持。     

酚-黄复方微乳的体外透皮特性考察

目的:制备酚-黄复方微乳并考察其体外透皮扩散特性。方法:以油酸乙酯为油相,聚山梨酯-80(tween-80)为乳化剂,聚乙二醇-400(PEG-400)为助乳化剂,水相逐步加入到含药混合溶液中,制备酚-黄复方微乳。测定微乳的外观形态、粒径分布及稳定性。采用Franze扩散池法,考察补骨脂酚、黄芩苷在溶液和微乳中经皮渗透特性。结果:制备的微乳澄清透明,呈近球形,平均粒径(17.30±0.17)nm,稳定性较好。黄芩苷在溶液和微乳中24 h累积透过量(Q24 h)分别为(208.80±5.26),(232.38±15.07)μg·cm-2,经皮渗透特性无显著性差异。补骨脂酚24 h累积透过量由溶液中(324.81±56.63)μg·cm-2增加至微乳中的(721.30±108.88)μg·cm-2,增加了2.22倍,稳态渗透速率和表观渗透系数为溶液组的5.0倍,均有显著提高。结论:微乳经皮给药系统具有很好的促透作用,为酚-黄复方皮肤给药新制剂的设计提供实验依据。