摘要
目的:了解汗衣台抗HBV有效成分及对HepG2.2.15细胞HBVcccDNA和HBVDNA的影响。方法:对汗衣台分离提取及HPLC-ESI-MS/MS测定,发现6种含量较高的有效成分:古伦宾、异古伦宾、掌叶防己碱、蝙蝠葛碱、非洲防己碱、药根碱。予各有效成分标准品不同浓度作用于2.2.15细胞,孵育9天,取上清ELISA法测定HBsAg和HBeAg含量;荧光定量PCR法检测HBVDNA;提取细胞总DNA并以PSAD酶消化后选择性荧光定量PCR法检测HBVcccDNA;后两者抑制率做相关性分析。结果:全部浓度的异古伦宾(10~160μmol/L)、非洲防己碱(10~160μmol/L)、蝙蝠葛碱(0.25~10μmol/L)、药根碱(20~320μmol/L)以及40~160μmol/L浓度的古伦宾、50~400μmol/L掌叶防己碱对HBsAg有明显抑制作用,40~160μmol/L浓度非洲防己碱、5μmol/L蝙蝠葛碱、80~320μmol/L药根碱及200~400μmol/L的掌叶防己碱对HBeAg有明显抑制作用;全部浓度的异古伦宾、药根碱,20~160μmol/L的古伦宾、160μmol/L非洲防己碱及400μmol/L浓度掌叶防己碱、5~10μmol/L蝙蝠葛碱对HBVDNA具有抑制作用,抑制率均不高;所有有效成分在较低浓度以上对HBVcccDNA均具有抑制作用,抑制率普遍较高,呈剂量依赖性,160μmol/L的非洲防己碱抑制率39.55%超过干扰素21.44%,200μmol/L的掌叶防己碱抑制率29.55%超过干扰素及拉米夫定24.6%;HBVDNA与HBVcccDNA相关性比较情况复杂,趋势一致但程度差异较大。结论:汗衣台6种主要成分均具有不同程度的抗HBV作用,均可视为抗HBV的有效成分。6种有效成分不同程度抑制HBVDNA复制,对HBVcccDNA的抑制呈剂量依赖性;上清HBVDNA检测并不能完全代表细胞内HBVcccDNA情况。
Objective： To discover effective components of HanYiTai plant on anti-HBV and research the effect of these components on HBVcccDNA and HBVDNA in HepG2. 2. 15 cells. Methods： The separation and purification of Han Yi Tai and HPLC-ESI-MS/MS determination were performed,six main effective components were found： Columbin,Isocolumbin,Palmatine,Dauricine,Columbamine,Jatrorrhizine. The different concentrations of each component＇s standard products were incubated in HepG2. 2. 15 cells for 9 days,contents of HBs Ag and HBeAg in the supernatant were determined by ELISA and HBVDNA was detected by fluorescence quantitative PCR. The total DNA of cells was extracted and then HBVcccDNA was detected by selective fluorescence quantitative PCR after digestion. The correlation analysis of the inhibition rate between HBVDNA and HBVcccDNA was done. Results： All the concentrations of Isocolumbin（10 ～ 160μmol/L）,Columbamine（10 ～ 160μmol/L）,Dauricine（0. 25 ～ 10μmol/L）,Jatrorrhizine（20 ～ 320μmol/L）,40 ～ 160μmol/L Columbin and 50 ～ 400μmol/L Palmatine significantly inhibited HBs Ag. 40 ～ 160μmol/L Columbamine,5μmol/L Dauricine and 80 ～ 320μmol/L Palmatine showed the significant inhibition on HBeAg. All concentrations of Isocolumbin, Jatrorrhizine,20 ～ 160μmol/L Columbin,160μmol/L Columbamine,400μmol/L Palmatine and 5 ～ 10μmol/L Dauricine effectually inhibited HBVDNA（P〈0. 05）,but inhibition rates were low. All active ingredients showed the inhibitory effect on HBVcccDNA in lower concentrations（P〈0. 05） when inhibition rates were higher,in a dose-dependent manner. The inhibition rate of 160μmol/L Columbamine was higher more than that of interferon（P = 0. 012）,and that of 200μmol/L Palmatine was higher more than those of interferon（P = 0. 000） and lamivudine（P = 0. 000）. The correlation between HBVDNA and HBVcccDNA was complex,trends was consistent,but the data was different. Conclusion： All of the six main components of Han Yi Tai have got the function of anti-HBV respectively,can be regarded as the effective components on anti-HBV. Six effective components inhibite HBVDNA replication,at the same time,it also inhibite HBVcccDNA replication in dose dependent manner. The concentration of HBVDNA in supernatant does not completely represent the situation of HBVcccDNA in nuclear.
作者
林才志
张贇赟
段雪琳
马静
范卫红
胡乃强
张林英
廖丹
Lin Caizhi;Zhang Yunyun;Duan Xuelin;Ma Jing;Fan Weihong;Hu Naiqiang;Zhang Linying;Liao Dan(Ren Ai Hospital of First Affiliated Hospital of Guangxi Chinese Medicine University, Nanning 530001;Guangxi Institute Of Chinese Medicine and Pharmaceutical Science, Nanning 530022;Base Medicine College Of Guangxi Chinese Medicine University, Nanning 530001)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2018年第1期57-62,共6页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然科学基金地区科学基金项目(81560678)
广西自然科学基金面上项目(2014GXNSFAA118211)
广西教育厅科研立项项目(KY2015LX166)
