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目的:对辽东楤木叶总皂苷进行HPLC分析并确定不同的极性部位,利用C18反相硅胶(ODS)柱分离获得不同极性部位,比较不同部位的体外抗肿瘤作用效果。方法:辽东楤木叶经70%乙醇提取后通过AB-8型大孔树脂柱,获得总皂苷,用不同体积分数甲醇经ODS柱洗脱分离获得经HPLC确定的不同部位。利用噻唑蓝(MTT)比色法测试不同部位对人肺腺癌细胞株(A549),人宫颈癌细胞株(He La)和人结肠癌细胞株(HT-29)的体外生长抑制作用。结果:由总皂苷HPLC的保留时间确定了5个极性部位,分别为A(保留时间20 min以前的部位,ODS柱上0~25%甲醇洗脱组分),B(保留时间20~45 min,ODS柱上30%~45%甲醇洗脱组分),C(保留时间50~75 min,ODS柱上50%~65%甲醇洗脱组分),D(保留时间80~110 min,ODS柱上70%~75%甲醇洗脱组分)和E(保留时间>110 min,ODS柱上80%~100%甲醇洗脱组分)。5个部位给药48 h后对3种细胞的抑制作用明显不同,A部位对3种癌细胞均无抑制作用;B部位对3种癌细胞抑制作用微弱;C,D,E部位对3种癌细胞表现出较强的抑制作用,且明显强于总皂苷,最佳的D部位对A549,Hella和HT-29抑制作用的半抑制浓度分别为4.06,4.29,3.60 mg·L~(-1)。结论:辽东楤木叶总皂苷经HPLC分析后,利用ODS柱进行分离,可获得其抗肿瘤作用的有效部位及最佳部位。
OBJECTIVE: To determine the different polar sites of total saponins of Alnus liiandica by HPLC and to obtain the antitumor effects of different sites by comparing the different polar sites with C18 reverse phase silica gel (ODS) column. Methods: The total saponin was obtained from the leaves of Alnus johnsonii after being extracted with 70% ethanol through AB-8 macroporous resin column. The different fractions determined by HPLC were eluted and separated with different volume fraction of methanol by ODS column. In vitro growth inhibition of human lung adenocarcinoma cell line (A549), human cervical cancer cell line (He La) and human colon cancer cell line (HT-29) was tested by MTT colorimetric assay. Results: Five polar sites were identified by the retention time of total saponin HPLC, which were A (retention time of 20 minutes ago, 0-25% methanol elution on ODS column), B (retention time 20 ~ 45 min, 30% -45% methanol on ODS column), C (retention time 50-75 min, 50% -65% methanol on ODS column), D (retention time 80-110 min, ODS column 70% ~ 75% methanol eluting components) and E (retention time> 110 min, 80% ~ 100% methanol eluting components on ODS column). The inhibitory effects of 5 sites on the three kinds of cells were obviously different after 48 h administration. The A site showed no inhibitory effect on all the three kinds of cancer cells. The inhibitory effect on the three kinds of cancer cells was weak at the site B, The inhibitory effect of the best D site on A549, Hella and HT-29 was 4.06, 4.29 and 3.60 mg · L -1, respectively ). Conclusion: The total saponins from Alder leaves of Liaodong were separated by ODS column after HPLC analysis, and the effective sites and optimal sites of antitumor activities were obtained.