北豆根抗肿瘤有效成分的分离、纯化和活性分析

Isolation and Purification of Antitumor Component in Rhizoma Menispermi and Analysis of Its Activity

背景与目的:提取、纯化和鉴定北豆根抗肿瘤有效成分,并对纯化的成分作进一步的活性研究。材料与方法:常规方法制备北豆根水提液(RMW)和醇提液(RME),对RME以3%HCl溶解、有机溶剂萃取和水相浓缩得到纯化提取物1、2、3(PE1、PE2、PE3)。用MTT法测定各提取物的抑瘤活性。根据抑瘤实验结果,通过凝胶和硅胶柱层析将PE2进一步分离得到两个单体化合物PF1和PF2。提取RME后的北豆根药渣用水回流提取,去除蛋白和鞣酸,再经乙醇沉淀和冷冻干燥得到多糖成分(RMP)。有效成分采用薄层层析和改良碘化铋钾显色对其进行鉴定分析。用电喷雾质谱ESI-MS检测单体化合物PF1和PF2的分子量。用紫外分光光度计检测RMP的纯度。结果:RMW和RME对K562、BGC823及TE13三种肿瘤细胞的增殖反应均有明显的抑制作用(P<0.01),并有良好的量效及时效关系,RMP无此作用。进一步将RME应用3步法纯化得到PE1、PE2、PE3。PE1和PE2对上述3种细胞的增殖反应均有明显的抑制作用(提高了近10倍)(P<0.01),PE3无此抑制作用。有效成分分析结果显示,PE2主要含一系列生物碱成分。对PE2进一步分离得到两个化合物PF1和PF2,分子量分别为624和610。PF1和PF2对K562、BGC823及TE13细胞均有显著抑制作用(P<0.01),PF2的抑制作用更强,PF2可导致上述细胞的形态学改变,表现为细胞体积变小,形态不规则等。PF2对肿瘤组织原代培养细胞增殖反应有较强抑制作用(P<0.01)。结论:北豆根水提物和醇提物在体外均有很强的抗肿瘤作用,醇提物的作用高于水提物。PE2是醇提物抗肿瘤的有效部位,其化学成分为生物碱;PF1和PF2均是醇提物抗肿瘤的有效成分,可能为蝙蝠葛碱和蝙蝠葛苏林碱,PF2对肿瘤组织原代培养细胞也有较强的杀伤作用。北豆根多糖成分对K562等3种肿瘤细胞均无抑制作用。

BACKGROUND ＆ AIM： Isolate and purify active component in Rhizoma Menispermi extract and analyze the activity of the active component. MATERIAL AND METHODS： The suppressive effects of Rhizoma Menipermi extracts on the proliferation of tumor cells were assayed in vitro using MTY colorimetric method. Rhizoma Menipermi extract with ethanol was preliminarily purified using three steps method. PE2 was isolated and purified further by Pole Chromatography. The components were identified by Thin Layer Chromatography, ESI-MS and Ultraviolet Spectrophotometry, RESULTS： Rhizoma Menipermi extracts RMW and RME inhibited markedly the proliferation of tumor cells such as K562, BGC823 and TE13 in a dose-and time-dependent manner（P〈0.01） But Rhizoma Menipermi extract RMP had no inhibitory effect. The two compounts, PE1 and PE2 seperated from RME, also had very strong inhibitory effect（P 〈 0.01）. Through active component analysis, PE2 was found to contain some alkloids. PE2 was purified further and then PF1 and PF2 were isolated, with molecular weights of 624 and 610, respectively. PF1 and PF2 inhibited markedly the proliferation of tumor cells such as K562, BGC823 and TE13 （P〈0.01） . PF2 had stronger inhibitory effect than PF1. PF2 could induce morphologic changes of K562, BGC823 and TE13 cells. PF2 also inhibited the proliferation of tumor cells from primary culture of tumor tissues of patients （P 〈 0.01）. CONCLUSION： Both RMW and RME had very strong antitumor effects in vitro .The effect of RME was stronger than RMW. PE2 was the active antitumor part of RME. PF1 and PF2 were the active antitumor components of PE2, and were probably Dauricine and Daurisoline,respectively. RMP had no inhibitory effect on tuner cells.