Sobhani Armin Madadkar, Ebrahimi Sultan-Ahmad, Mahmoudian Massoud
Department of Pharmacology, Iran University of Medical Sciences, Tehran, Iran.
J Pharm Pharm Sci. 2002 Jan-Apr;5(1):19-23.
Peganum harmala L. (Zygophyllaceae) seeds extract is one of the main components of an ethnobotanical preparation used in the treatment of neoplasms in Iran. Cytotoxic effects of P. harmala extract on cancerous cell-lines have been reported before. beta-carbolines like harmaline and harmine are the major alkaloids present in the seeds of the P. harmala. Considering reports concerning DNA topoisomerase inhibition by other beta-carbolines like harmane, we have used DNA relaxation assays to investigate topoisomerase I inhibitory activity of P. harmala seeds extract and its beta-carboline alkaloids to further inspecting the mechanism of its cytotoxic activity.
Harmine and harmaline contents of the extract were determined using an HPTLC method. DNA topoisomerase I enzyme needed for investigating inhibitory effect of the compounds using DNA relaxation assay, was partially purified from the human placenta. DNA relaxation assay is based on the conversion of a supercoiled plasmid substrate to its relaxed form by the catalytic activity of the enzyme. The supercoiled substrate and its relaxed product can be easily distinguished using agarose gel electrophoresis, since the relaxed topological isomers of DNA migrate more slowly than supercoiled species.
Using HPTLC method, it was found that each gram of dried extract contained 55.5 and 79.0 mg of harmine and harmaline respectively. In the DNA relaxation assay, order of potency was harmine > harmane > harmaline > extract. The most active compound was harmine with IC50 value of 13.5 +/- 1.7 microg/ml.
Our in vitro findings demonstrate that P. harmala seeds extract do inhibit human DNA topoisomerase I and based on the results of HPTLC analysis, it appears that the biological activity of the extract can be explained by its beta-carboline content.
骆驼蓬（蒺藜科）种子提取物是伊朗一种用于治疗肿瘤的民族植物制剂的主要成分之一。此前已有报道称骆驼蓬提取物对癌细胞系具有细胞毒性作用。像骆驼蓬碱和骆驼蓬灵这样的β-咔啉是骆驼蓬种子中的主要生物碱。鉴于有报道称其他β-咔啉如哈尔满可抑制DNA拓扑异构酶，我们采用DNA松弛试验来研究骆驼蓬种子提取物及其β-咔啉生物碱对拓扑异构酶I的抑制活性，以进一步探究其细胞毒性活性的机制。
采用高效薄层色谱法测定提取物中骆驼蓬灵和骆驼蓬碱的含量。用于通过DNA松弛试验研究化合物抑制作用所需的DNA拓扑异构酶I酶，是从人胎盘中部分纯化得到的。DNA松弛试验基于超螺旋质粒底物通过该酶的催化活性转化为其松弛形式。由于DNA的松弛拓扑异构体比超螺旋形式迁移得更慢，因此使用琼脂糖凝胶电泳可以很容易地区分超螺旋底物及其松弛产物。
采用高效薄层色谱法发现，每克干燥提取物分别含有55.5毫克和79.0毫克的骆驼蓬灵和骆驼蓬碱。在DNA松弛试验中，效力顺序为骆驼蓬灵＞哈尔满＞骆驼蓬碱＞提取物。活性最强的化合物是骆驼蓬灵，IC50值为13.5±1.7微克/毫升。
我们的体外研究结果表明，骆驼蓬种子提取物确实能抑制人DNA拓扑异构酶I，并且根据高效薄层色谱分析结果，提取物的生物活性似乎可以用其β-咔啉含量来解释。
