Suzuki Ryuichiro, Matsushima Yasuaki, Okudaira Noriyuki, Sakagami Hiroshi, Shirataki Yoshiaki
Faculty of Pharmaceutical Sciences, Josai University, Sakado, Japan.
Division of Pharmacology, Meikai University School of Dentistry, Sakado, Japan.
Anticancer Res. 2016 Nov;36(11):5931-5935. doi: 10.21873/anticanres.11180.
The 5-year survival rate of patients with oral cancer has remained approximately 50% during the past 30 years, possibly due to the poor tumor selectivity of conventional anticancer drugs. This prompted us to search for new candidates for anticancer drugs that have higher cytotoxicity and tumor selectivity.
Dried leaves of Andrographis paniculata were supplied from a market in Shanghai. The methanolic fraction of A. paniculata was further fractionated to identify cytotoxic principles by spectroscopic analysis and comparison with literature values. Viable cell number was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method, and tumor specificity was calculated by relative cytotoxicity against oral squamous cell carcinoma cell lines compared to that against normal oral cells. Apoptosis induction was detected by cleaved poly (ADP-ribose) polymerase and caspase-3 on western blot analysis.
Major cytotoxicity in the methanol extract of a leaf of A. paniculata was recovered by partitioning with EtOAc, followed by silica gel chromatography. Further purification with reversed-phase high-performance liquid chromatography led to isolation of four known cytotoxic compounds, 14-deoxyandrographolide, andrographolide, neoandrographolide and deoxyandrographiside. Among them, andrographolide had the greatest cytotoxicity and tumor specificity, also inducing caspase-3 activation of HSC-2 oral squamous cell carcinoma cells.
The present study identified andrographolide as a major antitumor principle in the methanolic extract of leaves of A. paniculata.
在过去30年中，口腔癌患者的5年生存率一直维持在约50%，这可能是由于传统抗癌药物对肿瘤的选择性较差。这促使我们寻找具有更高细胞毒性和肿瘤选择性的新型抗癌药物候选物。
穿心莲干叶购自上海某市场。对穿心莲的甲醇提取物进行进一步分离，通过光谱分析并与文献值比较来确定细胞毒性成分。采用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐法测定活细胞数量，并通过与正常口腔细胞相比对口腔鳞状细胞癌细胞系的相对细胞毒性来计算肿瘤特异性。通过蛋白质免疫印迹分析检测裂解的聚（ADP-核糖）聚合酶和半胱天冬酶-3来检测凋亡诱导情况。
穿心莲叶甲醇提取物中的主要细胞毒性通过用乙酸乙酯分配，随后进行硅胶柱色谱得以回收。通过反相高效液相色谱进一步纯化，分离出四种已知的细胞毒性化合物，14-去氧穿心莲内酯、穿心莲内酯、新穿心莲内酯和去氧穿心莲苷。其中，穿心莲内酯具有最大的细胞毒性和肿瘤特异性，还能诱导HSC-2口腔鳞状细胞癌细胞的半胱天冬酶-3激活。
本研究确定穿心莲内酯是穿心莲叶甲醇提取物中的主要抗肿瘤成分。
