Doctor Hari Singh Gour Vishwavidyalaya, Sagar, Madhya Pradesh, India.
Integr Cancer Ther. 2013 May;12(3):236-47. doi: 10.1177/1534735412451997. Epub 2012 Aug 22.
The study was designed to screen Sphaeranthus indicus, Ganoderma lucidum, and Urtica dioica for their anticancer activity against human cancer cell lines. Phytochemical screening of active extracts was also planned.
Petroleum ether, ethanolic, and aqueous extracts of S indicus Linn, G lucidum P Karst, and U dioica Linn were subjected to cytotoxicity studies using 7 different cancer cell lines. Potent cytotoxicity was noted in petroleum ether extract of S indicus (SIP), which inhibited proliferation of various cancer cell lines. Growth inhibition was determined by sulforhodamine B assay. Two biochemical markers, namely β-sitosterol and 7-hydroxyfrullanolide were isolated and characterized using high-performance thin layer chromatography, melting point, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and mass analysis. Cytotoxicity of isolated β-sitosterol and 7-hydroxyfrullanolide were also determined. The IC(50) of SIP was calculated in the HL-60 cells and was found to be 53 µg/mL. Furthermore, SIP induced apoptosis in human leukemia HL-60 cells as measured by several biological end points. Cell cycle analysis and change in mitochondrial membrane potential was quantified by flow cytometry. Subsequently, using annexin V/PI assay, proportion of cells actively undergoing apoptosis was determined. Changes in DNA were observed by DNA ladder assay.
SIP induced apoptotic bodies formation, induced DNA laddering, enhanced annexin-V-FITC binding of the cells, increased sub-G(0) DNA fraction, and induced loss of mitochondrial membrane potential (ΔΨm) in HL-60 cells. SIP also elevated the caspase 3 and caspase 9 levels in the HL-60 cells, which clearly indicates the involvement of the intrinsic proteins in inducing apoptosis.
All the above parameters revealed that SIP induced apoptosis through the mitochondrial-dependent pathway in HL-60 cells. The criterion for anticancer activity in cytotoxicity assay was ≥70% growth inhibition at 100 µg/mL against at least 4 cell lines. As G lucidum and U dioica did not exhibit appreciable inhibitory activity against human cancer cell lines (less than 50%), they were not included in the study thereafter. The results established that SIP has apoptosis-inducing effect against HL-60 cells in vitro and is a promising candidate for further anticancer study. β-Sitosterol and 7-hydroxyfrullanolide can be considered to be potent anticancer compounds isolated from SIP on the basis of present studies.
本研究旨在筛选土槿皮、灵芝和荨麻，以寻找其对人癌细胞系的抗癌活性。还计划对活性提取物进行植物化学筛选。
对土槿皮、灵芝和荨麻的石油醚、乙醇和水提取物进行了 7 种不同癌细胞系的细胞毒性研究。土槿皮石油醚提取物（SIP）具有显著的细胞毒性，能抑制多种癌细胞系的增殖。采用磺基罗丹明 B 法测定生长抑制率。采用高效薄层色谱、熔点、傅里叶变换红外光谱、核磁共振光谱和质谱分析分离和鉴定两种生化标志物，即β-谷甾醇和 7-羟基苍术醇。还测定了分离出的β-谷甾醇和 7-羟基苍术醇的细胞毒性。计算了 SIP 在 HL-60 细胞中的 IC50，发现为 53μg/ml。此外，SIP 通过几种生物学终点在人白血病 HL-60 细胞中诱导细胞凋亡。通过流式细胞术定量分析细胞周期和线粒体膜电位变化。随后，通过 Annexin V/PI 测定法，测定细胞主动凋亡的比例。通过 DNA 梯状电泳观察 DNA 变化。
SIP 诱导 HL-60 细胞形成凋亡小体，诱导 DNA 梯状形成，增强细胞的 Annexin-V-FITC 结合，增加亚 G0 DNA 片段，并诱导 HL-60 细胞中线粒体膜电位（ΔΨm）丧失。SIP 还提高了 HL-60 细胞中的 caspase 3 和 caspase 9 水平，这清楚地表明内在蛋白参与诱导细胞凋亡。
所有上述参数表明，SIP 通过 HL-60 细胞中的线粒体依赖性途径诱导细胞凋亡。细胞毒性测定中抗癌活性的标准是在 100μg/ml 时对至少 4 种细胞系的生长抑制率≥70%。由于灵芝和荨麻对人癌细胞系没有表现出明显的抑制活性（小于 50%），因此在此后研究中未将其包括在内。结果表明，SIP 在体外对 HL-60 细胞具有诱导凋亡作用，是进一步抗癌研究的有前途的候选药物。基于目前的研究，β-谷甾醇和 7-羟基苍术醇可被认为是 SIP 中分离出的有效抗癌化合物。
