Saini Prasanta, Gayen Prajna, Kumar Deepak, Nayak Ananya, Mukherjee Niladri, Mukherjee Suprabhat, Pal Bikas C, Babu Santi P Sinha
Parasitology Laboratory, Department of Zoology, Centre for Advanced Studies, Visva-Bharati University, Santiniketan 731235, West Bengal, India.
National Institute of Pharmaceutical Education and Research (NIPER), 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
Parasitol Int. 2014 Oct;63(5):717-28. doi: 10.1016/j.parint.2014.06.008. Epub 2014 Jul 5.
A bio-assay guided fractionation and purification approach was used to examine in vitro antifilarial activities of the crude methanolic extract of Nyctanthes arbortristis as well as fractions and isolated compound. From ethyl-acetate fraction we isolated and identified a triterpenoid compound which has been characterized as ursolic acid (UA) by HPLC and NMR data. We are reporting for the first time isolation and identification of UA from the leaves of N. arbortristis. The crude extract and UA showed significant micro- as well as macrofilaricidal activities against the oocyte, microfilaria and adult of Setaria cervi (S. cervi) by dye exclusion test and MTT reduction assay. Significant microfilaricidal activity of UA was further proved against mf of W. bancrofti by viability assay. The findings thus provide a new lead for development of a suitable filaricide from natural products. The molecular mechanism of UA was investigated by performing TUNEL, Hoechst staining, Annexin V-Cy3, flow cytometric analysis and DNA fragmentation assay. Differential expressions of pro- and anti-apoptotic genes were observed at the transcription and translational levels in a dose-dependent manner. Depletion in the worm GSH level and elevation in the parasite GST, SOD and super oxide anion indicated the generation of ROS. In this investigation we are reporting for the first time that UA acts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants like GSH, GST and SOD of S. cervi.
采用生物测定引导的分离纯化方法，研究了夜花树粗甲醇提取物及其各馏分和分离出的化合物的体外抗丝虫活性。从乙酸乙酯馏分中，我们分离并鉴定出一种三萜类化合物，通过高效液相色谱（HPLC）和核磁共振（NMR）数据将其表征为熊果酸（UA）。我们首次报道了从夜花树叶片中分离鉴定出UA。通过染料排斥试验和MTT还原试验，粗提取物和UA对鹿丝状线虫（S. cervi）的卵母细胞、微丝蚴和成虫均表现出显著的微丝蚴杀灭和成虫杀灭活性。通过活力测定进一步证明了UA对班氏吴策线虫微丝蚴具有显著的杀微丝蚴活性。因此，这些发现为从天然产物开发合适的杀丝虫剂提供了新线索。通过进行TUNEL、Hoechst染色、膜联蛋白V-Cy3、流式细胞术分析和DNA片段化试验，研究了UA的分子机制。在转录和翻译水平上观察到促凋亡和抗凋亡基因的差异表达呈剂量依赖性。线虫谷胱甘肽（GSH）水平的降低以及寄生虫谷胱甘肽S-转移酶（GST）、超氧化物歧化酶（SOD）和超氧阴离子的升高表明活性氧（ROS）的产生。在本研究中，我们首次报道UA通过诱导细胞凋亡以及下调和改变鹿丝状线虫的一些关键抗氧化剂如GSH、GST和SOD的水平来发挥其抗丝虫作用。
