Senathilake K S, Karunanayake E H, Samarakoon S R, Tennekoon K H, de Silva E D, Adhikari A
Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Colombo 03, Sri Lanka.
Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Colombo 03, Sri Lanka.
Exp Parasitol. 2017 Jun;177:13-21. doi: 10.1016/j.exppara.2017.03.007. Epub 2017 Mar 27.
Absence of a drug that kills adult filarial parasites remains the major challenge in eliminating human lymphatic filariasis (LF); the second leading cause of long-term and permanent disability. Thus, the discovery of novel antifilarial natural products with potent adulticidal activity is an urgent need. In the present study, methanol extracts of leaves, bark and winged seeds of Dipterocarpus zeylanicus (Dipterocarpaceae) were investigated for macro and microfilaricidal activity. Two antifilarial triterpene saponins were isolated from winged seed extracts by bioactivity guided chromatographic separation and identified using Nuclear Magnetic Resonance and mass spectroscopic analysis as oleanolic acid 3-O-β-D- glucopyranoside (1) (IC50 = 20.54 μM for adult worms, 19.71 μM for microfilariae ) and oleanolic acid 3-O-α-L-arabinopyranoside (2) (IC50 = 29.02 μM for adult worms, 25.99 μM for microfilariae). Acid hydrolysis of both compounds yielded oleanolic acid (3) which was non or least toxic to human peripheral blood mono nuclear cells (Selectivity index = >10) while retaining similar macrofilaricidal (IC50 = 38.4 μM) and microfilaricidal (IC50 = 35.6 μM) activities. In adult female worms treated with 50 and 100 μM doses of oleanolic acid, condensation of nuclear DNA, apoptotic body formation and tissue damage was observed by using Hoechst 33342 staining, TUNEL assay and Hematoxylin and Eosin staining respectively. A dose dependent increase in caspase 3/CED3 activity and decrease in total protein content were also observed in these parasites. A dose dependant DNA fragmentation was observed in adult parasites and microfilariae. Decreased levels of reduced glutathione (GSH) and elevated levels of glutathione S transferase (GST), superoxide dismutase (SOD) and reactive oxygen species (ROS) were also observed in parasites treated with oleanolic acid indicating an oxidative stress mediated apoptotic event. Compound 3/oleanolic acid was thus identified as a potent and safe antifilarial compound in vitro.
缺乏能够杀死成年丝虫寄生虫的药物仍然是消除人类淋巴丝虫病（LF）的主要挑战，淋巴丝虫病是导致长期和永久性残疾的第二大原因。因此，迫切需要发现具有强大杀成虫活性的新型抗丝虫天然产物。在本研究中，对龙脑香科植物羯布罗香（Dipterocarpus zeylanicus）的叶、树皮和带翅种子的甲醇提取物进行了抗成虫和抗微丝蚴活性研究。通过生物活性导向色谱分离从带翅种子提取物中分离出两种抗丝虫三萜皂苷，并利用核磁共振和质谱分析鉴定为齐墩果酸3 - O - β - D - 吡喃葡萄糖苷（1）（对成虫的IC50 = 20.54 μM，对微丝蚴的IC50 = 19.71 μM）和齐墩果酸3 - O - α - L - 阿拉伯吡喃糖苷（2）（对成虫的IC50 = 29.02 μM，对微丝蚴的IC50 = 25.99 μM）。两种化合物的酸水解均产生齐墩果酸（3），其对人外周血单个核细胞无毒或毒性最小（选择性指数>10），同时保留了相似的抗成虫（IC50 = 38.4 μM）和抗微丝蚴（IC50 = 35.6 μM）活性。在用50和100 μM剂量的齐墩果酸处理的成年雌虫中，分别使用Hoechst 33342染色、TUNEL检测和苏木精 - 伊红染色观察到核DNA凝聚、凋亡小体形成和组织损伤。在这些寄生虫中还观察到半胱天冬酶3/CED3活性的剂量依赖性增加和总蛋白含量的降低。在成年寄生虫和微丝蚴中观察到剂量依赖性DNA片段化。在用齐墩果酸处理的寄生虫中还观察到还原型谷胱甘肽（GSH）水平降低，谷胱甘肽S - 转移酶（GST）、超氧化物歧化酶（SOD）和活性氧（ROS）水平升高，表明存在氧化应激介导的凋亡事件。因此，化合物3/齐墩果酸被鉴定为一种体外有效的安全抗丝虫化合物。
