Faculty of Pharmacy, Sri Ramachandra University, Chennai, India.
Faculty of Pharmacy, Sri Ramachandra University, Chennai, India.
J Ethnopharmacol. 2018 Jan 10;210:408-416. doi: 10.1016/j.jep.2017.09.001. Epub 2017 Sep 8.
Roots of Inula racemosa are used as a cardio protective in Ayurveda in India, being prescribed as a medicine for precordial chest pain, cough and dyspnoea, both singly and as a poly herbal.
Evaluation of Phytoestrogenic activity of the root extracts of Inula racemosa and compounds isolated therefrom in vivo, in silico and in vitro.
Alcohol (IrA) and hexane (IrH) extracts characterized by HPTLC/GC-MS analysis respectively and processed for compound isolation were evaluated for estrogenic activity (100 & 250mg/kg bw) by the Immature rat uterotrophic assay using ethinylestradiol (EE -30µg/kg bw) as standard drug. Alantolactone (ALT), Isoalantolactone (IALT) and Stigmasterolglucoside (SG) isolated from the extracts were characterized and screened in silico for ERα, ERβ binding affinity, assessed in vitro for growth modulatory effects on MCF-7 cells by MTT assay and cell cycle distribution analysis using Flow cytometry. RT-PCR analysis evaluated the mRNA expression of pS2 in these cells post exposure to ALT, IALT and SG.
In the IrA treated groups there has been a statistically significant increase (P < 0.05) in absolute and normalised uterine weight, uterine diameter, endometrial thickness, luminal epithelial cell height,diameter of ovary and in the number of primary and secondary ovarian follicles relative to untreated controls. Presence of ciliated epithelial cells in the oviduct, elevated number of early growing follicles characterized by an increased oocyte diameter, and signs of vascularization in the cortex of ovarian sections in this group relative to EE treated group are indicative of pervasive activation of follicular growth and initiation. Virtual docking demonstrated ERα affinity for IALT, ERβ affinity for ALT, while SG showed a high binding affinity to both with a relatively greater ERβ binding affinity. Dose dependent decrease in cell viability mediated by IALT and SG in the MTT assay is corroborated by a statistically significant increase (p < 0.05) in sub G0-G1 cells by SG at 200 and 400µM in cell cycle analysis and there has been an induction of pS2 by IALT and SG in the ER regulated MCF-7 cells.
Demonstration of classical morphological changes induced by estrogen stimulation mediated by IrA in vivo at both the tested doses, isolation of the antioxidant SG from IrA and its dose dependent growth inhibitory effect on estrogen sensitive MCF-7 cells through apoptotic induction and an up regulation of pS2 are suggestive of an anti-estrogenic effect through estrogen receptor binding affinity, typical of phytoestrogens that bind to ER but do not elicit a full estrogenic response. The observed estrogenic effect of IrA suggests a multi mechanistic molecular action involving antioxidant as well as redox signalling pathways acting in consonance with their anti-estrogenic effects owing to the weak estrogen like competitive receptor binding of SG.
在印度的阿育吠陀医学中，旋覆花的根被用作心脏保护剂，被开为治疗胸痛、咳嗽和呼吸困难的药物，单独使用或作为复方草药使用。
评估旋覆花根提取物及其分离化合物的植物雌激素活性，包括体内、体外和计算机模拟研究。
采用 HPTLC/GC-MS 分析分别对醇提物（IrA）和正己烷提物（IrH）进行了特征描述，并对其进行了化合物分离处理，然后采用以乙炔雌二醇（EE-30μg/kg bw）为标准药物的未成熟大鼠子宫增重试验，评估其雌激素活性（100 和 250mg/kg bw）。从提取物中分离得到的土木香内酯（ALT）、异土木香内酯（IALT）和豆甾烷苷（SG）进行了特征描述和计算机模拟 ERα、ERβ 结合亲和力筛选，并通过 MTT 测定和流式细胞术分析细胞周期分布评估其对 MCF-7 细胞的生长调节作用。RT-PCR 分析评估了这些细胞在暴露于 ALT、IALT 和 SG 后 pS2 的 mRNA 表达。
在 IrA 处理组中，与未处理对照组相比，绝对和正常化子宫重量、子宫直径、子宫内膜厚度、腔上皮细胞高度、卵巢直径以及初级和次级卵泡数量均有统计学意义上的显著增加（P<0.05）。在该组中，输卵管中存在纤毛上皮细胞，早期生长的卵泡数量增加，卵母细胞直径增大，卵巢切片皮质中的血管化迹象表明卵泡生长和启动的普遍激活。虚拟对接显示，IALT 对 ERα 具有亲和力，ALT 对 ERβ 具有亲和力，而 SG 对两者均具有高结合亲和力，且对 ERβ 的结合亲和力相对更高。MTT 试验中 IALT 和 SG 介导的细胞活力剂量依赖性下降，通过 SG 在细胞周期分析中在 200 和 400μM 时在 sub G0-G1 细胞中统计学上显著增加（p<0.05），并且 IALT 和 SG 在 ER 调节的 MCF-7 细胞中诱导了 pS2。
IrA 在体内的两种测试剂量均能诱导经典的雌激素刺激引起的形态学变化，从 IrA 中分离出抗氧化剂 SG，并通过诱导凋亡和上调 pS2，对雌激素敏感的 MCF-7 细胞产生剂量依赖性的生长抑制作用，这表明其具有通过与雌激素受体结合亲和力发挥的抗雌激素作用，这是植物雌激素的典型特征，它们与 ER 结合但不会引起完全的雌激素反应。IrA 的观察到的雌激素作用表明存在一种多机制的分子作用，涉及抗氧化和氧化还原信号通路，这些作用与它们的抗雌激素作用一致，这归因于 SG 对雌激素受体的弱雌激素样竞争性结合。
