Table_2_Identification of Steroidogenic Components Derived From Gardenia jasminoides Ellis Potentially Useful for Treating Postmenopausal Syndrome.docx

Estrogen-stimulating principles have been demonstrated to relieve postmenopausal syndrome effectively. Gardenia jasminoides Ellis (GJE) is an herbal medicine possessing multiple pharmacological effects on human health with low toxicity. However, the therapeutic effects of GJE on the management of postmenopausal syndrome and its mechanism of action have not been fully elucidated. In this study, network pharmacology-based approaches were employed to examine steroidogenesis under the influence of GJE. In addition, the possibility of toxicity of GJE was ruled out and four probable active compounds were predicted. In parallel, a chromatographic fraction of GJE with estrogen-stimulating effect was identified and nine major compounds were isolated from this active fraction. Among the nine compounds, four of them were identified by network pharmacology, validating the use of network pharmacology to predict active compounds. Then the phenotypic approaches were utilized to verify that rutin, chlorogenic acid (CGA) and geniposidic acid (GA) exerted an estrogen-stimulating effect on ovarian granulosa cells. Furthermore, the results of target-based approaches indicated that rutin, CGA, and GA could up-regulate the FSHR-aromatase pathway in ovarian granulosa cells. The stimulation of estrogen production by rat ovarian granulosa cells under the influence of the three compounds underwent a decline when the follicle-stimulating hormone receptor (FSHR) was blocked by antibodies against the receptor, indicating the involvement of FSHR in the estradiol-stimulating activity of the three compounds. The effects of the three compounds on estrogen biosynthesis- related gene expression level were further confirmed by Western blot assay. Importantly, the MTT results showed that exposure of breast cancer cells to the three compounds resulted in reduction of cell viability, demonstrating the cytotoxicity of the three compounds. Collectively, rutin, chlorogenic acid and geniposidic acid may contribute to the therapeutic potential of GJE for the treatment of postmenopausal syndrome.

雌激素样活性成分已被证实可有效缓解绝经后综合征。栀子（Gardenia jasminoides Ellis, GJE）是一种对人体健康具有多种药理活性且毒性较低的中药材，然而其用于改善绝经后综合征的治疗效果及作用机制尚未完全阐明。本研究采用基于网络药理学（network pharmacology）的方法，探究了栀子提取物干预下的类固醇生成过程；此外，本研究排除了栀子提取物的潜在毒性，并预测出四种潜在活性成分。与此同时，本研究鉴定出具有雌激素样活性的栀子提取物色谱组分，并从该活性组分中分离得到9种主要成分，其中4种可通过网络药理学方法鉴定得到，验证了网络药理学用于预测活性成分的可行性。随后，本研究采用表型研究方法验证了芦丁（rutin）、绿原酸（chlorogenic acid, CGA）以及京尼平苷酸（geniposidic acid, GA）可对卵巢颗粒细胞产生雌激素样激活作用。基于靶点的研究结果显示，芦丁、CGA与GA可上调卵巢颗粒细胞中的卵泡刺激素受体（follicle-stimulating hormone receptor, FSHR）-芳香化酶（aromatase）通路；当使用抗FSHR抗体阻断该受体后，三种化合物对大鼠卵巢颗粒细胞雌激素生成的刺激作用显著下降，表明FSHR参与了三种化合物的雌二醇激活活性。蛋白质印迹（Western blot）实验进一步证实了三种化合物对雌激素生物合成相关基因表达水平的调控作用。值得注意的是，MTT实验结果显示，三种化合物处理乳腺癌细胞后可降低细胞存活率，证实了这三种化合物具有细胞毒性。综上，芦丁、绿原酸与京尼平苷酸可能是栀子提取物（GJE）用于治疗绝经后综合征的潜在活性物质基础。