Department of Food Science and Biotechnology, Sejong University, Gwangjin-gu, Seoul 05006, Republic of Korea.
Graduate School of Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea.
Phytomedicine. 2020 Jan;66:153129. doi: 10.1016/j.phymed.2019.153129. Epub 2019 Oct 31.
BACKGROUND: Phyllanthus emblica L. (Indian gooseberry) is widely used in the Ayurveda for thousands of years to treat health complications including disorders of the immune system, diabetes, and obesity. PURPOSE: For the first time, our study aims to demonstrate the molecular mechanisms of the fruit extract of Phyllanthus emblica (PEFE) involved in the promotion of fat cell apoptosis and alleviation of adipogenesis. METHODS: The active constituents from PEFE were identified using high performance liquid chromatography-mass spectrometry (HPLC-MS). We carried out the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effects of PEFE using 3T3-L1 pre-adipocytes. The colonogenic assay was carried out to determine the inhibitory effect of 3T3-L1 adipocytes after PEFE treatment. In addition, inhibition of pancreatic lipase activity was performed and the lipolytic activity of PEFE and digallic acid was compared with the well-known standard drug orlistat. Besides, the molecular interaction and ligand optimization between digallic and adipogenesis/apoptosis markers were also carried out. Furthermore, to confirm fat cell apoptosis we have used several detection methods that includes Hoechst staining, PI staining, Oil staining and qPCR respectively. RESULTS: Digallic acid was identified as a major component in the PEFE. The IC values of digallic acid and PEFE were found to be 3.82 µg/ml and 21.85 µg/ml respectively. PEFE and digallic acid showed significant anti-lipolytic activity compared to the standard drug orlistat. In the mature adipocytes, PEFE significantly decreased triglyceride accumulation by downregulating adiponectin, PPARγ, cEBPα, and FABP4 respectively. We further analyzed the expression of apoptosis related genes upon PEFE treatment. Apoptotic process initiated through upregulation of BAX and downregulation of BCL2 resulting in an increased caspase-3 activity. In addition, we have also confirmed the apoptosis and DNA fragmentation in 3T3-L1 cells using Hoechst, PI and TUNEL assays. CONCLUSION: PEFE negatively regulates adipogenesis by initiating fat cell apoptosis and therefore it can be considered as a potential herbal medicinal product for treating obesity.
背景：余甘子（印度醋栗）在印度阿育吠陀中被广泛使用了数千年，用于治疗包括免疫系统紊乱、糖尿病和肥胖症在内的健康并发症。
目的：我们的研究首次旨在展示余甘子果提取物（PEFE）促进脂肪细胞凋亡和减轻脂肪生成的分子机制。
方法：使用高效液相色谱-质谱联用技术（HPLC-MS）鉴定 PEFE 中的活性成分。我们使用 3T3-L1 前脂肪细胞进行 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定，以评估 PEFE 的细胞毒性作用。进行集落形成测定以确定 PEFE 处理后 3T3-L1 脂肪细胞的抑制作用。此外，还进行了胰腺脂肪酶活性抑制测定，并将 PEFE 和没食子酸的脂肪分解活性与知名标准药物奥利司他进行了比较。此外，还进行了没食子酸与脂肪生成/凋亡标志物之间的分子相互作用和配体优化。此外，为了确认脂肪细胞凋亡，我们使用了几种检测方法，包括 Hoechst 染色、PI 染色、油染色和 qPCR。
结果：鉴定出没食子酸为 PEFE 的主要成分。没食子酸和 PEFE 的 IC 值分别为 3.82μg/ml 和 21.85μg/ml。PEFE 和没食子酸与标准药物奥利司他相比显示出显著的抗脂肪分解活性。在成熟脂肪细胞中，PEFE 通过下调脂联素、PPARγ、cEBPα 和 FABP4 显著减少甘油三酯积累。我们进一步分析了 PEFE 处理后与凋亡相关的基因表达。通过上调 BAX 和下调 BCL2 启动凋亡过程，导致 caspase-3 活性增加。此外，我们还使用 Hoechst、PI 和 TUNEL 测定法在 3T3-L1 细胞中证实了凋亡和 DNA 片段化。
结论：PEFE 通过启动脂肪细胞凋亡来负调节脂肪生成，因此它可以被认为是治疗肥胖症的潜在草药药物。
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