Effects of Anoectochilus formosanus Hayata on TNF-α induced insulin resistance FL83B cells

根據世界衛生組織統計，全球有約346萬名糖尿病病患，且數量將在2030年增加一倍。台灣金線蓮為蘭科植物，過去已有初步指出其具抗糖尿病之作用，但針對糖尿病的細胞研究，仍未被報導。本實驗以熱水、水與乙醇混合(50:50, v/v)及乙醇，分別萃取金線連根部、莖及葉部位，並進行化學成分分析。根據實驗結果，根部的總酚含量最多，其中以熱水水萃和混合萃取物的總酚類為最高。經由phenol-sulfuric acid assay測試萃取物之醣類含量，以根部熱水水萃醣類含量為最多。另外，不同萃取物對於小鼠肝臟細胞FL83B存活率影響，以根部熱水水萃的毒性為最低。經由40 ng/mL TNF-α處理12小時的細胞會產生胰島素抗性的現象，同時細胞對葡萄糖吸收能力有下降的現象。以低濃度根部熱水水萃物處理細胞，可以增加胰島素抗性細胞對葡萄糖吸收能力。最後，以高濃度根部熱水水萃物與抗糖尿病藥物（Pioglitazone）處理胰島素抗性細胞，可以增加細胞對葡萄糖攝取能力，而且兩者皆可達到同樣效果。綜合以上結果，金線連根部熱水水萃物具有最佳降低細胞胰島素抗性的能力，增加葡萄糖吸收能力效果。Anoectochilus formosanus Hayata (Orchidaceac) is an indigenous and valuable Taiwanese medicinal plant which usually used as a folk medicine for liver fibrosis, diabetes and treatment of cardiovascular diseases. Although, studies have pointed out that it possesses antidiabetic properties, not much study in cell culture has been conducted. The main purpose of this study was to investigate the anti-insulin resistance effects of three different extracts (hot water extract, distilled water and ethanol (50:50, v/v) extract and ethanol extract) from leaf, stem and root of A. formosanus Hayata towards mouse normal liver cells (FL83B) which induced by using tumor necrosis factor-α (TNF-α). In this study, DPPH antioxidant activity, total phenolic content and carbohydrate analysis were carried out to estimate the chemical content of different extracts from leaves, stems and roots of A. formosanus. Yet, hot water extract from the root showed relatively higher total phenolic content and carbohydrate content than the other extracts. Meanwhile, the cytotoxicity of different part of the plant was also evaluated by using MTT ((3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Hot water extracted from root at concentration of 0.125 mg/mL showed no toxicity towards the mouse liver normal cell line FL83B. Additionally, in order to investigate effects of extracts on restoration of insulin resistant FL83B cells, the cells were co-treated with 40 ng/mL of TNF-α and different extract from A. formosanus for 12 hours. Then the cells was incubated at 37 °C water bath for 1 hour with a fluorescent D-glucose analog 2- [N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl) amino]-2deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. The results showed that water extract from the root displayed higher glucose uptake ability as compared to control group. Furthermore, the glucose uptake ability of FL83B cells increased as increased the concentration of hot water extract from root. In conclusion, hot water extract from root of A. formosanus Hayata significantly improved insulin sensitivity in the TNF-α induced FL83B cells