消渴草改善小鼠FL83B 肝臟細胞株葡萄糖攝入活性成分之鑑定

糖尿病已經連續數年位居國內前五大死因，平均每小時會有一人死於糖尿病，糖尿病已成為國人不可忽視的重要議題。第二型糖尿病(Type 2 diabetes mellitus, T2DM)的病因主要是體內胰島素分泌不足或細胞對胰島素無法產生反應，使葡萄糖無法進入細胞內被利用而產生高血糖的現象。消渴草(Ruellia tuberosa Linn.)屬爵床科(Acanthaceae)蘆利草屬植物，為坊間常用來治療糖尿病的傳統中草藥，文獻指出其具有消炎、止痛、消渴、利尿及解毒之功效，本實驗室先前的研究也已證實消渴草具有改善以高脂飲食及STZ誘導糖尿病大鼠之高血醣、胰島素阻抗及肝臟脂肪變性等症狀；另外也發現，消渴草之乙酸乙酯區分物具有改善有胰島素阻抗之小鼠肝臟細胞葡萄糖攝取及減少肝臟細胞脂肪堆積的能力。本研究欲進一步進行消渴草之乙酸乙酯區分物有效成分之分離及純化，以Tumor necrosis factor-alpha (TNF-α)誘導成胰島素阻抗之小鼠FL83B肝臟細胞株為篩選平臺，評估純化後之區分物對細胞葡萄糖攝取之影響，並以液相色層分析(High Performance Liquid Chromatography, HPLC)進行分析。結果顯示，消渴草之乙酸乙酯層以Sephadex LH20 gel進行劃分可得到8個區分物(fraction)，經由MTT assay評估在濃度25µg/ml以下8個fraction皆不會明顯抑制細胞生長，以此濃度進行葡萄糖攝取實驗，結果發現EAF5區分物提升胰島素阻抗小鼠FL83B肝臟細胞株葡萄糖攝入之效果最佳。進一步將EAF5以MCI gel進行管柱層析，再將分離出之區分物以MTT assay評估其對細胞之毒性，接著以濃度25µg/ml區分物處理胰島素阻抗肝臟細胞進行葡萄糖攝入實驗，結果發現EAF5-5提高胰島素阻抗肝臟細胞葡萄糖攝入之效果最佳。最後以HPLC分析經由Sephadex LH20 gel、MCI gel管柱分離後之區分物組成分，透過HPLC圖譜比對，推測消渴草EAF5-5區分物之活性成分可能包括syringic acid和p-Coumaric acid等兩種酚酸以及類黃酮Cirsimaritin。

關鍵字

消渴草； FL83B小鼠肝臟細胞；胰島素阻抗；高效能液相層析

並列摘要

Recently, Type 2 diabetes mellitus (T2DM) is the 5th of the top ten causes of death in Taiwain, approximately one person died per hour causes by T2DM. Therefore, the treatment of diabetes is an importent issue that requires more attention. T2DM is characterized by insulin resistance, caused by insufficent production of insulin or by the ineffectiveness of insulin activity. Ruellia tuberosa L. (RTL) is an herb plant and has been used as folk medicine to cure diabetes in Asian countries for decades. RTL was reported to have anti-inflammatory, analgesic, anti-diabetes, diuretic and detoxifying effects. Previous studies in our laboratory showed that RTL may alleviate hyperglycemia, insulin resistance and liver steatosis in STZ-induced DM rats. In addition, we also found the ethyl acetate fraction from RTL improved glucose uptake and reduced lipid accumulation in TNF-α treated FL83B hepatocytes. The aim of this study was to investigate the the active compounds with improve insulin resistance potential in RTL-EA using glucose uptake assay in TNF-α treated FL83B hepatocytes. The results showed that eight sub-fractions were isolated from RTL-EA (EAF) by Sephadex LH20 gel, and no significant difference on cytotoxicity effect was observed for eight EAFs at the concentration of 25 μg/ml in FL83B cells. Moreover, EAF5 significantly enhances the uptake of 2-NBDG in TNF-α induced-insulin resistant FL83B cells. Furthermore, EAF5 was separated into five sub-fractions by MCI gel and no significant cytotoxicity effect was found for these fractions at the concentration of 25 μg/ml in FL83B cells. Among them, EAF5-5 significantly enhanced the uptake of 2-NBDG in TNF-α treated FL83B cells. According to HPLC analysis, the possible active compounds were identified includes syringic acid, p-coumaric acid and cirsimaritin.