- 學位論文
白鶴靈芝草葉片萃取物之抗氧化活性及對人類肝癌細胞 HepG2 抑制活性之探討
Investigation of antioxidant activity and inhibitory effects on human hepatoma cells (HepG2) of extracts from the leaf of Rhinacanthus nasutus
摘要
氧化壓力為一種與許多病理情況相關之現象，包括癌症、糖尿病、發炎和神經退化性疾病等。植物之酚類化合物，為天然之抗氧化劑及抗癌劑之潛在來源。白鶴靈芝草 (Rhinacanthus nasutus) 為一種中草藥，屬於爵床科 (Acanthaceae) 之灌木，研究顯示白鶴靈芝草可分離出許多具有抗氧化特性之生物活性物質，因此，本研究使用95℃ 熱水、甲醇和不同濃度乙醇 （25、50、75、95%）針對葉片進行萃取，測定其總酚 (TPC)、總類黃酮化合物含量 (TFC) 和抗氧化活性，包括DPPH˙、ABTS·+自由基清除活性和還原力，並透過高效能液相層析儀 (HPLC) 分析生物活性物質。於抗癌活性中，評估萃取物處理對人類肝癌細胞 (HepG2) 存活率、細胞週期與細胞凋亡之影響，並利用西方墨點法分析細胞凋亡途徑及相關蛋白之表現量。結果顯示75%乙醇萃取物呈現最高含量之TPC (154.58 mg/g DW) 和TFC (171.37 mg/g DW)。於HPLC 分析中，發現七種酚類化合物，分別為沒食子酸、兒茶素、綠原酸、咖啡酸、芸香苷、楊梅素和槲皮素，此外，75%乙醇萃取物有最豐富之酚類化合物。75%乙醇萃取物有最佳之DPPH˙自由基清除活性和還原力，顯示最低之 EC50值，分別為 61.22 和 694.36 μg/mL，而ABTS·+自由基清除活性為甲醇萃取物最佳，EC50值為 221.40 μg/mL。於抗癌活性分析中，75%乙醇萃取物對HepG2細胞有最強之抑制作用，於72小時之EC50值為1454.29 μg /mL，細胞存活率為36.2%，且能將細胞週期停滯於G0/G1期或S期，並阻止細胞進入G2/M期，同時增加Sub-G1期之細胞數量，誘導HepG2細胞透過細胞凋亡走向死亡。細胞凋亡之機制涉及外在途徑及內在途徑，75%乙醇萃取物顯示以劑量依賴性下調Bcl-2以及上調FADD蛋白，使Pro-caspase -8與 -9裂解，促使Cleaved-caspase - 9之生成，進而使Pro-caspase -7及-3 裂解，最終引發細胞凋亡。綜上所述，白鶴靈芝草葉片中天然生物活性成分具有抗氧化及抗癌活性，有潛力可作為保健食品及治療藥物，預防氧化壓力相關疾病之發生與肝癌之治療。
並列摘要
Oxidative stress is a phenomenon that is related to the development of many pathological conditions, including cancer, diabetes, inflammatory, and neurodegenerative disorders, among others. Phenolic compounds in plants are potential sources of natural antioxidants and anticancer agents. Rhinacanthus nasutus is a medicinal herb belonging to the family Acanthaceae of shrubs. Previous studies demonstrated it can isolate many bioactive compounds that possess antioxidant property. Therefore, the leaves were extracted using hot water, methanol, and varying concentrations of ethanol (25, 50, 75, 95%). The total phenolic (TPC), total flavonoids contents (TFC) and antioxidant activity were measured, including DPPH˙ and ABTS·+ free radical scavenging activities as well as reducing power. The bioactive compounds were analyzed using high-performance liquid chromatography (HPLC). In anticancer activity, evaluation of the effects of extracts treatment on the cell viability, cell cycle, and apoptosis of human hepatoma cells (HepG2). Furthermore, western blot analysis was performed to investigate the apoptotic pathways and expression levels of related proteins. The results indicated that 75% ethanol extract presented the highest content of TPC (154.58 mg/ g DW) and TFC (171.37 mg/ g DW). In the HPLC analysis, seven phenolic compounds were found, including gallic acid, catechin, chlorogenic acid, caffeic acid, quercetin, myricetin, and quercetin. Moreover, the 75% ethanol extract showed the highest abundance of phenolic compounds. The 75% ethanol extract exhibited the best DPPH˙ free radical scavenging activity and reducing power, demonstrating the lowest EC50 values of 61.22 and 694.36 μg/mL, respectively. The methanol extract demonstrated the strongest ABTS·+ free radical scavenging activity, with an EC50 value of 221.40 μg/mL. In anticancer activity assays, the 75% ethanol extract exhibited the strongest inhibitory effect on HepG2 cells, with an EC50 value of 1454.29 μg/mL at 72 hours, resulting in a cell viability of 36.2%. In addition, 75% ethanol extract was able to arrest the cell cycle at the G0/G1 or S phase, to prevent G2/M phase entry and increased the population of cells in the Sub-G1 phase, induced cell death by apoptosis. The 75% ethanol extract exhibited a dose-dependent downregulation of Bcl-2 and downregulation of FADD proteins, pro-caspase-8 and -9 were cleaved, leading to the generation of cleaved-caspase-9. This further resulted in the cleavage of pro-caspase-7 and -3, ultimately inducing cell apoptosis. In summary, the natural bioactive compounds found in R. nasutus leaf exhibited antioxidant and anticancer activities, showing potential as functional foods and therapeutic drug for preventing oxidative stress-related diseases and treating liver cancer.