- 學位論文
中草藥及其芽孢桿菌發酵產物之抗氧化和抗衰老功效
Antioxidant and Anti-Senescence Effects of Chinese Herbal Medicines and Fermentation Products of Bacillus subtilis
摘要
中草藥作為傳統藥物已有數千年歷史，其具有免疫調節、抗氧化、抗發炎等功效。中草藥發酵技術為一種可以用來提升中草藥療效及降低中草藥毒性之方法。本研究目的為探討七種中草藥之抗氧化及抗老化活性，包含梔子、黃芩、黃連、甘草、菊花、柴胡及板藍根，分析中草藥與枯草芽孢桿菌共培養前後之抗氧化及抗老化活性是否有改變，並利用高效液相層析儀分析中草藥共培養前後之活性成分變化。中草藥抗氧化分析包含兩大部分，一為清除DPPH自由基能力測定，另一為清除氫氧自由基能力測定。早期衰老細胞模式之建立，係以過氧化氫誘導人類胚肺成纖維細胞MRC-5建立細胞衰老，並以流氏細胞儀分析細胞週期。在過氧化氫誘導細胞老化前給予中草藥及其與枯草桿菌之共培養物以提供保護作用，分析氧化損傷的主要指標，包括超氧化物歧化酶及穀胱甘肽過氧化物酶。結果顯示，七種中草藥在共培養前後其清除DPPH自由基能力皆有顯著提升；而清除氫氧自由基能力則只有梔子、黃芩及甘草有顯著提升。中草藥活性成分分析以黃芩、甘草及柴胡在共培養後有顯著提升。利用過氧化氫誘導氧化損傷會使細胞之細胞週期發生G2停滯現象。酵素分析方面，板藍根在共培養前其超氧化物歧化酶之活性最佳，但板藍根在共培養後，其活性卻最低；黃芩在共培養前其穀胱甘肽過氧化物酶之活性最佳，而柴胡在共培養後其活性最佳。綜上所述，中草藥具有清除自由基的分子，且富含抗氧化活性，其藥用價值可經由微生物共培養發酵後提升，期望未來在動物實驗上能有好的表現並發展為保健食品。
並列摘要
Chinese herbal medicines (CHMs), which possess immunomodulatory, anti-oxidant, and anti-inflammatory activities, have been used as traditional medicines for thousands of years. Fermentation is a traditional technology for improving the efficacy or reducing the adverse effects of CHMs. The objective of this study is to investigate the antioxidant and anti-senescence activities of the CHMs, including Gardenia jasminoides, Scutellaria baicalensis, Coptidis rhizome, Glycyrrhizae radix et rhizome, Chrysanthemi flos, Bupleuri radix, and Baphicacanthi cusiae Rhizoma et radix. The antioxidant and anti-senescence activities of the CHMs fermented by Bacillus subtilis were also studied. The CHMs were inoculated with B. subtilis AC and were incubated at 37°C for 24 h. The active ingredients of the CHMs before and after fermentation were analyzed by using high performance liquid chromatography (HPLC). The antioxidant activities of the CHMs and their fermentation products were measured by DPPH radical photometric assay and deoxyribose assay. For the establishment of premature senescence cellular model, human lung fibroblast MRC-5 cells were treated with H2O2, and the cell cycle was analyzed by flow cytometry. The MRC-5 cell before hydrogen peroxide (H2O2)-induced oxidative damage were pretreated with CHMs or the fermentation products of CHMs, and then the major indexes of oxidative damage, including superoxide dismutase (SOD) and glutathione peroxidase (GPx), were quantified by biochemical assays. The results indicated that the DPPH scavenging activities of Gardenia jasminoides, Scutellaria baicalensis, Coptidis rhizome, Glycyrrhizae radix et rhizome, Chrysanthemi flos, Bupleuri radix, and Baphicacanthi cusiae Rhizoma et radix were elevated by fermentation. For deoxyribose assay, only Gardenia jasminoides, Scutellaria baicalensis, and Glycyrrhizae radix et rhizome showed significantly difference after fermentation (p<0.05). The active ingredients of Scutellaria baicalensis, Glycyrrhizae radix et rhizome, and Bupleuri radix were elevated by fermentation (p<0.05). On the other hand, the results of cell cycle assay showed that H2O2-induced oxidative damage, leading to G2 arrest of the cell cycle. Gardenia jasminoides and Glycyrrhizae radix et rhizome have best capacity to relieve G2 arrest before fermentation, while Scutellaria baicalensis, Chrysanthemi flos, Bupleuri radix, and Baphicacanthi cusiae Rhizoma et radix have best capacity after fermentation. Furthermore, the results of SOD activity assay showed that Baphicacanthi cusiae Rhizoma et radix has best activity before fermentation, however, Baphicacanthi cusiae Rhizoma et radix has the lowest activity after fermentation. Moreover, the results of GPx activity showed that Scutellaria baicalensis has best activity before fermentation, while Chrysanthemi flos and Bupleuri radix have best activity after fermentation. To sum up, the CHMs may contain a wide variety of free radical scavenging molecules, which are rich in antioxidant activity. The medicinal value can be enhanced by microbial fermentation. Last but not least, we are looking forward to conducting animal experiment and developing health food.