銀耳萃取物之抗氧化功效及其對腸道保健功能之研究

The Studies of the Antioxidant Activities and the Intestinal Health Functions of Tremella fuciformis Extracts

林俊旭(Chun-Hsu Lin)

指導教授 ： 施養佳

亞洲大學/健康學院/生物科技學系碩士班/碩士(2011年)

摘要

銀耳為天然食用及藥用真菌，含有豐富的多醣體、三帖類、蛋白質、膳食纖維、維生素與幾丁質，科學研究中顯示大部分菇類熱水萃取液都有很高的抗腫瘤活性及提升人體免疫力之功能，其有效成分主要是經由酸水解後產生的β-葡聚醣(β-D-glucan)。臺灣的氣候與地理環境並不適合大量種植銀耳，所以大多從中國大陸進口，但經由檢驗發現大陸銀耳有大量的農藥殘留。針對此情況，本論文中所使用之材料是由本校健康學院林俊義院長以環控栽培生產無農藥殘留之銀耳(品系LT1& LT6) 進行研究。本論文之研究目的有四項: (1) 探討銀耳萃取物之最佳萃取條件與成分分析；(2) 評估銀耳萃取物改善腸胃道菌相之功效；(3)分析銀耳萃取物之抗氧化成分與功效；(4) 探討銀耳萃取物保護與修復H2O2對視網膜色素上皮細胞(ARPE-19)細胞氧化傷害之功效。根據研究結果顯示: (1)銀耳萃取物以1:60的比例(銀耳粉末 g : 水 ml)，5小時以75℃熱水萃取5小時，其萃取率可達35-40%。 銀耳品系LT1和 LT6之萃取物其β-葡聚醣之含量分別為10.65% 和6.14%。此外由測定結果顯示LT1與LT6 品系分別具有高含量之膳食纖維，LT1含66.42% 而LT6含46.23%。(2) 以不同劑量之銀耳萃取物分組餵食40隻S.D. 大鼠，經28天後，實驗組與對照組之大鼠其體重呈相同直線上升之趨勢，表示銀耳萃取物無毒性，不會對動物生長造成影響。由實驗結果發現，依大鼠每公斤體重餵食40 mg之銀耳萃取物，顯示有明顯增加腸道指標好菌-雙叉乳桿菌之菌數，而腸道壞菌-產氣夾膜桿菌完全沒有生成，因此證實銀耳萃取物的確有改善大鼠腸道菌相之功效。(3) 經抗氧化之實驗結果顯示，銀耳萃取物濃度為10 mg/ml時，其抗氧化能力為銀耳品系LT1(52%) > LT6品系(32%)。 清除DPPH自由基能力為銀耳品系LT1 (57%) > LT6品系(33%)。螯合亞鐵離子能力為銀耳品系LT1(31%) > LT6品系(27%) 。還原力銀耳LT1品系和LT6品系均為O.D. 0.1, 表示銀耳萃取物之還原能力不佳。當銀耳LT1品系濃度為3 mg/ml時，其清除超氧陰離子自由基能力超過100%，比LT6品系在5 mg/ml時之清除率還要更好。此外，清除一氧化氮(NO-)能力之實驗結果顯示，LT1清除能力比LT6高，由以上試驗可知LT1的抗氧化能力比LT6好。(4)由細胞存活率之試驗結果顯示，銀耳萃取物對視網膜色素上皮細胞(ARPE-19)無毒性且有促進細胞增生的效果。其次，銀耳萃取物在濃度0.1mg/ml時即能保護ARPE-19細胞免於被H2O2氧化傷害。而濃度0.5~2 mg/ml之銀耳萃取物皆有顯著的保護細胞效果。此外，銀耳萃取物濃度為0.5 mg/ml時，具有修復被H2O2氧化傷害之ARPE-19細胞功效，且濃度2 mg/ml之銀耳萃取物有顯著的修復效果。綜合上述之實驗結果，銀耳萃取物含有豐富β-glucan和膳食纖維，具有改善腸道菌相之功效和良好清除自由基之能力，且可以保護與修護視網膜色素上皮細胞免受被H2O2氧化傷害之影響。後續可更深入研究銀耳萃取物保護及修復氧化傷害細胞之機制。根據本論文之研究證實，銀耳萃取物具有良好的抗氧化活性與改善腸道菌相之功效，將來可以進一步開發成多種保健食品，例如：天然的抗氧化劑、整腸健胃產品及預防視網膜病變等之天然機能性保健食品，更可以增加銀耳之產業利用性並提升其產品之附加價值。

關鍵字

銀耳 ； 葡聚醣 ； 膳食纖維 ； 腸道菌相 ； 清除DPPH自由基 ； 還原力 ； 螯合亞鐵離子 ； 抗氧化力 ； 清除超氧陰離子 ； 清除一氧化氮 ； 視網膜色素上皮細胞

並列摘要

Tremella fuciformis is a natural edible and medicinal fungi, which is rich in polysaccharides, triterpenoids, protein, dietary fiber, vitamins and chitin. The previous researches presented that most of the mushrooms extracts by hot water were able to enhance anti-tumor activities of human immunity, and the active ingredient of these mushrooms were β-D-glucan which produced by acid hydrolysis. Taiwan's climate and geographical environment are not suitable for the cultivation of T. fuciformis, so most of T. fuciformis are imported from China. However, the problems were the imported T. fuciformis were detected the pesticide residues inside. In this thesis we used the non-toxic T. fuciformis which comes from the environment-controlled cultivation by Professor Lin, the Dean of the Health Collage in Asia University. The four purposes of this study were: (1) To examine the best extraction conditions and composition analysis of T. fuciformis; (2) To evaluate the effects of T. fuciformis extract to improve the gastrointestinal microflora of rats; (3) To analyze the antioxidant abilities and components of T. fuciformis extracts; (4) To investigate the protection and restoration effects of T. fuciformis extract to the retinal pigment epithelial cells (ARPE-19) cells by H2O2 oxidative damage. The results indicated as follows: T. fuciformis was extracted by hot water as the ratio of 1:60 (T. fuciformis powder : water) for 5 hours at 75 ℃. The extraction rate reached 35-40%. LT1 and LT6 T. uciformis extract contained β-glucan were 10.65% and 6.14%. LT1 and LT6 demonstrated a high content of dietary fibers which were 66.42% and 46.23%. (2) After 28 days experiments of feeding different dosage of T. fuciformis extracts to 40 S.D. rats, the weight of rats presented linear upward trend in both control and experiments, which means T. fuciformis extracts were non-toxic to the rats and no harmful impact to them as well. In addition, feeding 40 mg of T. fuciformis extracts per kg of rat weight showed the enhancement of the growth of Bifidobacterium, and the pathogen Clostridium perfringens were not detected in rats at all. These indicated T. fuciformis extracts was able to improve gastrointestinal microflora of rats. (3) The antioxidant abilities and components of T. fuciformis extracts were proved by the different experiments. The results indicated that 10 mg/ml of T. fuciformis extracts presented that the antioxidant capacity was LT1(52%)>LT6(32%); DPPH radical removal ability was LT1(57%)>LT6(33%); ferrousion chelating ability was LT1 (31%)>LT6(27%); LT1 and LT6 had low reducing power (OD 0.1). The removal ability of superoxide anion of 3 mg/ml LT1 strain presented better results (>100% clearance rate) than 5 mg/ml LT6 strain. Moreover, LT1 showed the better nitric oxide removal ability than LT6 strain. (4) In cell survival experiments, T. fuciformis extract was harmless to ARPE-19 cells and even was able to increase the cell proliferation. According to the results, 0.1mg/ml of T. fuciformis extract was able to prevent the ARPE-19 cells from the H2O2 oxidative damage. The concentrations of T. fuciformis extract between 0.5-2 mg/ml showed the significant protective effects to ARPE-19 cells. In addition, 0.5 mg/ml of T. fuciformis extract was able to help the repairing of ARPE-19 cells after H2O2 damaging, and 2mg/ml of extract showed the significant effect. In summary, T. fuciformis extract was rich in β-glucan and dietary fiber, and it presented the effects of improving the rat intestinal microflora and antioxidant ability. Furthermore, T. fuciformis extract was able to protect and repair the retinal pigment epithelial cells by H2O2 damage. Follow-up can be in-depth study of the mechanism of antioxidant effects of T. fuciformis extract. Based on the results of this study, T. fuciformis extract can be further developed to different functional health food such as natural antioxidant, stomach and intestinal improvement products or the prevention of retinopathy of products, which can increase the industrial utilities and enhance the value-added products of T. fuciformis in the future.

並列關鍵字

Tremella fuciformis ； β-glucan ； Dietary fiber ； intestinal microflora ； DPPH radical ； Reducing power ； Ferrous ion chelating ； Antioxidant ； Superoxide anion ； Nitric oxide removal ； Retinal pigment epithelial cells

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