臺灣博碩士論文加值系統

樟芝(Antrodia cinnamomea)為台灣特有的藥用菇類，已知主要的有效成分為多醣體、固醇類及三萜類，其生理活性成分及功能仍是目前研究重點。本研究主要以天然穀物做為基質進行固態發酵以及添加不同水萃液之液態發酵，分析其生理活性成分，且將所獲得之菌絲體經由甲醇萃取後，探討抗氧化及抗腫瘤能力分析。結果發現液態培養中添加薏仁水萃液具有較佳的抗氧化與抗腫瘤能力，在抗氧化力(共軛雙烯法)方面EC50 為1.13 mg/ml；在抗腫瘤能力方面，濃度400 ppm時，對MCF-7與HepG2腫瘤細胞其細胞存活率可降至41.89%與38.3%。固態培養中以燕麥為基質具有較佳的抗氧化及抗腫瘤能力，且對不同腫瘤細胞有著不同的毒殺效果，在抗氧化力(共軛雙烯法)方面EC50 為0.57 mg/ml；捕捉DPPH能力EC50 為1.07 mg/ml；還原力EC50 為0.31 mg/ml。在抗腫瘤能力方面，濃度150 ppm時，MCF-7腫瘤細胞其細胞存活率降至28.72%，但HepG2腫瘤細胞其細胞存活率尚有75.90%。而樟芝經由固態發酵後，可產生較多之二次代謝產物，在相同濃度下固態發酵之菌絲體甲醇萃取物抗腫瘤及抗氧化能力均比液態高。研究結果希望可供日後樟芝相關治療藥物或保健食品之開發與應用的參考。

Antrodia cinnamomea is an endemic medical mushroom in Taiwan. It is well known that the major effective components in this medical fungus are polysaccharides, steroids and triterpenoids. The bioactive and efficacy of A. cinnamomea are still the main focus in many researches. In this research different kinds of natural grains were used as a substrate for solid state fermentation and different water extracts were added in liquid fermentation to study their effects on the formation of bioactive components. Moreover, antioxidant properties and anti-tumor activities of mycelia were also investigated.
The results showed that when liquid fermentation media contained barley water extract, mycelia product has better antioxidant and anti-tumor capacities. The antioxidant activity (conjugated diene method) of EC50 value was 1.13 mg/ml. About the anti-tumor activity, the cell viabilities of MCF-7 and HepG2 cancer cells were 41.89% and 38.3% respectively at the extract concentration of 400 ppm. Concerning solid state fermentation, when oats were used as a substrate, the mycelia product had stronger antioxidant properties and different extract revealed various levels of inhibitory activity to cancer cell lines. The levels of EC50 for the antioxidant activity (conjugated diene method), the DPPH radicals scavenging ability and reducing power were estimated to be around 0.57 mg/ml, 1.07 mg/ml and 0.31 mg/ml, respectively. About the anti-tumor activity, the resulted showed that the cell viability of MCF-7 cancer cell was 28.72%, but the treatment for HepG2 was less effective and the cell viability reached to 75.90%. In general solid state fermentation of A. cinnamomea could produce more of the secondary metabolities and their methanolic extracts showed stronger antioxidant and anti-tumor activities than extracts obtained from liquid fermentation at the same concentration. The results are expected to be used for the development of new functional food in the future.

目錄
中文摘要 I
Abstract II
目錄 IV
圖目錄 X
表目錄 XII
第一章 緒論 1
1-1 前言 1
1-2 研究動機與目的 2
第二章 文獻回顧 3
2-1 樟芝的介紹 3
2-1-1 樟芝的分類 3
2-1-2 樟芝的命名 3
2-1-3 樟芝生理活性成分 6
2-1-3-1 多醣體（polysaccharides） 6
2-1-3-2 三萜類（triterpenoids） 7
2-1-4 樟芝生理活性成分研究及應用 8
2-1-4-1 抗氧化功效 9
2-1-4-2 毒殺腫瘤細胞能力 10
2-1-4-3 提升免疫力 11
2-1-4-4 保肝功能 11
2-1-4-5 降血脂功能 11
2-1-4-6 抗發炎反應 12
2-2 自由基與活性氧對生物體之影響 13
2-2-1 自由基 13
2-2-2 活性氧 14
2-2-3 脂質自氧化作用 14
2-2-4 抗氧化劑之作用機制 17
2-2-4-1 自由基終止劑 17
2-2-4-2 還原劑、清除劑 17
2-2-4-3 金屬螯合劑 18
2-2-4-4 單重態氧抑制劑 18
2-3 固態發酵 18
2-3-1固態發酵與液態發酵之差異 19
2-3-2 固態發酵之優缺點 19
2-3-3 固態發酵之應用 21
2-3-3-1 固態發酵應用於工業方面 21
2-3-3-2 固態發酵應用於農業方面 21
2-3-3-3 固態發酵應用於食品方面 22
第三章 實驗材料與分法 23
3-1 實驗菌株及細胞 23
3-1-1 樟芝菌株 23
3-1-2 動物細胞株 23
3-2 實驗藥品 24
3-3 實驗儀器與設備 25
3-4 分析方法 27
3-4-1菌體濃度 27
3-4-2 澱粉分析方法 27
3-4-3 總多酚類化合物含量測定 28
3-4-4 多醣濃度測定 28
3-4-5 胞內粗三萜含量測定 29
3-4-6 抗氧化性質之測定 29
3-4-6-1 抗氧化能力之測定 29
3-4-6-2 捕捉DPPH自由基能力測定 30
3-4-6-3 還原力測定 30
3-4-7細胞增生與毒性測試 31
3-5 實驗方法 33
3-5-1 實驗架構 33
3-5-2 樟芝菌種培養與保存 34
3-5-2-1菌種斜面試管保存 34
3-5-2-2 培養皿平面培養與接菌活化 34
3-5-2-3 種菌的製備 34
3-5-3 三角瓶液態培養試驗 35
3-5-3-1 水萃液製備 35
3-5-4樟芝胞內多醣萃取 35
3-5-5 三角瓶固態培養試驗 36
3-5-6 樟芝菌絲體甲醇萃取物之製備 36
3-5-7細胞培養與繼代 37
3-5-8 抑制腫瘤細胞生長試驗 38
第四章 結果與討論 39
4-1 樟芝三角瓶液態培養試驗 39
4-1-1 不同水萃液對樟芝液態發酵菌絲體生長曲線之影響 39
4-1-2 不同水萃液對樟芝液態發酵菌絲體生理活性成分含量影響 42
4-1-2-1 不同水萃液對樟芝液態發酵菌絲體總酚含量影響 42
4-1-2-2 不同水萃液對樟芝液態發酵菌絲體胞內多醣含量影響 43
4-1-2-3 不同水萃液對樟芝液態發酵菌絲體粗三萜含量影響 44
4-1-3 不同水萃液對液態發酵菌絲體甲醇萃取物抗氧化活性測定 47
4-1-3-1 菌絲體甲醇萃取物之抗氧化力 47
4-1-3-2菌絲體甲醇萃取物捕捉DPPH之能力 49
4-1-3-3菌絲體甲醇萃取物之還原力 51
4-1-4 菌絲體甲醇萃取物抗氧化活性之EC50 52
4-2 樟芝三角瓶固態培養試驗 54
4-2-1 探討不同基質對樟芝固態發酵菌絲體生理活性成分含量影響 54
4-2-1-1 不同基質對樟芝固態發酵菌絲體總酚含量影響 54
4-2-1-2不同基質對樟芝固態發酵其發酵物粗三萜含量影響 55
4-2-2 未發酵基質與發酵基質甲醇萃取物抗氧化活性比較 57
4-2-2-1未發酵基質與發酵基質甲醇萃取物捕捉DPPH之能力 57
4-2-2-2未發酵基質與發酵基質甲醇萃取物之還原力 58
4-2-3 不同基質對樟芝固態發酵甲醇萃取物抗氧化活性測定 59
4-2-3-1 固態發酵甲醇萃取物之抗氧化力 59
4-2-3-2固態發酵甲醇萃取物捕捉DPPH之能力 60
4-2-3-3固態發酵甲醇萃取物之還原力 61
4-2-4 固態發酵甲醇萃取物抗氧化活性之EC50 62
4-3 樟芝甲醇萃取物對正常細胞毒性測試 64
4-3-1 液態發酵甲醇萃取物對正常細胞毒性測試 64
4-3-2 固態發酵甲醇萃取物對正常細胞毒性測試 65
4-4 樟芝甲醇萃取物對腫瘤細胞存活率之影響 66
4-4-1 液態發酵甲醇萃取物對腫瘤細胞存活率之影響 67
4-4-2固態發酵甲醇萃取物對腫瘤細胞存活率之影響 70
第五章 結論與未來展望 74
5-1 結論 74
5-2 未來展望 76
參考文獻 77


圖目錄
圖2-1 野生樟芝子實體 4
圖2-2 樟芝木屑培養 4
圖2-3 樟芝固態平板培養 5
圖2-4 樟芝固態培養 5
圖2-5 樟芝液態培養之菌絲球 5
圖2-6 脂質自氧化反應途徑及產物 16
圖3-1 實驗架構 33
圖4-1 不同水萃液對樟芝液態發酵菌絲體生長曲線之影響 41
圖4-2 不同水萃液對樟芝液態發酵菌絲體總酚含量之影響 43
圖4-3 不同水萃液對樟芝液態發酵菌絲體胞內多醣含量之影響 44
圖4-4 不同水萃液對樟芝液態發酵菌絲體粗三萜含量之影響 45
圖4-5 不同水萃液對樟芝液態發酵菌絲體甲醇萃取物之抗氧化力 48
圖4-6 不同水萃液對樟芝液態發酵菌絲體甲醇萃取物捕捉DPPH能力 50
圖4-7 不同水萃液對樟芝液態發酵菌絲體甲醇萃取物之還原力 52
圖4-8 不同基質對樟芝固態發酵菌絲體總酚含量之影響 55
圖4-9 不同基質對樟芝固態發酵菌絲體粗三萜含量之影響 56
圖4-10 未發酵基質與發酵基質甲醇萃取物捕捉DPPH之能力 58
圖4-11 未發酵基質發酵基質甲醇萃取物之還原力 59
圖4-12 不同基質對樟芝固態發酵菌絲體甲醇萃取物之抗氧化力 60
圖4-13 不同基質對樟芝固態發酵菌絲體甲醇萃取物捕捉DPPH能力 61
圖4-14 不同基質對樟芝固態發酵菌絲體甲醇萃取物還原力 62
圖4-15 液態發酵菌絲體甲醇萃取物對正常細胞(3T3)存活率之影響 65
圖4-16 固態發酵菌絲體甲醇萃取物對正常細胞(3T3)存活率之影響 66
圖4-17 液態發酵菌絲體甲醇萃取物對MCF-7腫瘤細胞存活率之影響 69
圖4-18 液態發酵菌絲體甲醇萃取物對HepG2腫瘤細胞存活率之影響 69
圖4-19 固態發酵菌絲體甲醇萃取物對MCF-7腫瘤細胞存活率之影響 73
圖4-20 固態發酵菌絲體甲醇萃取物對HepG2腫瘤細胞存活率之影響 73




表目錄
表3-1 實驗藥品清單 24
表3-2 實驗儀器清單 25
表3-3 3T3、HepG2細胞培養基組成 37
表3-4 MCF-7細胞培養基組成 37
表4-1 不同水萃液對液態發酵菌絲體乾重、胞內多醣、總酚及粗三萜含量之影響 46
表4-2 不同水萃液對樟芝液態發酵菌絲體甲醇萃取物抗氧化活性之EC50 53
表4-3 不同基質對樟芝固態發酵菌絲體甲醇萃取物抗氧化活性之EC50 63

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