臺灣博碩士論文加值系統

牛樟芝（Antrodia camphorata）為台灣特有之藥用真菌，生長於台灣保育樹種牛樟的腐朽心材上，然而其野生子實體的生長速度緩慢，因此造成醫療及研究上材料不足。由於野外寄主牛樟（Cinnamomun kanehirai）為保育植物，故本研究以針葉樹種取代寄主樹種，將七種針葉樹之萃取物添加於液態培養基中，以生成之生物量及菌絲體代謝活性為依據，篩選能促進牛樟芝快速生長之因子，並對培養之菌絲體進行化學成分研究。篩選結果顯示，添加香杉（Cunninghamia konishii）葉部之多醣體於3 g/l之劑量下，菌絲體乾重可達5.29 ± 0.16 g/l，相較於對照組2.69 ± 0.13 g/l有明顯促進生長效果（** P < 0.01），其菌絲體甲醇萃取物在濃度2 mg/ml時對於α,α-diphenyl-β-picrylhydrazyl（DPPH）自由基清除能力為95.48 ± 0.20 %，高於基礎培養基組的69.94 ± 4.32 %及子實體的79.22%。分析香杉葉部之多醣體，結果顯示所含單醣之重量百分比為半乳糖胺（Galactosamine）5.15 ± 0.13 %、半乳糖（Galactose）3.75 ± 0.11 %、葡萄糖（Glucose）2.00 ± 0.14 %、甘露糖（Mannose）1.86 ± 0.24 %、肌醇（myo-inositol）0.48 ± 0.06 %、岩藻醣（Fucose）0.47 ± 0.05 %、山梨醇（Sorbitol）0.27 ± 0.05 %。針對培養於基礎培養基之牛樟芝菌絲體之酒精萃取物進行分離純化，得到四個羊毛甾酸化合物（lanostane-type compound）分別為dehydroeburicoic acid，eburicoic acid，dehydrosulphurenic acid及sulphurenic acid，這類化合物亦存在子實體中。

Antrodia camphorata is a peculiar medicinal fungus in Taiwan, which grows on the moribund heart wood of the endemic tree species, Cinnamomum kanehirai. Because of its slow growth-rate in nature and host plant endangered, the material is insufficient on medical treatment and research. This research screened for the hyphal growth factor of A. camphorata from seven coniferous trees (Cunninghamia konishii, Cryptomeria japonica, Taiwania cryptomerioides, Pinus taiwanensis, Pinus armandi, Tsugo chinensis, Taxus sumatrana) based on biomass and mycelial growth-related metabolic activity. Chemical constituents of cultured A. camphorata mycelia were also investigated. The results showed that A. camphorata fed with polysaccharide (PS) of C. konishii leaves at the dose of 3 g/l exhibited the highest growth effect on the biomass of 5.29 ± 0.16 g/l, significantly higher than that of control (2.69 ± 0.13 g/l) (** P < 0.01). The scavenging activity on α,α-diphenyl-β-picrylhydrazyl (DPPH) of 2 mg/ml methanolic extracts of PS-cultured mycelia was 95.48 ± 0.20 %, higher than both of the control (69.94 ± 4.32 %) and fruiting body (79.22 %). The compositional analysis of C. konishii leaves PS was carried out by high-performance anion-exchange chromatography. The result showed that C. konishii leaves PS composed of galactosamine (5.15 ± 0.13), galactose (3.75 ± 0.11), glucose (2.00 ± 0.14), mannose (1.86 ± 0.24), myo-inositol (0.48 ± 0.06), fucose (0.47 ± 0.05), and sorbitol (0.27 ± 0.05 %). Four lanostane-type compounds: dehydroeburicoic acid, eburicoic acid, dehydrosulphurenic acid, sulphurenic acid were purified from the ethanolic extracts of cultured A. camphorata mycelia. These compounds also exist in the fruiting body.

目錄
頁次
致謝…………………………………………………………………… I
中文摘要……………………………………………………………… II
英文摘要……………………………………………………………… III
目錄…………………………………………………………………… IV
圖表目錄……………………………………………………………… VII
名稱縮寫……………………………………………………………… X
牛樟芝菌絲體之化合物構造………………………………………… XI
壹、緒論
一、研究背景與目的………………………………………………. 1
二、牛樟芝之相關研究……………………………………………. 2
（一）牛樟芝簡介………………………………………………… 2
（二）培養技術…………………………………………………… 6
（三）成分探索…………………………………………………… 7
（四）藥理活性…………………………………………………… 15
（五）安全性評估………………………………………………… 18
貳、實驗結果
一、牛樟芝菌絲體生長促進因子探討…………………………… 20
（一）牛樟芝菌絲體生長曲線…………………………………… 20
（二）針葉樹種對牛樟芝生長促進之探討……………………… 23
（三）香杉葉不同溶媒萃取物對牛樟芝生長促進之探討……… 35
（四）香杉葉部水萃取物對牛樟芝生長促進之探討…………… 43
（五）香杉葉部多醣體對牛樟芝生長促進之探討……………… 49
（六）香杉葉部多醣體組成分析………………………………… 49
二、牛樟芝菌絲體及子實體比較………………………………… 58
（一）牛樟芝粗萃取物產率比較………………………………… 58
（二）牛樟芝多醣體之比較……………………………………… 59
（三）牛樟芝甲醇萃取物抗氧化能力之比較…………………… 60
（四）牛樟芝中Dehydrosulphurenic acid、15-Acetyl-dehydro- sulphurenic acid及Dehydroeburicoic acid成分之定量…..
63
三、牛樟芝菌絲體成分分離………………………………………… 67
（一）AC-1：Dehydroeburicoic acid……………………………. 68
（二）AC-2：Eburicoic acid……………………………………… 83
（三）AC-3：Dehydrosulphurenic acid………………………….. 96
（四）AC-4：Sulphurenic acid…………………………………… 100
參、討論與結論
（一）培養技術………………………………………………....... 117
（二）牛樟芝多醣體之單醣組成………………………………… 118
（三）牛樟芝二次代謝物………………………………………… 118
（四）未來發展………………………………………………….... 120
肆、 實驗部分
第一部份：材料
一、試驗菌種……………………………………………………… 121
二、針葉樹種……………………………………………………… 121
三、培養基………………………………………………………… 121
四、溶媒及試藥…………………………………………………… 122
五、管柱層析……………………………………………………… 122
六、儀器…………………………………………………………. 122
第二部份：方法
一、牛樟芝菌絲體培養…………………………………………… 124
二、菌絲體生長指標……………………………………………… 124
（一）生物量測定………………………………………………… 124
（二）菌絲體代謝活性…………………………………………… 125
三、生長曲線……………………………………………………… 125
四、針葉樹種萃取物對牛樟芝菌絲體生長之影響……………… 125
（一）樹種篩選…………………………………………………… 125
（二）香杉葉不同溶煤萃取物對牛樟芝菌絲體生長之影響…… 126
（三）香杉葉部水萃取物對牛樟芝菌絲體生長之影響………… 126
（四）香杉葉部多醣體對牛樟芝菌絲體生長之影響…………… 126
五、多醣體分析…………………………………………………… 128
（一）多醣體分子量分佈分析…………………………………… 128
（二）單醣組成分析……………………………………………… 129
六、牛樟芝粗萃取物之比較……………………………………… 130
（一）牛樟芝水萃取物及甲醇萃取物產率比較………………… 130
（二）牛樟芝中Dehydrosulphurenic acid、15-Acetyl-dehydro- sulphurenic acid及Dehydroeburicoic acid成分之定量…..
130
（三）牛樟芝甲醇萃取物抗氧化能力之測定…………………… 132
七、牛樟芝菌絲體成分分離……………………………………… 133
第三部份：化合物之物理性質
AC-1: Dehydroeburicoic acid……………………………………… 135
AC-2: Eburicoic acid………………………………………………. 136
AC-3: Dehydrosulphurenic acid…………………………………… 137
AC-4: Sulphurenic acid……………………………………………. 138
伍、參考文獻……………………………………………………….... 139
圖表目錄
頁次
圖1 牛樟芝（全生型）……………………………………………..... 4
圖2 牛樟芝子實體………………………………………………........ 5
圖3 不同培養天數之牛樟芝菌絲體外表型…………………............ 21
圖4 牛樟芝菌絲體液態培養生長曲線………………........................ 22
圖5 針葉樹樹幹部水萃取物對牛樟芝菌絲體乾重生成之影響…… 25
圖6 針葉樹樹幹部水萃取物（10 g wood/l）培養之牛樟芝菌絲體外表型……………………………………………………………
26
圖7 針葉樹樹幹部水萃取物（20 g wood/l）培養之牛樟芝菌絲體外表型……………………………………………………………
27
圖8 針葉樹樹幹部水萃取物（30 g wood/l）培養之牛樟芝菌絲體外表型……………………………………………………………
28
圖9 針葉樹樹幹部水萃取物（40 g wood/l）培養之牛樟芝菌絲體外表型……………………………………………………………
29
圖10 針葉樹葉部水萃取物對牛樟芝菌絲體乾重生成之影響……… 30
圖11 針葉樹葉部水萃取物（10 g leaves/l）培養之牛樟芝菌絲體外表型………………………………………………………………
31
圖12 針葉樹葉部水萃取物（20 g leaves/l）培養之牛樟芝菌絲體外表型………………………………………………………………
32
圖13 針葉樹葉部水萃取物（30 g leaves/l）培養之牛樟芝菌絲體外表型………………………………………………………………
33
圖14 針葉樹葉部水萃取物（40 g leaves/l）培養之牛樟芝菌絲體外表型………………………………………………………………
34
圖15 香杉葉部不同溶媒萃取物對牛樟芝菌絲體乾重生成之影響… 36
圖16 香杉葉部不同溶媒萃取物對牛樟芝菌絲體代謝活性之影響… 37
圖17 外加葡萄糖培養之牛樟芝菌絲體外表型……………………… 38
圖18 香杉葉部正己烷萃取物培養之牛樟芝菌絲體外表型………… 39
圖19 香杉葉部丙酮萃取物培養之牛樟芝菌絲體外表型…………… 40
圖20 香杉葉部甲醇萃取物培養之牛樟芝菌絲體外表型…………… 41
圖21 香杉葉部水萃取物培養之牛樟芝菌絲體外表型……………… 42
圖22 香杉葉部水萃取物對牛樟芝菌絲體乾重生成之影響………… 44
圖23 香杉葉水萃取物對牛樟芝菌絲體代謝活性之影響…………… 45
圖24 香杉葉部水萃取物培養之牛樟芝菌絲體外表型……………… 46
圖25 香杉葉部水萃取物多醣體層培養之牛樟芝菌絲體外表型…… 47
圖26 香杉葉部水萃取物非多醣體層培養之牛樟芝菌絲體外表型… 48
圖27 多醣體標準品校正曲線………………………………………… 51
圖28 香杉多醣體分子篩滲透層析分析圖…………………………… 52
圖29 香杉葉部多醣體經分子篩管柱分離圖………………………… 53
圖30 香杉葉部多醣體對牛樟芝菌絲體乾重生成之影響…………… 54
圖31 香杉葉部多醣體對牛樟芝菌絲體代謝活性之影響…………… 55
圖32 香杉多醣體之單醣組成分析圖………………………………… 56
圖33 牛樟芝甲醇萃取物清除DPPH自由基之能力………………… 62
圖34 牛樟芝甲醇萃取物之高壓液相層析分析結果………………… 65
圖35 1H-NMR spectrum of dehydroeburicoic acid………………….... 71
圖36 1H-NMR spectrum of dehydroeburicoic acid局部放大………… 72
圖37 1H-NMR spectrum of dehydroeburicoic acid局部放大………… 73
圖38 13C-NMR spectrum of dehydroeburicoic acid.…………………... 74
圖39 13C-NMR spectrum of dehydroeburicoic acid局部放大………... 75
圖40 DEPT spectrum of dehydroeburicoic acid………………….……. 76
圖41 DEPT spectrum of dehydroeburicoic acid局部放大…………..... 77
圖42 HMBC spectrum of dehydroeburicoic acid.……………………... 78
圖43 HMQC spectrum of dehydroeburicoic acid.……………………... 79
圖44 1H-1H COSY spectrum of dehydroeburicoic acid........................... 80
圖45 1H-1H COSY spectrum of dehydroeburicoic acid局部放大….…. 81
圖46 APCI-MS spectrum of dehydroeburicoic acid….………………... 82
圖47 1H-NMR spectrum of eburicoic acid..………………………........ 85
圖48 1H-NMR spectrum of eburicoic acid局部放大………………..… 86
圖49 1H-NMR spectrum of eburicoic acid局部放大………………..… 87
圖50 13C-NMR spectrum of eburicoic acid..…………………………... 88
圖51 13C-NMR spectrum of eburicoic acid局部放大……………..….. 89
圖52 DEPT spectrum of eburicoic acid……………………..…………. 90
圖53 DEPT spectrum of eburicoic acid局部放大…………………..… 91
圖54 HMBC spectrum of eburicoic acid..……………………………... 92
圖55 HMQC spectrum of eburicoic acid..……………………………... 93
圖56 1H-1H COSY spectrum of eburicoic acid........................................ 94
圖57 APCI-MS spectrum of eburicoic acid..…………………………... 95
圖58 1H-NMR spectrum of dehydrosulphurenic acid……….……........ 98
圖59 1H-NMR spectrum of dehydrosulphurenic acid局部放大………. 99
圖60 13C-NMR spectrum of dehydrosulphurenic acid.………………... 100
圖61 DEPT spectrum of dehydrosulphurenic acid.……………………. 101
圖62 HMBC spectrum of dehydrosulphurenic acid.…………………... 102
圖63 HMQC spectrum of dehydrosulphurenic acid.…………………... 103
圖64 1H-1H COSY spectrum of dehydrosulphurenic acid....................... 104
圖65 APCI-MS spectrum of AC-3……………………………………... 105
圖66 1H-NMR spectrum of sulphurenic acid…………………..…........ 108
圖67 1H-NMR spectrum of sulphurenic acid局部放大……………..… 109
圖68 13C-NMR spectrum of sulphurenic acid..………………………... 110
圖69 DEPT spectrum of sulphurenic acid..……………………………. 111
圖70 HMBC spectrum of sulphurenic acid..…………………………... 112
圖71 HMQC spectrum of sulphurenic acid..…………………………... 113
圖72 1H-1H COSY spectrum of sulphurenic acid.................................... 114
圖73 APCI-MS spectrum of sulphurenic acid..………………………... 115
圖74 牛樟芝生長因子追蹤流程…………………………………........ 127
圖75 牛樟芝菌絲體成分分離流程圖……………………………........ 134
表1 香杉葉部多醣體之單醣組成…………………………………… 57
表2 牛樟芝粗萃取物含量…………………………………………… 58
表3 牛樟芝多醣體之單醣組成……………………………………… 60
表4 牛樟芝甲醇萃取物清除DPPH自由基之能力………………… 61
表5 牛樟芝樣品中三個羊毛甾酸化合物之含量…………………… 64
表6 13C-NMR spectra data of AC 1-4 (pyridine-d5, 125 MHz) ……… 116

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