臺灣博碩士論文加值系統

粉防己鹼(Tetrandrine)為bisbenzylisoquinoline類之生物鹼，萃取自中草藥植物粉防己(Stephania tetrandra S. MOORE)的根，具有抗發炎、抗氧化及降血壓等功效，並能有效抑制結腸癌細胞生長與和血小板凝集之作用；本研究利用農桿根群菌(Agrobacterium rhizogenes)菌株ATCC 15834及 ATCC 43057轉殖粉防己所誘導的毛狀根(Hairy roots)，於液態培養基中建立二次代謝產物粉防己鹼之生產系統，提供了有效生產二次代謝產物的另一途徑。所獲得毛狀根經PCR鑑定證實為轉殖根系，挑選高生質量及高粉防己鹼含量根系HR10-1, HR11-1經固態培養基培養一個月後，其生質量分別為 0.137, 0.068 g dw/petri-dish，經HPLC分析其粉防己鹼含量分別為 8.57, 11.66 mg/g，其值高於市售藥材(4.81 mg/g)；比較毛狀根在不同基礎鹽類配方(MS, 1/2MS, B5, WPM, N6)之固態培養基中生長狀況，可發現WPM培養基最適合粉防己毛狀根生長；粉防己毛狀根於WPM液態培養之生長曲線試驗，於第五週可達最高生質量為 6.5 g/L，粉防己鹼產量為 68.69 mg/L；以WPM鹽類配方為基礎培養基，進一步利用反應曲面試驗設計法(Response Surface Methodology, RSM)，針對培養基內蔗糖、硝酸銨和硝酸鈣等組成進行調整，尋求其對生質量、粉防己鹼含量及產量之最佳濃度組成，於二階層試驗結果顯示，提高硝酸銨與硝酸鈣可得較高之粉防己鹼含量與產量，分別為控制組的1.8倍與1.5倍，針對生質量的陡升路徑試驗中，結果於第5組的處理，可得最高之生質量，中心混成之生質量實驗設計試驗之結果顯示，三個因子最佳濃度分別為硝酸銨 564.34 mg/L、硝酸鈣 624.14 mg/L、蔗糖 41.24 g/L，毛狀根生質量之預估最佳值為 8.09 g/L，此外，以中心混成之粉防己鹼產量實驗設計試驗之結果顯示，三個因子最佳濃度分別為硝酸銨555.48 mg/L、硝酸鈣880.41 mg/L、蔗糖29.32 g/L，毛狀根粉防己鹼產量之預估最佳值為 65.74 mg/L。毛狀根利用氣泡塔生物反應器及噴霧滴流式生物反應器培養於WPM培養基五週後，結果顯示，粉防己毛狀根之生質量、粉防己鹼含量及產量經氣泡塔生物反應器培養都可得較高值。
關鍵字：粉防己、毛狀根、粉防己鹼、反應曲面試驗設計法、生物反應器

Stephania tetrandra S. MOORE is an important Chinese medicinal herb and it has been traditionally used as anti-allergic, anti-inflammatory, anti-cancer and against coronary diseases. Tetrandrine, a bis-benzylisoquinoline alkaloid has been identified as an active ingredient in S. tetrandrae and it is mainly exists in the root. Extraction of these metabolites from plants grown in the natural habitat is tedious and several factors can alter their yield. In vitro hairy root culture can be the probable solution to extract this alkaloid. The present study describes the consistent production of tetrandrine, achieved by Agrobacterium mediated transformation using A. rhizogenes strains ATCC 15834 and ATCC 43057. WPM was the most suitable media for in vitro hairy root culture. The integration of foreign gene into the genome was confirmed by PCR using rolB and rolC gene specific primers in different transgenic lines. There was variation in the growth rate and alkaloid content within different transgenic hairy root lines. Two transgenic lines HR10-1 and HR11-1 were selected and their HPLC analysis showed that the content of tetrandrine was 8.57 mg g-1 and 11.66 mg g-1, respectively. The tetrandrine contents were higher than that in the marketed crude drug (4.81 mg g-1). Furthermore, the HR10-1 transgenic line produced maximum biomass (6.5 g L-1) and tetrandrine (68.69 mg L-1) after 5 weeks cultivation. Affect three additives total biomass Response Surface Methodology (RSM). For media optimization were used to the higher biomass by alterity three different component of WPM media ie (NH4NO3, Ca(NO3)2 and sucrose). According to the central composite design experiment and mathematic calculation, the optimal concentration of NH4NO3, Ca(NO3)2 and sucrose was 564.34 mg L-1, 624.14 mg L-1, 41.24 g L-1, respectively, and the best hairy root biomass was 8.09 g L-1.Our results indicated that transgenic lines of S. tetrandra hairy root culture exhibited potential for mass production of tetrandrine.
Key words: Stephania tetrandra S. MOORE, Hairy root, Tetrandrine, RSM, Bioreactor

目 錄
中文摘要........................................................................................................... I
Summary………………………………………………..…………………….. III
表目錄……………………………………………...……………………. VII
圖目錄……………………………………………...………………….......... VII
第一章、前言……………………………………………………………. 1
第二章、前人研究..................................................................................... 5
2.1 粉防己簡介……………………………………………………… 5
2.2 粉防己藥理活性……………………………………………….....5
2.3 毛狀根形成與特性……………………………………………….8
2.4 培養基組成之影響…………………………………..………….10
2.4.1碳水化合物(carbohydrates)…………………….......…….………...10
2.4.2氮(nitrogen)…………………………………………………11
2.5 實驗設計法(Statistical experimental design)…………...……...……...12
2.6 反應曲面試驗設計法(Response Surface Methodology,
RSM)……………………………...…………………………………..15
2.6.1 二階層因子設計法(Two-level factorial design)……….………...16
2.6.2 陡升路徑法(Method of path steepest ascent)………..……..……20
2.6.3 中心混成試驗設計(Central composite design, CCD)……….…..21
2.7 建立適合粉防己毛狀根培養之生物反應器及其操作條件…………23
第三章、材料與方法................................................................................27
3.1 試驗材料……………………………………………………...27
3.2 粉防己毛狀根系統之建立…………………………………...27
3.2.1 植株培植體預培養之試驗……………………..…………27
3.2.2 農桿根群菌(A. rhizogenes)之活化及菌液……………..…28
3.2.3 農桿根群菌之接種及共培養…………...……………..…………28
3.2.4 毛狀根誘導之觀察及繼代培養……………..……………29
3.3 篩選具有高產率潛力之毛狀根根系……………….………...29
3.3.1 轉殖株之鑑定……………………………………………29
3.3.2 指標成分之分析………………………..………..………………30
3.3.2.1標準品.....................................................................................30
3.3.2.2 檢品之製備...........................................................................30
3.3.2.3 標準曲線之繪製....................................................................31
3.4 統計分析方法……………………………………..…………...31
3.5 粉防己毛狀根時序培養試驗…………………………….…...32
3.5.1試驗材料………………………………………………….32
3.5.2 固態培養條件……………………………………………32
3.5.3 液態培養條件……………………………………………32
3.5.4 粉防己毛狀根乾重之測定………………………………33
3.5.5 粉防己毛狀根培養液之pH值及導電度測量………….33
3.5.6 粉防己毛狀根培養液中殘醣含量測定………………..…..……33
3.5.7 粉防己毛狀根培養基中陰、陽離子濃度測定………..…..…….34
3.5.8 粉防己毛狀根中粉防己鹼產量之計算………..……………..…34
3.6 單一培養基成分濃度對粉防己毛狀根生長與粉防己鹼含量
影響試驗…………………………………………………...………..35
3.6.1 硝酸銨濃度對粉防己毛狀根生長、粉防己鹼含量及產
量之影響試驗……………………...……………..………………35
3.6.2 硝酸鈣濃度對粉防己毛狀根生長、粉防己鹼含量及產
量之影響試驗……………………...………………………..……35
3.6.3 蔗糖濃度對粉防己毛狀根生長、粉防己鹼含量及產量
之影響試驗……………………...………………..………………35
3.6.4 水解酪蛋白(casein hydrolysate)濃度對粉防己毛狀根
生長、粉防己鹼含量及產量之影響試驗………..………………36
3.7 粉防己毛狀根於第三週進行饋料批次培養(fed-batch
culture)之試驗………………………..…..…………………………..36
3.8 利用反應曲面試驗設計法探討最佳培養基組成
試驗………………………………………………...………………....37
3.8.1 二階層因子試驗設計……………………..………………..……37
3.8.2 陡升路徑試驗設計……………………………...….……………38
3.8.3中心混成試驗設計……………………………..…………………38
3.9 粉防己毛狀根應用生物反應器之培養試驗……………………44
3.9.1 生物反應器附屬設備…………………………………………..44
3.9.2 氣泡塔生物反應器(Bubble Column Bioreactor)培養方式…...44
3.9.3 噴霧滴流式生物反應器(Mist Trickling Bioreactor)培養方式….44
第四章、結果與討論………………………………………………….47
4.1 毛狀根轉殖系統之建立…………………………………………..47
4.2 毛狀根轉殖根系之鑑定..................................................................46
4.3 毛狀根轉殖根系的篩選及增生…………………………………..47
4.4 毛狀根於不同固態培養基之試驗………………………………..54
4.5 粉防己毛狀根時序培養試驗……………………………………..56
4.6 培養基成分對粉防己毛狀根生長與粉防己鹼含量及產量影響
試驗…………………………………………..………………………..63
4.6.1 硝酸銨濃度對粉防己毛狀根生長與粉防己鹼含量及產量影
響試驗……………………………………………………………63
4.6.2 硝酸鈣濃度對粉防己毛狀根生長與粉防己鹼含量及產量影
響試驗……………………………..……………………………..64
4.6.3 蔗糖濃度對粉防己毛狀根生長與粉防己鹼含量及產量影響
試驗……………………………………………………………..69
4.6.4 水解酪蛋白濃度對粉防己毛狀根生長與粉防己鹼含量及產
量影響試驗……………………………………………………..72
4.7 饋料批次因子對粉防己毛狀根生長與粉防己鹼含量及產量影響
試驗…………………………………………………………………73
4.7.1 粉防己毛狀根於第三週進行饋料批次培養之試驗……………73
4.8 反應曲面試驗設計………………………………………………...77
4.8.1 二階層因子試驗設計……………………………………………77
4.8.2 陡升路徑之生質量試驗設計……………………………………82
4.8.3 中心混成之生質量試驗設計……………………………………85
4.8.4 中心混成之生質量試驗設計驗證…………………….……….94
4.8.5 陡升路徑試驗設計之粉防己鹼產量……………………………96
4.8.6 中心混成試驗設計之粉防己鹼產量………...……………….99
4.9生物反應器之培養(一)…………………...……………………….….108
4.10 生物反應器之培養(二)……………………………………………..116
第五章、結論…………………………………………………………121
參考文獻………………………………………………………………122
附錄一、不同基本鹽類培養基之組成………………………………131
附錄二、植物DNA之抽取步驟……………………………………..132
附錄三、PCR檢測轉殖基因rolB與rolC使用之引子..............................133
附錄四、粉防己鹼標準品迴歸檢量線…………………………………….134
附錄五、防己諾林鹼標準品迴歸檢量線…………………………….135
附錄六、果糖標準品迴歸檢量線…………………………………….136
附錄七、葡萄糖標準品迴歸檢量線…………………………………137
附錄八、蔗糖標準品迴歸檢量線……………………………………138
附錄九、陰、陽離子測試範圍及其溶離液與化學抑制器用去除液
之配法………………………………………………………139
簡歷........................................................................................................140


表目錄
表1. 利用毛狀根培養生產生物鹼之植物名稱及其產物................................9
表2. 利用毛狀根培養生產生物鹼之反應器種類及其植物名稱與產物.......25
表3. 二階層因子試驗設計中參試之三種培養基成分及其使用之濃度，
以及利用部分因子設計法之各處理組成...............................................39
表4. 培養基中三種成分利用陡升路徑設計法之生質量迴歸公式計算之
爬升距離以及爬升後新處理之各濃度組成..........................................40
表5. 培養基中三種成分利用陡升路徑設計法之粉防己鹼產量迴歸公式
計算之爬升距離以及爬升後新處理之各濃度組成..............................41
表6. 三種參試成分在中心混成之生質量試驗設計中使用之濃度...............42
表7. 三種參試成分在中心混成之粉防己鹼產量試驗設計中使用之濃度...43
表8. 不同毛狀根根系生質量與粉防己鹼含量之比較...................................53
表9. 硝酸銨濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾林鹼含
量及粉防己鹼產量之影響......................................................................62
表10. 硝酸鈣濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾林鹼
含量及粉防己鹼產量之影響..................................................................65
表11. 蔗糖濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾林鹼含
量及粉防己鹼產量之影響....................................................................68
表12. 水解酪蛋白濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾
林鹼含量及粉防己鹼產量之影響........................................................71
表13. 粉防己毛狀根於第三週進行饋料批次培養五週之影響(一)..............75
表14. 粉防己毛狀根於第三週進行饋料批次培養五週之影響(二)..............76
表15. 粉防己毛狀根以三因子之二階層試驗設計之結果.............................80
表16. 粉防己毛狀根以三因子進行陡升路徑試驗之生質量結果.................83
表17. 粉防己毛狀根以三因子進行中心混成試驗之生質量結果.................86
表18. 生質量複迴歸分析之結果.....................................................................88
表19. 二階RSM模式之變異係數分析(ANOVA)..........................................89
表20. 中心混成試驗預估粉防己毛狀根產生最大生質量之培養基組成.....90
表21. 粉防己毛狀根以WPM及modifiled WPM(mWPMB)培養基於
125 mL三角錐型瓶培養五週之比較...................................................95
表22. 粉防己毛狀根以三因子進行陡升路徑試驗之粉防己鹼產量結果.....97
表23. 粉防己毛狀根以三因子進行中心混成試驗之粉防己鹼產量結果...100
表24. 粉防己鹼產量複迴歸分析之結果.......................................................102
表25. 二階RSM模式之變異係數分析(ANOVA)........................................103
表26. 中心混成試驗預估粉防己毛狀根產生最大粉防己鹼產量之培養
基組成..................................................................................................104
表27. 粉防己毛狀根以氣泡塔生物反應器及噴霧滴流式生物反應器
培養五週之影響..................................................................................115
表28. 粉防己毛狀根以氣泡塔生物反應器及噴霧滴流式生物反應器
培養八週之影響..................................................................................118


圖目錄
圖1. 粉防己毛狀根培養系統建立之架構........................................................4
圖2. 粉防己市售藥材飲片................................................................................7
圖3. 粉防己鹼與防己諾林己鹼化學結構........................................................7
圖4. 三種求得極值之試驗方法(a)單次單變數法(b)矩陣(c)實驗設計法......14
圖5. 反應曲面試驗設計法流程圖...................................................................18
圖6. 二階層因子設計法之概念圖...................................................................19
圖7. 陡升路徑法之概念圖...............................................................................22
圖8. 粉防己組培苗...........................................................................................27
圖9. 粉防己培植體(A)未經轉殖, 經菌株(B)ATCC 15834, (C)ATCC
43057培養14天之情形；培植體經菌株(D)ATCC 15834, (E)ATCC 43057培養30天之情形..........................................................................50
圖10. 粉防己轉殖毛狀根系以(A)rolB及(B)rolC基因片段為引子之PCR
檢測........................................................................................................51
圖11. 粉防己毛狀根不同根系(A)HR10-1, (B)HR10-2, (C)HR10-3, (D)HR10-4, (E)HR10-5, (F)HR11-1, (G)HR11-3, (H)HR11-4於
WPM培養30天之情形.........................................................................52
圖12. 粉防己毛狀根(HR10-1)於(A)MS, (B)N6, (C)B5, (D)1/2MS, (E)WPM
不同培養基生長 30天之影響.............................................................55

圖13. 不同基礎鹽類固態培養基對毛狀根(HR10-1)生質量與粉防己鹼
含量之影響............................................................................................55
圖14. 粉防己毛狀根時序培養，培養七週之生質量、pH值及導電度.......59
圖15. 粉防己毛狀根時序培養，培養七週之粉防己鹼含量及產量.............59
圖16. 粉防己毛狀根時序培養，培養七週培養液中硝酸態氮與銨態氮
含量........................................................................................................60
圖17. 粉防己毛狀根時序培養，培養七週之培養液中殘糖含量.................60
圖18. 硝酸銨濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾林鹼
含量及粉防己鹼產量之影響................................................................62
圖19. 粉防己毛狀根於不同濃度硝酸銨培養基中培養5週之情形.............63
圖20. 硝酸鈣濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾林鹼
含量及粉防己鹼產量之影響................................................................65
圖21. 粉防己毛狀根於不同濃度硝酸鈣培養基中培養5週之情形.............66
圖22. 蔗糖濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾林鹼
含量及粉防己鹼產量之影響................................................................68
圖23. 粉防己毛狀根於不同濃度蔗糖培養基中培養5週之情形.................69
圖24. 水解酪蛋白濃度對粉防己毛狀根生長、粉防己鹼含量、防己諾
林鹼含量及粉防己鹼產量之影響........................................................71
圖25. 粉防己毛狀根於不同濃度水解酪蛋白培養基中培養5週之情形.....72
圖26. 粉防己毛狀根於三因子二階層試驗設計培養5週之情形.................81
圖27. 粉防己毛狀根以三因子進行陡升路徑培養5週之情形.....................84
圖28. 粉防己毛狀根以三因子進行生質量中心混成試驗培養5週之情形..87
圖29. 硝酸銨濃度對硝酸鈣濃度之生質量曲面圖.........................................91
圖30. 硝酸銨濃度對蔗糖濃度之生質量曲面圖.............................................92
圖31. 硝酸鈣濃度對蔗糖濃度之生質量曲面圖.............................................93
圖32. 粉防己毛狀根以三因子進行粉防己鹼產量陡升路徑培養5週之
情形........................................................................................................98
圖33. 粉防己毛狀根以三因子進行中心混成之粉防己鹼產量試驗培養
5週之情形...........................................................................................101
圖34. 硝酸銨濃度對硝酸鈣濃度之黺防己鹼產量曲面圖...........................105
圖35. 硝酸銨濃度對蔗糖濃度之粉防己鹼產量曲面圖...............................106
圖36. 硝酸鈣濃度對蔗糖濃度之粉防己鹼產量曲面圖.............................. 107
圖37. 粉防己毛狀根以WPM培養基於4 L氣泡塔生物反應器培養五週
之情形 (A)一, (B)二, (C)三, (D)四, (E)五週.....................................110
圖38. 粉防己毛狀根以WPM培養基於4 L噴霧滴流式生物反應器培養
五週之情形(A)一, (B)二, (C)三, (D)四, (E)五週...............................111
圖39. 粉防己毛狀根培養於4 L氣泡塔生物反應器及4 L噴霧滴流式生
物反應器之時序培養，培養五週之pH值.........................................112
圖40. 粉防己毛狀根培養於4 L氣泡塔生物反應器及4 L噴霧滴流式生
物反應器之時序培養，培養五週之導電度.......................................112
圖41. 粉防己毛狀根培養於4 L氣泡塔生物反應器之時序培養，培養
五週培養液中硝酸態氮與銨態氮含量..............................................113
圖42. 粉防己毛狀根培養於4 L噴霧滴流式生物反應器之時序培養，
培養五週培養液中硝酸態氮與銨態氮含量......................................113
圖43. 粉防己毛狀根培養於4 L氣泡塔生物反應器之時序培養，培養
五週之培養液中殘糖含量..................................................................114
圖44. 粉防己毛狀根培養於4 L噴霧滴流式生物反應器之時序培養，
培養五週之培養液中殘糖含量..........................................................114
圖45. 粉防己毛狀根以氣泡塔生物反應器培養八週之情形(A)反應器
培養粉防己毛狀根外觀(B)近照(C, D)根叢俯視...............................117
圖46. 粉防己毛狀根以噴霧滴流式生物反應器培養八週之情形...............118

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