臺灣博碩士論文加值系統

粉防己為防已科千金藤屬草本植物，田間栽培5-8年之粉防己地下塊根可供藥材使用，粉防己鹼（Tetrandrine）為中草藥植物粉防己（Stephania tetrandra S. MOORE）特有二次代謝物，具有抗發炎、抗氧化等功效，並能有效抑制結腸癌細胞生長與抗血小板凝集之作用。本研究室誘導所得生長快速之粉防己毛狀根經培養5週後，其粉防己鹼含量可高於市售藥材（4.52 mg/g）達6.75 mg/g。初步於時序培養過程中探討培養基之導電度對線上監測毛狀根生質量之可行性，結果可由時序培養得知增加的生質量與減少的導電度之關係式推得之關係式R2為0.9987，表示導電度的下降可準確作為線上監測毛狀根的生質量之根據；由生長勢知3-5週為毛狀根之對數生長期，進一步於三角錐形瓶中培養毛狀根5週，於第4週添加誘引劑尋求對粉防己鹼含量之顯著因子，結果顯示，於第4週添加ABA（1 ppm）、L-Proline（10 mM）粉防己鹼含量與粉防己鹼產量皆達最高，粉防己鹼含量為10.96與10.59 mg/g，分別為對照組之1.31 倍與 1.26 倍，粉防己鹼產量為82.26與80.44 mg/L，分別為對照組之1.26 倍與1.23 倍。於5 L氣泡塔生物反應器（ bubble column bioreactor）探討初始接種量（1, 3, 5及7 g）對毛狀根生質量、粉防己鹼含量及產量之影響。接種不同重量之毛狀根於內含3.5 L WPM培養基之5 L氣泡生物反應器中培養5週，固定通氣量為50 mL/min進行試驗，分析其生質量、粉防己鹼含量與計算增長倍數（GI值）。其接種量為1和7 g fw/batch，有最低及最高生質量，分別為21.41 g fw/L（1.87 g dw/L）與61.42 g fw/L（10.73 g dw/L），反之，接種量為1和7 g fw/batch，則有最高及最低增長倍數（GI），分別為69.63與26.90。

Stephania tetrandra S. Moore, an important Chinese medicinal plant, belongs to the family Menispermance and it has long been used in folk medicine for the treatment of edema, gastric ulcer, duodenal ulcer, hypertension, gastroenteritis, dysentery, snake bite, boils, furuncles, oliguria, genitourinary tract infection, parotitis, tonsillitis, beriberi, leucorrhea, neuralgia and rheumatic arthritis. Tetrandrine is a main active compound of S. tetrandra and accumulated in the tuberous roots. These tuberous roots have been harvested (3-5 years) for medicinal uses in China for centuries. Agrobacterium rhizogenes mediated hairy root cultures is an alternative technique to conventional plant cultivation for producing pharmaceutically important secondary metabolites. In this study, the hairy root culture was initiated by infecting leaf explants of S. tetrandra with A. rhizogenes strain ATCC 15834. The hairy root line (HR10-1) was established in Woody Plant medium(WPM). The HPLC analysis results showed the transgenic hairy root lines (HR10-1) produced more tetrandrine (6.75 mg/L) than that marketed crude drug (4.81 mg/L). Furthermore, to evaluate the relationship between growth of the hairy roots and their tetrandrine production in the bioreactor, the correlation between growth (ΔX) and conductivity (ΔC) during the time course of the cultures was first studied in conical flasks. Resulting, an inverse linear relationship (R2=0.9987) between hairy root biomass growth and conductivity was clearly observed. Conductivity declined gradually while the biomass was increasing. The result indicates that the decrease of medium conductivity could be an indicator for the online monitoring the biomass of hairy roots in bioreactor. Moreover, to enhance the tetrandrine content and biomass of hairy root culture, we investigated the effect of elicitors, precursors and amino acids on 4th week of hairy root cultures. It was found that the WPM supplemented with ABA (1 ppm) and L-Proline (10 mM) produced the maximum tetradrine content (10.96 and 10.59 mg/g) and production (82.26 and 80.44 mg/L) than control hairy root culture (8. 38 mg/g and 65.28 mg/L). Further, we examined the effects of initial inoculums densities (1, 3, 5 and 7 g of hairy roots per batch), growth index value (GI), tetrandrine content and production of hairy root culture in 5 L bubble column bioreactor containing 3.5 L WPM with 50 mL/min of aeration rate. After 5 weeks of cultivation, the results indicate that the growth index values have reverse tendency with the biomass of hairy roots. It was found that the minimum and maximum biomass of hairy roots (1.87 and 10.73 g dw/L) in bioreactor were obtained when the initial inoculum densities were 1 and 7 g respectively.

目錄

中文摘要……………………………………………………………………………….I
英文摘要……………………………………………………………………………..III
目錄…………………………………………………………………………………..IV
表目錄…………………………………………………………………………..…. ..IX
圖目錄………………………………………………………………...……………...XI
第一章 前言………………………………………………………………..…………1
第二章 前人研究…………………………………………………….……………….8
2.1 粉防己簡介……………………………………………………………….………8
2.2 粉防己藥理活性……………………………………………………………….…9
2.2.1 生物鹼（Alkaliod）………………………………………………………..…10
2.2.1.1 物理化學性質……………………………………………………….…13
2.2.2 二次代謝物（Secondary metabolite）……………………………………….14
2.2.3 植物組織培養………………………………………………………………18
2.3 毛狀根形成與特性……………………………………………………………...20
2.4 培養基組成之影響……………………………………………………………...26
2.4.1 氮（Nitrogen）………………………………………………………………..26
2.4.2 磷（Phosphorus）…………………………………………………………….27
2.4.3 鉀（Potassium）……………………………………………………………...28
2.5 誘引劑與前驅物………………………………………………………………...28
2.5.1 誘引劑（Elicitors）…………………………………………………………..29
2.5.2 前驅物（Precursors）………………………………………………………...30
2.6 粉防己毛狀根應用於氣泡塔生物反應器-吸附回收粉防己鹼之培養試驗…..35
2.6.1 刺激粉防己毛狀根中粉防己鹼外泌試驗…………………………………35
2.6.1.1 吸附樹脂（Adsorbent）…………………………………………………35
2.6.1.2 鈣離子拮抗劑………………………………………………………….37
2.6.1.3培養基pH值…………………………………………………………….37
2.6.2 建立氣泡塔生物反應器中適合粉防己毛狀根生長之條件………………37
第三章 材料方法……………………………………………………………………43
3.1 試驗材料………………………………………………………………………...43
3.2 固態培養條件…………………………………………………………………...43
3.3 液態培養條件…………………………………………………………………...43
3.3.1 粉防己毛狀根鮮、乾重之測定……………………………………………..43
3.3.2 指標成分分析………………………………………………………………43
3.3.2.1 標準品………………………………………………………………….44
3.3.2.2 檢品製備……………………………………………………………….44
3.3.2.3 標準曲線之繪製……………………………………………………….44
3.3.2.4 粉防己毛狀根中粉防己鹼產量之計算……………………………….44
3.3.2.5 粉防己毛狀根培養液pH值及導電度之測定………………………...45
3.3.2.6 粉防己毛狀根培養液中殘醣含量之測定…………………………….45
3.3.2.7 粉防己毛狀根培養液中陰、陽離子濃度之測定……………………...45
3.3.2.8 統計分析方法………………………………………………………….46
3.4 建立線上估算粉防己毛狀根生質量之數學模式……………………………...46
3.4.1線上預估毛狀根生質量方法之建立……………………………………….46
3.4.2 硝酸銨對粉防己毛狀根生質量、粉防己鹼含量及產量之影響試驗……..46
3.4.3 硝酸鈣對粉防己毛狀根生質量、粉防己鹼含量及產量之影響試驗……..47
3.4.4 不同濃度磷酸二氫鉀對粉防己毛狀根生質量、粉防己鹼含量及產量之影
響試驗………………………………………………………………………47
3.5 誘引劑、前驅物、營養物之添加對粉防己毛狀生長、粉防己鹼含量與產量之影響試驗..............................................................................................................47
3.5.1 離層酸（Abscisic Acid, ABA）添加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……………………………………………………48
3.5.2 茉莉酸甲酯（Methyl jasmonate, MeJA）添加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…………………………………………48
3.5.3 幾丁質（Chitosan）添加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…………………………………………………………………48
3.5.4 醋酸鈉（Sodium acetate trihydrate, NaOAc）添加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……………………………………..48
3.5.5 谷胺酸（L-Glutamic acid）添加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…………………………………………………………49
3.5.6 脯胺酸（L-Proline）添加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………………………………………………………………49
3.5.7 苯丙胺酸（L-Phenylalanine）添加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……………………………………………………49
3.5.8 色胺酸（L-Tryptophan）添加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……………………………………………………………49
3.6 粉防己毛狀根應用於氣泡塔生物反應器-吸附回收粉防己鹼之培養試驗…..50
3.6.1吸附樹脂篩選……………………………………………………………….51
3.6.1.1 脫附溶劑篩選………………………………………………………….54
3.6.1.2 吸、脫附流速篩選……………………………………………………...54
3.6.2 刺激粉防己毛狀根中粉防己鹼外泌試驗…………………………………55
3.6.2.1 吸附樹脂之添加……………………………………………………….56
3.6.2.2 鈣離子拮抗劑之添加………………………………………………….56
3.6.2.3 改變培養基中pH值…………………………………………………...57
3.6.3 粉防己毛狀根於氣泡塔生物反應器之培養試驗…………………………57
3.6.3.1 氣泡塔生物反應器（bubble column bioreactor）初始接種量對粉防己
毛狀根生質量、粉防己鹼含量與產量之影響試驗…………..………..57
第四章 結果與討論…………………………………………………………………59
4.4.1 線上預估毛狀根生質量方法之建立………………………………………59
4.4.2 氮源對粉防己毛狀根生質量、粉防己鹼含量及產量之影響試驗………..64
4.4.3 不同濃度磷酸二氫鉀對粉防己毛狀根生質量、粉防己鹼含量及產量之影
響試驗………………………………………………………………………69
4.5 誘引劑、前驅物的添加對粉防己毛狀生長、粉防己鹼含量與產量之影響試驗………………………………………………………………………………..76
4.5.1 不同濃度離層酸的添加對粉防己毛狀生長、粉防己鹼含量與產量之影響
試驗…………………………………………………………………………76
4.5.2 不同濃度茉莉酸甲酯的添加對粉防己毛狀生長、粉防己鹼含量與產量之
影響試驗……………………………………………………………………80
4.5.3 不同濃度幾丁質的添加對粉防己毛狀生長、粉防己鹼含量與產量之影響
試驗…………………………………………………………………………82
4.5.4 不同濃度醋酸鈉的添加對粉防己毛狀生長、粉防己鹼含量與產量之影響
試驗…………………………………………………………………………84
4.5.5 不同濃度谷胺酸的添加對粉防己毛狀生長、粉防己鹼含量與產量之影響
試驗…………………………………………………………………………86
4.5.6 不同濃度脯胺酸的添加對粉防己毛狀生長、粉防己鹼含量與產量之影響
試驗…………………………………………………………………………88
4.5.7 不同濃度苯丙胺酸的添加對粉防己毛狀生長、粉防己鹼含量與產量之影
響試驗………………………………………………………………………91
4.5.8 不同濃度色胺酸的添加對粉防己毛狀生長、粉防己鹼含量與產量之影
響試驗………………………………………………………………………93
4.6 氣泡塔生物反應器培養粉防己毛狀根及粉防己鹼吸附回收試驗…………...96
4.6.1 吸附樹脂篩選………………………………………………………………96
4.6.1.1 脫附溶劑篩選………………………………………………………….97
4.6.1.2 吸、脫附流速篩選……………………………………………………...99
4.6.2 刺激粉防己毛狀根中粉防己鹼外泌試驗………………………………..102
4.6.2.1吸附樹脂之添加………………………………………………………102
4.6.2.2 鈣離子拮抗劑之添加………………………………………………...107
4.6.2.3 改變培養基之pH值………………………………………………….111
4.6.3 氣泡塔生物反應器（bubble column bioreactor）中初始接種量對粉防己毛
根生質量、粉防己鹼含量與產量之影響試驗……………………………114
第五章 結論………………………………………………………………….…….120
第六章 參考文獻…………………………………………………………………..121

表目錄

表1. 利用植物細胞培養生產食品添加劑之實例…………………….…………….4
表2. 毛狀根培養生產醫藥產品的實例……………………………………………..6
表3. 利用毛狀根培養進行生物轉化的藥品………………………………………..7
表4. 植物體中生物鹼的分類………………………………………………………11
表5. 利用植物細胞培養生產具生物活性二次生代謝物實例……………………15
表6. 提高植物細胞培養生產二次生代謝產物之策略……………………………19
表7. 利用毛狀根培養生產二次代謝物之實例……………………………………21
表8. 利用誘引劑刺激二次代謝物生產之實例……………………………………32
表9. 吸附樹脂應用於植物細胞培養之實例………………………………………36
表10. 利用生物反應器培養毛狀根生產二次代謝物之實例……………………..39
表11. 利用不同類型生物反應器培養植物細胞實例……………………………...41
表12. 氣泡塔反應器應用於生化研究之實例……………………………………..41
表13. 應用於粉防己鹼吸附之吸附樹脂種類……………………………………..51
表14. WPM基礎鹽類配方中各別元素之導電度………………………………….61
表15. 利用WPM基礎鹽類培養基培養粉防己毛狀根後不同時期之生質量、粉防
己鹼含量及培養基導電度…………………………………..........................62
表16. 硝酸銨對粉防己毛狀根生質量、粉防己鹼含量與培養基導電度變化之影
響……………………………………………………………..………………65
表17. 硝酸鈣對粉防己毛狀根生質量、粉防己鹼含量與培養基導電度變化之影響……………………………………………………………………..………67
表18. 磷酸二氫鉀濃度為0 mg/L對粉防己毛狀根生質量、粉防己鹼含量之影響……………………………………………………………………………..70
表19. 磷酸二氫鉀濃度為50 mg/L對粉防己毛狀根生質量、粉防己鹼含量之影
響……………………………………………………………………………..72
表20. 磷酸二氫鉀濃度為100 mg/L對粉防己毛狀根生質量、粉防己鹼含量之影 響……………………………………………………………………………..74
表21. 不同濃度離層酸對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…78
表22. 不同濃度茉莉酸甲酯對粉防己毛狀根生質量、粉防己鹼含量及產量之影 響…………………………………………………………….……………….81
表23. 不同濃度幾丁質對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…83
表24. 不同濃度醋酸鈉對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…85
表25. 不同濃度谷胺酸對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…87
表26. 不同濃度脯胺酸對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…89
表27. 不同濃度苯丙胺酸對粉防己毛狀根生質量、粉防己鹼含量及產量之影響. …………………………………………..………………………………..92
表28. 不同濃度色胺酸對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…94
表29. 不同吸附樹脂吸附之粉防己鹼吸附量.……………………...…………… .96
表30. 不同吸附樹脂吸、脫附之粉防己鹼吸附量………………………………....98
表31. 不同流速對吸附粉防己鹼之影響………………………………………... 100
表32. 不同流速對脫附粉防己鹼之脫附率、回收率影響………………………..101
表33. 吸附樹脂SP70於不同時間添加對粉防己毛狀根生質量、粉防己鹼含量
及產量之影響………………………………………………………………103
表34. 吸附樹脂SP70添加量的多寡對粉防己毛狀根生質量、粉防己鹼含量及產
量之影響……………………………………………………………………105
表35. 鈣離子拮抗劑Verapamil之添加對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…………………………………………………………………108
表36. 拮抗劑EGTA之添加對毛狀根生質量、粉防己鹼含量及產量之影響…...109
表37. 培養基pH值對毛狀根生質量、粉防己鹼含量及產量之影響…………….112
表38. 不同粉防己毛狀根接種量於3.5 L氣泡式生物反應器培養5週之影響…118

圖目錄

圖1. 粉防己毛狀根培養生產粉防己鹼之架構……………………………………..3
圖2. 粉防己市售藥材飲片…………………………………………………………..8
圖3. 粉防己鹼與防己諾林鹼之化學結構式………………………………………10
圖4. 各類型生物鹼結構圖…………………………………………………………12
圖5. 植物細胞阻止病原體感染所產生的防禦機制簡圖…………………………30
圖6. 生物反應器培養粉防己毛狀根中粉防己鹼之生產與回收流程圖…………42
圖7. 生物反應器培養粉防己毛狀根中粉防己鹼之生產與回收流程圖…………50
圖8. 初步篩選吸附樹脂示意流程圖………………………………………………52
圖9. 吸附樹脂篩選流程圖…………………………………………………………53
圖10. 吸、脫附流速試驗流程圖……………………………………………………55
圖11. WPM基礎鹽類培養基培養粉防己毛狀根於接種後第（A）5;（B）10;（C）
15;（D）20天之生長情形…………………………..………………………...60
圖12. 粉防己毛狀根時序培養，培養20天之（A）乾濕重比的變化;（B）陰、
陽離子的變化;（C）生質量、粉防己鹼含量及導電度之變化;（D）乾重與
導電度的線性相關變化之動力參數;（E）生質量增長量與減少之導電度的
關係式………………………………………………………………………..63
圖13. 粉防己毛狀根於硝酸銨為氮源時之WPM培養基中培養（A）5;（B）10;
（C）15; （D）20天之生長情形……………………………………….…….65
圖14. 硝酸銨對粉防己毛狀根生質量、粉防己鹼含量與培養基導電度變化之影
響……………………………………………………………………………. 66
圖15. 粉防己毛狀根於硝酸鈣為氮源時之WPM培養基中培養（A）5;（B）10;
（C）15; （D）20天之生長情形……………………………………………..67
圖16. 硝酸鈣對粉防己毛狀根生質量、粉防己鹼含量與培養基導電度變化之影
響…………………………………………………………..…………………68
圖17. 粉防己毛狀根於磷酸二氫鉀濃度為0 mg/L之WPM培養基中培養（A）5;（B）10; （C）15; （D）20天之生長情形…………………………………….70
圖18. 磷酸二氫鉀濃度為0 mg/L對粉防己毛狀根生質量、粉防己鹼含量與培養
基導電度變化之影響……………………………..…………………………71
圖19. 粉防己毛狀根於磷酸二氫鉀濃度為50 mg/L之WPM培養基中培養（A）
5; （B）10; （C）15; （D）20天之生長情形………………………………...72
圖20. 磷酸二氫鉀濃度為50 mg/L對粉防己毛狀根生質量、粉防己鹼含量與培養
基導電度變化之影響………………………………………………………..73
圖21. 粉防己毛狀根於磷酸二氫鉀濃度為100 mg/L之WPM培養基中培養（A）5; （B）10; （C）15; （D）20天之生長情形………………………………......74
圖22. 磷酸二氫鉀濃度為100 mg/L對粉防己毛狀根生質量、粉防己鹼含量與培養基導電度變化之影響……………………………………………………..75
圖23. 粉防己毛狀根培養4週後添加不同濃度離層酸於WPM液態培養基中共培養1週之生長情形…………………………………………………………...78
圖24. 不同濃度離層酸對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…79
圖25. 粉防己毛狀根培養4週後添加不同濃度茉莉酸甲酯於WPM液態培養基中共培養1週之生長情形………………………………..…………………….81
圖26. 粉防己毛狀根培養4週後添加不同濃度幾丁質於WPM液態培養基中共培養1週之生長情形………………………………………………………..….83
圖27. 粉防己毛狀根培養4週後添加不同濃度醋酸鈉於WPM液態培養基中共培養1週之生長情形………………………………………...…………………85
圖28. 粉防己毛狀根培養4週後添加不同濃度谷胺酸於WPM液態培養基中共培
養1週之生長情形…………………………………………………………...87
圖29. 粉防己毛狀根培養4週後添加不同濃度脯胺酸於WPM液態培養基中共培養1週之生長情形………………………………...…………………………89
圖30. 不同濃度脯胺酸對粉防己毛狀根生質量、粉防己鹼含量及產量之影響…90
圖31. 粉防己毛狀根培養4週後添加不同濃度苯丙胺酸於WPM液態培養基中共培養1週之生長情形………………………………………..……………….92
圖32. 粉防己毛狀根培養4週後添加不同濃度色胺酸於WPM液態培養基中共培
養1週之生長情形………………………………………...............................94
圖33. 利用有機溶劑從不同類型吸附劑回收粉防己鹼…………………………..98
圖34. 吸附樹脂SP70於不同時間（A）對照組;（B）初始;（C）第1週;（D）第2週;（E）第3週;（F）第4週添加，對粉防己毛狀根生質量、粉防己鹼含量及產量之影響………………………………………………………103
圖35. 吸附樹脂SP70於不同時間添加對粉防己毛狀根生質量、粉防己鹼含量
及產量之影響………………………………………………………………104
圖36. 吸附樹脂SP70量多寡於初始添加（A）對照組;（B）0.150 g;（C）0.300 g;
（D）0.450 g，對粉防己毛狀根生質量、粉防己鹼含量及產量之影……...105
圖37. 吸附樹脂SP70添加量的多寡對粉防己毛狀根生質量、粉防己鹼含量及產
量之影響…………………………….……………………………………...106
圖38. 於第4週添加不同濃度Verapamil（A）對照組;（B）DMSO;（C）1 μM;（D）10 μM;（E）100 μM;（F）1000 μM培養1週，對粉防己毛狀根生質
量、粉防己鹼含量及產量之影響…………………………………………..108
圖39. 第4週添加EGTA（A）對照組;（B）1 μM;（C）10 μM;（D）100 μM;（E）1000 μM培養1週，於第5週取樣對粉防己毛狀根生質量、粉防己鹼
含量及產量之影響…………………………………………………………109
圖40. 鈣離子拮抗劑對毛狀根中總生物鹼之影響……………………………….110
圖41. 第4週更換不同pH值之培養基（A）對照組;（B）3;（C）7;（D）9，培
養粉防己毛狀根1週，於第五週取樣對粉防己毛狀根生質量、粉防己鹼含
量及產量之影響……………………………………………………………112
圖42. 培養基pH值對毛狀根生質量、粉防己鹼含量及產量之影響…………….113
圖43. 粉防己毛狀根接種量為1 g時於5 L氣泡塔生物反應器中之生長情形（A）
1; （B）2; （C）3; （D）4; （E）5週…………………………………………..115
圖44. 粉防己毛狀根接種量為3 g時於5 L氣泡塔生物反應器中之生長情形（A）
1; （B）2; （C）3; （D）4; （E）5週…………………………………………..115
圖45. 粉防己毛狀根接種量為5 g時於5 L氣泡塔生物反應器中之生長情形（A）
1; （B）2; （C）3; （D）4; （E）5週…………………………………………..116
圖46. 粉防己毛狀根接種量為7 g時於5 L氣泡塔生物反應器中之生長情形（A）
1; （B）2; （C）3; （D）4; （E）5週……………………………………..……116
圖47. 不同粉防己毛狀根初始接種量培養5週後，培養液中pH值變化之情形..117
圖48. 不同粉防己毛狀根初始接種量培養5週後，培養液中導電度變化之情形……………………………………………………………………………117
圖49. 不同粉防己毛狀根接種量於3.5 L氣泡塔生物反應器培養5週之影響…118
圖50. 不同粉防己毛狀根接種量於3.5 L氣泡塔生物反應器培養5週之影響…119

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