臺灣博碩士論文加值系統

丹參(Salvia miltiorrhiza)為唇形科(Lamiaceae)鼠尾草屬多年生草本植物，其根部含二萜類的丹參酮化合物，對治療心血管疾病有良好之功效。本研究以農桿根群菌(Agrobacterium rhizogenes) R1601感染丹參葉片所得之毛狀根為材料，比較以單因子試驗設計，調整培養基中單一成分及其濃度與反應曲面試驗設計法調整培養基內之多種組成分及其濃度，對提高毛狀根之生質量及丹參酮產量之影響。試驗以B5基本鹽類配方為基礎培養基，首先進行培養基內各別成分之最佳濃度測試，進一步利用反應曲面試驗設計法(Response Surface Methodology, RSM)，進行培養基內多種成分及其濃度的同時調整，尋求對生質量、丹參酮(隱丹參酮、丹參酮I、丹參酮IIA)含量及三種丹參酮總產量最佳之培養基組成。
在單一成分試驗中對生質量之影響，結果顯示降低B5培養基中硫酸銨濃度至0 mg/L，毛狀根之最佳生質量為10.25 g/L；磷酸二氫鈉濃度提高至300 mg/L時，可得最佳生質量為9.45 g/L；硫酸銅濃度提高至75 μg/L時，可得最佳生質量為10.03 g/L。在丹參酮的含量及產量方面，調整培養基中之蔗糖、硫酸銨及硝酸鉀的濃度可提高毛狀根的丹參酮含量及產量，其中提高蔗糖濃度至60 g/L，丹參酮產量為56.93 mg/L；提高硝酸鉀濃度至3750 mg/L，丹參酮產量為53.42 mg/L；降低硫酸銨濃度至67 mg/L，丹參酮產量為60.94 mg/L。
選取六種培養基成分以反應曲面設計試驗進行培養基組成最適化研究，六種成分分別為硝酸鉀、硫酸銨、硫酸銅、氯化鈷、磷酸二氫鈉及蔗糖，由於在反應曲面設計試驗最終之中心混成試驗中未能預估出個別成分之最適值，因此減少試驗之因子數量，選擇以影響較顯著之硝酸鉀、硫酸銨及蔗糖三個因子，重新進行反應曲面試驗設計。所得之結果顯示，毛狀根生質量之預估最佳值為10.55 g/L，三個因子之最佳濃度分別為硝酸鉀5.9 g/L、硫酸銨0 mg/L以及蔗糖48.3 g/L。

Salvia miltiorrhiza Bunge, a perennial plant belongs to Lamiaceae Family. Its roots contain diterpene compounds of tanshinones, which are important medicine for cardiovascular disease. Hairy roots derived from leaves of S. miltiorrhiza infected by Agrobacterium rhizogenes were used to produce tanshinone compounds. A salt formula of B5 medium was taken as basal culture medium and statistic methods of single factor experimental design for single medium component and response surface methodology (RSM) design for multiple medium components were conducted separately on optimization medium composition for tanshinone (cryptotanshinone, tanshinone I, tanshinone IIA) production in this study.
In single factor design experiments, decreasing ammonium sulfate concentration (0 mg/L), increasing sodium dihydrogen phosphate (300 mg/L) and sulfate copper concentration (75 μg/L) of the B5 medium enhanced hairy root biomass production to 10.25 g/L, 9.45 g/L and 10.03 g/L, respectively. Meanwhile, increasing concentration of sucrose (60 g/L), ammonium sulfate (67 g/L) and potassium nitrate (3750 mg/L) was found to enhance tanshinone content and three tanshinones production of hairy root was up to 56.93 mg/L, 60.94 mg/L, and 53.42 mg/L, respectively.
Six components of potassium nitrate, ammonium sulfate, copper sulfate, cobalt chloride, sodium dihydrogen phosphate and sucrose were tested in proceeding of optimization medium composition using response surface methodology. Since the of central composite design failed to estimate the optimal value, three components factors of potassium nitrate, ammonium sulfate and sucrose which with more influential on tanshinone production were chosen to conduct RSM again. According to the mathematic calculation, the optimal hairy root biomass was 10.55 g/L, and the best concentration of potassium nitrate, ammonium sulfate and sucrose was 5.9 g/L, 0 mg/L and 48.3 g/L, respectively.

表目錄 i
圖目錄 iv
中文摘要 1
英文摘要 3
第一章、前言 5
第二章、前人研究 8
第三章、材料方法 22
第四章、結果與討論 41
參考文獻 80
表1. 利用植物毛狀根進行二次代謝產物生產之研究................................... 11
表2. 利用添加誘引劑提高丹參毛狀根培養生產二次代謝產物之研究......12
表3. 二階層因子試驗設計中參試之6 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...................................................32
表4. 培養基中5 種成分利用陡升路徑設計法計算之爬升距離以及爬升
後新處理之各濃度組成...........................................................................33
表5. 五種參試成分在中心混成試驗設計中使用之濃度...............................34
表6. 二階層因子試驗設計中參試之3 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...................................................38
表7. 培養基中三種成分利用陡升路徑設計法計算之爬升距離以及爬升
後新處理之各濃度組成...........................................................................39
表8. 三種參試成分在中心混成試驗設計中使用之濃度...............................40
表9. 蔗糖濃度對丹參毛狀根生長、三種丹參酮含量及丹參酮總產量之影
響...............................................................................................................45
表10. 硫酸銨濃度對丹參毛狀根生長、三種丹參酮含量及丹參酮總產量
之影響.....................................................................................................47
ii
表11. 硝酸鉀濃度對丹參毛狀根生長、三種丹參酮含量及丹參酮總產量
之影響.....................................................................................................49
表12. 磷酸二氫鈉濃度對丹參毛狀根生長、三種丹參酮含量及總丹參酮
產量之影響.............................................................................................52
表13. 硫酸銅濃度對丹參毛狀根生長、三種丹參酮含量及總丹參酮產量
之影響.....................................................................................................54
表14. 氯化鈷濃度對丹參毛狀根生長、三種丹參酮含量及總丹參酮產量
之影響.....................................................................................................56
表15. 在培養丹參毛狀根的過程中添加硫酸銨對毛狀根生質量、三種丹
參酮含量與總丹參酮產量的影響........................................................58
表16. 利用6 種參試因子以二階層試驗設計對丹參毛狀根生長及丹參酮
生產之影響.............................................................................................60
表17. 陡升路徑試驗設計之結果.....................................................................63
表18. 丹參毛狀根利用5 種參試因子以中心混成試驗設計進行8 週培養
之結果.....................................................................................................66
表19. 生質量複迴歸分析之結果.....................................................................67
表20. 總丹參酮含量複迴歸分析之結果.........................................................68
表21. 總丹參酮產量複迴歸分析之結果.........................................................69
表22. 丹參毛狀根以三因子之二階層試驗設計之生質量結果.....................71
iii
表23. 丹參毛狀根以三因子進行陡升路徑試驗之生質量結果.....................72
表24. 丹參毛狀根以三因子進行中心混成試驗之生質量結果.....................73
表25. 中心混成試驗預估最大生質量之培養基組成.....................................74
iv
圖目錄
圖1. 丹參毛狀根培養生產丹參酮試驗之架構................................................. 7
圖2. 反應曲面試驗設計法流程圖...................................................................17
圖3. 二階層因子設計法之概念圖...................................................................18
圖4. 陡升路徑法之概念圖...............................................................................21
圖5. 隱丹參酮標準品迴歸檢量線...................................................................25
圖6. 丹參酮I 標準品迴歸檢量線...................................................................26
圖7. 丹參酮IIA 標準品迴歸檢量線...............................................................27
圖8. 丹參毛狀根時序培養，培養10 週之 (a)生質量、總丹參酮含量與總丹
參酮產量 (b)培養液之導電度 (c)培養液中殘醣含量.........................43
圖9. 丹參毛狀根於不同濃度蔗糖培養基中培養8 週之情形.......................45
圖10. 丹參毛狀根於不同濃度硫酸銨培養基中培養8 週之情形。.............47
圖11. 丹參毛狀根於不同濃度硝酸鉀培養基中培養8 週之情形.................50
圖12. 丹參毛狀根於不同濃度硫酸銅培養基中培養8 週之情形.................54
圖13. 丹參毛狀根於培養第4 週進行硫酸銨添加，丹參毛狀根培養至第8
週之情形................................................................................................58
圖 14. 硝酸鉀濃度對硫酸銨濃度之生質量曲面圖........................................75
圖15. 硝酸鉀濃度對蔗糖濃度之生質量曲面圖76
v
圖16. 酸酸銨濃度對硫酸銨濃度之生質量曲面圖.........................................77

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