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研究生:黃百鳴
研究生(外文):Bai-Ming Huang
論文名稱:利用液態培養丹參毛狀根生產丹參酚酸B之研究
論文名稱(外文):Studies on production of salvianolic acid B from hairy root of Salvia miltiorrhiza in liquid culture medium
指導教授:詹効松
指導教授(外文):Hsiao-Sung Chan
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:應用化學系生化科技碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:72
中文關鍵詞:銅離子銨態氮丹參酚酸B毛狀根丹參
外文關鍵詞:Copper ionsAmmoniacal nitrogenSalvianolic acid B (SAB)Hairy rootsSalvia Miltiorrhiza
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丹參(Salvia miltiorrhiza Bunge)為唇形科(Labiatae)、鼠尾草屬(Salvia)植物,其藥用部為根部含有脂溶性丹參酮類及水溶性之酚酸類化合物,近年來用於治療心血管疾病具有良好之功效。本研究以農桿根群菌(Agrobacterium rhizogenes) BCRC15010感染丹參葉片所得之毛狀根作為試驗材料於液態培養系統生產丹參酚酸B,探討基礎鹽類培養基(½MS、B5、½B5、WPM及½WPM)種類以及B5培養基之組成成分對丹參毛狀根生長與丹參酚酸B累積之影響,並針對B5培養基主要組成成分(蔗糖、硝酸鉀、硫酸銨、硫酸銅、氯化鈷及磷酸二氫鈉等)濃度試驗經4週培養。所得結果顯示,丹參毛狀根不適宜於以½MS培養基進行培養;以WPM培養基(1x, ½x)進行培養3週可得較高之生質量(6.36 g dw/L)與酚酸B 含量(33.6 mg/g),酚酸B含量於培養過程中呈現先增後降之情形;以B5培養基(1x, ½x)進行培養,8週後可得最高之生質量(7.87 g dw/L),酚酸B含量於培養過程中均呈現下降之情形。B5鹽類培養基中單一成分之調整試驗結果顯示,硫酸銨濃度在0-2.0 mM之間培養4週後,各試驗組之毛狀根生質量無顯著性差異,然而毛狀根之丹參酚酸B含量會隨硫酸銨濃度提升而下降,於不含硫酸銨之試驗組其丹參酚酸B含量為對照組(1 mM)的3.1倍。於硫酸銅濃度試驗結果顯示,提高培養基中硫酸銅濃度(0-75 µg/L)可加速毛狀根生長,硫酸銅濃度50 µg/L試驗組之毛狀根生質量為對照組(25 µg/L)的1.5倍。此外,提高B5培養基中硫酸銅濃度0至75 µg/L,有助於提高丹參酚酸B含量。綜合不同鹽類培養基與B5培養基單一成分調整試驗的結果後推測,銨態氮的濃度與銅離子對毛狀根生長及丹參酚酸B累積有關鍵性的影響。為進一步證實銨態氮的存在對於毛狀根生長有所影響,以不含硫酸銨B5培養基進行毛狀根培養,並於培養第3週進行不同濃度之硫酸銨饋料添加試驗。實驗結果顯示,培養第3週於B5培養基(對照組)中毛狀根生質量高於不含硫酸銨之試驗組;但隨培養週數增長(4-6週),毛狀根生質量與增加趨勢改變,以不含硫酸銨試驗組較對照組為高。在培養第3週時於初始不含硫酸銨試驗組中各別添加0、1及2 mM硫酸銨,觀察其第4-6週之生質量變化並與對照組比較,顯示於培養第4週,試驗組之毛狀根生質量增加情形與添加之硫酸銨量呈現正比關係(2 mM > 1 mM > 0 mM >對照組);於培養第5、6週之毛狀根生質量變化顯示,饋料添加硫酸銨(1、2 mM)之試驗組,其生質量增加趨勢均減緩,顯示添加硫酸銨雖可於添加初期增加毛狀根生質量,但卻會妨礙後期毛狀根之生長。綜合上述,本研究證實提高培養基中銅離子濃度會促進丹參毛狀根生長與酚酸B含量累積,並釐清銨態氮對於丹參毛狀根生長與丹參酚酸B累積之影響,推測此現象為銨態氮的利用會損耗培養基中銅離子所致。
Salvia miltiorrhiza Bunge, a perennial plant belongs to Lamiaceae Family. Its roots contain tanshinones and phenolic acids. In the recent years, roots of S. miltiorrhiza have been used for the treatment of cardiovascular diseases. In the present study, hairy roots were induced in leaves of S. miltiorrhiza by infecting with Agrobacterium rhizogenes. In a liquid culture system, these hairy roots were cultured for the production of Salvianolic acid B (SAB). Three basal salt media (MS, B5, and WPM) in half (½x) and full (1x) strengths were evaluated for hairy root growth and SAB accumulation. Among these three basal salt media, B5 was selected because here nitrogen source in form of NO3- and NH4+ are separate and can be controlled to investigate their influence on production of hairy root biomass and SAB. Thus, different concentrations of six medium components viz. sucrose, KNO3, (NH4)2SO4, CuSO4, CoCl2 and NaH2PO4 were investigated for hairy roots biomass and SAB content. At 4 weeks of culture, results showed that ½ strength of MS basal medium gave the least hairy root biomass and was not suitable for the hairy roots culture of S. miltiorrhiza. In case of WPM (1x and ½x), production of the maximum root biomass (6.36 g dw/L) and SAB content (33.6 mg/g) was obtained after 3 weeks of culture. Thereafter, there was a decreasing trend in SAB content in the hairy roots. In the B5 medium (1x and ½x), the maximum production of root biomass (7.87 g dw/L) was obtained at 8 weeks of culture. Thereafter, there was a decreasing trend in the SAB content. It was found that there was no significant difference in biomass when hairy roots were cultured in the B5 basal medium containing different concentrations of (NH4)2SO4 from 0 to 2.0 mM for 4 weeks. However, with the increasing concentrations of (NH4)2SO4, there was a decreasing trend in the SAB content. The SAB content was 3.1-fold higher in the medium without (NH4)2SO4 compared to the medium with 1.0 mM (NH4)2SO4 indicating the influence of (NH4)2SO4 on SAB production. Results showed that growth of hairy roots varied with concentration of CuSO4 (0-75 µg/L) in the medium. B5 medium containing 50 µg/L CuSO4 produced 1.5-fold higher root biomass compared to medium with 25 µg/L CuSO4 demonstrating its influence on root biomass of S. miltiorrhiza. Thus, the present study demonstrates that concentrations of NH4+ and Cu+ in the culture medium effect hairy root production and its SAB content. To verify the effect of NH4+ ions on growth of hairy roots, a further experiment was carried out. The hairy roots were grown in the B5 medium without (NH4)2SO4 for 3 weeks, before adding different concentrations of (NH4)2SO4 in the medium. Results showed that production of biomass was directly proportional to concentration of (NH4)2SO4 (2 mM > 1 mM > 0 mM > CK) at the 4th week of evaluation. However, further culture of hairy roots beyond 4 weeks retarded the biomass production.
In conclusion, this study demonstrates that the type of basal medium, concentrations of Cu+ ions and (NH4)2SO4 in the culture medium significantly influence the biomass and SAB production in hairy root culture of S. miltiorrhiza, thus, needing culture medium optimization for the optimum results.
中文摘要 I
Summary III
表目錄 IX
圖目錄 XI
第一章、前言 1
第二章、前人研究 8
2.1 丹參簡介 8
2.2 丹參藥理活性 9
2.3 丹參酚酸B之生合成 9
2.4 毛狀根形成與特性 12
2.5 丹參毛狀培養生產酚酸類化合物之現況 13
2.6 培養基組成之影響 13
2.6.1 碳水化合物(carbohydrates) 14
2.6.2 氮(nitrogen) 14
2.6.3 磷(phosphorus) 15
2.6.4 銅(copper) 16
2.6.5 鈷(cobalt) 16
第三章、材料與方法 22
3.1 試驗材料 22
3.2 丹參植株培養條件 22
3.3 丹參毛狀根培養條件 22
3.3.1 固態培養條件 22
3.3.2 液態培養條件 22
3.3.3 丹參毛狀根乾重之測定 22
3.3.4 丹參毛狀根培養液之pH值及導電度測量 23
3.3.5 指標成分丹參酚酸B含量之分析 23
3.3.5.1 丹參毛狀根中丹參酚酸B之萃取 23
3.3.5.2 丹參毛狀根中丹參酚酸B含量及產量之計算 23
3.3.5.3 HPLC分析丹參酚酸B之條件 24
3.3.5.4 標準品 24
3.3.5.5 丹參酚酸B檢量線製作 24
3.4 統計分析方法 24
3.5 固態基礎鹽類培養基時序培養之試驗 25
3.6 液態基礎鹽類培養基時序培養試驗 25
3.7 培養基中單一成分及其濃度改變試驗 25
3.7.1 蔗糖(sucrose)濃度試驗 25
3.7.2 硝酸鉀(KNO3)濃度試驗 26
3.7.3 硫酸銨((NH4)2SO4)濃度試驗 26
3.7.4 硫酸銅(CuSO4)濃度試驗 26
3.7.5 氯化鈷(CoCl2)濃度試驗 26
3.7.6 磷酸二氫鈉(NaH2PO4)濃度試驗 26
3.8饋料添加培養試驗 27
第四章、結果與討論 30
4.1 丹參毛狀根於固態B5基礎鹽類培養基之時序培養試驗 30
4.2 丹參毛狀根於不同液態基礎鹽類培養基之時序培養試驗 33
4.3 培養基成分對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 40
4.3.1 蔗糖濃度對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 40
4.3.2 硝酸鉀濃度對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 43
4.3.3 硫酸銨濃度對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 45
4.3.4 硫酸銅濃度對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 47
4.3.5 氯化鈷濃度對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 49
4.3.6 磷酸二氫鈉濃度對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 51
4.4 饋料添加硫酸銨對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 53
4.4.1 丹參毛狀根於第3週進行饋料添加,培養至第4週對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 53
4.4.2 丹參毛狀根於第3週進行饋料添加,培養至第5週對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 56
4.4.3 丹參毛狀根於第3週進行饋料添加,培養至第6週對丹參毛狀根生長、丹參酚酸B含量及產量影響試驗 58
第五章、結論 63
參考文獻 65
附錄1. 不同基礎鹽類培養基之組成 72

表目錄
表 1. 利用植物細胞培養生產食品添加劑之研究實例 ............................... 5
表 2. 毛狀根培養生產醫藥用成分之研究實例 .......................................... 7
表 3. 利用毛狀根培養生產二次代謝產物之研究實例 ............................. 18
表 4. 利用添加誘引劑提高丹參毛狀根培養生產酚酸類化合物之研究.. 21
表 5. 丹參化學成分溶媒移動相之梯度沖堤表 ........................................ 28
表 6. 丹參毛狀根與瓶苗不同組織部位中丹參酚酸B 含量之比較 ......... 32
表 7. 不同基礎鹽類培養基之總氮源含量及銨態氮(NH4+)與硝酸態(NO3-)
含量比例分析 .................................................................................. 39
表 8. 蔗糖濃度對丹參毛狀根生長、丹參酚酸B 含量及丹參酚酸B 產量
之影響 ............................................................................................. 42
表 9. 硝酸鉀濃度對丹參毛狀根生長、丹參酚酸B 含量及丹參酚酸B 產
量之影響 .......................................................................................... 44
表 10. 硫酸銨濃度對丹參毛狀根生長、丹參酚酸B 含量及產量之影響 ...
......................................................................................................... 46
表 11. 硫酸銅濃度對丹參毛狀根生長、丹參酚酸B 含量及丹參酚酸B 產
量之影響 .......................................................................................... 48
表 12. 氯化鈷濃度對丹參毛狀根生長、丹參酚酸B 含量及丹參酚酸B 產
量之影響 .......................................................................................... 50
表 13. 磷酸二氫鈉濃度對丹參毛狀根生長、丹參酚酸B 含量及丹參酚酸
B 產量之影響 .................................................................................. 52
表 14. 利用培養期間添加硫酸銨,培養至第4 週對丹參毛狀根生質量、
丹參酚酸B 含量及產量之影響 ...................................................... 55
表 15. 利用培養期間添加硫酸銨,培養至第5 週對丹參毛狀根生質量、
丹參酚酸B 含量及產量之影響、丹參酚酸B 含量及產量之影響 ...
......................................................................................................... 57
表 16. 利用培養期間添加硫酸銨,培養至第6 週對丹參毛狀根生質量、
丹參酚酸B 含量及產量之影響 ...................................................... 60
表 17. 饋料添加硫酸銨後丹參毛狀根於培養不同週數後之比生長速率 ....
......................................................................................................... 62
表 18. 饋料添加硫酸銨後丹參毛狀根於不同硫酸銨添加濃度在不同培養
週數之丹參酚酸B 產率 .................................................................. 62

圖目錄
圖 1. 丹參毛狀根培養系統生產丹參酚酸B 建立之架構 .......................... 4
圖 2. 丹參植物及丹參藥材(A)丹參植株 及(B)丹參之乾燥根部 .............. 8
圖 3. 丹參酚酸B 結構式 ........................................................................... 10
圖 4. 丹參酚酸B 生合成路徑 ................................................................... 11
圖 5. 丹參酚酸B 標準品回歸檢量線 ....................................................... 29
圖 6. 丹參毛狀根於B5 固態培養基中培養2 至8 週之情形(A) 2, (B) 4, (C)
6, (D) 8 週 ....................................................................................... 31
圖 7. 丹參毛狀根於B5 基礎鹽類培養基時序培養8 週之生質量及丹參酚
酸B 含量 ......................................................................................... 31
圖 8. 丹參毛狀根接種於液態不同種類鹽類基礎培養基中培養不同週數
毛狀根生長之情形。(A, B, C 及D) ½MS 培養2, 4, 6 及8 週、(E, F,
G 及H) B5 培養2, 4, 6 及8 週、(I, J, K 及L) ½B5 培養2, 4, 6 及8
週、(M, N, O 及P) WPM 培養2, 4, 6 及8 週及(Q, R, S 及T) ½WPM
培養2, 4, 6 及8 週 .......................................................................... 37
圖 9. 丹參毛狀根於液態不同鹽類基礎鹽類培養基中時序培養8 週之生
質量、導電度及pH 值 ................................................................... 38
圖 10. 丹參毛狀根於液態不同鹽類基礎鹽類培養基中時序培養8 週之生
質量及丹參酚酸B 含量 .................................................................. 39
圖 11. 丹參毛狀根於不同蔗糖濃度培養基中培養4 週之情形(A) 30, (B)
50, (C) 70, (D) 90 g/L ....................................................................... 42
圖 12. 丹參毛狀根於不同硝酸鉀濃度培養基中培養4 週之情形(A) 0, (B)
6.2, (C) 12.4, (D) 24.7, (E) 37.1 mM ................................................ 44
圖 13. 丹參毛狀根於不同硫酸銨濃度培養基中培養4 週之情形(A) 0, (B)
0.5, (C) 1.0, (D) 1.5, (E) 2.0 mM ...................................................... 46
圖 14. 丹參毛狀根於不同硫酸銅濃度培養基中培養4 週之情形(A) 0, (B)
12.5, (C) 25, (D) 50, (E) 75 μg/L ...................................................... 48
圖 15. 丹參毛狀根於不同氯化鈷濃度培養基中培養4 週之情形(A) 0, (B)
12.5, (C) 25, (D) 50, (E) 75 μg/L ...................................................... 50
圖 16. 丹參毛狀根於不同磷酸二氫鈉濃度培養基中培養4 週之情形(A) 0,
(B) 75, (C) 150, (D) 225, (E) 300 mg/L ............................................ 52
圖 17. 丹參毛狀根培養於不同濃度硫酸銨之B5 培養基中生長至第4 週之
情形。培養基中硫酸銨濃度分別為(A) 1 mM 及(B) 0 mM,及毛狀
根於不含硫酸銨之培養基中培養3 週後再添加硫酸銨(C) 1 mM 及
(D) 2 mM ......................................................................................... 55
圖 18. 丹參毛狀根培養於不同濃度硫酸銨之B5 培養基中生長至第5 週之
情形。培養基中硫酸銨濃度分別為(A) 1 mM 及(B) 0 mM,及毛狀
根於不含硫酸銨之培養基中培養3 週後再添加硫酸銨(C) 1 mM 及
(D) 2 mM ......................................................................................... 57
圖 19. 丹參毛狀根培養於不同濃度硫酸銨之B5 培養基中生長至第6 週之
情形。培養基中硫酸銨濃度分別為(A) 1 mM 及(B) 0 mM,及毛狀
根於不含硫酸銨之培養基中培養3 週後再添加硫酸銨(C) 1 mM 及
(D) 2 mM ......................................................................................... 60
圖 20. 培養3 週後添加硫酸銨對於丹參毛狀根生質量及丹參酚酸B 含量
之影響 ............................................................................................. 61
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