臺灣博碩士論文加值系統

粗糙龍膽(Gentiana scabra Bunge)為龍膽科(Gentianaceae)、龍膽屬(Gentiana)植物，藥用部位為根部，為臺灣中藥典收載龍膽之基原植物，其主要活性成分為gentiopicroside (龍膽苦苷) 與swertiamarin (當藥苦苷)。由於龍膽藥材多來自於中國，其藥材真偽、安全性及活性成分含量具有潛在性的問題。本研究的重點為建立龍膽毛狀根液態培養生產活性成分，係本研究室所保存的粗糙龍膽(G. scabra Bunge) 經葉片培植體以農桿根群菌(Agrobacterium rhizogenes)BCRC15010感染所得之5個根系(HR 32、35、36、39、49) ，經固態WPM培養基培養30天，挑選生質量最佳之HR-32(2.74 g dw/petri-dish)為試驗材料，在液態MS、WPM、B5、N6培養基於4週培養，可發現在液態B5培養基其生質量為最佳(4.56 g dw / L) ， gentiopicroside與swertiamarin分別為29.0 mg/g及2.2 mg/g；然而在毛狀根繼代培養與試驗過程中，發現生質量變化較大，因此建立穩定的毛狀根材料來源供液態培養試驗所用，極有必要。本研究進行固態、液態交替培養觀察生長與生質量之變化，結果顯示經過液態培養的毛狀根作為試驗接種材料在生長與生質量上均有較佳之表現；於生長曲線試驗中觀察於第4週gentiopicroside與swertiamarin含量達最高，分別為39.8 mg/g及2.3 mg/g。個別調整B5培養基中組成成分(蔗糖、硝酸鉀、硫酸銨及硫酸銅)之濃度進行4週培養，結果顯示改變硫酸銨濃度0-2 mM 與硫酸銅濃度 0-75 μg/L 對於生質量之增加並沒有顯著性差異；於硝酸鉀濃度低於24.7 mM 時對於毛狀根生長阻抑極為明顯。以上結果顯示以龍膽毛狀根經過液態培養後作為實驗材料的接種來源較為穩定，且液態B5培養基適合培養龍膽毛狀根，並且具有擴大規模培養大量生產活性成分之潛力。

Gentiana scabra Bunge, commonly known as ‘Longdan’ is a perennial plant belonging to Gentianaceae family. In traditional Chinese medicine, roots of ‘Londan’ are used to prevent indigestion, anorexia and gastric infections. Due to an extensive medicinal use, wild G. scabra plants are in huge demand resulting in its over exploitation and decline in wild populations. Hence, it is necessary to find alternative method for production of active compounds without destruction of wild plants. The aim of the present study is to optimize hairy root culture system in order to improve production of root biomass and pharmaceutically important compounds of G. scabra.
Hairy roots obtained from leaf explants of G. scabra infected with Agrobacterium rhizogenes were used as starting material. The rapidly growing hairy roots of five transgenic lines HR-32, HR-35, HR-36, HR-39, and HR-49 were cultured in semi-solid Woody Plant Medium (WPM) for four weeks. Of these five, line HR-32 yielded the highest root biomass (2.74 g dw/petri-dish), hence, it was selected for further experiments. Hairy roots of HR-32 were cultured in liquid medium of five different basal media like Murashige and Skoog (MS), WPM, B5, 1/2B5, and N6. After four weeks of culture, the highest root biomass (4.56 g dw/L) was obtained in B5 liquid medium. Also, it produced maximum Gentiopicroside (29.0 mg dw/g) and Swertiamain (2.2 mg/g dw), therefore, all further experiments were carried out with B5 medium alone.
In order to determine whether liquid or semi-solid medium is better for production of root biomass and bioactive compounds, experiments were performed with liquid and semi-solid media or alternate subculture on solid to liquid or solid to solid medium. Also, certain medium components play an important role in production of root biomass and active compounds. Therefore, further experiments were carried out to optimize components like sucrose (10 – 70 g/L), Potassium nitrate (6.2, 12.4, 24.7 and 37.1 mM), ammonium sulfate (05, 1, 1.5 and 2 mM) and copper sulfate (12.5, 25, 50 and 75 μg/L) in the B5 medium. Root biomass in both semi-solid medium and liquid cultures was recorded. Production of Gentiopicroside and Swertiamarin in semi-solid and
liquid cultures was estimated by High Performance Liquid Chromatography (HPLC) analysis.
Among different sucrose concentrations in the medium, a supplement of 50g/L sucrose resulted in the highest biomass (5.49 g dw/L) production. While, sucrose at 70g/L supported maximum Gentiopicroside (29.2 mg/g) and Swertiamarin (1.6 mg/g) production. Potassium nitrate at 24.7mM in the medium supported the highest biomass of 8.51 g dw/L with Gentiopicroside and Swertiamarin contents as 49.2 mg/g and 3.1 mg/g, respectively. The maximum root biomass (6.68g d.w/L) among different concentrations of Ammonium sulfate was recorded at 2mM. The contents of Gentiopicroside and Swertiamarin were 56.6 mg/g and 2.9 mg/g, respectively. There was a marginal difference in biomass production at different levels of Copper sulfate, however, Copper sulfate at 75 μg/L in the medium supported highest contents of Gentiopicroside (40.0 mg/g) and Swertiamarin (2.1 mg/g).
The results obtained in present study indicate that the concentration of carbon source (sucrose) and nitrogen level in the medium affect the hairy root growth and production of Swertiamarin and Gentiopicroside in cultures of G. scabra. Also, present study affirms that there is a possibility of producing the bioactive compounds through hairy root cultures without sacrificing the wild plants of G. scabra Bunge.

中文摘要................................................................................................................I
Abstract................................................................................................................III
表目錄................................................................................................................ XI
圖目錄................................................................................................................XII
縮寫表……………………………………………………………………….XIV
第一章、前言.........................................................................................................1
第二章、前人研究.................................................................................................4
2.1 粗糙龍膽植物形態....................................................................................4
2.2 粗糙龍膽活性成分藥理考察…................................................................6
2.3 毛狀根形成與特性....................................................................................8
2.4 培養基組成之影響....................................................................................9
2.4.1 碳水化合物(carbohydrates)..............................................................10
2.4.2 氮(nitrogen).......................................................................................11
2.4.4 銅(copper)..........................................................................................12
第三章、材料與方法...........................................................................................17
3.1 試驗材料..................................................................................................17
3.2 培養條件.............………….....................................................................17
3.2.1 固態培養條件...................................................................................17
3.2.2 液態培養條件...................................................................................17
3.2.3 粗糙龍膽毛狀根乾、溼重之測定.....................................................17
3.2.4 粗糙龍膽毛狀根培養液之pH值及導電度測量.............................18
3.2.5 粗糙龍膽毛狀根指標成分含量之分析...........................................18
3.2.5.1指標成分之萃取......................................................................18
3.2.5.2 HPLC分析之條件..................................................................18
3.2.5.3指標成分含量及產量之計算..................................................19
3.2.5.4 粗糙龍膽指標成分之標準品................................................19
3.2.5.5 標準品檢量線之製作............................................................20
3.3 統計分析方法..........................................................................................20
3.4 毛狀根根系之篩選..................................................................................20
3.5液態基礎培養基之試驗............................................................................20
3.6 粗糙龍膽毛狀根時序培養之試驗..........................................................21
3.7毛狀根接種材料之穩定性試驗…………………………........................21
3.8 培養基成分對粗糙龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.....................................................................................................22
3.8.1蔗糖濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.............................................................................................22
3.8.2硝酸鉀濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.............................................................................................22
3.8.3硫酸銨濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.............................................................................................22
3.6.4硫酸銅濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.............................................................................................23
第四章、結果與討論...........................................................................................24
4.1 毛狀根根系之篩選……………..............................................................24
4.2液態培養基之試驗....................................................................................26
4.3 粗糙龍膽毛狀根時序培養之試驗..........................................................32
4.4毛狀根接種材料之穩定性試驗................................................................36
4.5 培養基成份對粗糙龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量 之影響……………………………………………………………….....43
4.5.1蔗糖濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響…….....................................................................................43

4.5.2硝酸鉀濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.............................................................................................46
4.5.3硫酸銨濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.............................................................................................49
4.5.4硫酸銅濃度對龍膽毛狀根產生龍膽苦苷及當藥苦苷含量與產量
之影響.............................................................................................52
第五章、結論.......................................................................................................54
參考文獻.............................................................................................................56
附錄1. 不同基礎鹽類培養基之組成................................................................61









表目錄
表 1. 利用毛狀根生產二次代謝產物之實例..................................................13
表 2. 不同粗糙龍膽毛狀根於固態WPM培養基培養30天之生質量
與龍膽苦苷及當藥苦苷含量及產量之影響………………………….25
表 3. 龍膽毛狀根第一到三代生質量之影響…………………………….….39
表 4. 龍膽毛狀根經固態培養第一到三代生長變化之顯著性與百分率
影響…………………………………………………………………….41
表 5. 龍膽毛狀根經液態培養第一到三代之生長變化…………….……….42
表 6. 蔗糖濃度之B5培養基對龍膽毛狀根生長、龍膽苦苷與當藥
苦苷含量及產量之影響……………………………………………….45
表 7. 硝酸鉀濃度之B5培養基對龍膽毛狀根生長、龍膽苦苷與當藥
苦苷含量及產量之影響……………………………………………….48
表 9. 硫酸銨濃度之B5培養基對龍膽毛狀根生長、龍膽苦苷與當藥
苦苷含量及產量之影響……………………………………………….51
表 10. 硫酸銅濃度之B5培養基對龍膽毛狀根生長、龍膽苦苷與當藥
苦苷含量及產量之影響……………………………………………….53

圖目錄
圖 1.龍膽毛狀根培養系統建立之架構..............................................................3
圖 2. 粗糙龍膽植物(A)粗糙龍膽瓶苗植株 (B)粗糙龍膽野外植株 (C)粗糙龍膽之原植物 (D)粗糙龍膽藥材根部.....................................................5
圖 3. 龍膽苦苷及當藥苦苷生合成路徑............................................................7
圖 4. 粗糙龍膽毛狀根5個不同根系(A)HR-32, (B)HR-35, (C)HR-36, (D)HR-39, (E)HR-49於固態WPM基礎培養基培養30天之情形.........25
圖 5. 粗糙龍膽毛狀根(HR-32)接種於不同成分之液態培養基中培養
不同週數之生長情況...............................................................................29
圖 6. 粗糙龍膽毛狀根(HR-32)培養4週和6週於不同液態培養基
之生質量…...............................................................................................30
圖 7. 粗糙龍膽毛狀根(HR-32)培養4週於不同液態培養基之龍膽苦苷及當藥苦苷含量與產量...................................................................................30
圖 8. 粗糙龍膽毛狀根(HR-32)培養6週於不同液態培養基之龍膽苦苷及當藥苦苷含量與產量...................................................................................31
圖 9. 粗糙龍膽毛狀根(HR-32)時序培養，培養於125 ml錐形瓶(含20 ml medium 5 roots tips)不同週數之生長情況..............................................34
圖 10. 粗糙龍膽毛狀根時序培養，培養8週之生質量、pH值
及導電度….............................................................................................34
圖 11. 粗糙龍膽毛狀根時序培養，培養8週之龍膽苦苷
含量與產量.............................................................................................35
圖 12. 粗糙龍膽毛狀根時序培養，培養8週之當藥苦苷
含量與產量.............................................................................................35
圖 13. 粗糙龍膽毛狀根接種材料之穩定性試驗示意圖................................38
圖 14. 龍膽毛狀根第一代生長於(A) SS (B) SL 培養基4週
之生長情形.............................................................................................40
圖 15. 龍膽毛狀根第二代生長於(C) SSS(D) SSL(E) SLS (F) SLL培養基
4週之生長情形.......................................................................................40
圖 16. 龍膽毛狀根第三代生長於(G) SSSS(H) SSSL(I) SSLS(J) SSLL (K) SLSS(L) SLSL (M) SLLS (N) SLLL培養基4週之生長情形…..........41
圖 17. 龍膽毛狀根於不同濃度蔗糖之B5培養基中培養4週之情形...........45
圖 18. 龍膽毛狀根於不同濃度硝酸鉀之B5培養基中培養4週之情形.......48
圖 19. 龍膽毛狀根於不同濃度硫酸銨之B5培養基中培養4週之情形…...51
圖 20. 龍膽毛狀根於不同濃度硫酸銅之B5培養基中培養4週之情形…...53

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