粗糙龍膽毛狀根液態培養生產龍膽苦苷與當藥苦苷之研究

Studies on the production of gentiopicroside and swertiamarin in hairy root liquid culture of Gentiana scabra Bunge.

廖哲鋐(Che-Hung Liao)

指導教授 ： 詹効松

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2015年)

摘要

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。

關鍵字

當藥苦苷 ； 龍膽苦苷 ； 毛狀根 ； 粗糙龍膽

並列摘要

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.

並列關鍵字

gentiopicroside ； hairy root ； Gentiana scabra Bunge ； swertiamarin

參考文獻

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