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  • 學位論文

粉防己毛狀根大量生產粉防己鹼之研究

Study on Mass Production of Tetrandrine in Stephania tetrandra S. Moore Hairy root

指導教授 : 詹効松
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摘要


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。

並列摘要


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.

參考文獻


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