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研究生:黃士倫
研究生(外文):Shih-Lun Huang
論文名稱:牛膝毛狀根之建立及其藥效成分齊墩果酸型皂甙生產之研究
論文名稱(外文):Studies on the hairy root cultures of Achyranthes bidentata and the production of oleanane-type saponins
指導教授:陳建源陳建源引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:112
中文關鍵詞:牛膝齊墩果酸型皂甙毛狀根激發因子細胞毒性
外文關鍵詞:Achyranthes bidentataoleanane-type saponinshairy rootselicitorscytotoxicity
相關次數:
  • 被引用被引用:1
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牛膝 (Achyranthes bidentata Blume) 為多年生草本植物,含有以齊墩果酸 (oleanolic acid) 為甙元之皂甙類 (saponins) 藥效成分。本研究以農桿根群菌 (Agrobacterium rhizogenes) 對牛膝外植體進行感染,成功誘導生成牛膝之毛狀根 (hairy roots) ,繼續探討基礎培養基中各主要成分對牛膝毛狀根生長及皂甙合成之影響,發現蔗糖濃度除了能夠影響毛狀根的生物量,同時也會影響累積於毛狀根中之皂甙含量。
激發因子 (elicitors) 能夠誘導植物產生植物防禦素 (phytoalexins) ,進而誘導植物二次代謝物之生合成,因此成為有效增加毛狀根二次代謝物產量的方法。本研究亦檢討不同類型之激發因子對牛膝毛狀根皂甙累積量之影響,結果以茉莉酸 (jasmonic acid) 的誘導效果最佳,其最適作用濃度為250 μM,毛狀根在添加茉莉酸之三天後可得到較對照組提高2.8倍之皂甙含量。
以兩種人類癌細胞株HepG2及WiDr,測試牛膝毛狀根之甲醇萃取物是否能夠對癌細胞產生細胞毒性 (cytotoxicity) 之作用。結果顯示毛狀根萃取物能夠使細胞形態出現明顯變化,包括細胞形狀較不規則、細胞膜皺縮、細胞核裂解等,並且顯著抑制WiDr細胞的生長。
Achyeanthes bidentata Blume, a perennial herbaceous plant, contains the oleanane-type saponins as one of the main pharmacologically active compounds. Hairy roots of A. bidentata were induced by the infection of Agrobacterium rhizogenes. The main constituents of basal medium significantly affected the growth of hairy roots, and the saponin content was also increased by higher concentration of sucrose.
The addition of elicictors to the hairy root cultures is an effective method for inducing the biosynthesis of plant secondary metabolites by increasing the accumulation of phytoalexins in plant cells. In this study, using different types of elicitors to investigate the effect of the saponin content in A. bidentata hairy roots and found that the most effective one was jasmonic acid. Three days after the addition of jasmonic acid to the hairy root cultures by final concentration of 250 μM, the saponin content was 2.8-fold increase compared with the controls.
To analyze the cytotoxicity of the hairy root methanolic extracts, using two human carcinoma cell lines, HepG2 and WiDr cells. As the result, treating with the hairy root extracts, significantly inhibiting the proliferation of WiDr cells. Furthermore, the morphological changes in HepG2 and WiDr cells were observed when exposed to the hairy root extracts, including membrane shrinking, and nuclear fragmentation. The further isolation and purification of hairy root extracts may be needed for the future work to find new sources of anti-cancer drugs.
第一章 前言 1
第一節 牛膝 1
一、牛膝之本草考證 1
二、型態與特徵 2
三、植物資源 2
第二節 牛膝之成分與藥理研究 3
一、牛膝之化學組成 3
二、牛膝藥理作用之研究 5
第三節 植物二次代謝物 7
第四節 毛狀根系統之建立 10
一、農桿根群菌治病結構及功能 10
二、農桿根群菌之感染機制 15
三、毛狀根之建立與培養 19
四、毛狀根之特性及應用 21
第五節 激發因子對毛狀根生長與二物代謝物合成之
影響 24
一、激發因子之定義與分類 24
二、激發因子之作用機制 25
三、常用之激發因子 27
第六節 研究目的 30

第二章 材料與方法 33
第一節 牛膝無菌植株 33
第二節 牛膝毛狀根之誘導與培養 34
第三節 牛膝毛狀根之確認 36
第四節 牛膝毛狀根之藥效成分分析 38
第五節 培養基組成之最適化探討 41
第六節 激發因子對二物代謝物產量之影響 45
第七節 牛膝毛狀根萃取一對人類癌細胞生長之影響 47

第三章 結果與討論 49
第一節 牛膝無菌植株之培養 49
第二節 牛膝毛狀根之培養與誘導 51
第三節 牛膝毛狀根之確認與藥效成分分析 54
一、毛狀根基因轉型之確認 54
二、牛膝毛狀根藥效成分分析 56
第四節 牛膝毛狀根生長動力學之探討 60
一、生物量與總皂甙含量之變化          60
二、培養液pH與導電度之變化          65
   第五節 牛膝毛狀根培養條件之最適化探討 66
一、最適接種量 66
二、不同培養基對毛狀根生長之影響 69
三、碳源之影響 69
四、氮源之影響 74
五、鉀離子之影響 79
六、磷酸鹽之影響 80
第六節 激發因子對牛膝毛狀根之影響 84
第七節 牛膝毛狀根萃取物之細胞毒性測試 89

第四章 結論 95

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