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

Abstract

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.