地黃毛狀根培養之調控與放大

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Title:	地黃毛狀根培養之調控與放大 Regulation and amplification on hairy root cultures of Rehmannia glutinosa
Authors:	黃于軒;Yu-Hsuan Huang
Contributors:	中國藥學暨中藥資源學系碩士班
Keywords:	地黃;毛狀根培養;誘引劑;生物反應器;抗氧化活性;毛蕊花糖?;Rehmannia glutinosa;Hairy root culture;Elicitors;Bioreactor;Verbascoside
Date:	2013-07-31
Issue Date:	2013-10-02 09:34:31 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	地黃（Rehmannia glutinosa (Gaert.) Libosch.）為玄參科（Scrophulariaceae）多年生草本植物，其根部為傳統中醫藥常用藥材。地黃中含有多種?類成分，包含梓醇（catalpol）、地黃?A、B、C、D（rehmanniosides A-D）、毛蕊花糖?（verbascoside）、水蘇糖（stachyose）等，其?類成分對血液系統，免疫系統，內分泌系統，心血管系統和神經系統，抗腫瘤，抗衰老等皆具有藥理作用。本研究針對提高地黃毛狀根之毛蕊花糖?的生產，利用光照與植物誘引劑（茉莉酸甲酯、茉莉酸、水楊酸、幾丁質）之不同處理進行探討，藉以刺激地黃毛狀根之成分累積。結果顯示，地黃毛狀根之毛蕊花糖?含量與產量，在第28天時達到最高，在光照培養下之含量與產量分別為4.08 mg/g與61.64 mg/L，且為對照組(暗培養)之3.00倍與3.13倍，表示光照可刺激毛蕊花糖?之生合成。進一步利用攪拌式生物反應器大量培養，在黑暗與光照培養下，生質量分別為接種量之10.30倍與5.15倍。添加茉莉酸甲酯（MJ, 20 mg/mL）、水楊酸（SA, 40 mg/mL）、幾丁質（CH, 40 mg/mL）誘引劑之地黃毛狀根培養中，毛蕊花糖?含量與產量皆達到最高，其產量分別為對照組之3.17倍、3.15倍與1.85倍。進一步探討茉莉酸甲酯（MJ, 20 mg/mL）之不同添加時間對地黃毛狀根之影響，以共培養天數為十四天並培養十四天之毛狀根的毛蕊花糖?產量（0.10 mg/L）較高，且為對照組之1.65倍。另一方面，針對地黃毛狀根甲醇萃取物之藥理活性作探討，其清除DPPH自由基能力，在光照培養的清除DPPH自由基能力為黑暗培養之1.45倍，並推測地黃毛狀根之清除DPPH自由基能力為藉由其生產之毛蕊花糖?而產生活性。而其測定樹突細胞活性能力，其中以MJ、SA與CH誘導之毛狀根甲醇萃取物具有促進樹突細胞活性的能力，而以JA誘導與光照刺激之毛狀根甲醇萃取物對樹突細胞活性有抑制的能力。綜合以上結果，顯示光照與誘引劑可促進地黃毛狀根中毛蕊花糖?的產量，且地黃毛狀根甲醇萃取物具有抗氧化活性與免疫系統有調節的作用。 Rehmannia glutinosa Libosch. (Scrophulariaceae), known as Di Huang, is widely used in Chinese medicine with hypoglycemic property. Its mainly biological active compounds are glycosides (catalpol, rehmanniosides A-D, verbascoside and stachyose). The activities of these compounds include anti-tumoral, anti-inﬂammatory, cancer-chemopreventive and antioxidant effects. In this study, in order to enhance the verbascoside content and biomass from hairy root culture of R. glutinosa, the effects of light and elicitors, including salicylic acid (SA), chitosan (CH), jasmonic acid (JA) and methyl jasmonate (MJ), have been investigated . The results show that the highest verbascoside content and production in R. glutinosa hairy root culture was on the 28th day during the culture. The highest content (4.08 mg/g) and production (61.64 mg/L) were achieved under light condition, and compared with the control group (dark culture) were 3.00 times and 3.13 times, respectively. It means that light is the role to stimulate verbascoside biosynthesis in R. glutinosa hairy root cultures. In addition, the R. glutinosa hairy roots were incubated in 10 L stirred tank bioreactors under light and dark conditions. The biomass for each condition were achieved 10.30 times and 5.15 times compared with the initial embedded weight, respectively. Moreover, The added methyl jasmonate (MJ, 20 mg/mL), salicylic acid (SA, 40 mg/mL) and chitinase (CH, 40 mg/mL) in the hairy root cultures of R. glutinosa were able to produce the highest verbascoside content (4.54, 6.85 and 2.15 mg/g ) and production ( 47.62, 47.27 and 27.71 mg/L ) than control one. Furthermore, different exposure time of methyl jasmonate (MJ, 20mg/mL) on hairy root cultures of R. glutinosa was examined. . The highest production of verbascoside (0.10 mg/L ) was achieved on the 14th day and reached to 1.65 times with the control group. In the meanwhile, the methanol extract of hairy root of R. glutinosa showed the radical scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) bioassay. The radical scavenging activity of DPPH from the light cultured hairy root of R. glutinosa was higher than the dark cultured one with 1.45 times. This result may be caused by the production of verbascoside in the hairy root cultures of R. glutinosa. In addition, the methanol extract of hairy root of R. glutinosa showed the activity of DCs as well. The methanol extract of hairy root cultures of R. glutinosa with the treatment of MJ, SA and CH had activating effects on DCs. Contrary to activation, that the methanol extract from light condition and JA treatment had inhibitory effect on DCs. In conclusion, the light and elicitors can enhance the verbascoside content on the hairy root culture of R. glutinosa. Meanwhile, its methanol extract also had antioxidant activity and immunomodulatory effects, respectively.
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