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研究生:林怡辰
研究生(外文):Yi-Chen Lin
論文名稱:掌葉大黃毛狀根之建立及其藥用蒽醌成份之生產條件檢討
論文名稱(外文):The Establishment Of Rheum palmatum L. Hairy Root Culture And The Production Of Anthraquinone By The Culture Thereof
指導教授:陳建源陳建源引用關係
指導教授(外文):Chien-Yuan Chen
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:2004
畢業學年度:92
語文別:中文
論文頁數:95
中文關鍵詞:大黃酸毛狀根掌葉大黃
外文關鍵詞:RheinRheum palmatum L.Hairy Root
相關次數:
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利用農桿根群菌(Agrobacterium rhizogenes) A4 野生型菌株感染掌葉大黃外植體誘導毛狀根以建立掌葉大黃毛狀根的培養系統,再經由PCR方法進行確認。檢討該毛狀根之最適培養基組成,並且檢討擴大培養之最適培養條件。分別利用比色法、薄層層析法和高效能液相層析法分析毛狀根中游離蒽醌化合物的含量。
本研究以大黃酸作為標準品,在搖瓶培養中測試其最適培養基組成。由實驗結果得知,以B5培養基含2%蔗糖、20.25 mM硝酸鉀、6.75 mM硫酸銨和0.75 mM磷酸二氫鈉為最適培養基組成分。利用最適化之B5培養基進行掌葉大黃毛狀根的培養,培養42天後可得到最高大黃酸產量10.06 mg/L,與搖瓶培養所得之7.264 ± 0.06 mg/L相比較,大黃酸產量增加了1.38倍。擴大培養係利用1公升之氣泡塔式生物反應器(Bubble column reactor),以B5培養基為基礎培養基培養49 天,可得到的大黃酸產量為17.245 mg/L,與搖瓶培養所得之7.264 ± 0.06 mg/L相比較,大黃酸產量增加了2.37倍。


Culture of Rheum palmatum L. hairy roots were established after transformation with Agrobacterium rhizogenese A4, and the transformation was confirmed by PCR for the presence of a T-DNA sequence in their genomes. Quantitative determination of anthraquinones were performed by colorimetry, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Among these anthraquinones produced by Rheum palmatum L., emodin and rhein were considered more important. In the studies, rhein is taken as the reference standard. Root growth and production of rhein were investigated in various basal media. It was found that B5 medium was superior to MS medium in terms of both root growth and rhein production. The results indicate that Rheum palmatum L. hairy root can be cultivated in the B5 medium enriched with 2 % sucrose, 20.25 mM KNO3, 6.75 mM NH4NO3 and 0.75 mM NaH2PO4. The rhein production is 10.06 mg/L in a 42 days` culture. The productivity is 1.38 folds as that in normal flask culture. A scale up culture of Rheum palmatum L. hairy root from 125 mL flask to 1 L bubble column reactor was conducted. The rhein production is 17.245 mg/L in a 49 days` culture. The productivity is 2.37 folds as that in flask culture.

中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ
目錄……………………………………………………………………Ⅲ
表次……………………………………………………………………Ⅴ
圖次……………………………………………………………………Ⅵ

第一章 前言…………………………………………………………1
第一節 掌葉大黃…………………………………………………1
第二節 掌葉大黃有效成份之介紹………………………………6
第三節 植物二次代謝物…………………………………………8
第四節 毛狀根……………………………………………………13
第五節 研究目的…………………………………………………28

第二章 材料與方法…………………………………………………29
第一節 掌葉大黃無菌植株………………………………………29
第二節 掌葉大黃毛狀根系統的建立……………………………32
第三節 掌葉大黃毛狀根系統的確認……………………………34
第四節 掌葉大黃毛狀根及其培養液之分析……………………36
第五節 培養基組成之最適化探討………………………………39
第六節 生物反應器之培養………………………………………40
第七節 實驗儀器…………………………………………………41

第三章 結果與討論…………………………………………………43
第一節 掌葉大黃無菌植株………………………………………43
第二節 掌葉大黃毛狀根系統的建立……………………………44
第三節 掌葉大黃毛狀根系統的確認……………………………48
第四節 掌葉大黃毛狀根及其培養液之分析……………………50
第五節 培養基組成之最適化探討………………………………56
第六節 生物反應器之培養………………………………………78

第四章 結論………………………………………………………… 85
參考資料………………………………………………………………87


Agarwal S. K., Singh S. S., Verma S., Kumar S. (2000) Antifungal
activity of anthraquinone derivatives from Rheum emodi. J. of
Ethnopharm. 72, 43-46.
Aitken-Christie J., Singh A. P., Horgan K. J. and Thorpe T. A. (1985)
Explant development state and shoot formation in Pinus radiate
cotyledons. Bot. Gaz. 46, 196-203.
Alfermann, A. W. and Petersen M. (1995) Natural product formation by
plant cell biotechnology - results and perspectives. Plant Cell Tiss.
Org. Cult. 43, 199-205.
Bieleski, R. L. and Ferguson, I. B. (1983) Physiology and metabolism of
phosphate and its compounds. In: Lauchli, A. and Bieleski R.L.
(Eds) Inorganic Plant Nutrition. New Series Vol. 15A Springer,
Berlin, pp. 422-449.
Bouchez D. and Tourneur J. (1991) Organization of the agropine
synthesis region of the T-DNA of the Ri plasmid from
Agrobacterium rhizogenes. Plasmid 25, 27-39.
Bourgaud F., Vani S., Shanks JV. (1993) Production of plant secondary
metabolites: a historical perspective. Plant science 161, 839-851.
Bibikova T. N., Langen A. A. N. and Heidstra R., Tramper J. (1991)
Growth and thiophene production by hairy root cultures of Tagetus
patula in various two liquid phase bioreactors. Enzyme Microbiol.
Technol. 13, 487-494.
Cacho M., Moran M.,Herrera T., Fernandez-Tarrago and Corchete M. P.
(1991) Morphogenesis in leaf, hypocotyls and explants of Digitalis
thapsi L. cultures in vitro. Plant Cell Tiss. Org. Cult. 25, 117-123.
Chattopadhyay S., Datta S. K. and Mahato S.B. (1994) Production of
L-DOPA from cell suspension culture of Mucuna pruriens f.
pruriens. Plant Cell Rep. 13, 519-522.
Christen P., Roberts M. F., Phillipson J. D. and Evans W.C. (1989) High
yield production of tropane alkaloids by hairy-root cultures of a
Datura candida hybrid. Plant Cell Rep. 8, 75-77.
Cresswell R. C., Fowler M. W., Stafford A. and Sarkissian G. S. (1989)
Input and output: primary substrates and secondary metabolism. In:
Kurz, W. G. and W. (Eds.), Primary and Secondary Metabolism of
Plant Cell CultureⅡ., Springer, Berlin, pp.14-26.
Dubois M. et al.(1956) Colorimetric method for determination of sugers
and related substances. Analytical Chemistry 28(3), 350-356.
Flores H. E., Hoy M. W. and Pikard J. J. (1987) Secondary metabolites
from root cultures. Trends Biotechnol. 5, 64-68.
Fowler MW. (1982) Substrate utilization by plant-cell cultures. J. Chem.
Tech. Biotechnol. 32, 338-346.
Gamborg O. L., Miller R. A.,and Ojima K. (1968) Nutrient requirements
of suspension cultures of soybean root cells. Exp. Cell Res. 50,
151-158.
Giri A., and Narasu M. L. (2000) Transgenic hairy roots: recent trends
and applications. Biotechnol. Advances 18, 1-22.
Godwin I., Todd G., Ford-Lloyd B. and Newbury H. J. (1991) The effects
of acetosyringone and pH on Agrobacterium-mediated
transformation vary according to plant species. Plant Cell Rep. 9,
671-675.
Hactor E. J., Hoy M. W., and Pickard J. J. (1987) Secondary metabolites
from root cultures. TIBTECH. 5, 64-68.
Hamill J. D., Parr A. J., Robins R. J. and Rhodes M. J. C. (1986)
Secondary product formation by cultures of Beta vulgaris and
Nicotiana rustica transformed with Agrobacterium rhizogenes.
Plant Cell Rep. 5, 111-114.
Hamill J. D., Robins R. J. and Rhodes M. J. C. (1989) Alkaloid
production by .transformed root cultures of Cinchona ledgeriana.
Planta Med. 55, 354-357.
Hilton M. G. and Rhodes M. J. C. (1990) Growth and hyoscyamine
production of hairy root cultures of Datura stramonium in a
modified stirred tank reactorm. Microbiol. Biotechnol. 33, 132-138.
Humphrey A. (1998) Shake flask to fermentor. What have we learned?
Biotechnol. Prog. 14, 3-7.
Ibrahim RK. (1987) Regulation of synthesis of phenolics. In: Constabel F. and Vasil I.L. (Eds), Cell Culture and Somatic Cell Genetics of Plants, Vol. 4. Academic Press Inc., New York, pp77-95.
Jouanin L. (1984) Restriction map of an agropine-type Ri plasmid and its
homologies with Ti plasmids. Plasmid 12, 91-102.
Jouanin L., Guerche P., Pamboukdjian N., Tourneur C., Casse-Delbart F.
and Tourneur J. (1987) Structure of T-DNA in plants regenerated
from roots transformed by Agrobacterium rhizogenes strain A4.
Mol. Gen. Genet. 206, 387-392.
Jurgen E. T. (1995) Agrobacterium-Mediated Transformation of stem
disks from oilseed rape. In: Keven M. A. G. and Michael R. D.
(Eds), Agrobacterium protocols, chapter 9. Humana Press, New
Jersey, pp. 81-82.
Kamada H., Oakmara N., Harada M. S. and Shimomura K. (1986)
Alkaloid production by hairy root cultures in Atropa belladonna.
Plant Cell Rep. 5, 239-242.
Kanokwaree K. and Doran PM. (1997) Effect of inoculum size on
growth of Atropa belladonna hairy roots in shake flasks. J. Ferment.
Bioeng. 84, 378-381.
Kassanis B. (1957) The use of tissue cultures to produce virus-free
clones from infected potato varieties. Ann. Appl. Biol. 45, 422-427.
Kondo O., Honda H., Taya M., and Kobayashi T. (1989) Comparison of
growth properties of carrot hairy root in various bioreactors. Appl.
Microbiol. Biotechnol. 32, 291-294.
Lewin B. (1990) In: Gene IV. Oxford University press, Cambridge,
Mass. pp. 744-749.
Martin S. M., and Rose D. (1976) Growth of plant cell (Ipomoea)
suspension cultures at controlled pH levels. Can. J. Bot. 54,
1264-1270.
Miura Y., Hirata K. Kurano N., Miyamoto K. and Uchida K. (1988)
Formation of vinblastine in multiple shoot culture of Catharanthus
roseus. Planta Med. 54, 18-20.
Moreno-Valenzuela O., Coello-Coello J., Loyola-Vargas VM.,
Vazquez-Flota F. (1999) Nutrient consumption and alkaloid
accumulation in a hairy root line of Catharanthus roseus.
Biotechnol. Lett. 21, 1017-1021.
Mukundan U. and Hjortso MA. (1991) Growth and thiophene
accumulation by hairy root cultures of Tagetes patula in media of
varying initial pH. Plant Cell Rep. 9, 627-630.
Murashige T. and Skoog F. (1962) Arevised medium for rapid growth
and bioassays with tobacco tissue culture. Physiol. Plant 15,
437-439.
Murashige T. (1977) Manipulation of orange initiation in plant tissue
culture. Bot. Bull. Acad. Sin. 18, 1-24.
Nigra H. M., Caro O. H. and Giulietti A. M. (1987) Production of
solasodine by calli from dofferent parts of Solanum eleagnifolium
Cav. plant. Plant Cell Rep. 6, 135-137.
Nuutila A. M., Toivonen L., and Kauppinen V. (1994) Bioreactor studies
on hairy root cultures of Catharanthus roseus: compassion of three
bioreactor types. Biotechnol. Tech. 8, 61-66.
Ozeki Y. and Komamine A. (1985) Effect of inoculum density, zeatin and
sucrose on anthocyanin accumulation in a carrot suspension culture.
Plant Cell Tissu. Org. cult. 5, 45-53.
Petit A., David C., Dahl G. A., Ellis J. G., and Guyon P. (1983) Further
extension of the opine concept: plasmids in Agrobacterium
rhizogenes cooperate for opine degradation. Mol. Gen. Genet. 190,
204-214.
Porter J. R. (1991) Host range and implications of plant infection by
Agrobacterium rhizogenes. Critical Reviews in Plant Science 10,
387-421.
Rhodes M. J. C., Hilton M., Parr A. J., Hamill J. D. and Robins R. J.
(1986) Nicotine production by hairy root cultures of Nicotiana
rustica:Fermentation and product recovery. Biotechnol. Lett. 8,
415-420.
Rhodes M. J., Robins R. J., Lindsay E., Aird H., Payne J., Parr A. J. and
Walton N. J. (1989) Regulation of secondary metabolism in
transformed root cultures. In Primary and Secondary Metabolism
of Plant Cell Cultures Ⅱ; Kurz, W. G. W., Ed, SPRINGER-Verlag:
Berlin, pp. 58-72.
Slightom J. L., Durand-Tardif M., Jouanin L., and Tepfer D. (1986)
Nucleotide sequence analysis of TL-DNA of Agrobacterium
rhizogenes agropine-type plasmid. J. Biol. Chem. 261, 108-121.
Sim S. J. and Chang H. N. (1993) Increased shikonin production by hairy
roots of Lithospermum erythrorhizon in two phase bubble column
reactor. Biotechnol Lett. 15, 145-150.
Tanaka H., Uemura M., Kaneko Y., and Aoyagi H. (1993) Estimation of
cell biomass in plant cell suspensions by the osmotic pressure
measurement of culture broth. J . Ferment. Bioeng. 76, 501-504.
Taya M., Yoyama A., Kondo O., and Kobayashi T. (1989) Growth
characteristics of plant hairy roots and their cultures in bioreactors.
J. of Chemical Engineering of Japan 22 (1), 84-89.
Taya M., Sato H., Kino Oka M. and Tone S. (1994) Characterization of
pakbung green hairy roots. J .Ferment. Bioeng. 77, 215-217.
Toivonen L., Ojala M., and Kauppinen V. (1990) Indole alkaloid
production by hairy root cultures of Catharanthus roseus: Growth
kinetics and fermentations. Biotechnol. Lett. 12, 519-524.
Toivonen L. (1993) Utilization of hairy root cultures for production of
secondary metabolites. Biotech. Prog. 9, 12-20.
Ullrich WR. (1992) Transport of nitrate and ammonium through plant
membranes. In:Mengel K, Pilbeam DJ, eds. Nitrogen Metabolism
in Plants. London: Clarendon Press, pp. 121-137.
Weathers P. J., Wyslouzil B. E. 1nd Whioole M. (1997) Laboratory–scale
studies of nutrient mist reactor for culturing hairy roots, In “Hairy
Roots: Culture and Application,” Doran P. M. Ed., Harwood
Academic Publishers, The Netherland, pp. 191-199.
Whitney P. J. (1992)Novel bioreactors for the growth of roots
transformed by Agrobacterium rhizogenes. Enzyme Microbiol.
Technol. 14, 13-17.
Wichers H. J., Wijnsma R., Visser J. F., Malingre T. M. and Huizing H. J.
(1985) Production of L-DOPA by cell suspension cultures of
Mucuna pruriens. Ⅱ. Effect of environmental parameyers on the production of L-DOPA. Plant Cell Tiss. Org. Cult. 4, 75-82.
Wilson P. D. G. (1997) The pilot scale cultivation of transformed roots.
In: Doran P. M., ed. Hairy Roots – Culture and Application.
Amsterdam: Harwood Academic Publishers, pp. 179-190.
Yoshikawa T. and Furuya T. (1987) Saponin production by cultures of
Panax ginseng transformed with Agrobacterium rhizogenes. Plant
Cell Rep. 6, 449-453.
朱祥生、陶雨梁、王秀蓮 (1997) 複方大黃軟膏外治單純性肥胖症的臨床療效觀察。中國實驗方劑學雜誌,3(2):47-48。
江蘇新醫學院 (1977) 大黃。中藥大辭典。上海科學技術出版社。
pp.102-108。
余傳隆、黃泰康、丁志遵、高宣亮、張智徳 (1993) 大黃。中藥辭海(第一卷)。中國醫藥科技出版社。pp.323-331。
吳宛勳 (2000) 枸杞之組織細胞培養研究,國立台灣大學藥學研究所碩士論文。
宋麟祥 (2000) 藜豆轉植培養系統生產L-DOPA最佳化研究即其生
物反應器之探討,國立台灣大學化學工程學研究所博士論文。
李玉環、魏新梅、王曉萌 (1997) 大黃塗膜劑促進燒傷面修復作用的實驗與臨床觀察。河北中醫,19(3):13-14。
阮琴芳 (1998) 大黃黃蓮油治療口咽部黏膜燒傷。浙江中醫雜誌,33:310。
林李昌 (2003) 何首烏之組織培養,國立中興大學農藝學研究所碩士論文。
林宗目、林宗平、林景松 (1996) 中藥藥理學。華香園出版社,
pp.163-173。
張光 (1997) 大黃在救治急性心肌梗塞方面的應用。河南中醫,14(4):223。
張蘭昌 (1981) 大黃。中藥大辭典。昭人出版社。pp.185-191。
曹利民 (1998) 大黃羚羊散為主治療急性出血性中風45例。浙江中醫雜誌,33(8):359。
郭巧生、周子靜、林安平 (1993) 大黃。周榮漢主編,中藥資源學。
中國醫藥科技出版社,pp.255-264。
郭澄、李學忠、李曉梅 (1996) 減肥降酯片的研製及臨床應用。醫學雜誌,6(4):40-42。
黃文理、劉麗飛 (1998) 植物細胞培養過程中碳水化合物之代謝。科學農業,46: 227-235。
廉南、曾曉蓉、曹均告 (1997) 趙國良選用捍衛通便法治療老年冠狀心病經驗。成都中醫藥大學學報,20(4):12-13。
楊瑞森 (1993) 展望植物細胞培養生產二級代謝物。生物產業,4:
51-57。
詹明才 (1991) 植物二次代謝產物生合成之新工具。科學農業,39:
201-205。
詹明才,張新維 (1991) 農桿菌轉殖系統之影響因素。科學農業,39:249。
劉小琴、彭玉萍、曹細芳 (1998) 生大黃片治療腦動脈硬化正62例臨床觀察。湖南中醫雜誌,3:7-9。
劉春朝、王玉春、歐陽藩 (1991) 植物組織培養生產有用次生代謝
產物的研究進展。生物技術通報,5:1-7。
劉新裕、林義恭、賴瑞聲、胡敏夫、高瑞隆、王昭月 (2001) 藥用植物大黃之開發與利用。農業世界雜誌,210:63-67。
劉興祥 (1992) 大黃的藥理作用及其臨床研究。中國中西醫結合雜誌,12:571-573。

冉先德 (1993) 中華藥海。哈爾濱出版社,pp.530-537。



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