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研究生:林俊良
研究生(外文):Chun-Liang
論文名稱:北冬蟲夏草無菌栽培研究及動物實驗
論文名稱(外文):The Culture of Cordyceps militaris and Animal Experiments
指導教授:錢佑陳志豪陳志豪引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:55
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冬蟲夏草 (Cordyceps sinensis) 自古有云「冬蟲夏草、甘平、保肺益腎、止血化痰、止勞咳…」,《中醫藥典》認為產於青康藏高原的冬蟲夏草確有改善呼吸道疾患之功效,因其稀少不易取得,價格居高不下。北冬蟲夏草 (Cordyceps militaris) 又名北蟲草或蛹蟲草,是子囊菌亞門,麥角菌目,麥角菌科,蟲草屬的模式種,有著與天然冬蟲夏草同樣的生活史,與Cordyceps sinensis極其近似,是冬蟲夏草唯一的替代品,但在自然界北蟲草亦是一種罕見而難以採收的草藥,本實驗室取天然成熟北蟲草之子實體利用人工培育的方式成功地培養出子實體,約七十天即可成熟採收。接著進行動物實驗,利用實驗室採收之北蟲草子實體,首先進行急性毒性及慢性毒性動物實驗,根據實驗中之小白鼠均未有死亡現象,並於犧牲解剖後檢查所有組織正常,證明北蟲草並不具有毒性。
由於北蟲草與冬蟲夏草性質相近,我們想要證實北蟲草是否對於呼吸道緩解具有療效,因而進行人工誘發氣喘之動物試驗。在急性氣喘試驗中,我們以蒸餾水作為陰性對照組;麻杏甘石湯作為陽性對照組;北蟲草液為實驗組,各液體進行腹腔注射後誘發氣喘,結果發現:注射麻杏甘石湯及北蟲草液的小鼠,對於氣喘發作具有較長時間的耐受性,注射蒸餾水平均耐受性為 96.6±7.6秒;注射麻杏石甘湯為172.8±37.4秒;注射北蟲草液1ml、2ml與3ml其耐受性分別為174.4±25.0;177.6±25.1 及 196.0±8.9秒 (P<0.05) 。在長期餵食北蟲草之氣喘試驗中,將6周齡之BALB/c小鼠餵食北蟲草滿四週後,再使用吸入性組織胺引發氣喘反應,最後犧牲小鼠取其氣管作病理切片及細胞計數,結果發現無餵食北蟲草之對照組小鼠的氣管徑四隻中有三隻明顯收縮變形;而有餵食北蟲草的實驗組的四隻小鼠之氣管均無明顯收縮現象。鏡檢發現,無餵食北蟲草之小鼠的嗜伊紅性白血球有明顯增多 (>5/HPF) 而有餵食組平均為2/HPF (P<0.05) 。由以上兩組動物實驗清楚顯示餵食北蟲草後之實驗組動物對人工誘發之氣喘均可產生緩解的功能。所以我們已經可以成功地人工培養出北蟲草子實體,也可以從實驗中得知北蟲草對於氣喘確實具有改善之功效也可以印證中醫藥典上對於北蟲草功效的論述。


Cordyceps belongs to the family of numerous mushrooms, which is, actually, parasitic fungi, growing wildly on the caterpillars in the high plateaus of China, Nepal, and Tibet. It has the ability to enhances the immune system and improves the lung function from the ancestral codex. But Cordyceps sinensis are rare and difficult to collect, most of the available commercial products belong to the mycelium production, since the real mature mechanism are in the carpophore stage. According to the official published Chinese medicine book, the only substitute herb is Cordyceps militaris. We have developed a cultivating method in laboratory for the growing of the carpophore stage of C. militaris. Several papers have been proved the anti-oxidative function of C. militaris and some traditional medicine researches also mention the improvement of the human asthma condition. In this study, we have done the acute and chronic C. militaris toxicity tests for the mice and could not find out any adversities nor any side effects from the results. In acute asthma test, we used the extract of C. militaris to inject into the peritoneum of the mice and use the Ma Hsing Kan Shin Tang (M.H.K.S.T) injected mice as the positive control. The results have been shown the C. militaris injected mice and the positive control groups which have the longer ability to stand with the histamine challenge, if they compare with the normal saline injected mice (normal saline injected mice: 96.6±7.6 seconds; M.H.K.S.T mice : 172.8±37.4 seconds; C. militaris mice: 1ml- 174.4±25.0 seconds; 2ml- 177.6±25.1 seconds and 3ml- 196.0±8.9 seconds (P<0.05) . And we also fed C. militaris exrtract for the mice a period and sensitized with ova-albumin at the same time, then compared with the control group (non-sensitized mice) to observe the effects of C. militaris to the airway hypersensitivity. The result showed the sensitized mice without C. militaris feeding have prominent airway contraction about 75%; however, all sensitized mice feeding with C. militaris had no airway constriction. Furthermore, the sensitized mice without extract feeding have found more eosinophil counted under the high power field microscopy (>5-6/HPF) compared with the extract feeding mice (2/HPF) (P<0.05). In conclusion, these experiments were shown the laboratory cultured C. militaris has improved the asthma and airway condition in tested animals, that’s to says in the future we still need to do more extensive researches.

目錄
中文摘要Ⅰ
英文摘要Ⅲ
目錄Ⅴ
圖次Ⅷ
表次Ⅸ
壹、前言與文獻探討1
(一) 冬蟲夏草與北冬蟲夏草1
(二) 北蟲草的生活史3
(三) 北蟲草之生活條件4
(四) 北蟲草菌種的主要特性7
(1) 北蟲草母種的主要特徵7
(2) 原種和栽培種的主要特徵7
(五) 北蟲草菌種的保存8
(六) 北蟲草與呼吸道疾病-氣喘探討9
貳、材料與方法12
一、材料設備12
(一) 配料設備12
(二) 滅菌設備12
(三) 接種設備12
(四) 培養設備13
二、培養基的製備與培養條件14
(一) 母種培養基 (斜面培養基) 配方14
(二) 固體培養 (子實體培養)14
(三) 北蟲草的液體培養15
參、實驗方法17
(一) 北蟲草之培育過程實驗17
(二) 北蟲草動物餵食實驗19
1. 北蟲草毒性動物實驗19
a. 北蟲草急性毒性動物實驗19
b. 北蟲草長期餵食毒性動物實驗19
2. 北蟲草對誘發氣喘之動物實驗20
a. 北蟲草即時誘發氣喘動物實驗20
b. 北蟲草餵食後誘發氣喘動物實驗21
肆、結果23
1. 北蟲草毒性動物實驗23
a. 北蟲草急性毒性動物實驗23
b. 北蟲草長期餵食毒性動物實驗23
2. 北蟲草對誘發氣喘之動物實驗23
a. 北蟲草即時誘發氣喘動物實驗23
b. 北蟲草餵食後誘發氣喘動物實驗24
伍、討論26
陸、參考文獻30
圖次
圖一、北蟲草子實體的型態40
圖二、北蟲草在固體培養基培養之菌絲團階段41
圖三、北蟲草在固體培養基培養之小子實體42
圖四、北蟲草之子實體43
圖五、北蟲草之成熟子實體44
圖六、北蟲草之成熟即將採收之子實體45
圖七、北蟲草餵食後誘發氣喘動物實驗46
圖八、北蟲草餵食後誘發氣喘動物實驗 (無致敏小鼠)47
圖九、北蟲草餵食後誘發氣喘動物實驗 (有致敏小鼠)48
圖十、北蟲草餵食後誘發氣喘動物實驗 (餵食去離子水)48
圖十一、北蟲草餵食後誘發氣喘動物實驗 (無致敏組氣管組織切片)
49
圖十二、北蟲草餵食後誘發氣喘動物實驗 (致敏組氣管組織切片)
50
圖十三、實驗動物餵食北蟲草溶液給藥之每日最大耐受量之平均體重變化曲線圖52
圖十四、北蟲草腹腔注射誘發氣喘動物實驗結果之柱狀圖53
表次
表一、北蟲草腹腔注射對誘發氣喘耐受程度之各組小鼠氣喘發作時間 之結果54
表二、北蟲草餵食後誘發氣喘小鼠之支氣管沖洗液,進行嗜伊紅性白血球計數之結果55


丁恆山等,1993,中國藥用孢子植物編寫組,中國藥用孢子植物,上海科學技術出版社,上海,345-346。
中藥寶典,1985,滿庭芳有限公司印製,民國74三月初,1539-1540。
甘偉松,1981,藥用植物學,國立中國醫藥研究所,民國70年八月增訂,85。
冉翠香等,2001,人工培植蛹蟲草子實體原基的誘發形成,食用菌,第4期,9-10。
行政院衛生署中醫藥委員會編,1985,中華民國中藥典範,民國74年十月十日,669-670。
全聯會中藥研究發展委員會,1991,中華民國藥師公會全國聯合會,民國80年七月印行,24。
林石源,2004,蛹蟲草子實體培養條件與活性成分組成之研究,大葉大學生物產業技術學系碩士論文,21-25。
林清淵,2003,是蟲?是草?冬蟲夏草,科學發展,第364期,6-9。
周佳賢,2004,蛹蟲草固態培養研究探討,朝陽科技大學生物技術所碩士論文,32-42。
武進謝,1981,中國醫學大辭典,台灣商務印書館,民國70年三月台十三版,668-669。
吳雲鶴等,1996,北冬蟲夏草的人工培養條件及子實體成份分析,食用菌學報,第3卷第2期,59~61。
姚淑玲,2005,培養條件對北蟲草 (Cordyceps militaris) 菌絲生長之影響,東海大學化學工程研究所碩士論文,3-21。
張緒璋,1998,北蟲草的人工培植及其營養成份分析,中國食用菌,第22卷第2期,19-21。
莊曉莉等,2003,蠶蛹蟲草之特點與安全性,中華真菌學會會刊,18: 151-161。
莊曉莉等,1990,中國藥典編寫組,中國藥典,人民衛生出版社,北京,731。
黃仕政,2005,蛹蟲草之培養和其呈味性質及經γ-照射之巴西蘑菇子實體和樟芝菌絲體之抗氧化性質,國立中興大學食品科學系博士論文,51-57。
陳炳輝,2000,類胡蘿蔔素的特性與應用,科學發展月刊,第28卷第8期,599-604。
葉宗仁,1998,麻杏石甘湯及其減方對過敏原激發氣喘天竺鼠呼吸道阻力與發炎細胞之研究及組織病理學之觀察,中國醫藥學院中國醫學研究所博士論文,1-24。
劉慈欣,2004,液態培養環境對北蟲草 (Cordyceps militaris) 菌絲體生長及其機能性成分之影響,東海大學食品科學系碩士論文,2-108。
蔡秉芸,2005,氣喘診療指引,台灣胸腔暨重症加護醫學會。取自http://health.edu.tw/health/upload/tbteaching_kb/ 20060616104457_file1.pdf。
謝明村,1988,中國藥材學,民國77年,正中書局,593。
戴新民,1985,中草藥學,啟業書局有限公司,民國74年五月再版,546-547。
蕭恆毅,2007,中醫氣喘觀,取自:振興復健醫學中心全球資訊網http:// news.epochtimes.com.tw/8/4/2/80949.htm。
Barnes, P. J. (1987) New concepts in the pathogenesis of bronchial hyper-responsiveness. Agents and Actions Supplements. 21:225-237.
Cho, H. J. ,Cho, J. Y., Rhee, M. H., Park, H. J. (2007) Cordycepin (3''-deoxyadenosine) inhibits human platelet aggregation in a cyclic AMP- and cyclic GMP-dependent manner. European Journal of Pharmacology. 558 (1-3) :43-51.
Chuang, H. L., Lee, X.L., Huang, T.C. (2003) Extract of Tsarm Yung Chung Tsao which exhibit remarkably antioxidative an free-radical scavenging activities. Journal of Taiwan Normal University. 48 (1,2) : 13-24.
Furuya, T., Hirotani, M., and Matsuzawa, M. (1983) N6(2-hydroxyethyl) adenosine, a bio-logically active compound from cultured mycelial of Cordyceps and Isaria speciesl. Phytochemistry. 22:2509-2512.
Global strategy for asthma management and prevention. (1995) Global Initiative for Asthma. Retrieved from http://home.123india. com/dr_patraskidshealth/managepreventionasthma.html.
Gu, Y.X., Song, Y.W., Fan, L.Q., Yuan, Q.S. (2007) Antioxidant activity of natural and cultured Cordyceps sp. antioxidant activity of natural and cultured. China Journal of Chinese Materia Medica. 32 (11) :1028-31.
Hamburger, M. (2007) Comment on comparison of protective effects between cultured Cordyceps militaris and natural Cordyceps sinensis against oxidative damage. Journal of Agricultural and Food Chemistry. 55:7213-7214.
Huang, L.F., Guo, F.Q., Liang, Y.Z., Chen, B.M. (2004) Determination of adenosine and cordycepin in Cordyceps sinensis and C. militaris with HPLC-ESI-MS. China Journal of Chinese Materia Medica. 29 (8) :762-4.
Huand, B.M., Hsu, C.C., Tsai, S.J., Sheu, C.C., and Leu, S.F. (2001) Effects of Cordyceps sinensis on testosterone producion in normal mouse Leydig cells. Life Sciences. 69:2593-2602.
Hwang, I.K., Lim, S.S., Yoo, K.Y., Lee, Y.S., Kim, H.G., Kang, I.J., Kwon, H.J., Park, J., Choi, S.Y., Won, M.H. (2008)
A Phytochemically characterized extract of Cordyceps militaris and cordycepin protect hippocampal neurons from ischemic injury in gerbils. Planta Medica. 74 (2) :114-9.
Kim, H.G., Shrestha, B., Lim, S. Y., Yoon, D.H., Chang, W.C., Shin, D.J., Han, S.K., Park, S.M., Park, J.H., Park, H., Sung, J.M., Jang, Y., Chung, N., Hwang, K.C., Kim, T.W. (2006). Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells. European Journal of Pharmacology. 545 (2-3) :192-9.
Kim, J.S., Sapkota, K., Park, S.E., Choi, B.S.,Kim, S., Nguyen, T.H., Kim, C.S., Choi, H.S., Kim, M.K., Chun, H.S., Park, Y., Kim, S.J. (2006) A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris. Journal of Microbiology. 44 (6) :622-31.
Kim, J., Remick, D.G. (2007) Tumor necrosis factor inhibitors for the treatment of asthma. Current Allergy and Asthma Reports. 7 (2) : 151-6.
Kuo, Y.C., Lin, C.Y., Tsai, W.J., Wu, C.L., Chen, C.F., and Shiao, M.S. (1994) Growth inhibitors against tumor cells in Cordyceps sinensis other than cordycepin and polysaccharides. Cancel- Investigation 12:611-615.
Kuo, Y.C., Tsai, W.J., Wang, J.Y., Chang, S.C., Lin, C.Y., Shiao, M.S. (2001) Regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from Cordyceps sinensis. Life Sciences. 68 (9) :1067-82.
Lee, H., Kim, Y.J., Kim, H.W., Lee, D.H., Sung, M.K., Park, T. (2006) Induction of apoptosis by Cordyceps militaris through activation of caspase-3 in leukemia HL-60 cells. Biological & Pharmaceutical Bulletin. 29 (4) :670-4.
Li, F.H., Liu, P., Xiong, W.G., Xu, G.F. (2006) Effects of corydyceps polysaccharide on liver fibrosis induced by DMN in rats.
China Journal of Chinese Materia Medica. 31 (23) :1968-71.
Li, S P., Zhang, G H., Zeng, Q., Huang, Z G., Wang, Y T., Dong, T. T.T., Tsim, K.W.K. (2006) Hypoglycemic activity of polysaccharide, with antioxidation, isolated from cultured Cordyceps mycelia.
Phytomedicine. 13 (6) :428-33.
Lin, C.Y. (2006) The apoptotic effect of cordycepin on MA-10 mouse Leydig tumor cell line. Master Thesis . Institute of Cell Biology and Anatomy. 13-17
Lin, W.H., Tsai, M.T., Chen, Y.S., Hou, R.C.W., Hung, H.F., Li, C.H., Wang, H.K., Lai, M.N., Jeng, K.C.G. (2007) Improvement of sperm production in subfertile boars by Cordyceps militaris supplement. The American Journal of Chinese Medicine. 35 (4) :631-41.
Lo, H.C., Tu, S.T., Lin, K.C., Lin, S.C. (2004) The anti-hyperglycemic activity of the fruiting body of Cordyceps in diabetic rats induced by nicotinamide and streptozotocin. Life Sciences. 74 (23) :2897-908.
Manabe, N., Sugimoto, M., Azuma, Y., Take-tomo, N., Yamashita, A., Tsuboi, H., Tsunco, A., Kinjo, N., Huang ,N.L., and Miyamoto, H. (1996) Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism in the mouse. Japanese Journal of Pharmacology. 70:85-88.
Nauta, A.J., Engels, F., Knippels, L.M., Garssen, J., Nijkamp, F.P. Redegeld, F.A. (2008) Mechanisms of allergy and asthma. European Journal of Clinical Pharmacology 18 .
Ohmori, T., Tamura, K., Tsuru, S., and Nomoto, K. (1986) Antitumor activity of protein bound polysaccharide from Cordyceps ophioglos- soides in mice. Japanese Journal of Cancer Research. 77:1256-1263.
Ohta, Y., Lee, J.B., Hayashi, K.,Fujita, A., Park, D.K., Hayashi, T. (2007) In vivo anti-influenza virus activity of an immunomodulatory acidic polysaccharide isolated from Cordyceps militaris grown on germinated soybeans. Journal of Agricultural & Food Chemistry. 55 (25) :10194-9.
Park, C., Hong, S.H., Lee, J.Y., Kim, G.Y., Choi, B.T., Lee, Y.T., Park, D. Park, Y.M., Jeong, Y.K., Choi, Y.H. (2005) Growth inhibition of U937 leukemia cells by aqueous extract of Cordyceps militaris through induction of apoptosis. Oncology Reports. 13 (6) :1211-6.
Park, N.S., Lee, K.S., Sohn, H.D., Kim, D.H., Lee, S.M., Park, E., Kim, I., Je, Y.H., Jin, B.R. (2005) Molecular cloning, expression, and characterization of the Cu, Zn superoxide dismutase (SOD1) gene from the entomopathogenic fungus Cordyceps militaris. Mycologia. 97 (1) :130-8.
Reuter, S., Taube, C. (2008) Mast cells and the development of allergic airway disease. Journal of Occupational Medicine And Toxicology. 27 (3) Retrieved from http://www.occup-med.com/content/pdf/1745-6673-3-S1-S2.pdf.
Wang, Z.S., Gu, Y.X., Yuan, Q.S., (2006) Effect of nutrition factors on the synthesis of superoxide dismutase, catalase, and membrane lipid peroxide levels in Cordyceps militaris mycelium. Current Microbiology. 52 (1) :74-9.
Won, S.Y., Park, E.H. (2005) Anti-inflammatory and related
pharmacological activities of cultured mycelia and fruiting bodies of
Cordyceps militaris. Journal of Ethnopharmacology. 96 (3) :555-61.
Wu, Z.L., Wang, X.X., and Cheng, W.Y. (2000) Inhibitory effect of Cordyceps sinensis and Cordyceps militaris on human glomerular mesan-gial cell proliferation induced by native LDL. Cell Biochemistry and Function. 18:93-97.
Yoo, H.S., Shin, J.W., Cho, J.H., Son, C.G., Lee, Y.W., Park, S.Y.,
Cho, C.K. (2004) Effects of Cordyceps militaris extract on angiogenesis and tumor growth. Acta Pharmacologica Sinica. 25 (5) : 657-65.
Zhang, G.Q., Huang, Y.D., Bian, Y., Wong, J. H., Wang, H. (2006) Hypoglycemic activity of the fungi Cordyceps militaris, Cordyceps sinensis, Tricholoma mongolicum, and Omphalia lapidescens in streptozotocin-induced diabetic rats. Applied Microbiology and Biotechnology. 72: 1152–1156.
Zhu, J.S., Halpem, G.M., and Jones, K. (1998) The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis. part I. Journal of Alternative and Complementary Medicine. 4 (3) :289-303.


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