臺灣博碩士論文加值系統

由前人研究，牛樟芝編號CCRC 35396具有生長速度快、含量豐富的adenosine，且被證實具有預防因缺乏血清而引起細胞凋亡的功效(Lu, 2006)。因此在本實驗中，選擇CCRC 35396 牛樟芝品系，探討其在不同濃度含硫鹽類逆境下，對菌體型態、菌絲體乾重、多醣體、乙醇萃取物、甲醇萃取物、多醣體分子量分布以及含硫多醣產量的影響及其在細胞保護方面的生理活性。實驗結果發現多醣體的累積，並沒有依生長期間而呈現相關性；相反地，在乙醇萃取物產量方面，卻是與生長時間呈現負相關。MgSO4 0.025% 以及0.25% 的濃度處理下可以促進菌絲的生長；而MnSO4處理下，則是在濃度於0.025%、0.05%、0.10%與0.25%，會抑制菌絲體的生長。以MnSO4 0.25%濃度處理之下，多醣體可以得到最高的產量(8.82 ± 0.79 %)。在乙醇萃取物方面，以MnSO4 0.10%處理為最佳，可達到39.00 ± 2.24 %。三萜含量方面在各個鹽類處理的條件下皆不盡相同，但在不添加鹽類處理的生長曲線之下，三萜的含量隨著培養天數增加而累積。在細胞保護方面，選擇MTT作為分析方式，結果顯示Serum-free狀態下，MnSO4 0.50% 處理時產生的含硫多醣體在30μg/ml即具有明顯保護性。此外，MPP+(1-methyl- 4-phenylpyridine)可抑制呼吸鏈的complex I，導致細胞ATP量快速下滑，同時增加電子從complex I漏損的量，因此提高了粒線體superoxide的產量，對細胞造成毒害與破壞。在MPP+(1-methyl- 4-phenylpyridine)1.5mM處理狀態下，MnSO4 0.05%、0.10%、0.25%處理所產生的含硫多醣體，在100μg/ml皆有保護細胞的效果。

In previous study, the strains CCRC 35396 of A. cinnamomea grew fastest among all the strains in our laboratory, and rich in adenosine. Also the adenosine has been proved that it could prevent cells from serum deprivation-induced apoptosis (Lu, 2006). In this study, strain CCRC 35396 was used to investigate the effects of sulfated-salt stress on the chemical constituents of A. cinnamomea and anti-apoptosis effect. Four fractions, sulfated- polysaccharides(SPS), polysaccharides (PS), ethanolic extracts, and triterpene were analyzed. The PS accumulation was not growth-associated and there was a negative correlation between growth and the yield of NPS. Mycelia were treated with MgSO4 or MnSO4 at the concentrations of 0.025％, 0.05％, 0.1％, 0.25％ and 0.5％. From the results, treatment of MgSO4 in the concentration of 0.025％ & 0.25％ could enhance the mycelial growth. Whereas MnSO4 could inhibit the mycelial growth beyond concentration of 0.025％. The treatment of MnSO4 in the concentration of 0.25％ showed the maximal yield of PS among all the tested concentrations at the value of 8.82 ± 0.79 %. MnSO4 at 0.10％ showed the highest yield of NPS at the value of 39.00 ± 2.24 %. The contents of triterpenoids were all different by series sulfated-salt treatment. The accumulation of triterpenoids were time-dependent without sulfated-salt treatment. The cell protectivity of cell was performed by the MTT test. The result showed that by SPS (30μg/ml) isolated from 0.50% MnSO4 treated mycelia exhibited protectivity significantly in Serum-free test. Meanwhile, in the presence of MPP+1.5mM, all the MnSO4 0.05%、0.10%、0.25% treated mycelia SPS showed the protectivity.

致謝 1
目錄 2
縮寫字全名對照 4
中文摘要 6
英文摘要 8
圖表目錄 10
序論 14
前人研究 16
1. 牛樟芝的介紹 16
1.1. 名稱變革 16
1.2. 分類地位 17
1.3. 型態構造與分佈 17
2. 牛樟芝的成分 18
3. 牛樟芝的生物活性 19
3.1. 多醣體 (polysaccharides) 19
3.2. 乙醇萃取物 19
3.3. 甲醇萃取物 19
材料與方法 20
1. 材料 20
1.1. 菌株 20
1.2. 鹽類處理 20
2. 方法 21
2.1. 培養基之配製方法 21
2.2. 牛樟芝菌絲培養方法 21
2.3. 生長曲線測定 21
2.4. 菌絲體萃取物製備流程 22
2.5. 含硫多醣體之萃取流程………………………………………………...23
2.6. 牛樟芝中dehydrosulphurenic acid、15a-acetyl-dehydro sulphurenic acid及dehydroeburicoic acid成分之定量………………………………………..24
2.7. 牛樟芝乙醇萃取物中cytidine、adenosine、inosine及thymidine成分之定量…………………………………………………………………………….26
2.8. 多醣體分子量分佈分析 27
2.9. 水解及單醣的組成分析 28
2.10. 細胞培養方法 29
2.11. MTT分析 29

結果 30
1. 生長曲線 30
2. 菌絲外表型態 34
3. MgSO4及MnSO4處理下，對菌絲乾重產率及多醣體、乙醇萃取物、 含硫多醣之影響……………………………………………………..39
4. 牛樟芝中三萜類dehydrosulphurenic acid、15a-acetyl-dehydro sulphurenic acid及dehydroeburicoic acid成分之定量與比較 47
5. 牛樟芝乙醇萃取物中adenosine及inosine成分之定量 59
6. 多醣體分子量分析 69
7. 多醣體單醣組成分析 91
8. 含硫多醣體對PC12細胞之保護性探討 102

討論 114
結論 117
參考文獻 119

丁懷謙 2000. 食藥用菇多醣體之免疫生理活性。食品工業 32:28-42.
水野卓、川合正允 1997. 菇類的化學生化學。國立編譯館。
王伯徹 2002. 綜論保健用菇之研發。食品工業 34:1-2.
王伯徹、陳啟楨、華傑．1998．食藥用菇類的培養與應用．食品工業發展研究所報告．第M-87-019號．187頁．新竹．台灣。
張中姿 2002. 樟芝菌絲體之甲醇萃取部份對人類肝癌細胞株 (Hep G2) 生長抑制作用的機轉探討。國立台灣大學醫學院生物化學暨分子生物學研究所碩士論文。
張怡潔 2003. 樟屬植物之牛樟芝菌絲體生長促進因子。台北醫學大學生藥學研究所碩士論文。
陳清農、許勝傑、陳勁初 2000. 紅色旋風勢不可擋再談台灣樟芝。鄉間小路 26:48-50.
黃惟敏 1999. 樟芝微量成分的研究。靜宜大學應用化學研究所碩士論文。
黃鈴娟 2000. 樟芝與姬松茸之抗氧化性質及其多醣組成分析。國立中興大學食品科學系碩士論文。
楊書威 1990. 中藥樟菇活性成分之研究。國立台灣大學藥學研究所碩士論文。
葉怡真 2002. 樟芝對血管內皮細胞之影響及保心血管疾病之機制探討。中國醫藥學院營養研究所碩士論文。
趙世彬、鄒煦杕、蔡東磯、陳河吉、周大中、李文齡 1997. 生物化學。藝軒圖書出版社。
劉國柱 1990. 現代科學看靈芝。
蔡雁暉 2002. 樟芝深層培養液及其多醣體之抗氧化特性。國立中興大學食品科學系研究所碩士論文。
蔡敬民 2001. 樟芝菌絲體及醱酵液對於血糖及血脂的影響。行政院國家科學委員會九十年度保健食品研究開發計畫。
簡秋源、姜宏哲、陳淑貞 1997. 牛樟菇培養性狀及其三萜類成分分析之研究。牛樟研討會論文集，林業叢刊第72號:133-137.
Akihisa, T., Y. Mizushina, M. Ukiya, M. Oshikubo, S. Kondo, Y. Kimura, T. Suzuki, and T. Tai. 2004. Dehydrotrametenonic acid and dehydroeburiconic acid from Poria cocos and their inhibitory effects on eukaryotic DNA polymerase alpha and beta. Biosci. Biotechnol. Biochem. 68:448-450.
Albano, R., and P. Mourão. 1986. Isolation, fractionation, and preliminary characterization of a novel class of sulfated glycans from the tunic of styela plicata (Chordata Tunicata). J. Biol. Chem. 261:758-765.
Chang, T.T., and W.N. Chou. 1995. Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan. Mycol. Res. 99:756-758.
Chang, T.T., and W.N. Chou. 2004. Antrodia cinnamomea reconsidered and A. salmonea sp. nov. on Cunninghamia konishii in Taiwan. Bot. Bull. Acad. Sin. 45:347-352.
Chang, T.T., and W.R. Wang. 2005. Basidiomatal formation of Antrodia cinnamomea on artificial agar media. Bot. Bull. Acad. Sin. 46:151-154.
Chen, C.H., S.W. Yang, and Y.C. Shen. 1995. New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. J. Nat. Prod. 58:1655-1661.
Chen, S.H., M.K Lu, J.J. Cheng, and D.L. Wang. 2005. Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life Sci. 76: 3029-3042. (SCI).
Chen, C.J., C.H. Su, and M.H. Lan. 2001a. Study on solid cultivation and bioactivity of Antrodia camphorata. Fung. Sci. 16:65-72.
Chen, C.J., W.H. Lin, C.N. Chen, S.J. Sheu, S.J. Huang, and Y.L. Chen. 2001b. Development of Antrodia camphorata mycelium with submerge culture. Fung. Sci. 16:7-22.
Cheng, J.J., N.K. Huang, T.T. Chang, D.L. Wang, and M.K. Lu. 2005a. Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life Sci. 76:3029-3042.
Cheng, J.J., C.J. Yang, C.H. Cheng, Y.T. Wang, N.K. Huang, and M.K. Lu. 2005b. Characterization and functional study of Antrodia camphorata lipopolysaccharide. J. Agric. Food Chem. 53:469-474.
Cherng, I.H., D.P. Wu, and H.C. Chiang. 1996. Triterpenoids from Antrodia cinnamomea. Phytochem. 41:263-267.
Cherng, I.H., H.C. Chiang, M.C. Cheng, and Y. Wang. 1995. Three new triterpenoids from Antrodia cinnamomea. J. Nat. Prod. 58:365-371.
Chihara, G. 1992. Recent progress in immunopharmacology and therapeutic effects of polysaccharides. Dev. Biol. Stand. 77:191-197.
Chihara, G., J. Hamuro, Y.Y. Maeda, T. Shiio, T. Suga, N. Takasuka, and T. Sasaki. 1987. Antitumor and metastasis-inhibitory activities of lentinan as an immunomodulator: An overview. Cancer Detect. Prev. Suppl. 1:423-443.
Fujimiya, Y., Y. Suzuki, K. Oshiman, H. Kobori, K. Moriguchi, H. Nakashima, Y. Matumoto, S. Takahara, T. Ebina, and R. Katakura. 1998. Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis. Cancer Immunol. Immunother. 46:147-159.
Hotta, T., A. Enomoto, C. Yoshikumi, M. Ohara, and S. Ueno. 1981. Protein-bound polysaccharides. US Patent 4,271,151 1981.
Hseu, Y.C., W.C. Chang, Y.T. Hseu, C.Y. Lee, Y.J. Yech, P.C. Chen, J.Y. Chen, and H.L. Yang. 2002. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Sci. 71:469-482.
Hsiao, G., M.Y. Shen, K.H. Lin, M.H. Lan, L.Y. Wu, D.S. Chou, C.H. Lin, C.H. Su, and J.R. Sheu. 2003. Antioxidative and hepatoprotective effects of Antrodia camphorata extract. J. Agric. Food Chem. 51:3302-3308.
Hsu, Y.L., Y.C. Kuo, P.L. Kuo, L.T. Ng, Y.H. Kuo, and C.C. Lin. 2005. Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines. Cancer Lett. 221:77-89.
Huang, N.K., J.J. Cheng, W.L. Lai, and M.K. Lu. 2005. Antrodia camphorata prevents rat pheochromocytoma cells from serum deprivation-induced apoptosis. FEMS Microbiol. Lett. 244:213-219.
Lee, I.H., R.L. Huang, C.T. Chen, H.C. Chen, W.C. Hsu, and M.K. Lu. 2002. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol. Lett. 209:63-67.
Liao, J.C. 1996. in Flora of Taiwan; Lauraceae, 2 nd ed., Editorial Committee of the Flora of Taiwan, Taipei. vol II:437-448.
Liu, J.J., T.S. Huang, M.L. Hsu, C.C. Chen, W.S. Lin, F.J. Lu, and W.H. Chang. 2004. Antitumor effects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action. Toxicol. Appl. Pharmacol. 201:186-193.
Lu, M.K., J.J. Cheng, W.L. Lai, and N.K. Hung. 2006. Adenosine as an active component of Antrodia cinnamomea that prevents rat PC12 cells from serum deprivation-induced apoptosis through the activation of adenosine A2A receptors. Life sci. 79:252-258.
Mizuno, T. 1995. Bioactive biomolecules of mushrooms: Food function and medicinal effect of mushroom fungi. Food Rev. Int. 11:7-21.
Mizuno, T. 1996. A development of antitumor polysaccharides from mushroom fungi. Foods Food Ingred. J. Jpn. 167: 69-85
Mizuno, T. 1999a. Bioactive substances in Hericium erinaceus (Bull.: Fr.) Pers. (Yamabushitake), and its medicinal utilization. Int. J. Med. Mushrooms. 1:105-119.
Mizuno, T. 1999b. The extraction and development of antitumoractive polysaccharides from medicinal mushrooms in Japan. Int. J. Med. Mushrooms. 1:9-29.
Mizushina, Y., T. Akihisa, M. Ukiya, C. Murakami, I. Kuriyama, X. Xu, H. Yoshida, and K. Sakaguchi. 2004. A novel DNA topoisomerase inhibitor: dehydroebriconic acid, one of the lanostane-type triterpene acids from Poria cocos. Cancer Sci. 95:354-360.
Nakamura, N., A. Hirakawa, J.J. Gao, H. Kakuda, M. Shiro, Y. Komatsu, C.C. Sheu, and M. Hattori. 2004. Five new maleic and succinic acid derivatives from the mycelium of Antrodia camphorata and their cytotoxic effects on LLC tumor cell line. J. Nat. Prod. 67:46-48.
Parish, C.R., F. Craig, K. J. Brown, D. J. Francis, and W. B. Cowden. 1999. Identification of Sulfated oligosaccharide-based Inhibitors of tumor growth and metastasis using novel in Vitro assays for angiogenesis and heparanase activity1. Cancer Res. 59:3433-3441.
Shen, C.C., Y.C. Kao, R.L. Huang, L.C. Lin, M.J. Don, T.T. Chang, and C.J. Chou. 2003a. New ergostane and lanostane from Antrodia camphorata. Chin. Med. J. 14:247-258.
Shen, Y.C., C.F. Chen, Y.H. Wang, T.T. Chang, and C.J. Chou. 2003b. Evaluation of the immuno-modulating activity of some active principles isolated from the fruiting bodies of Antrodia camphorata. Chin. Pharm. J. 55:313-318.
Shen, Y.C., S.W. Yang, C.S. Lin, C.H. Chen, Y.H. Kao, and C.F. Chen. 1997. Zhankuic acid F: A new metabolite from fungus Antrodia cinnamomea. Planta Med. 63:86-88.
Shen, Y.C., C.J. Chou, Y.H. Wang, C.F. Chen, Y.C. Chou, and M.K. Lu. 2004a. Anti-inflammatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble fractions from five different Cinnamomum species. FEMS Microbiol. Lett. 231:137-143.
Shen, Y.C., Y.H. Wang, Y.C. Chou, C.F. Chen, L.C. Lin, T.T. Chang, J.H. Tien, and C.J. Chou. 2004b. Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata. Planta Med. 70:310-314.
Shu, C.H., and M.Y. Lung. 2004.
Effect of pH on the production and molecular weight distribution of
exopolysaccharide by Antrodia camphorata in batch cultures.
Process Biochem. 39: 931-937.
Song, T.Y., and G.C. Yen. 2002. Antioxidant properties of Antrodia camphorata in submerged culture. J. Agric. Food Chem. 50:3322-3327.
Song, T.Y., and G.C. Yen. 2003. Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in rats. J. Agric. Food Chem. 51:1571-1577.
Tai, T., A. Akahori, and T. Shingu. 1993. Triterpenes of Poria cocos. Phytochem. 32:1239-1244.
Wang, G.J., H.W. Tseng, C.J. Chou, T.H. Tsai, C.T. Chen, and M.K. Lu. 2003. The vasorelaxation of Antrodia camphorata mycelia: involvement of endothelial Ca2+-NO-cGMP pathway. Life Sci. 73:2769-2783.
Wang, S.Y., M.L. Hsu, H.C. Hsu, C.H. Tzeng, S.S. Lee, M.S. Shiao, and C.K. Ho. 1997. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from actived macrophages and T lymphocytes. Int. J. Cancer 70:699-705.
Wang, Y.Y., K.H. Khoo, S.T. Chen, C.C. Lin, C.H. Wong, and C.H. Lin. 2002. Studies on the immuno-modulating and antitumor activities of Ganoderma lucidum (Reishi) polysaccharides: Functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities. Bioorg. Med. Chem. 10:1057-1062.
Wasser, S.P. 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60:258-274.
Wu, D.P., and H.C. Chiang. 1995. Constituents of Antrodia cinnamomea. J. Chin. Chem. Soc. 42:797-800.
Wu, S.H., L. Ryvarden, and T.T. Chang. 1997. Antrodia camphorata ("niu-chang- chih"), new combination of a medicinal fungus in Taiwan. Bot. Bull. Acad. Sin. 38:273-275.
Yang, S.W., Y.C. Shen, and C.H. Chen. 1996. Steroids and triterpenoids of Antrodia cinnamomea-a fungus parasitic on Cinnamomum micranthum. Phytochem. 41:1389-1392.
Young, D.S., H.C. Chiang, and L.K. Liu. 1998. Identification of bioactive components in Antrodia cinnamomea by MS/MS via EI ionization. J. Chin. Chem. Soc. 45:123-129.
Zang, M., and C.H. Su. 1990. Ganoderma camphoratum, a new taxon in genus Ganoderma from Taiwan, China. Acta Bot. Yunnanica. 12:395-396.
Zhang, J., G. Wang, H. Li, C. Zhuang, T. Mizuno, H. Ito, h. Mayuzumi, H. Okamoto, and J. Li. 1994. Antitumor active protein-containing glycans from the chinese mushroom Songshan Lingzhi, Ganoderma tsugae mycelium. Biosci. Biotechnol. Biochem. 58:1202-1205.
Zierer, M., and P. Mourão. 2000. A wide diversity of sulfated polysaccharides are synthesized by different species of marine sponges. Carbohydr. Res. 328:209-216.