臺灣博碩士論文加值系統

摘要
中藥厚朴中有效成分厚朴酚magnolol已被證實是一具有包括抗氧化性、抗發炎及抗癌等活性的新木脂素類化合物，本研究擬利用酵素方法( HRP/H2O2 )來合成此一具藥理活性之天然物厚朴酚，探討不同來源的peroxidases、不同反應條件（pH、有機溶液、酵素基質比例）對產物及產率的影響，另一方面進一步探討magnolol的化學結構與抗發炎及抗癌活性的關係。本研究成功地以酵素方法合成厚朴酚，雖然在此合成反應中產物magnolol產生的同時，仍會被酵素作用繼續反應而消耗，而往更高分子量之產物進行，造成提高產率的困難度，然而在改變不同之反應條件後，發現以10% Methanol/ pH 6.0磷酸buffer作為反應溶液，在適當條件下(100 units HRP/0.1mmol substrate, H2O2 0.54eq/18min )，可以將產率提高至40.4 %，此結果顯示適量比率之有機溶劑有助於提高低分子量產物之產率，並適當調整酵素與過氧化氫添加量後，反應有較高產率。另外分離出副產物AP02 (9.6 %)、AP03 (2.2 %) AP04 (7.4% ) AP05 (2.1 %)。由Magnolol及其結構類似物之抗炎及抗肝癌活性的結果顯示，magnolol若沒有側鏈及-OH基時，幾乎不具有抗炎及抗癌活性，而側鏈上之雙鍵位置與存在與否，對活性沒有太大之影響，而在其類似物中，以honokiol的抗炎及抗癌活性均較magnolol來的強，顯示雖然-OH基的數目增加沒有加強活性，但是-OH基的位置改變，卻有使得活性增強的現象，可能是立體構形改變，使得其與作用之目標分子的結合增強之故。

Abstract

Magnolol is an active neolignan isolated from Magnolia officinalis. Recent studies indicate that magnolol and related neolignans show anti-oxidation, anti-inflammation and anti-tumor activities. Enzymatic synthesis of magnolol was studied in this thesis. The effects of different conditions on the yields of magnolol including different enzymes, organic solvents, buffers, ratios of enzyme/substrate and addition rates of hydrogen peroxide were studied. In addition, the structure-bioactivity relationships of magnolol and its analogs were confirmed by comparing their anti-inflammation and anti-tumor activities. The magnolol have been successfully synthesized in this study. Satisfactory yield ( 40.4% ) of magnolol was obtained when the reaction was performed in 10% methanol/ phosphate buffer ( pH 6.0 ) and suitable conditions ( 1000 units HRP/ 1mmol substrate, H2O2 0.54 eq/ 18 min ).
A mmole scale reaction has been perforemed. Magnolol and by-products like AP02, AP04 and AP05 were isolated and identified. Twelve analogs have been synthesized and studied their structure-bioactivity relationships by comparing the anti-inflammatory and anti-tumor activities. The hydroxy group and the allyl group of magnolol were necessary for the activities. The position of the allyl group in the magnolol have significantly affected the activity. Among these analogs, honokiol has highest activity than other compounds in inhibition of LOX activity and anti Hep-G2 growth. On the other hand, magnolol has the highest inhibition activity against COX.

目 錄
中文摘要 I
英文摘要 II

壹、前言 1
貳、文獻整理 3
一、厚朴在傳統中醫之應用及近代研究 3
1. 古籍記載 3
2. 近代研究 3
二、厚朴酚的生合成 5
三、酵素合成方法 6
1.發展 6
2.過氧化酶於合成多酚化合物的重要性 8
3. 以HRP進行C–C coupling反應 12
四、發炎反應 14
1. 環氧合酶（cyclooxygenase, COX）作用 14
2. 5-脂氧合酶（lipooxygenase, 5-LOX）作用 15
叁、材料與方法 18
一、實驗材料 18
二、實驗方法 21
1. 酵素合成厚朴酚 22
2. 厚朴酚結構與生化活性之關係 24
2.1厚朴酚類似物之合成 24
2.2抗癌活性測試 28
2.3抗發炎活性測試 30
肆、結果與討論 31
一、 酵素方法合成厚朴酚 31
1. 4-allylphenol的製備 31
2. 以HRP催化4-allylphenol合成magnolol 32
3. 反應條件對產率之影響 33
4. 大量製備與副產物分離 43
二、 厚朴酚結構與生化活性之關係 45
1. 厚朴酚類似物之合成 47
2. 厚朴酚及其類似物之抗炎、抗癌活性 51
伍、結論 53
陸、參考文獻 54

圖目錄
圖一、以HPLC監測反應進程 32
圖二、起始物、產物濃度與H2O2添加之變化 33
圖三、化合物4-allylphenol 1H-NMR圖譜 63
圖四、化合物Magnolol 1H-NMR圖譜 64
圖五、化合物Pummerer’s ketone (AP02) 1H-NMR圖譜 65
圖六、化合物o,O-coupled dimer (AP04) 1H-NMR圖譜 66
圖七、化合物o,o-coupled trimer (AP05) 1H-NMR圖譜 67
圖八、化合物Dimethoxymagnolol 1H-NMR圖譜 68
圖九、化合物bis-eugenol 1H-NMR圖譜 69
圖十、化合物Demethylated bis-eugenol 1H-NMR圖譜 70
圖十一、化合物tetrahydromagnolol 1H-NMR圖譜 71
圖十二、化合物isomagnolol 1H-NMR圖譜 72
圖十三、化合物o,o'-Biphenol, 6,6'-diallyl 1H-NMR圖譜 73

表目錄
表一、不同來源之peroxidase對magnolol產率之影響 35
表二、不同過氧化氫添加速率對magnolol產率之影響 36
表三、不同比例之MeOH對magnolol產率之影響 37
表四、10%不同有機溶劑對magnolol產率之影響 39
表五、50 %不互溶之不同有機溶劑對magnolol產率之影響 40
表六、不同HRP / substrate比值對magnolol產率之影響 41
表七、不同有機溶劑、pH及buffer對magnolol產率之影響 42
表八、厚朴酚與副產物之結構、色層分析與產率 45
表九、厚朴酚及其類似物對抑制脂氧合酶、環氧合酶及人類肝癌
細胞(Hep-G2)細胞存活率之IC50 52
表十、化合物之1H-NMR δH 74

參考文獻
何東英、曹治勤、陳敦禮及陳清發。1985。木蘭醇之合成。化學，43: 1-3.
陸蘊如。1995。香豆素與木脂素。pp.84-88。中藥化學。學苑出版社。北京。
許桂森、林明忠及蕭哲志。2001。抗發炎藥物。pp. 479-500。簡明圖解藥理學。藝軒出版社。台北。
鄭虎占、董澤宏及余靖。1998。厚朴。pp.3280-3304。中藥現代研究與應用。第四卷。學苑出版社。北京。
Adam, W., Lazarus, M., Saha-Moller, C.R., Weichold, O., Hoch, U., Haring, D. and Schreier, P. 1999. Biotransformations with peroxidases. Adv. Biochem. Eng. Biotechnol. 63: 74–108.
Agharahimi, M.R. and N.A. LeBel. 1995. Synthesis of (-)-monoterpenylmagnolol and magnolol. J. Org. Chem. 60: 1856-1863.
Atsumi T, Fujisawa S, Satoh K, Sakagami H, Iwakura I, Ueha T, Sugita Y and Yokoe I. 2000. Cytotoxicity and radical intensity of eugenol, isoeugenol or related dimers. Anticancer Res. 20: 2519-2524.
Ayyagari, M.S., Kaplan, D.L., Chatterjeea, S., Walker, J.E. and Akkara, J.A. 2002. Solvent effects in horseradish peroxidase-catalyzed polyphenol synthesis. Enzym. Microb. Technol. 30: 3-9.
Azevado, A.M., Prazeres, D.M.F., Cabral, J.M.S. and Fonseca, L.P. 2001. Stability of free and immobilized peroxidase in aqueous-organic solvents mixtures. J. Mol. Catal. B: Enzym. 15: 147-153.
Berglund, G.I., Carlsson, G.H., Smith, A.T., Szöke, H., Henriksen, A. and Hajdu, J. 2002. The catalytic pathway of horseradish peroxidase at high resolution. Nature. 417: 463-468.
Chen Y.L., Lin K.F., Shiao M.S., Chen Y.T., Hong C.Y. and Lin S.J. 2001. Magnolol, a potent antioxidant from Magnolia officinalis, attenuates intimal thickening and MCP-1 expression after balloon injury of the aorta in cholesterol-fed rabbits. Basic Res. Cardiol. 96: 353-63.
Colonna, S., Gaggero N., Richelmi C. and Pasta P. 1999. Recent biotechnological developments in the use of peroxidases. Trends Biotechnol. 17: 163-168.
Dannhardt, G. and W. Kiefer. 2001. Cyclooxygenase inhibitors-current status and future prospects. Eur. J. Med. Chem. 36: 109-126.
Faber, K. 1997. Biotransformations in organic chemistry. pp.336-337. Springer-Verlag. Berlin.
Fujita, M., Itokawa, H. and Sashida, Y. 1972. Honokiol, a new phenolic compound isolated from the bark of Magnolia obovata Thunb. Chem. Pharm. bull. 20: 212-213.
Gierse, J.K., C.M. Koboldt, M.C. Walker, K.Seibert and P.C. Isakson. 1999. Kinetic basis for selective inhibition of cyclo-oxygenases. Biochem. J. 339: 607-614.
Goodwin, D.C., L.M. Landino and L.J. Marnett. 1999. Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis. FASEB J. 13: 1121-1136.
Guo, Zhi-Wei, Koji Machiya, You-An Ma and C.J. Sih. 1998. Enzymatic oxidative coupling of hydroxyphenylglycine derivatives. Tetrahedron Lett. 39: 5679-5682.
Harris, R.R., G.W. Carter, R.L. Bell, J.L. Moore and D.W. Brooks. 1995. Clinical activity of leukotriene inhibitors. Int. J. Immunopharmac. 17: 147-156.
Henderson, W.R. 1994. The role of leukotrienes in inflammation. Ann. Intern. Med. 121: 684-697.
Hirano T., Gotoh M. and Oka K. 1994. Natural flavonoids and lignans are potent cytostatic agents against human leukemic HL-60 cells. Life Sci. 55: 1061-1069.
Ho K.Y., Tsai C.C., Chen C.P., Huang J.S. and Lin C.C. 2001. Antimicrobial activity of honokiol and magnolol isolated from Magnolia officinalis. Phytotherapy research. 15: 139-41.
Jing, Xiaobi, Wenxin Gu, Xinfeng Ren, Pingyan Bie and Xinfu Pan. 2001. Total synthesis of (±)-eusiderin G and (±)-eusiderin M. J. Chin. Chem. Soc. 48: 59-63.
Koeller, K.M. and Wong, Chi-Huey. 2001. Enzymes for chemical synthesis. Nature. 409: 232-240.
Kim, Y.K. and Ryu Y.U. 1999. Cytotoxic components from stem bark of Magnolia obovata. Planta Med. 65: 291-292.
Klibanov, A.M. 2001. Improving enzymes by using them in organic solvents. Nature. 409: 241-246.
Kobayashi, A., Yutaka Koguchi, Hiroshi Kanzaki and Shin-ichiro Kajiyama. 1994. Production of a new type of bioactive phenolic compound. Biosci. Biotech. Biochem. 58: 133-134.
Kondo, Y., Y. Kawai, T. Miyazawa, H. Matsui and J. Mizutani. 1994. An assay for lipoxygenase activity by chemiluminescence. Biosci. Biotech. Biochem. 58: 421-422.
Krawczyk, A.R., Ewa Lipkowska and Wrόbel J.T. 1991. Horseradish peroxidase-mediated preparation of dimmers from eugenol and isoeugenol. Collect. Czech. Chem. Common. 56: 1147-1150.
Kuribara H., Kishi E., Hattori N., Okada M. and Maruyama Y. 2000. The anxiolytic effect of two oriental herbal drugs in Japan attributed to honokiol from Magnolia bark. J. Pharm. Pharmacol. 52: 1425-1429.
Li C.Y., Wang Y. and Hu M.K. 2003. Allylmagnolol, a novel magnolol derivative as potent antioxidant. Bioorg Med Chem. 11: 3665-3671.
Lin S.Y., Chang Y.T., Liu J.D., Yu C.H., Ho Y.S., Lee, Y.H. and Lee W.S. 2001. Molecular mechanisms of apoptosis induced by magnolol in colon and liver caner cells. Mol.Carcinog. 32: 73-83.
Lin, S.Y., Liu J.D., Chang H.C., Yeh S.D., Lin C.H. and Lee W.S. 2002. Magnolol suppresses proliferation of cultured human colon and liver cancer cells by inhibiting DNA synthesis and acticating apoptosis. J. Cell. Biochem. 84: 532-544.
Ma, You-An, Zhi-Wei Guo and C.J. Sih. 1998. Enzymatic oxidative coupling of monohalogenated tyrosine derivatives. Tetrahedron Lett. 39: 9357-9360.
Naruyoshi M., N. Maruichi, H. Tonami, R. Nagahata, S. Tawaki, H. Uyama, and S. Kobayashi. 2003. Enzymatic Oxidative Polymerization of p-t-Butylphenol and Characterization of the Product Polymer. Bull. Chem. Soc. Jpn. 76: 375–379.
Oguchi, T., Wakisaka A., Tawaki S., Tonami H., Uyama, H. and S. Kobayashi. 2002. Self-association of m-cresol in aqueous organic solvents: relation to enzymatic polymerization reaction. J. Phys. Chem. B. 106: 1421-1429.
Ouk, S., Thiebaud S., Borredon E., Legars P. and Lecomte, L. 2002. O-Methylation of phenolic compounds with dimethyl carbonate under solid/liquid phase transfer system. Tetrahedron Lett. 43: 2661-2663.
Ryu, K. and Dordick J.S. 1992. How do organic solvents affect peroxidase structure and function. Biochemistry. 31: 2588-2598.
Stantucci, R., Laurenti, E., Sinibaldi, F. and Ferrari, R.P. 2002. Effect of dimethyl sulfoxide on the structure and the functional properties of horseradish peroxidase as observed by spectroscopy and cyclic voltammetry. Biochimica et Biophysica Acta. 1596: 225-233.
Sridhar, M., S.K. Vadivel and U.T. Bhalerao. 1997. Novel Horseradish peroxidase catalysed enantioselective oxidation of 2-naphthols to 1,1'-binaphthyl-2,2'-didls. Tetrahedron Lett. 38: 5695-5696.
Takahisa O., S. Tawaki, H. Uyama, and S. Kobayashi. 2000. Enzymatic synthesis of soluble polyphenol. Bull. Chem. Soc. Jpn. 73: 1389-1396.
Teng C.M., Ko F.N., Wang J.P., Lin C.N., Wu T.S., Chen C.C. and Huang T.F. 1991. Antihaemostatic and antithrombotic effect of some antiplatelet agents isolated from Chinese herbs. J. Pharma. Pharmaco. 43: 667-669.
Torres, E., Siminovich, B., Barzana, E. and Vazquez-Duhalt, R. 1998. Thermodynamic hydrophobicity of aqueous mixtures of water-miscible organic solvents predicts peroxidase activity. J. Mol. Catal. B: Enzym. 4: 155-159.
Wang J.P., Hsu M.F., Raung S.L., Chen C.C., Kuo J.S. and Teng C.M. 1992. Anti inflammatory and analgesic effects of magnolol. Naunyn-Schmiedeberg's Arch. Pharmacol. 346: 707-712.
Wang, J.P., Ho T.F., Chang L.C. and Chen C.C. 1995. Anti-inflammatory effect of magnolol, isolated from Magnolia officinalis, on A23187-induced pleurisy in mice. J. Pharma. Pharmacol. 47: 857-860.
Yamamoto, S. 1992. Mammalian lipoxygenase: molecular structures and functions. Biochem. Biophys. Acta., 1128: 117-131.