臺灣博碩士論文加值系統

Stevioside (1) 可由菊科植物甜菊(Stevia rebaudiana (Bertoni) Bertoni (Compositae) 葉片中萃取而得，一般作為甜味劑，目前在日本及巴西等地區被廣泛的當成代糖使用。Isosteviol (2; ent-16-ketobeyeran-19-oic acid) 為stevioside經由酸水解後所得之四環二萜類，屬於ent-beyerane結構的化合物。研究報告顯示其具有抑制liver mitochondria function，降低glucose 產生及抑制oxygen uptake，抑制 D-glucose 及 D-fructose 穿透細胞膜，抑制腫瘤生長，在動物實驗中，藉由打開自發性高血壓老鼠身上鉀離子管道來抑制鈣離子回流，使血壓降低，預防缺血再灌流對心臟的傷害及降低糖尿病老鼠之血糖等作用。為了取得更多的四環二萜類衍生物以進行生物活性試驗及其機轉探討，因此選擇isosteviol (2)為受質進行微生物轉換。
經過30株菌種篩選isosteviol (2)，選擇Mucor recurvatus、Absidia pseudocylindrospora及 Aspergillus niger進行大量發酵培養，再經由抽取、分離及純化，由Mucor recurvatus得到ent-7a-hydroxy- 16-ketobeyeran-19-oic acid (3), ent-7b-hydroxy-16-ketobeyeran-19-oic acid (4), ent-12a,15a-dihydroxy-16-ketobeyeran-19-oic acid (5), ent-7a, 15a-dihydroxy-16-ketobeyeran-19-oic acid (6) 以及ent-15a-hydroxy- 16-ketobeyeran-19-oic acid (7)；從Absidia pseudo- cylindrospora得到ent-12a,17-dihydroxy-16-ketobeyeran-19-oic acid (8), ent-9a,17-di- hydroxy-16-ketobeyeran-19-oic acid (9), ent-12b,17-dihydroxy-16- ketobeyeran-19-oic acid (10), ent-7b,12a-dihydroxy-16-ketobeyeran-19- oic acid (11), ent-7b,17-dihydroxy-16-ketobeyeran-19-oic acid (12) 以及 ent-7b,12a,17-trihydroxy-16-ketobeyeran-19-oic acid (13)；從Aspergillus niger得到化合物3、ent-1b-hydroxy-16-ketobeyeran-19-oic acid (14), ent-1b,7a-dihydroxy-16-ketobeyeran-19-oic acid (15), ent-7a, 11a-dihydroxy-16-ketobeyeran-19-oic acid (16), ent-7a-hydroxy-1,16- diketobeyeran-19-oic acid (17), ent-1b-hydroxy-7,16-diketobeyeran- 19-oic acid (18), ent-11a-hydroxy-7,16-diketobeyeran-19-oic acid (19)以及ent-1b,6b,7a-trihydroxy-16-ketobeyeran-19-oic acid (20)，其中化合物7、11、13、14及16-20為新化合物，所得化合物均經由低解析、高解析質譜、紅外線光譜及一維、二維核磁共振光譜等鑑定其結構。由於四環二萜類化合物與類固醇結構類似，因此接著將isosteviol (2)及所得代謝物(2-19)進行GRE (glucocorticoid-responsive element)及ARE (androgen-responsive element)-mediated luciferase reporter gene assays之生物活性試驗，經由GRE-mediated luciferase reporter gene assay篩選，發現化合物9、14、16及18均呈現有意義結果，但活性比methylprednisolone及dexamethasone弱；另經由ARE-mediated luciferase reporter gene assay篩選，發現化合物4、7、13、14、15及16均呈現有意義結果，尤其4、13、15及16的活性比testosterone強，未來將進一步探討有效化合物的作用機轉。

Stevioside (1) is a sweet-tasting glycoside occurring abundantly in the leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae), which has been popularly used as a sugar substitute in Japan and Brazil for decades. Hydrolysis of stevioside in acid solution produces isosteviol (2; ent-16-ketobeyeran-19-oic acid), a tetracyclic diterpenoid with an ent-beyerane skeleton. Several biological activities of 2 have been reported including the inhibition of rat liver mitochondria functions, decrease of glucose production and inhibition of oxygen uptake in the isolated rat renal tubules, inhibition of D-glucose and D-fructose transport across the cell membrane in the isolated perfused rat liver, lowering blood pressure in spontaneously hypertensive rats through opening of the K+ channel to inhibit calcium influx, potent inhibition of tumor promoters, cardioprotective effect on rats with heart ischemia-reperfusion injury, and reducing plasma glucose levels in a diabetic animal model. As a part of a series studies on ways concerning the production of novel tetracyclic diterpenoid derivatives for biological testings, structural modification of isosteviol (2) by microbial transformations was carried out.
By screening thirty microorganisms, Mucor recurvatus, Absidia pseudocylindrospora, and Aspergillus niger were selected for the biotransformations of isosteviol (2) at this time. Preparative-scale fermentation of isosteviol (2) with Mucor recurvatus produced ent-7a-hydroxy-16-ketobeyeran-19-oic acid (3), ent-7b-hydroxy-16-keto-
beyeran-19-oic acid (4), ent-12a,15a-dihydroxy-16-ketobeyeran-19-oic acid (5), ent-7a,15a-dihydroxy-16-ketobeyeran-19-oic acid (6), and ent-15a-hydroxy-16-ketobeyeran-19-oic acid (7). Preparative-scale fermentation of isosteviol (2) with Absidia pseudocylindrospora produced ent-12a,17-dihydroxy-16-ketobeyeran-19-oic acid (8), ent-9a,17-dihydr-
oxy-16-ketobeyeran-19-oic acid (9), ent-12b,17-dihydroxy-16-keto-
beyeran-19-oic acid (10), ent-7b,12a-dihydroxy-16-ketobeyeran-19-oic acid (11), ent-7b,17-dihydroxy-16-ketobeyeran-19-oic acid (12), and ent-7b,12a,17-trihydroxy-16-ketobeyeran-19-oic acid (13). Preparative-
scale fermentation of isosteviol (2) with Aspergillus niger produced ent-7a-hydroxy-16-ketobeyeran-19-oic acid (3), ent-1b-hydroxy-16-keto-
beyeran-19-oic acid (14), ent-1b,7a-dihydroxy-16-ketobeyeran-19-oic acid (15), ent-7a,11a-dihydroxy-16-ketobeyeran-19-oic acid (16), ent-7a-hydroxy-1,16-diketobeyeran-19-oic acid (17), ent-1b-hydroxy-
7,16-diketobeyeran-19-oic acid (18), ent-11a-hydroxy-7,16-diketo-
beyeran-19-oic acid (19), and ent-1b,6b,7a-trihydroxy-16-ketobeyeran-
19-oic acid (20). Among them, metabolites (7), (11), (13), (14), and (16-20) are the new compounds. The structures of metabolites were established on the basis of HRFABMS, IR, and 1D and 2D NMR. Tetracyclic diterpenoids possess a formal similarity to steroids. Thus, both GRE (glucocorticoid-responsive element) and ARE (androgen-responsive element)-mediated luciferase assays were used to initially screen for the biological activities of isosteviol (2) and biotransformation products (2-19) as glucocorticoid and androgen agonists. By using a GRE-mediated luciferase reporter gene assay, compounds 9, 14, 16, and 18 were found to be significant, but less active than the reference compounds of methylprednisolone and dexamethasone. By using an ARE-mediated luciferase reporter gene assay, compounds 4, 7, 13, 14, 15, and 16 were found to be significant. In particular, 4, 13, 15, and 16 are more active than the reference compound of testosterone.

目錄　i
附表目錄　iii
附圖目錄　iv
流程目錄　viii
詞彙　1
中文摘要　2
英文摘要　4
第一章　緒論
　研究背景　7
　研究目的　14
第二章　結果與討論
　受質 isosteviol (2)之製備與結構鑑定　17
　Isosteviol (2)與 Mucor recurvatus 之微生物轉換及代謝物結構判定　18
　Isosteviol (2)與 Absidia pseudocylindrospora 之微生物轉換及代謝物結構
　判定　21
　Isosteviol (2)與 Aspergillus niger 之微生物轉換及代謝物結構判定　26
　結論　34
第三章　Isosteviol (2)及其代謝物之生物活性試驗研究背景　36
　Isosteviol (2)及其代謝物進行 Glucocorticoid Receptor-Mediated Assay 與
　Androgen Receptor-Mediated Assay 結果　38　　　
　結論　40
第四章　實驗部分
　實驗儀器　41
　管柱　41
　溶劑與試劑　41
　微生物　42
　受質 isosteviol (2)之製備　44
　微生物轉換實驗
　培養及發酵程序　45
　Isosteviol (2)之微生物轉換實驗　48　
　Mucor recurvatus 轉換 isosteviol (2)　48
　Absidia pseudocylindrospora 轉換 isosteviol (2)　51
　Aspergillus niger 轉換 isosteviol (2)　56
　Transfection Procedures and Reporter Gene Assays　64
產率附表　66
參考文獻　159

1.Davies, H. G.; Green, R. H.; Kelly, D. R.; Roberts, S. M. Biotransformations in preparative organic chemistry; Academic Press : London, p. 1, 1989
2.Pasteur L. Compt. Rend., 45, 913, 1857
3.Büchner, E. Ber. Dysch. Chem. Ges. 30, 117, 1897
4.蘇遠志及黃世佑, 應用微生物學；華香園出版社, 台北, 1999, 初版
5.Ptersone, D. H.; Murry, H. C.Microbiological oxygenation of steroids at carbon 11. J. Am. Chem. Soc., 74, 1872-1872, 1952
6.Kurt, F. Biotransformations in organic chemistry. Chapter 1, Springer, Germany, 2000
7.Venisetty, R. K.; Ciddi, V. Application of microbial biotransformation for the new drug discovery using natural drugs as substrates. Curr. Pharm. Biotech., 4, 153-167, 2003
8.Srisilam, K.; Veeresham, C. Biotransformation of drugs by microbial cultures for predicting mammalian drug metabolism. Biotech. Adv., 21, 3-39, 2003
9.Hanc, O.; Capek, A.; Tadra, M.; Macek, K.; Simek, A. Microbial transformation of steroids. III. Direct conversion of progesterone into testosterone. Arzneimittelforschung, 7, 175-176, 1957
10.Rosi, D.; Peruzzotti, G.; Dennis, E. W.; Berberian, D. A.; Freele, H. B.; Tullar, F.; Archer, S. Hycanthone, a new active metabolite of lucanthone. J. Med. Chem., 10, 867-876, 1967
11.Marsheck, W. J.; Karim, A. Preparation of metabolites of spironolactone by microbial oxygenation. Appl. Microbiol., 25, 647-649, 1973
12.Smith, R. V.; Rosazza, J. P. Microbial models of mammalian metabolism. Aromatic hydroxylation. Arch. Biochem. Biophysic., 161, 551-558, 1974
13.Marshall, V. P.; McGovren, J. P.; Richard, F. A.; Richard, R. E.; Wiley, P. F. Microbial metabolism of anthracycline antibiotics daunomycin and adriamycin. J. Antibiot., 31, 336-342, 1987
14.Hufford, C. D.; Capiton, G. A.; Clark, A. M.; Baker, J. K. Metabolism of imipramine by microorganisms. J. Pharm. Sci., 70, 151-155, 1981
15.Davis, P. J.; Rizzo, J. D. Microbial transformations of warfarin: stereoselective reduction by Nocardia corallia and Arthrobacter species. Appl. Environ. Microbiol., 43, 884-890, 1982
16.Davis, P. J.; Guenthner, L. E. Sulindac oxidation/reduction by microbial cultures; microbial models of mammalian metabolism. Xenobiotica, 15, 845-857, 1985
17.Hezari, M.; Davis, P. J. Microbial models of mammalian metabolism. Furosemide glucoside formation using the fungus Cunninghamella elegans. Drug. Metab. Dispos., 21, 259-267, 1993
18.Abourashed, E. A.; Clark, A. M.; Hufford, C. D. Microbial models of mammalian metabolism of xenobiotics: and updated review. Curr. Med. Chem., 6, 359-374, 1999
19.Ishii, H.; Niwaguchi, T.; Nakahara, Y.; Hayashi, M. Studies on lysergic acid diethylamide and related compounds. Part 8. Structural identification of new metabolites of lysergic acid diethylamide obtained by microbial transformation using Streptomyces roseochromogenes. J. Chem. Soc. Perkin Trans I, 4, 902-905, 1980
20.Yang, W.; Davis, P J. Microbial models of mammalian metabolism. Biotransformations of N-methylcarbazole using the fungus Cunninghamella echinulata. Drug Metab. Dispos., 20, 38-46, 1992
21.Ye, M.; Qu, G.; Guo, H.; Guo, D. Specific 12β-hydroxylation of cinobufagin by filamentous fungi. Appl. Environ. Microbiol., 70, 3521-3527, 2004
22.Soejarto, D. D.; Kinghorn, A. D.; Farnsworth, N. R. Potential sweetening agents of plant origin. III. Organoleptic evaluation of Stevia leaf herbarium samples for sweetness. J. Nat. Prod., 45, 590-599, 1982
23.Wood, H. B.; Allerton, R.; Diehel, H. W.; Fletcher, H. G. Stevioside I. The structure of the glucose moieties. J. Org. Chem., 20, 875-883, 1955
24.Geuns, J. M. C. Stevioside. Phytochemistry, 64, 913-921, 2003
25.Fufita, H.; Edahiro, T. Safety and untilization of stevia sweetener. Food Ind., 22, 1-8, 1979
26.Plnaas, G. M.; Kuc, J. Contraceptive properties of Stevia rebaudiana. Science., 162, 1007, 1968
27.Oliverira-Filho, R. M.; Uehara, O. A.; Minetti, C. A. S. A.; Valle, L. B. S. Chronic administration of aqueous extract of Stevia rebaudiana (Bert.) Bertoni in rats: endocrine effects. Gen. Pharmacol., 20, 187-191, 1989
28.Yodyinggyrad, V.; Bunyawong, S. Effect of stevioside on growth and reproduction. Human Reproduction, 6, 158-165, 1991
29.Melis, M. S. Effects of chronic administration of Stevia rebaudiana on fertility in rats. J. of Ethnopharmacol., 67, 157-161, 1999
30.Tomita, T.; Sato, N.; Arai, T.; Shiraishi, H.; Sato, M.; Takeuchi, M.; Kamio, Y. Bactericidal activity of a fermented hot-water extract from Stevia rebaudiana Bertoni towards enterohemorrhagic Escherichia coli O157: H7 and other food-borne pathogenic bacteria. Microbiol. Immunol., 41, 1005-1009, 1997
31.Takahashi, K.; Matsuda, M.; Ohashi, K.; Taniguchi, K.; Nakagomi, O.; Abe, Y.; Mori, S.; Sato, N.; Okutani, K.; Shigeta, S. Analysis of anti-rotavirus activity of extract from Stevia rebaudiana. Antiviral Res., 49, 15-24, 2001
32.Curi, R.; Alvarez, M.; Bazotte, R. B.; Botion, L. M.; Godoy, J. L.; Bracht, A. Effect of Stevia rebaudiana on Glucose tolerance in normal adult humans. Brazil. J. Med. Biol. Res., 19, 771-774, 1986
33.Melis, M. S. Chronic administration of aqueous extract of Stevia rebaudiana in rats: renal effects. J. of Ethnopharmacol., 47, 129-134, 1995
34.Melis, M. S.; Sainati, A. R. Effect of calcium and verapamil on renal function of rats during treatment with stevioside, J. of Ethnopharmacol, 33, 257-262, 1991
35.Bracht, A. K.; Alvarez, M.; Bracht, A. Effects of Stevia rebaudiana Natural products on rats liver mitochondria. Biochem. Pharmacol., 34, 873-882, 1985
36.Pezzuto, J. M.; Compadre, C. M.; Swanson, S. M.; Nanayakkara, N. P. D.; Kinghorn, A. D. Metabolically activated steviol, the aglycone of stevioside, is mutagenic. Proc. Natl. Acad. Sci. USA, 82, 2478-2482, 1985
37. Pezzuto, J. M.; Nanayakkara, N. P. D.; Compadre, C. M.; Swanson, S. M.; Kinghorn, A. D.; Guenthner, T. M.; Sparnins, V. L.; Lam, L. K. T. Characterization of bacterial mutagenicity mediated by 13-hydroxy-ent-kaurenoic and (steviol) and several structurally-related derivatives and evaluation of potential to induce glutathion S-transferase in mice. Mutation Res., 169, 93-103, 1986
38. Matsui, M.; Matsui, K.; Kawasaki, Y.; Oda, Y.; Noguchi, T.; Kitogawa, Y.; Sawada, M; Hayashi, M.; Nohmi, t.;Yoshihira, K.; Ishidata, M.; Sofuni, T. Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays. Mutagenesis, 11, 573-579, 1996
39. Hsu, Y. H.; Liu, J.C.; Kao, P. F.; Lee, C. N.; Chen, Y. J. Hsieh, M. H.; Chan, P. Antihyperyensive effect of stevioside in different strains of hypertensive rats. Chin. Med. J., 65, 1-6, 2002
40. Lee, C. N.; Wong, K. L.; Liu, J. C.; Chen, Y. J.; Cheng, J. T.; Chan, P. Inhibitory effect of stevioside on calcium influx to produce antihypertension. Planta Med., 67, 796-799, 2001
41. Melis, M. S. Stevioside effect on renal function of normal and hypertensive rats. J. of Ethnopharmacol., 36, 213-217, 1992
42.Hubler, M. O.; Bracht, A.; Kelmer-Bracht, A. M. Influence of stevioside on hepatic glycogen levels in fasted rats. Res. Commun. in Chem. Pathol. Pharmacol., 84, 111-118, 1994
43.Jeppesen, P. B.; Gregersen, S.; Poulsen, C. R.; Hermansen, K.
Stevioside acts directly on pancreatic b cells to secrete insulin: Actions independent of cyclic adenosine monophosphate and adenosine triphosphate-sensitive K+-channel activity. Metabolism, 49, 208-214, 2000
44.Chaiwat, B.; Chaivat, T.; Molvibha, V. Anti-inflammatory and immunomodulatory activities of stevioside and its metabolite steviol on THP-1 Cells. J. Agric. Food Chem., 54, 785-789, 2006
45.Mosetting, E.; Nes, W. R. Stevioside. II. The structure of the aglucon. J. Org. Chem., 20, 884-889, 1955
46.Dolder, F.; Lichti, H.; Mosetting, E.; Quitt, P. The structure and stereochemistry of steviol and isosteviol. J. Am. Chem. Soc., 82, 246-247, 1960
47.Mosetting, E.; Quitt, P.;Beglinger, U.; Waters, J. A.; Vorbrueggen, H.; Djerassi, C. A direct correlation of the diterpene alkaloids and hydrocarbons of the phyllocladene group. Interconversion of garryfoline and steviol. J. Am. Chem. Soc., 83, 3163-3164, 1961
48.Mosetting, E.; Beglinger, U.; Dolder, F.; Lichti, H.; Quitt, P.; Waters, J. A. The absolute configuration of steviol and isosteviol. J. Am. Chem. Soc., 85, 2305-2309, 1963
49.Avent, A. G.; Hanson, J. R.; de Oliverira, B. H. Hydrolysis of the diterpenoid glycoside, stevioside. Phytochemistery, 29, 2712-2713, 1990
50.Bracht, A.K.; Alvarez, M.; Bracht, A. Effects of Stevia rebaudiana natural products on rats liver mitochondria. Biochem. Pharmacol., 34, 873-882, 1985
51.Yamamoto, N. S.; Kelmer Bracht, A. M.; Ishii, E. L.; Kemmelmeier, F. S.; Alvarez, M.; Bracht, A. Effect of steviol and its structural analogues on glucose production and oxygen uptake in rat renal tubes. Experientia, 41, 55-57, 1985
52.Ishii, E. L.; Schwab, A. J.; Bracht, A. Inhibition monosaccharide transport in the intact rat liver by stevioside. Biochem. Pharmacol., 36, 1417-1433, 1987
53.Akihisa, T.; Hamasaki, Y.; Tokuda, H.; Ukiya, M.; Kimura, Y.; Nishino, H.. Microbial transformation of isosteviol and inhibitory effects on Epstein-Barr virus activation of the transformation products. J. Nat. Prod., 67, 407-410, 2004
54.Wong, K. L.; Yang, H. Y.; Chan, P.; Cheng, T. H.; Liu, J. C.; Hsu, F. L.; Liu, I. M.; Cheng, Y. W.; Cheng, J. T. Isosteviol as a potassium channel opener to lower intracellular calcium concentrations in cultured aortic smooth muscle cells. Planta Med., 70, 108-112, 2004.
55.Xu, D.; Li, Y. F.; Wang, J. P.; Davery, A. K.; Zhang, S. J.; Evans, A,
M. The cardioprotective effect on rats with heart ischemia-reperfusion injury. Life Sci., 80, 269-274, 2007
56.Ma, J.; Ma, Z.; Wang, J.; Milne, R. W.; Xu, D.; Davey, A. K.; Evans,
A. M. The reducing plasma glucose levels in a diabetic animal model. Diabetes, obesity and metabolism, doi: 10.1111/j.1463-1325.2006. 00630.x., 2007
57.Hanson, J. R. The microbiological transformation of diterpenoids. Nat. Prod. Rep., 9, 139-151, 1992
58.Bearder, J. R.; Frydman, V. M.; Gaskin, P.; McMillan, J.; C. M.;
Phinney, B. O. Fungal products. Part XVI. Conversion of isosteviol and steviol acetate into gibberellin analogues by mutant B1-41 a Gibberella fujikuroi and the preparation of [3H] Gibberellin A20. J. Chem. Soc. Perkin Trans. I., 173-178, 1976
59.de Oliverira, B. H.; Strapasson, R. A. Biotransformation of isosteviol
by Fusarium verticilloides. Phytochemistry, 43, 393-395, 1996
60.de Oliverira, B. H.; Dos Santos, M. C.; M. C.; Leal, P. C. Biotransformation of diterpenoid, isosteviol by Aspergillus niger, Penicillium chrysogenum and Rhizopus arrhizus. Phytochemistry, 51, 737-741, 1999
61.Hsu, F. L.; Hou, C. C.; Yang, L. M.; Cheng, J. T.; Chi, T. C.; Liu, P. C.; Lin, S. J. Microbial transformation of isosteviol, J. Nat. Prod., 65, 273-277, 2002
62.Chang, S. F.; Yang, L. M.; Hsu, F. L.; Hsu, J. Y.; Liaw, J. H.; Lin, S. J. Transformation of Steviol-16a,17-epoxide by Streptomyces griseus and Cunninghamella bainieri, J. Nat. Prod., 69, 1450-1455, 2006
63.Pelaia, G.; Vatrella, A.; Cuda, G.; Maselli, R.; Marsico, S. A. Molecular mechanisms of corticosteroid actions in chronic inflammatory airway diseases, Life Sci., 72, 1549-1561, 2003
64.Lee, D. K.; Chang, C. S. Molecular communication between
androgen receptor and general transcription machinery. J. Steroid Biochem. Mol. Biol., 84, 41-49, 2003
65.Chen, J. Y.; Hwang, D. J.; Bohl, C. E.; Miller, D. D.; Dalton, J. T.A selective androgen receptor modulator for hormone male contraception. J. Pharmacol. Exp. Ther., 312, 546-553, 2005