| 摘要: | Steviol為stevioside的醣?配基，可直接作用於胰臟β cells而促進胰島素的分泌。我們曾利用Mucor recurvatus轉換steviol得到代謝物ent-13,16β,17-trihydroxy-kauran-19-oic acid (2)，並且得知代謝物2的降血糖活性較steviol佳。此外，ent-kaurane架構的化合物也具有抗HIV病毒及抗癌等多樣的生物活性。為了取得更多的代謝物2以進行活性試驗及其機轉探討，因此選擇以steviol-16α,17-epoxide (1)當作受質進行微生物轉換。經過15株菌種篩選，由Mucor recurvatus、Streptomyces griseus及Cunninghamella bainieri可得到不同的代謝物且具有再現性，因此選擇此三種菌種進行大量發酵培養，經由抽取、分離及純化由Mucor recurvatus得到ent-13,16β,17-trihydroxy-kauran- 19-oic acid (2)、ent-13,16β-dihydroxy-17-acetoxy-kauran-19-oic acid (3)、ent-11α,13,16α,17-tetrahydroxy-kauran-19-oic acid (4)、ent- 11β,13,16β,17-tetrahydroxy-kauran-19-oic acid (5)、ent-11β,13-di- hydroxy-16β,17-epoxy-kauran-19-oic acid (6)、ent-1β,17-dihydroxy-16- ketobeyeran-19-oic acid (7)以及ent-7α,17-dihydroxy-16-ketobeyeran- 19-oic acid (8)；從Streptomyces griseus得到化合物4、ent-13,17-dihydroxy-kaur-15-en-19-oic acid (9)、ent-17-hydroxy-16- ketobeyeran-19-oic acid (10)、ent-2α,17-dihydroxy-16-ketobeyeran- 19-oic acid (11)、ent-12α,17-dihydroxy-16-ketobeyeran-19-oic acid (12)、ent-12β,17-dihydroxy-16-ketobeyeran-19-oic acid (13)及ent- 14α,17-dihydroxy-16-ketobeyeran-19-oic acid (14)；從Cunninghamella bainieri得到化合物7、8、ent-7β,17-dihydroxy-16-ketobeyeran-19-oic acid (15)、ent-9α,13-dihydroxy-16β,17-epoxy-kauran-19-oic acid (16)、ent-9α,17-dihydroxy-16-ketobeyeran-19-oic acid (17)、ent-9α,13,16α,17- tetrahydroxy-kauran-19-oic acid (18)及ent-9α,13,16β,17-tetrahydroxy- kauran-19-oic acid (19)。其中化合物3-8及11-19為新化合物，所得化合物均經由一維、二維核磁共振光譜及低解析、高解析質譜等鑑定其結構。這是首度發現微生物具有將ent-kaurane架構進行重排反應而生成ent-beyerane架構的能力；此外，也發現Cunninghamella bainieri可以專一性的於steviol-16α,17-epoxide的C-9進行hydroxylation；同時，我們也推測Mucor recurvatus中應含有epoxidase及epoxide hydrolase，故能將steviol先氧化形成steviol-16α,17-epoxide (1)後，再進行水解反應產生化合物2；分離所得的代謝物其生物活性試驗目前正在進行中。
Steviol, the aglycone of stevioside, could stimulate insulin secretion via directly acting on β cells. Previously, we have isolated ent-13,16β,17- trihydroxy-kauran-19-oic acid (2) from transformation of steviol with Mucor recurvatus. The preliminary testing of antihyperglycemic effect showed that it was more potent than steviol. Besides, ent-kaurane possesses many biological activities, such as anti-HIV and anticancer activities. In order to generate more amount of 2 for further investigation of action mechanism and for biological testings, the microbial transformations of steviol-16α,17-epoxide (1) were conducted. By screening fifteen microorganisms, Mucor recurvatus, Streptomyces griseus and Cunninghamella bainieri were selected for preparative-scale transformations of steviol-16α,17-epoxide because they reproducibly formed metabolites. Microbial transformation of 1 with Mucor recurvatus produced ent-13,16β,17-trihydroxy-kauran-19-oic acid (2), ent-13,16β- dihydroxy-17-acetoxy-kauran-19-oic acid (3), ent-11α,13,16α,17-tetra- hydroxy-kauran-19-oic acid (4), ent-11β,13,16β,17-tetrahydroxy-kauran- 19-oic acid (5), ent-11β,13-dihydroxy-16β,17-epoxy-kauran-19-oic acid (6), ent-1β,17-dihydroxy-16-ketobeyeran-19-oic acid (7), and ent-7α,17- dihydroxy-16-ketobeyeran-19-oic acid (8). Microbial transformation of 1 with Streptomyces griseus produced 4, ent-13,17-dihydroxy-kaur-15-en- 19-oic acid (9), ent-17-hydroxy-16-ketobeyeran-19-oic acid (10), ent- 2α,17-dihydroxy-16-ketobeyeran-19-oic acid (11), ent-12α,17-dihydroxy- 16-ketobeyeran-19-oic acid (12), ent-12β,17-dihydroxy-16-ketobeyeran- 19-oic acid (13), and ent-14α,17-dihydroxy-16-ketobeyeran-19-oic acid (14). Microbial transformation of 1 with Cunninghamella bainieri produced 7, 8, ent-7β,17-dihydroxy-16-ketobeyeran-19-oic acid (15), ent-9α,13-dihydroxy-16β,17-epoxy-kauran-19-oic acid (16), ent-9α,17- dihydroxy-16-ketobeyeran-19-oic acid (17), ent-9α,13,16α,17-tetra- hydroxy-kauran-19-oic acid (18), and ent-9α,13,16β,17-tetrahydroxy- kauran-19-oic acid (19). Among them, metabolites 38 and 1119 are the new compounds. The structures of metabolites are established on the basis of HRFABMS, IR, and 1D and 2D NMR. This is the first report that microbe could process the rearrangement of ent-kaurane to ent-beyeran. We also found that Cunninghamella bainieri has the ability to regiospecific hydroxylation at C-9 position of steviol-16α,17-epoxide (1). Besides, the results suggested that steviol was transformed into 2 through 1. The biological testings of isolated metabolites are still in progress. |
