Biotransformation of dihydroisosteviol and the effects of transformed products on steroidogenic gene expressions.

The biotransformation of dihydroisosteviol with Absidia pseudocylindrospora ATCC 24169, Streptomyces griseus ATCC 10137, Mucor recurvatus MR36, and Aspergillus niger BCRC 31130 yielded 15 metabolites, eight of which were previously unknown. Structures of metabolites were established by 2D NMR techniques and HRMS data, two of which were further corroborated by chemical means, and another via single-crystal X-ray diffraction analysis. Subsequently, two steroidogenic cell lines (Y-1 mouse adrenal tumor and MA-10 mouse Leydig tumor cells) were used in a reverse transcription-PCR analysis to assess the effects of all compounds on steroidogenic gene expressions using forskolin as a positive control. The tested gene expressions included steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) enzyme. Gene expression profiles showed that ten of the tested compounds effectively suppressed P450SCC mRNA expression in both Y-1 and MA-10 cells. Several induced SF-1 gene expression and two enhanced StAR gene expression in Y-1 cells. By contrast, in MA-10 cells, one compound effectively suppressed StAR mRNA expression, whereas for others effectively suppressed SF-1 gene expression. The results suggest that analogs of dihydroisosteviol can be potential modulators to alter steroidogenic gene expressions and subsequent enzyme activities.

Oxygenated compounds from the bioconversion of isostevic acid and their inhibition of TNF-α and COX-2 expressions in LPS-stimulated RAW 264.7 cells.

Fourteen oxygenated compounds were isolated from the preparative-scale biotransformation of isostevic acid (ent-beyeran-19-oic acid). Incubation of it with Aspergillus niger BCRC 32720 produced eight metabolites, four with Bacillus megaterium ATCC 14581, and another four with Mortierella isabellina ATCC 38063. In addition to their structural elucidation by NMR spectroscopy and HRMS, structures of four of these were further confirmed by X-ray diffraction studies. Real-time reverse transcription PCR analysis found that 15 of these compounds displayed significant in vitro anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophages by reducing the levels of both TNF-α and COX-2 mRNA relative to control cells stimulated by LPS alone. The activity of one metabolite was similar to that of dexamethasone in inhibiting the expression of TNF-α mRNA, while all test compounds except two of them were more potent than dexamethasone in inhibiting the expression of the COX-2 mRNA.

Transformation of isosteviol lactam by fungi and the suppressive effects of its transformed products on LPS-induced iNOS expression in macrophages.

From the screening of 21 microbial strains, Absidia pseudocylindrospora ATCC 24169 and Aspergillus niger BCRC 32720 were found to reproducibly transform isosteviol lactam (4α-carboxy-13α-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam) (3) into various compounds. Preparative-scale transformation of 3 with Abs. pseudocylindrospora yielded two new hydroxylated compounds (4 and 5), with conservation of the lactam ring. Preparative-scale transformation of 3 with Asp. niger afforded seven new compounds, 6 and 9-14, together with the known compounds 7 and 8. A single-crystal X-ray diffraction experiment confirmed the structure of 14. The suppressive effects of compounds 1-14 on the lipopolysaccharide-induced expression of the inducible nitric oxide synthase gene in RAW 264.7 macrophages were examined by a reverse-transcription real-time PCR analysis. With the exception of 7, all other compounds significantly reduced levels of iNOS mRNA relative to control cells, which were induced by LPS alone. Compounds 2, 3, and 5 were similar in activity to dexamethasone, while 9 was more potent.

Fungal transformation of isosteviol lactone and its biological evaluation for inhibiting the AP-1 transcription factor.

A number of hydroxylated diterpenoids were obtained from the microbial transformation of isosteviol lactone (4alpha-carboxy-13alpha-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone) (2) with Mucorrecurvatus MR 36, Aspergillusniger BCRC 31130, and Absidiapseudocylindrospora ATCC 24169. Incubation of 2 with M. recurvatus and Asp.niger led to isolation of seven known compounds (1 and 3-8). Incubation of 2 with Abs. pseudocylindrospora produced 5 and six previously unreported compounds (9-14). The structures of these isolated compounds were deduced by high-field NMR techniques ((1)H, (13)C, DEPT, COSY, NOESY, HSQC, and HMBC), and those of 9 and 11 were further confirmed by X-ray crystallographic analyses. Subsequently, the inhibitory effects on activator protein-1 (AP-1) activation in lipopolysaccharide-stimulated RAW 264.7 macrophages of all of these compounds were evaluated. Compounds 2-5, 8, 9, 11, and 12 exhibited significant inhibitory activity, while 3 was more potent than the reference compound of dexamethasone.

Microbial transformation of isosteviol lactone and evaluation of the transformation products on androgen response element.

Two filamentous fungi, Cunninghamella bainieri ATCC 9244 and Aspergillus niger BCRC 32720, were used to investigate the biotransformation of isosteviol lactone (4alpha-carboxy-13alpha-hydroxy-13,16- seco-ent-19-norbeyeran-16-oic acid 13,16-lactone) ( 2), which was derived by reacting isosteviol ( ent-16-oxobeyeran-19-oic acid) ( 1) with m-chloroperbenzoic acid. Incubation of 2 with C. bainieri afforded metabolites 3- 6, which involved isomerization, hydroxylation, and ring cleavage reactions followed by oxidation and selective O-methylation. Incubation of 2 with A. niger afforded mono-, di-, and trihydroxylated metabolites 3, 4, and 7- 12. The structures of 3- 12 were elucidated on the basis of spectroscopic analyses, and structures 3, 4, and 6 were confirmed by X-ray crystallographic studies. Compounds 2- 6, 8- 10, and 12 were assayed as androgen agonists using an ARE (androgen response element)-mediated luciferase reporter gene assay. Compounds 3, 6, and 10 were significantly active, with 6 showing more activity than testosterone.

Microbial metabolism of steviol and steviol-16alpha,17-epoxide.

Steviol (2) possesses a blood glucose-lowering property. In order to produce potentially more- or less-active, toxic, or inactive metabolites compared to steviol (2), its microbial metabolism was investigated. Incubation of 2 with the microorganisms Bacillus megaterium ATCC 14581, Mucor recurvatus MR 36, and Aspergillus niger BCRC 32720 yielded one new metabolite, ent-7alpha,11beta,13-trihydroxykaur-16-en-19-oic acid (7), together with four known related biotransformation products, ent-7alpha,13-dihydroxykaur-16-en-19-oic acid (3), ent-13-hydroxykaur-16-en-19-alpha-d-glucopyranosyl ester (4), ent-13,16beta,17-trihydroxykauran-19-oic acid (5), and ent-13-hydroxy-7-ketokaur-16-en-19-oic acid (6). The preliminary testing of antihyperglycemic effects showed that 5 was more potent than the parent compound (2). Thus, the microbial metabolism of steviol-16alpha,17-epoxide (8) with M. recurvatus MR 36 was continued to produce higher amounts of 5 for future study of its action mechanism. Preparative-scale fermentation of 8 yielded 5, ent-11alpha,13,16alpha,17-tetrahydroxykauran-19-oic acid (10), ent-1beta,17-dihydroxy-16-ketobeyeran-19-oic acid (11), and ent-7alpha,17-dihydroxy-16-ketobeyeran-19-oic acid (13), together with three new metabolites: ent-13,16beta-dihydroxykauran-17-acetoxy-19-oic acid (9), ent-11beta,13-dihydroxy-16beta,17-epoxykauran-19-oic acid (12), and ent-11beta,13,16beta,17-tetrahydroxykauran-19-oic acid (14). The structures of the compounds were fully elucidated using 1D and 2D NMR spectroscopic techniques, as well as HRFABMS. In addition, a GRE (glucocorticoid responsive element)-mediated luciferase reporter assay was used to initially screen the compounds 3-5, and 7 as glucocorticoid agonists. Compounds 4, 5 and 7 showed significant effects.

Biotransformation of gallic acid by Beauveria sulfurescens ATCC 7159.

Preparative-scale fermentation of gallic acid (3,4,5-trihydroxybenzoic acid) (1) with Beauveria sulfurescens ATCC 7159 gave two new glucosidated compounds, 4-(3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yloxy)-3-hydroxy-5-methoxy-benzoic acid (4), 3-hydroxy-4,5-dimethoxy-benzoic acid 3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yl ester (7), along with four known compounds, 3-O-methylgallic acid (2), 4-O-methylgallic acid (3), 3,4-O-dimethylgallic acid (5), and 3,5-O-dimethylgallic acid (6). The new metabolite genistein 7-O-beta-D-4''-O-methyl-glucopyranoside (8) was also obtained as a byproduct due to the use of soybean meal in the fermentation medium. The structural elucidation of the metabolites was based primarily on 1D-, 2D-NMR, and HRFABMS analyses. Among these compounds, 2, 3, and 5 are metabolites of gallic acid in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, B. sulfurescens might be a useful tool for generating mammalian metabolites of related analogs of gallic acid (1) for complete structural identification and for further use in investigating pharmacological and toxicological properties in this series of compounds. In addition, a GRE (glucocorticoid response element)-mediated luciferase reporter gene assay was used to initially screen for the biological activity of the 6 compounds, 2-6 and 8, along with 1 and its chemical O-methylated derivatives 9-13. Among the 12 compounds tested, 11-13 were found to be significant, but less active than the reference compounds of methylprednisolone and dexamethasone.

Antioxidant iridoid glucosides from Wendlandia formosana.

Two new iridoid glucosides of 10-O-caffeoyl scandoside methyl ester (3), and 6-methoxy scandoside methyl ester (4) besides the known compounds of scandoside methyl ester (1), methyl deacetyl asperulosidate (2), 10-O-caffeoyl daphylloside (5), phytol (6), and ursolic acid (7) were isolated from the leaves of Wendlandia formosana. Structure elucidation of the new iridoid glucosides was based on interpretation of high-resolution 1D and 2D NMR spectral data and chemical conversions. Antioxidant activity of Compounds (1-5) against diphenylpicrylhydrazyl (DPPH) and hydroxyl radical, and peroxynitrite was reported.

Hydroxylation and glucosidation of ent-16beta-Hydroxybeyeran-19-oic acid by Bacillus megaterium and Aspergillus niger.

ent-16beta-Hydroxybeyeran-19-oic acid ( 1) has potential antihypertensive activity. To obtain novel and more-effective compounds, 1 was incubated with Bacillus megaterium ATCC 14 581 and Aspergillus niger CCRC 32 720. The structures of the metabolites were determined by HR-FAB-MS, 1D- and 2D-NMR spectral data, and enzymatic hydrolysis. Bacillus megaterium hydroxylated and glucosidated 1 to yield ent-7alpha,16beta-dihydroxybeyeran-19-oic acid ( 2), ent-16beta-hydroxybeyeran-19-oic acid alpha- D-glucopyranosyl ester ( 3), and ent-7alpha,16beta-dihydroxybeyeran-19-oic acid alpha- D-glucopyranosyl ester ( 4). Aspergillus niger hydroxylated 1 to yield ent-1beta,7alpha,16beta-trihydroxybeyeran-19-oic acid ( 5) and ent-1beta,7alpha-dihydroxy-16-oxobeyeran-19-oic acid ( 6). Metabolites 3 - 5 were characterized as new compounds. In addition, 2, 3, 5, and 6 were tested for antihypertensive effects, and we found that 5 and 6 were more potent than the parent compound 1.