Bing Feng

Enzymatic synthesis of α-glucosyl-timosaponin BII catalyzed by the extremely thermophilic enzyme: Toruzyme 3.0L

Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of (13)C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1-->4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100 degrees C. In addition, we also found that the alpha-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as gamma-amylase (GH15 family), had no such activity under the same reaction conditions.

Spirostanol saponins derivated from the seeds of Trigonella foenum-graecum by β-glucosidase hydrolysis and their inhibitory effects on rat platelet aggregation.

Nine spirostanol saponins (1-9) and seven mixtures of 25 R and 25 S spirostanol saponin isomers (10-16) were obtained from the seeds of Trigonella foenum-graecum after enzymatic hydrolysis of the furostanol saponin fraction by β-glucosidase. Their structures were determined by NMR and MS spectroscopy. Among them, 1- 4, 6, 8, and 9 were new compounds and five, 11B, 12A, 13B, 14A, and 14B, were new structures observed from seven mixtures. In addition, the inhibitory effects of all saponins on rat platelet aggregation were evaluated.

Hydrolysis of timosaponin BII by the crude enzyme from Aspergillus niger AS 3.0739

Timosaponin BII (1), a steroidal saponin showing potential anti-dementia activity, was regioselectively hydrolyzed into its deglycosyl derivatives by the crude enzyme from Aspergillus niger AS 3.0739. Three biotransformation products, timosaponin BII-a (2), timosaponin BII-b (3), and timosaponin BII-c (4), were purified and their structures were elucidated on the basis of 1D NMR, 2D NMR, FAB-MS, and HR-ESI-MS spectral data. Compounds 2 and 3 are new compounds.

Glucosylation of Steroidal Saponins by Cyclodextrin Glucanotransferase

It is known that the sugar chains of steroidal saponins play an important role in the biological and pharmacological activities. In order to synthesize steroidal saponins with novel sugar chains in one step for further studies on pharmacological activity, we here describe the glucosylation of steroidal saponins, and 5 compounds, timosaponin AIII (1), saponin Ta (2), saponin Tb (3), trillin (4) and cantalasaponin I (5), were converted into their glucosylated products by Toruzyme 3.0 L, a cyclodextrin glucanotransferase (CGTase). 12 glucosylated products were isolated and their structures elucidated on the basis of spectral data; they were all characterized as new compounds. The results showed that Toruzyme 3.0 L had the specific ability to add the α-D-glucopyranosyl group to the glucosyl group linked at the sugar chains of steroidal saponins, and the glucosyl group was the only acceptor. This is the first report of steroidal saponins with different degrees of glucosylation. The substrates and their glucosylated derivatives were evaluated for their cytotoxicity against HL-60 human promyelocytic leukemia cell by MTT assay. The substrates all exhibited high cytotoxicity (IC(50) < 10 µmol/L), excluding compound 5 (IC(50) > 150 µmol/L), and the cytotoxicity of most of the products showed no obvious changes compared with those of their substrates.

Selective hydrolysis of flavonoid glycosides by Curvularia lunata

Twelve flavonoid glycosides were involved in the biotransformation of the glycosyl moieties by Curvularia lunata 3.4381, and the products were analyzed by UPLC/PDA-Q-TOF-MS(E). Curvularia lunata displayed hydrolyzing activities on the terminal Rha or Glc units of some flavonoid glycosides. Terminal Rha with a 1 → 2 linkage of isorhamnetin-3-O-neohesperidoside and typhaneoside could be hydrolyzed by Curvularia lunata, but terminal Rha with a 1 → 6 linkage of rutin, typhaneoside, and quercetin-3-O-apiosyl-(1 → 2)-[rhamnosyl-(1 → 6)]-glucoside could not be hydrolyzed. Curvularia lunata could also hydrolyze the Glc of icariin, floramanoside B, and naringin. This is the first report of the hydrolysis of glycosyl units of flavonoid glycosides by Curvularia lunata. A new way to convert naringin to naringenin was found in this research.

Two new steroidal saponins from the biotransformation product of the rhizomes of Dioscorea nipponica

Two new steroidal saponins, dioscins E (1) and F (2), along with nine known steroidal saponins, were isolated from the biotransformation product of the rhizomes of Dioscorea nipponica using Aspergillus oryzae. The structures of new compounds were established as 25(R)-spirost-5-en-21β-methyl-3β-ol-3-O-α- l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranoside (1) and (25R)-spirost-5-en-3β-ol-7-one 3-O-α- l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranoside (2) by detailed spectroscopic analyses including 1D and 2D NMR spectral data (1H–1H COSY, HSQC, and HMBC) and MS spectrometry.

Biotransformation of Glycyrrhetinic Acid by Cunninghamella blakesleeana

Abstract Aim To study the hydroxylation in glycyrrhetinic acid (GA) by biocatalysis, and evaluate the antibacterial activities of the derivatives. Methods The hydroxylation was performed via incubating GA with a fungus, Cunninghamella blakesleeana (C. blakesleeana) AS 3.970 at 30 °C for 5 days. Results GA was converted into two major derivatives and four minor derivatives. The two major derivatives were isolated and elucidated as 3-oxo-7β-hydroxy-glycyrrhetinic acid (GA-1) and 7β-hydroxy-glycyrrhetinic acid (GA-2) based on their spectral data, and other derivatives (GA-3–GA-6) were identified by HR-ESI-MS. The antibacterial activities of substrate and two major derivatives were evaluated on four bacteria strains and one yeast strain. Conclusion Cunninghamella blakesleeana AS 3.970 has the capability of hydroxylation for GA. The two major derivatives and GA were found to have considerable activities against drug-resistant Enterococcus faecalis.

Biotransformation of glycyrrhizin by Aspergillus niger

Biotransformation of glycyrrhizin by Aspergillus niger was investigated and one new compound (1) and one known compound (2) were isolated and identified from the biotransformation products. These were 7β,15α-dihydroxy-3,11-dioxo-oleana-12-en-30-oic acid (1) and 15α-hydroxy-3,11-dione-oleana-12-en-30-oic acid (2). A biotransformation pathway was proposed from HPLC analyses at different reaction times. The biotransformation by A. niger included two stages: first, the two glucuronic acid residues at the C-3 position of glycyrrhizin were hydrolyzed to produce glycyrrhetic acid; and second, glycyrrhetic acid was oxidized and hydroxylated to compounds 1 and 2.

Structure elucidation and complete NMR spectral assignments of glucosylated saponins of cantalasaponin I

Five new glucosylated steroidal glycosides, cantalasaponin I‐B1 (1), I‐B2 (2), I‐B3 (3), I‐B4 (4) and I‐B5 (5), were isolated and purified from the transformed product of the cantalasaponin I by using Toruzyme 3.0 l as biocatalyst. Their structures were elucidated on the basis of high‐resolution electrospray ionization mass spectrometry, one‐dimensional (1H and 13C NMR) and two‐dimensional [COSY, heteronuclear single‐quantum correlation (HSQC), HMBC and HSQC‐TOCSY] NMR spectral analyses and chemical evidence. Copyright

Two new furostanol saponins from the seeds of Trigonella foenum-graecum

Two new furostanol saponins, together with two known steroidal saponins, were isolated from the seeds of Trigonella foenum-graecum L. The structures of the new compounds were determined by detailed analysis of 1D NMR, 2D NMR, MS spectra and chemical evidences as 26-O-β-d-glucopyranosyl-(25S)-5-en-furost-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 4)]-β-d-glucopyranoside (1) and 26-O-β-d-glucopyranosyl-(25R)-5-en-furost-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 4)]-β-d-glucopyranoside (2).