Highly regioselective biotransformation of ginsenoside Rb2 into compound Y and compound K by β-glycosidase purified from Armillaria mellea mycelia

Abstract

Background: The biological activities of ginseng saponins (ginsenosides) are associated with type, number, and position of sugar moieties linked to aglycone skeletons. Deglycosylated minor ginsenosides are known to be more biologically active than major ginsenosides. Accordingly, the deglycosylation of major ginsenosides can provide the multibioactive effects of ginsenosides. The purpose of this study was to transform ginsenoside Rb2, one of the protopanaxadiol-type major ginsenosides, into minor ginsenosides using β-glycosidase (BG-1) purified from Armillaria mellea mycelium.

Methods: Ginsenoside Rb2 was hydrolyzed by using BG-1; the hydrolytic properties of Rb2 by BG-1 were also characterized. In addition, the influence of reaction conditions such as reaction time, pH, and temperature, and transformation pathways of Rb2, Rd, F2, compound O (C-O), and C-Y by treatment with BG-1 were investigated.

Results: BG-1 first hydrolyzes 3-O-outer β-d-glucoside of Rb2, then 3-O-β-d-glucoside of C-O into C-Y. C-Y was gradually converted into C-K with a prolonged reaction time, but the pathway of Rb2 → Rd → F2 → C-K was not observed. The optimum reaction conditions for C-Y and C-K formation from Rb2 by BG-1 were pH 4.0-4.5, temperature 45-60°C, and reaction time 72-96 h.

Conclusion: β-Glycosidase purified from A. mellea mycelium can be efficiently used to transform Rb2 into C-Y and C-K. To our best knowledge, this is the first result of transformation from Rb2 into C-Y and C-K by basidiomycete mushroom enzyme.

Keywords: Armillaria mellea; biotransformation; compound Y; ginsenoside Rb2; β-glycosidase.

Fig. 1

Two main pathways of the transformation of ginsenoside Rb2 by microbial glycosidases . ara (p), α-l-arabinopyranosyl; C-K, compound K; C-O, compound O; C-Y, compound Y; Glc, β-d-glucopyranosyl.

Fig. 2

TLC analysis of reaction products of Rb2 and ammonium sulfate (30–80%) precipitates from mushroom mycelia. The reaction mixture contained Rb2 (2.0 mg) and ammonium sulfate (30–80%) precipitate containing 1.5 U as a β-glucosidase in 1.0 mL of 0.2M acetate buffer (pH 4.5), and distilled water to make a final volume of 2.0 mL was incubated for 96 h at 45°C, respectively. After heating for 10 min in boiling water, the reaction mixture was extracted with water-saturated n-butanol (2.0 mL × 2) and concentrated. The residue was dissolved in methanol (1.0 mL). S, ginsenoside standard mixture; AM, Armillaria mellea; GL, Ganoderma lucidum; PB, Phellinus baumii; GA, Ganoderma applanta; PO, Pleurotus ostreatus.

Fig. 3

Time course of transformation of Rb2 by β-glycosidase (BG-1) purified from Armillaria mellea mycelium. The reaction mixture containing Rb2 (20 mg) and BG-1 (15 U) in 10.0 mL of 0.2M acetate buffer (pH 4.5), and distilled water to make a final volume of 20 mL was incubated for 96 h at 45°C. The reaction mixture (2.0 mL) was taken at different time intervals. After heating for 10 min in boiling water, the reaction mixture was extracted with water-saturated n-butanol (2.0 mL × 2) and concentrated. The residue was dissolved in methanol (1.0 mL). S, ginsenoside standard mixture.

Fig. 4

HPLC analysis of the transformation products of Rb2 by BG-1 with different reaction times. The composition of the reaction mixture and the reaction conditions are the same as in Fig. 3. The reaction mixture was taken at different time intervals (0, 12, 48, and 96 h). C-K, compound K; C-O, compound O; C-Y, compound Y.

Fig. 5

MS spectra (negative ion mode) of the transformation products of Rb2 by BG-1. The reaction mixture (2.0 mL) contained Rb2 (2.0 mg) and BG-1 (1.5 U) in 2.0 mL of 0.1M sodium acetate buffer (pH 4.5) and was incubated for 24 h at 45°C. After heating for 10 min in boiling water, the reaction mixture was analyzed by UPLC/Q-TOF-MS. (A) C-O m/z 961.5005 [M-H + HCOOH ]^–; (B) C-Y m/z 799.4680 [M-H + HCOOH]^–; (C) C-K m/z 667.4415 [M-H + HCOOH]^–. C-K, compound K; C-O, compound O; C-Y, compound Y.

Fig. 6

Influence of pH on the transformation of Rb2 by BG-1. The reaction mixture (2.0 mL), containing Rb2 (2.0 mg) and BG-1 (1.5 U) in 2.0 mL of buffer solution (0.1M), was incubated for 96 h at 45°C at the given pH values (each at 0.1M); acetate (pH 4.0–5.5), sodium phosphate (pH 6.0 and 7.0), Tris–HCl (pH 8.0), and glycine–NaOH buffer (pH 9.0). Experiments were performed in triplicate, and results are shown as mean ± standard deviation. BG-1, β-glycosidase; C-K, compound K; C-O, compound O; C-Y, compound Y.

Fig. 7

Influence of temperature on the transformation of Rb2 by BG-1. The reaction mixture (2.0 mL), containing Rb2 (2.0 mg) and BG-1 (1.5 U) in 2.0 mL of 0.1M acetate buffer (pH 4.5), was incubated for 96 h at the given temperatures. Experiments were performed in triplicate, and results are shown as mean ± standard deviation. BG-1, β-glycosidase; C-K, compound K; C-O, compound O; C-Y, compound Y.

Fig. 8

TLC analysis of transformation products of Rb2, Rd, C-O, F2, and C-Y by BG-1. The reaction mixture (2.0 mL), containing ginsenoside (each 2.0 mg) in methanol (0.2 mL) and BG-1 (1.5 U) in 1.8 mL of 0.1M acetate buffer (pH 4.5), was incubated for 96 h at 45°C, respectively. BG-1, β-glycosidase; C-K, compound K; C-O, compound O; C-Y, compound Y.

Fig. 9

Proposed transformation pathways of Rb2, C-O, C-Y, Rd and F2 by BG-1. The Rf values on TLC of transformation products were compared with those of authentic standards. BG-1, β-glycosidase; C-K, compound K; C-O, compound O; C-Y, compound Y.