Glycosyltransformation of ginsenoside Rh2 into two novel ginsenosides using recombinant glycosyltransferase from Lactobacillus rhamnosus and its in vitro applications

Abstract

Background: Ginsenoside Rh2 is well known for many pharmacological activities, such as anticancer, antidiabetes, antiinflammatory, and antiobesity properties. Glycosyltransferases (GTs) are ubiquitous enzymes present in nature and are widely used for the synthesis of oligosaccharides, polysaccharides, glycoconjugates, and novel derivatives. We aimed to synthesize new ginsenosides from Rh2 using the recombinant GT enzyme and investigate its cytotoxicity with diverse cell lines.

Methods: We have used a GT gene with 1,224-bp gene sequence cloned from Lactobacillus rhamnosus (LRGT) and then expressed in Escherichia coli BL21 (DE3). The recombinant GT protein was purified and demonstrated to transform Rh2 into two novel ginsenosides, and they were characterized by nuclear magnetic resonance (NMR) techniques and evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay.

Results: Two novel ginsenosides with an additional glucopyranosyl (6→1) and two additional glucopyranosyl (6→1) linked with the C-3 position of the substrate Rh2 were synthesized, respectively. Cell viability assay in the lung cancer (A549) cell line showed that glucosyl ginsenoside Rh2 inhibited cell viability more potently than ginsenoside Rg3 and Rh2 at a concentration of 10 μM. Furthermore, glucosyl ginsenoside Rh2 did not exhibit any cytotoxic effect in murine macrophage cells (RAW264.7), mouse embryo fibroblasts cells (3T3-L1), and skin cells (B16BL6) at a concentration of 10 μM compared with ginsenoside Rh2 and Rg3.

Conclusion: This is the first report on the synthesis of two novel ginsenosides, namely, glucosyl ginsenoside Rh2 and diglucosyl ginsenoside Rh2 from Rh2 by using recombinant GT isolated from L. rhamnosus. Moreover, diglucosyl ginsenoside Rh2 might be a new candidate for treatment of inflammation, obesity, and skin whiting, and especially for anticancer.

Keywords: Cell viability; Ginsenoside Rh2; Glycosyltransferase; Novel ginsenosides.

Fig. 1

SDS-PAGE analysis of recombinant LRGT. M, molecular mass maker; 1, crude extract of induced recombinant BL 21 (DE3) cells carrying pMAL-LRGT; 2, pMAL-LRGT after purification with the MBP-bound agarose resin; MBP, maltose-binding protein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Fig. 2

Thin layer chromatography (TLC) analysis of glycosylated products transformed by LRGT. C1, Control 1—enzyme and Rh2; C2, Control 2—enzyme and UDP glucose; C3, Rh2 and UDP glucose; R, result of synthesis; S, ginsenoside standard; UDP,

Fig. 3

The kinetics of biosynthesis toward Metabolite 1 by LRGT were determined using the Michaelis–Menten model, the results of which are presented as follows: (A) HPLC spectra of the glycosylated metabolites transformed by LRGT. (B) The Lineweaver–Burk plot of LRGT. HPLC, high-performance liquid chromatography.

Fig. 4

Effect of glycosylated metabolites under diverse conditions including temperature, pH, and metal ions. (A) Effect of temperature on recombinant LRGT activity in synthesis of Metabolite 1. The most significant activity has been detected at the optimal temperature of 30°C. (B) Effect of pH on recombinant LRGT activity in synthesis of Metabolite 1. The optimal pH of LRGT activity was identified at pH 7, which was more effective while comparing catalytic activity with others. (C) Effect of metal ions on recombinant LRGT activity in synthesis of Metabolite 1. The optimal condition of metal ion regarding LRGT activity was intensively promoted by CuSO₄.

Fig. 5

MS spectrum of Metabolites 1 and 2 after transformation by recombinant LRGT. (A) Metabolite 1 molecular ion peak was m/z 783.4 ([M-H]^-, scaled for C₄₂H₇₁O₁₃, 783.4). (B) Metabolite 2 molecular ion peak was m/z 969.54014 ([M+Na]^-, scaled for C₄₈H₈₂O₁₈Na, 969.53934). MS, mass spectrometry.

Fig. 6

Biosynthesic pathway of ginsenoside Rh2 to glucosyl ginsenoside Rh2 and diglucosyl ginsenoside Rh2 using LRGT.

Fig. 7

Cytotoxicity assay of ginsenoside Rh2, Rg3, and glucosyl ginsenoside Rh2 on murine macrophage (RAW264.7), lung cancer (A549), preadipocyte (3T3-L1), and melanoma (B16BL6) cell lines. (A) Cell viability (%) of RAW264.7, exposed to ginsenoside Rh2, Rg3, and glucosyl ginsenoside Rh2. (B) Cell viability (%) of A549, exposed to ginsenoside Rh2, Rg3, and glucosyl ginsenoside Rh2. (C) Cell viability (%) of 3T3-L1, exposed to ginsenoside Rh2, Rg3, and glucosyl ginsenoside Rh2. (D) Cell viability (%) of B16BL6, exposed to ginsenoside Rh2, Rg3, and glucosyl ginsenoside Rh2. Such cell viability was determined by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are expressed as a percentage of sample-treated control and presented as mean ± SD of three separate experiments. *P < 0.05, **P < 0.01, and ***P ＜0.001 vs. control. SD, standard deviation.

Fig. 8

The predicted three-dimensional model using LRGT amino acid sequence as a template then by using the UCSF Chimera package.

Supplementary Fig. S1

A neighbor-joining–based phylogenetic tree was constructed using MEGA6 software with the bootstrap method. GT, glycosyltransferase.

Supplementary Fig. S2

Multiple alignments of LRGT and predicted conserved motifs on LRGT by using MEME. GT, glycosyltransferase.