ginsenoside-rh1 and protopanaxatriol

Bacillus subtilis CCTCC AB 2012913 can transform ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. Based on its genome sequence, strain B. subtilis 168 contains three UDP-glycosyltransferase genes. Here, we cloned the three UDP-glycosyltransferase genes (ydhE1, yojK1 and yjiC1) from B. subtilis CCTCC AB 2012913 and expressed in Escherichia coli BL21 (DE3) with His-tag. The crude enzyme extracts were assayed, respectively, for their activities to transform ginsenoside Rh1. Extracts containing enzymes YojK1 and YjiC1 could use ginsenoside Rh1 as a substrate to produce 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, which had an additional glucopyranosyl linked with C-3 over the substrate. Enzyme YjiC1 was purified by affinity chromatography on Ni-NTA His Binding resin. The molecular mass of purified YjiC1 was c. 47 kDa as determined by SDS-PAGE. This is the first report of an in vitro biotransformation of ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol using the recombinant UDP-glycosyltransferase.. The Chinese traditional medicinal plant Panax is reported to have multiple health benefits. Its main active ingredient is saponin, and different saponins have different activity spectrum. In the study, three UDP-glycosyltransferase genes, ydhE1, yojK1 and yjiC1, were cloned from Bacillus subtilis CCTCC AB2012913 and the three genes were expressed in Escherichia coli BL21 (DE3). The enzyme YjiC1 was purified and converted ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol in vitro. The compound is the first saponin possessing β-glucopyranosyl at both C-3 and C-6 sites. We showed that the in vitro biotransformation was effective, and the reaction condition was easy to control. Our research suggests that a diversity of saponins could be generated through efficient and directed enzymatic biotransformation.

Topics: Amino Acid Sequence; Bacillus subtilis; Bacterial Proteins; Biotransformation; Cloning, Molecular; Escherichia coli; Ginsenosides; Glycosylation; Glycosyltransferases; Molecular Sequence Data; Panax; Recombinant Proteins; Sapogenins; Saponins

A β-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg3 from Rb1 by the enzyme were pH 6.5, 30°C, 20 mg/ml enzyme, and 4 mg/ml Rb1. Under these conditions, G. terrae β-glucosidase completely converted Rb1 and Re to Rg3 and Rg2, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg1 to Rh1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg3, 1.47 mg/ml Rg2, and 1.17 mg/ml Rh1 from Rb1, Re, and Rg1, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30°C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg2, Rg3, and Rh1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg3, Rg2, and Rh1.

Topics: beta-Glucosidase; Escherichia coli; Ginsenosides; Gordonia Bacterium; Hydrogen-Ion Concentration; Molecular Weight; Panax; Plant Extracts; Plant Roots; Sapogenins; Substrate Specificity; Temperature

Ginsenosides, the main pharmacologically active natural compounds in ginseng (Panax ginseng), are mostly the glycosylated products of protopanaxadiol (PPD) and protopanaxatriol (PPT). No uridine diphosphate glycosyltransferase (UGT), which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPg1, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside F1. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity (>84%) with UGTPg1. We demonstrate that UGTPg100 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rh1, and UGTPg101 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rg1 from F1. However, UGTPg102 and UGTPg103 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rh1 by introducing the genetically engineered PPT-producing pathway and UGTPg1 or UGTPg100. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.

Topics: Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Biocatalysis; Cloning, Molecular; Genes, Plant; Genetic Engineering; Ginsenosides; Glycosyltransferases; Kinetics; Metabolic Engineering; Molecular Sequence Data; Mutant Proteins; Panax; Saccharomyces cerevisiae; Sapogenins; Substrate Specificity; Uridine Diphosphate

To study the pharmacokinetics of ginsenosides Rg1 and its metabolites after iv and oral administration in Wistar rats, the LC-MS/MS method was selected to determine ginsenosides Rg1 and its metabolites in plasma and their pharmacokinetic parameters were calculated. After oral administration of ginsenosides Rg1 to rats, ginsenosides Rg1, Rh1, F1 and protopanaxatriol (Ppt) could be detected in plasma. Their Tmax were 0.92, 3.64, 5.17, and 7.30 h, respectively; MRT were 2.68, 5.06, 6.65, and 5.33 h, respectively; AUC(o-t), were 2 363.5, 4 185.5, 3 774.3, and 396.2 ng x mL(-1) x h, respectively. After iv administration of ginsenosides Rg1 to rats, ginsenosides Rg1, Rh1 and FI could be detected in plasma. Their T1/2betaS were 3.12, 5.87, and 6.87 h, respectively; MRTs were 1.92, 5.99, and 7.13 h, respectively; AUCo-tS were 1 454.7, 597.5, and 805.6 ng x mL(-1) x h, respectively. So, it can be concluded that after oral administration, the amounts of metabolites were higher than the prototype in vivo, and the distribution and elimination of the metabolites were relatively slow. After iv administration, the amount of prototype were higher than that of the metabolites in vivo, and the distribution and elimination of the metabolites were relatively slow.

Topics: Administration, Oral; Animals; Area Under Curve; Chromatography, Liquid; Female; Ginsenosides; Injections, Intravenous; Male; Panax notoginseng; Plants, Medicinal; Random Allocation; Rats; Rats, Wistar; Sapogenins; Tandem Mass Spectrometry

Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method has been developed for the measurement of the concentrations of 20(S)-ginsenoside Rh1 and its aglycone 20(S)-protopanaxatriol in rat plasma with panaxatriol as internal standard. The method involved single liquid-liquid extraction of both 20(S)-ginsenoside Rh1 and 20(S)-protopanaxatriol from plasma samples with n-butanol. The limit of quantification (LOQ) was 5 ng mL(-1) for both compounds. The method was validated within the linear range 5-2000 ng.mL(-1) for both compounds. The correlation coefficient for the calibration regression line was 0.999 or better. Intra-day and inter-day accuracy were better than 15%. The method has been successfully used for the pharmacokinetic studies in rats. After intravenous administrations, the mean retention times of 20(S)-ginsenoside Rh1 and 20(S)-protopanaxatriol were 17.1 +/- 2.0 min and 3.46 +/- 0.33 h, respectively.

Topics: Animals; Atmospheric Pressure; Blood Chemical Analysis; Chromatography, Liquid; Ginsenosides; Mass Spectrometry; Molecular Structure; Rats; Rats, Sprague-Dawley; Sapogenins; Sensitivity and Specificity

Ginsenoside Rg1 has been reported to improve cognitive function in many memory-impaired animal models. However, little is known about the bioactivity of its metabolites in the central nervous system in vivo. In the present study, we employed the step through test and electrophysiological approach to investigate the effects of ginsenoside Rg1's primary metabolite ginsenoside Rh1 and end metabolite protopanaxatriol (Ppt) on learning and memory as well as hippocampal excitability. The behavioral study showed that both ginsenoside Rh1 and Ppt significantly ameliorated memory-impaired models induced by scopolamine in mice. Consistently, the electrophysiological work revealed that ginsenoside Rh1 and Ppt as well as their precursor ginsenoside Rg1 all increased hippocampal excitability in the dentate gyrus of anesthetized rats. These results demonstrated that both ginsenoside Rh1 and Ppt had similar but more potent actions than ginsenoside Rg1 in improving memory and hippocampal excitability, suggesting the role of ginsenoside's sugar moieties in biological activities is not as necessary as traditionally considered.

Topics: Animals; Avoidance Learning; Chromatography, High Pressure Liquid; Electrodes, Implanted; Electrophysiology; Evoked Potentials; Ginsenosides; Hippocampus; Injections, Intraventricular; Learning; Male; Memory; Mice; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Reaction Time; Sapogenins; Tandem Mass Spectrometry

Three 20(S)-protopanaxatriol-type saponins, ginsenoside-Rg1 (1), notoginsenoside-R1 (2), and ginsenoside-Re (3), were transformed by the fungus Absidia coerulea (AS 3.3389). Compound 1 was converted into five metabolites, ginsenoside-Rh4 (4), 3beta,2beta,25-trihydroxydammar-(E)-20(22)-ene-6-O-beta-D-glucopyranoside (5), 20(S)-ginsenoside-Rh1 (6), 20(R)-ginsenoside-Rh1 (7), and a mixture of 25-hydroxy-20(S)-ginsenoside-Rh1 and its C-20(R) epimer (8). Compound 2 was converted into 10 metabolites, 20(S)-notoginsenoside-R2 (9), 20(R)-notoginsenoside-R2 (10), 3beta,12beta,25-trihydroxydammar-(E)-20(22)-ene-6-O-beta-D-xylopyranosyl-(1-->2)-beta-D-glucopyranoside (11), 3beta,12beta-dihydroxydammar-(E)-20(22),24-diene-6-O-beta-D-xylopyranosyl-(1-->2)-beta-D-glucopyranoside (12), 3beta,12beta,20,25-tetrahydroxydammaran-6-O-beta-D-xylopyranosyl-(1-->2)-beta-D-glucopyranoside (13), and compounds 4-8. Compound 3 was metabolized to 20(S)-ginsenoside-Rg2 (14), 20(R)-ginsenoside-Rg2 (15), 3beta,12beta,25-trihydroxydammar-(E)-20(22)-ene-6-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside (16), 3beta,12beta-dihydroxydammar-(E)-20(22),24-diene-6-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside (17), 3beta,12beta,20,25-tetrahydroxydammaran-6-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside (18), and compounds 4-8. The structures of five new metabolites, 10-13 and 16, were established by spectroscopic methods.

Topics: Absidia; Biotransformation; Ginsenosides; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Panax; Plants, Medicinal; Sapogenins; Saponins; Triterpenes