ginsenoside-f1 and protopanaxatriol

Panax notoginseng saponins (PNS) are the major components of Panax notoginseng, with multiple pharmacological activities but poor oral bioavailability. PNS could be metabolized by gut microbiota in vitro, while the exact role of gut microbiota of PNS metabolism in vivo remains poorly understood. In this study, pseudo germ-free rat models were constructed by using broad-spectrum antibiotics to validate the gut microbiota-mediated transformation of PNS in vivo. Moreover, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was developed for quantitative analysis of four metabolites of PNS, including ginsenoside F1 (GF1), ginsenoside Rh2 (GRh2), ginsenoside compound K (GCK) and protopanaxatriol (PPT). The results showed that the four metabolites could be detected in the control rat plasma, while they could not be determined in pseudo germ-free rat plasma. The results implied that PNS could not be biotransformed effectively when gut microbiota was disrupted. In conclusion, gut microbiota plays an important role in biotransformation of PNS into metabolites in vivo.

Topics: Animals; Anti-Bacterial Agents; Biotransformation; Chromatography, High Pressure Liquid; Feces; Gastrointestinal Microbiome; Ginsenosides; Male; Panax notoginseng; Rats, Sprague-Dawley; Sapogenins; Saponins; Tandem Mass Spectrometry

Topics: Actinobacteria; Bacterial Proteins; beta-Glucosidase; Escherichia coli; Ginsenosides; Recombinant Proteins; Sapogenins

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