| Ginseng (Panax ginseng CAMeyer) is the root of araliaceae perennial herb ginseng, known as "king of herbs", is a traditional precious Chinese medicine. In China, Korea, Japan and other Asian countries has been widely used in nourishing health and medical care. Modern pharmacological studies show that ginseng has immunomodulatory, anti-cancer, anti-cerebral ischemia, diabetes, prevention of atherosclerosis and anti-inflammatory pharmacological activity. It also have found that the main active components of ginseng are ginsenosides, have isolated and identified structures of saponins monomer are more than50species. Ginsenosides are connected by aglycone and sugar together, belonging to triterpenoid saponins. Ginsenosides in accordance with the structure of the aglycone were divided into three categories:protopanaxadiol type ginsenosides (PPD), protopanaxatriol type ginsenosides (PPT) and oleanolic acid type ginsenosides. PPD and PPT type ginsenosides belong to dammarane tetracyclic triterpenoids, the majority of ginseng saponins, which are now considered as one of the main active components of ginseng. PPD-type ginsenosides such as ginsenosides Rb1, Rd, Rh2etc., and PPT-type ginsenosides such as ginsenosides Re, Rg1, Rg2, among which the content of ginsenosides Rb1, Rd, Re etc. are higher than other components, while the content of ginsenoside Rg3, Compound K, Rh2, Rh3and so on are extremely low. In the artificial cultivation of ginseng, the rare ginsenosides basic does not exist.After oral administration of ginsenosides or ginseng extract, ginsenosides would hydrolysis to deglycosylated metabolites under acidic gastric environment. While in the intestine, ginsenosides could also be deglycosylated by gut microbiota, which expressed by glycoside hydrolases (such as β-glucosidase, a-rhamnosidase, xylosidase enzymes etc.). The secondary metabolites or aglycone absorbed into the systemic circulation by the way of transmembrane transportation, and thus played the pharmacodynamics. The difference of ginsenosides mainly lies in the various numbers and types between the aglycone and glycosylation, as well as positions of glycosidic bond. Gastrointestinal metabolic disposition of saponins medicine had been comfirmed as an important node that affect the absorption and pharmacological activity. While glycoside hydrolase system expressed by the gut microbiota is the key factor which mediates the intestinal metabolism of ginsenosides.Gut microbiota as an important "microflora organ" for human, is an animate systems symbiosising with the host, and participates in a number of physiological processes. The gut microbiota dysbiosis gradually is recognized as an important pathological features of metabolic diseases and mental diseases, which is closely related to the occurrence and development of these diseases. Gut microbiota dysbiosis often exacerbate symptoms of the disease, while the disease process can also be counterproductive to the gut microbiota, increasing the degree of disorder of the gut microbiota. In addition, a variety of external factors can affect the steady-state balance of gut microbiota, such as stress and antibiotic abuse, which often lead to gut microbiota dysbiosis. Characterization of the system components and functions of the gut microbiota is an important means of researches.The subject was about the gut microbiota mediated metabolism of ginsenoside deglycosylation. Ginsenosides Rbi was applied to be a model drug which as one of protopanaxadiol-type representative components. The study was to reveal whether gut microbiota dysbiosis can affect their pharmacokinetics behavior of ginsenosides Rb1. If the differences exist, it also need to know whether the mechanism was realated to gut microbiota composition, numbers and changes of enzyme activity as well as other factors.The topic was first established a UPLC-MS/MS analytical methods to detect ginsenosides Rb1and its deglycosylated metabolites simultaneously. Orally given the rats80mg/kg ginsenosides Rb1and intravenous administration of ginsenoside Rb18mg/kg, then ginsenoside Rb1and deglycosylated metabolite plasma concentrations detected during72h. The results showed that the absolute bioavailability of ginsenoside Rb1was only2.01%, moreover the major metabolite ginsenoside Rd could also be measured in two groups of rats. Besides it could detect ginsenosides F2and C-K in rats plasma through oral administration, while ginsenoside Rg3and Rh2could be detected by the route of intravenous administration. We also hydrolysised ginsenoside Rb1in gut microbiota suspension from normal rats feces in vitro to describe the metabolic profiles of ginsenosides Rb1and deglycosylated metabolites in vitro.We use traditional antibiotic medicines lincomycin (5000mg/kg/day) to induce rats gut microbiota dysbiosis one week. Pharmacokinetic behavior study showed that AUC(0-∞) of ginsenoside Rb1in lincomycin group is (35568.85±11136.50)μg/L*h, while AUC(0-∞) of control group is (42475.46±21962.66)μg/L*h. And the ginsenoside Rd plasma concentration of in control group was significantly higher than lincomycin group. Two groups of gut microbiota suspension were prepared to incubate with ginsenoside Rb1in vitro. The results showed that Rd metabolic formation rate for control group was significantly higher than antibiotic-treated group. Deglycosylated reactions were mediated by β-D-glucosidase. And β-D-glucosidase activity study results illustrsted that after a week the activity of β-D-glucosidase of two groups were increasing significantly. The control β-glucosidase activity was approximately1.89times higher than lincomycin group.Based on the experimental results above, we can see that the gut microbiota indeed cause differences of metabolic behavior in vitro and vivo. Therefore, this study focused on restraint stress and antibiotics mixing with natamycin, neomycin and gramicidin to disorder model rats gut microbiota dysbiosis. Pharmacokinetic studies indicated that values of AUC(0-t), AUC(0-∞), Cmax for ginsenoside Rb1in restraint stress group were significantly lower than the values of control group. What’s more, Vz, Clz were both higher than control which also existed significant differences. The values of AUC(0-t), Cmax, T1/2for ginsenoside Rd in restraint stress group were higher than control. Due to the lower concentration of Rd in antibiotic-treated group, the differences between control group were more obvious.In the excretion experiment, even though we only depicted the cumulative excretion curve of ginsenoside Rb1and Rd, urinary excretion curves were interrelated with the trend of plasma concentration-time curves. Maximum cumulative of Rb1urinary excretion rate was (1.33±0.29)(?).We could detect ginsenoside Rb1, Rd, F2, Rg3and C-K in fecal excretion experiment. The cumulative fecal excretion of antibiotic-treated group for Rd was highest, while fecal excretions of F2, Rg3and C-K were lowest, from which we could infer metabolic pathways after Rd hydrolysis was inhibited. Cumulative fecal excretions of restraint stress group for F2and C-K were highest, which suggesting that it was more likely happened through the Rd→F2→C-K metabolic reaction. There was no big differences between three groups, cumulative excretion of Rb1was in the range of (24.17±3.19)%-(25.60±3.88)%.By means of ginsenosides Rb1incubation in vitro, we could observe metabolic differences in vitro. At the same time, we investigated the metabolic formation of ginsenoside Rd, F2and C-K. According to metabolic profiles and metabolic formation rate of each group, we could see each metabolic pathways was affected for antibiotic-treated group, resulting in slow response. Restraint stress made metabolite formation rate increased. It may stimulate the expression of enzyme which lead to differences in the metabolism of each component.This subject also investigated β-glucosidase and β-glucuronidase activity from one week model rats gut microbiota which showed β-glucosidase activity of restraint stress rats was higher than normal rats. Both of the glycosidase activity in antibiotic-treated group had lowest activity with significant differences. By the experimental results, we suggested β-glucosidase as an important enzyme mediating reactions of ginsenoside deglycosylated metabolism by gut microbiota, thereby affecting the intestinal absorption of drugs. Even though P-glucuronidase activity had been affected, this glucosidase might not have the obvious effect on ginsenoside Rb1and its metabolites intestinal metabolism.In the determination of short-chain fatty acids as a evaluation Index of intestinal mucosal barrier function, the results showed that six major short-chain fatty acids including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid. The concentrations of this six short-chain fatty acids in control group were higher than restraint stress group, which was significantly higher than antibiotic-treated group. The study indicated that the restraint stress and antibiotics can cause significant change of gut microbiota and its function.In summary, this paper systematically studied the pharmacokinetic behavior of model drug ginsenosides Rb1in rats with gut microbiota dysbiosis. We deeply explored the mechanism from associated glucosidase activity and function which will provide a common research direction for the study of glycoside drug metabolic law in rats with gut microbiota dysbiosis. |
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