| Chronic glomerular nephritis, a common disease caused by multiple etiologies, is globally growing in incidence and prevalence. As one of the primary causes of death in patients, chronic glomerular nephritis can ultimately lead to chronic renal failure and is usually accompanied by hypertension, hematuria, proteinuria, renal damage and other clinically symptoms. Traditional Chinese medicine (TCM) has been clinically used to prevent and treat chronic diseases for thousands of years. The couple of Rehmannia glutinosa Libosch and Cornus officinalis Sieb, derived from ’JinKuiYaoLue’, is a common drug pair applied for the treatment of chronic diseases. Loganin and morroniside are the two most abundant bioactive components in Cornus officinalis Sieb. While the main active ingredients of Rehmannia glutinosa Libosch are catalpol and acteoside. Generally, TCM is orally administered and its bioactive components will inevitable contact with the bacteria in the gut. The intestinal bacteria own multiple enzymes that can metabolize drugs and lead to chemical structure changes of the prototype drug, which may result in the variation of the bioavailabilities and pharmacological activities of the drug. In present paper, the interaction between the drug pair and intestinal bacteria in vitro and the disposition process of the drug pair in vivo were researched to study changes of the bioactive ingredients in the human body. It would offer scientific basis for the use of the drug pair for clinical treatment of diseases.1 Interactions between the drug pair and intestinal bacteriaAccording to Bergey’s manual of systematic bacteriology, about one hundred different types of bacterial colonies from Escherichia、Enterococcu and Bacillus were observed and initially identified based on the colony morphology, micro-morphology and comparative 16S rRNA gene sequence analysis. Monomers of the bioactive components, single drug extract and the drug pair extract were anaerobic incubated with the isolated intestinal bacteria. The ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) technology was used to search and identify the metabolites. On the other hand, the regulation effects of the drug extract on the growth of four typical intestinal flora (Bifidobacterium, Lactobacillus, Escherichia and Enterococcus) isolated from a healthy human feces were also studied.Compared with the blank sample, the metabolic profiles of the intestinal bacteria were acquired. Analysis of the UPLC/MS chromatography showed human intestinal bacteria had deglycosylation, hydrogenation, hydroxylation, methylation, acetylation towards bioactive components of Rehmannia glutinosa Libosch and Cornus officinalis Sieb. According the distributed regularity of intestinal bacteria with metabolic capability,56% of the isolated intestinal bacteria displayed deglycosylation effect on loganin, morroniside and catalpol. Deglycosylation reaction might be the major metabolic pathway of human intestinal bacteria. After loss of the glucose moiety, some gut bacteria could further biotransform the aglycones. For acteoside, de-caffeic acid moiety was the main metabolic pathway. About 55% of the intestinal bacteria showed this capability. Compared with the mixed intestinal bacteria, single strain had stronger metabolic ability. When used in couple, the amount of bioactive ingredients in the medicine extract and metabolites produced by intestinal bacteria were decreased.Regulation effects of Rehmannia glutinosa Libosch and Cornus officinalis Sieb extract on four typical human intestinal bacteria showed that the single drug and drug pair extracts all could promote the growth of probiotics like Bifidobacterium and Lactobacillus, and inhibit the growth of harmful such as Escherichia and Enterococcus. Compared with single drug, the drug pair showed stronger regulation effects on the growth of intestinal bacteria.2 Drug pair metabolism by rat intestinal bacteriaAfter a single intravenous injection of doxorubicin, the chronic nephritis model was successfully exhibited in rats. Then, Rehmannia glutinosa Libosch, Cornus officinalis Sieb and the drug pair were anaerobic incubated with the normal and model rat intestinal bacteria, reapectively. The UPLC-QTOF/MS technology was applied to detect and identify possible metabolites. Bioactive components in Cornus officinalis Sieb extract could be metabolited to be loganetin, hydrogenated loganetin, hydrogenated and demethylated loganetin, demethylated morronisid aglycone and dehydroxylated morronisid aglycone. While catalpol aglycone, hydrogenated catalpol aglycone, de-CA moiety acteoside, caffeic acid, methylated caffeic acid and hydroxytyrosol were acquired in the incubated sample of Rehmannia glutinosa Libosch.Intestinal bacteria from rats in pathological state showed same metabolic pathways as that from normal rats. However, metabolic capacity of model intestinal bacteria was reduced. The contents of metabolites were decreased and that of prototype compounds were increased. The rat intestinal bacteria displayed similar metabolic pathways with that from human gut. De-glucose moiety and de-caffeic acid moiety were the major intestinal bacteria metabolic pathways of iridoid glycosides and acteoside, respectively.3 Drug pair study in vivoDynamic contents of loganin, morroniside, catalpol and acteoside in normal and chronic glomerular nephritis rat plasma after oral administration of Rehmannia glutinosa Libosch, Cornus officinalis Sieb and the drug pair extracts were simultaneous detected using UPLC-MS method. On the other hand, the main bioactive ingredients and their metabolites in rat plasma, urine and feces were analyzed and identified. Additionally, the transmembrane capacities of the metabolites pruduecd by human intestinal bacteria were tested with the Caco-2 cell monolayer. Pharmacokinetic analysis showed that when used in couple, t1/2、AUC0't and AUC0-∞ of each bioactive ingredient were increased with the decresed CLz/F. Compared with normal rats, the active components in model rats had increased AUC0't, AUC0-∞, Cmax and decreased CLz/F, which indicated the increased bioavailabilities and extended efficacy of the medicines. The metabolites in vivo showed the bioactive components could take place sulfuric acid, glucuronide, methylation and hydrogenation reactions in rats. When used in couple, the contents of prototype compounds and their metabolites in vivo were decreased. Under pathological conditions, metabolic activities of enzymes were reduced which migh lead to the increase of prototype compounds and decrease of metabolites. Caco-2 cell experiments showed loganetin, hydrogenated loganetin, dehydroxylated morronisid aglycone, hydrogenated catalpol aglycone, caffeic acid, methylated caffeic acid and hydroxytyrosol of the Rehmannia glutinosa Libosch-Cornus officinalis Sieb drug pair metabolites produced by human intestinal bacteria could be absorbed through the cell membrane. These metabolites were also detected in the drug pair sample with less amount.In summary, the metabolism of Rehmannia glutinosa Libosch and Cornus officinalis Sieb by intestinal bacteria was closely related to the treatment function of chronic nephritis. On the one hand, drugs metabolized by intestinal bacteria would affect the absorption of the bioactive ingredients. On the other hand, drugs after oral administration could promote the growth of beneficial bacteria in the intestinal tract and inhibit the growth of harmful bacteria, which would decrease the content of inflammatory cytokines in the body and then reduce inflammation symptom in patients with chronic nephritis. This work laid an important foundation on the researches of effective substances, absorption, microbial metabolic mechanism and bioavailabilities of the drug pair in vivo. It might provide vital basis for the clinical application of the medicines and be helpful for the development of new drugs to treat chronic nephritis. |
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