| Dianbaizhu,a folk medicine made from Gaultheria leucocarpa Blume var.yunnanensis(Franch.)T.Z.Hsu&R.C.Fang(Ericaceae),is distributed in Sichuan,Yunnan,Tibet and other provinces and regions.It was used as an antirheumatic by nine ethnic groups in the southwest region.It was used for the treatment of rheumatoid arthritis,joint swelling and pain,bruises,cold flu and chronic bronchitis.Most of traditional Chinese medicines are orally administered.Drugs that exert their effects may include not only prototype components but also metabolites in vivo.Gastrointestinal digestion and absorption have important significance for the anti-inflammatory and analgesic activity and bioavailability of Dianbaizhu,but there are few studies on the metabolism of the anti-inflammation and analgesic active-fraction of Dianbaizhu in vitro.MSTG-A,MSTG-B and gaultherin have significant anti-inflammatory and analgesic activities,and the content of these compounds of the anti-inflammatory and analgesic active-fraction(ARF)is high.Among them,gaultherin is a methyl salicylate diglycoside whose structure is that the glucopyranose group is directly linked to the aglycone and the xylose group is linked to the-OH(6)of glucopyranose group.MSTG-A and MSTG-B are methyl salicylate triglycosides formed by xylose and glucosyl linked to the-OH(2)of glucopyranose group on the basis of the structure of gaultherin,respectively.Chlorogenic acid showed significant anti-inflammatory and analgesic activities in carrageenan-induced inflammation experiment and formalin-induced pain test.Therefore,in order to clarify the pharmacodynamics material basis of anti-inflammatory and analgesic effects of Dianbaizhu,the gastrointestinal metabolism and intestinal absorption of ARF of Dianbaizhu and MSTG-B,MSTG-A,gaultherin,chlorogenic acid were investigated in vitro.The experiments were carried out to investigate the compositional changes of ARF with human intestinal flora and summary the metabolic patterns.It may lay the foundation for the further study of the metabolic pathways and effective substances of ARF in Dianbaizhu.1.Literature review.Firstly,129 domestic and foreign literatures were reviewed systematically,including the progress in research of Dianbaizhu and the gastrointestinal metabolism of components in chinese medicines.The chemical composition and pharmacological activity of Dianbaizhu and the progress in research of artificial gastrointestinal fluids,intestinal bacteria transformation and intestinal absorption of the constituents of chinese medicine were summarized to be a reference for the gastrointestinal metabolism in vitro of ARF in Dianbaizhu.2.Quality control of anti-inflammatory and analgesic active-fraction(ARF)of DianbaizhuThe ARF of Dianbaizhu was prepared based on the earlier work,and HPLC fingerprint of ARF of the three batches of Dianbaizhu was established.MSTG-B was chosen as a reference peak,and the main common peaks were 14.The results of similarity calculation showed that the ARF of different batches of Dianbaizhu are similar,and the similarity of the ARF of the three batches of Dianbaizhu is greater than 0.900.The extraction process of ARF is stable and repeatable.What’s more,a method was established for the determination of MSTG-B,MSTG-A,gaultherin and chlorogenic acid of three batches of Dianbaizhu.The content of these four components in the ARF of Dianbaizhu are 23.608,41.973,8.282,and 2.673 mg/g,respectively.The methodological investigation results show that the method is stable and repeatable.3.Stability of ARF inartificial gastricand intestinal juiceIt was found in the experiment of simulated gastrointestinal environment in vitro that MSTG-B,MSTG-A,gaultherin and chlorogenic acid of ARF were stable in artificial gastric and intestinal juice.The contents of MSTG-B,MSTG-A,gaultherin and chlorogenic acid in the artificial gastric juice were varied from 3.0900%to 3.4806%,4.3242%to 4.6910%,0.4898%to 0.5778%,and 0.0450%to 0.0512%,respectively.And the contents of these four components in artificial intestinal juice varied from 3.0854%to 3.4028%,4.3622%to 4.7008%,0.4904%to 0.5310%,and 0.0376%to 0.0452%,respectively.The results show that the ARF of Dianbaizhu is stable in artificial gastrointestinal fluids.The results of this study were different from the views that glycoside bonds of phenolic glycosides and the ester bonds are hydrolyzed under acidic or basic conditions,possibly due to the stability of these four components is affected by other components of ARF.4.Transformation of ARF and active components in human intestinal bacteriaFresh human intestinal bacteria and the solution of human intestinal bacteria were anaerobically incubated with ARF of Dianbaizhu in vitro at 37℃ for 72 hours.The metabolites were determined by UPLC/MSn,and total 21 components were determined.Among them,there were 17 components in the samples before transformation,12 components in the samples after metabolism by fresh intestinal bacteria,and 15 components in the samples after metabolism by the solution of intestinal bacteria.The major changes that occured in the ARF of Dianbaizhu under the action of human intestinal flora were that methyl salicylate glycosides and flavonoid glycosides were hydrolyzed.And organic acids in human intestinal flora can be metabolized.It was found that the total number of chromatographic peaks of ARF and the peak area changed under the action of fresh intestinal bacteria.The content of MSTG-B,MSTG-A and chlorogenic acid in the ARF gradually decreased,and the metabolism rates were 41.95%,31.04%,and 100%,respectively.The content of gaultherin increased first and then decreased,and the metabolism rate was 100%.And methyl salicylate was detected.Both MSTG-A and gaultherin can be transformed into methyl salicylate,and MSTG-B can be metabolized to gaultherin and methyl salicylate with fresh intestinal bacteria.Chlorogenic acid can be metabolized into caffeic acid,ferulic acid,quinic acid,dihydro caffeic acid and 4-hydroxybenzoic acid.Under the action of the solution of intestinal bacteria,the content of the MSTG-B,MSTG-A and chlorogenic acids in the ARF gradually decreased,and the metabolism rates were 28.78%,25.45%,100%,respectively.The content of gaultherin gradually increased.A small amount of MSTG-A and gaultherin were transformed and MSTG-B can be transformed into gaultherin.Chlorogenic acid can be metabolized to ferulic acid,quinic acid and 4-hydroxybenzoic acid.The main reaction type of intestinal bacteria metabolism of MSTG-B,MSTG-A and gaultherin is hydrolysis reaction.The main reaction types of intestinal bacteria metabolism of chlorogenic acid are hydrolysis,reduction,demethoxylation and demethylation.In addition,transformation process is positively correlated with the activities of the bacteria,and the transformation of the monomer is faster than the the components of ARF.It was found that the metabolites produced by fresh intestinal bacteria and the solution of intestinal bacteria are different.MSTG-B can be metabolized to gaultherin and methyl salicylate with fresh intestinal bacteria,while MSTG-A was directly converted into methyl salicylate.The terminal xylose of MSTG-A was not hydrolyzed in the fresh intestinal bacteria and the solution of intestinal bacteria,speculating that xylose is more difficult to be metabolized by human intestinal bacteria.This study provides ideas for the metabolism of xylanosides in the intestinal bacteria.5.Intestinal absorption of ARF from DianbaizhuEverted intestinal sac model was established to study the absorptive characteristics of MSTG-B,MSTG-A,gaultherin and chlorogenic acid,which are the main active components of ARF in Dianbaizhu.It was found that these four components were well absorbed in the intestine(Papp>1×10-6 cm/s).The results showed that the absorption of these four components was linearity at different concentrations(R2>0.9),which was in consistent with zero order absorption rate.Ka of four components increased with increasing of concentration of ARF,which indicated that intestinal adsorption mechanism of these four components was passive diffusion. |
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