| Constipation is one of the most common gastrointestinal diseases,and its main clinical symptoms include reduced number of defecation,dry fecal knot,defecation difficulty and defecation insufficiency.In recent years,with the improvement of people’s consumption level and living conditions,coupled with the impact of environmental factors such as high work pressure,the number of chronic constipation in the population is on the rise year by year and has attracted more and more attention.Although constipation will not endanger people’s life,chronic constipation will bring a lot of inconvenience to people’s life,and increase the mortality rate of cardiovascular and cerebrovascular diseases such as hypertension and the prevalence of colon cancer.At present,the western medicine or Chinese patent medicine sold well on the market,such as lactilose and senna granules,often have strong dependence,and long-term use is easy to cause metabolic disorders,melanosis coli and other serious adverse reactions.Some emerging treatment methods in recent years,for most patients with constipation,the treatment effect is not ideal.Chinese herbs have the advantages of multi-channel and multi-target system to regulate diseases,and it has incomparable advantages over western medicine in reducing the occurrence of adverse reactions and overall regulation of the body,so it is of great significance to find a single Chinese herb or Chinese herbal compound preparation without anthraquinones which has less side effects and suitable for long-term administration.Aster tataricus has the effect of tonifying lung and moistening intestines,and is often combined with Semen coicix,Platycodon grandiflora,Almond,Fructus aurantii and Lepidii semen in clinical treatment of postoperative constipation and intestinal carbuncle.Both ancient books and modern pharmacological studies have also recorded that the lung treatment agents such as tataricus have the laxative activity,and traditional Chinese medicine believes that the drug theory of moistening intestines of Aster tataricus is related to the appearance and interior of lung and large intestine.Although there have been clinical reports on the biological activity of Aster tataricus for moistening intestines,the correlation and mechanism of action between the active components of Aster tataricus and the main pharmacodynamic effects still need to be studied comprehensively,systematically and deeply.Based on this,this study firstly conducted a pharmacodynamics study on laxative effect of Aster tataricus through in vivo and in vitro experiments,and from the experiments to explore its possible mechanisms in vivo and in vitro,then use UHPLC-Q-TOF-MS/MS technology and molecular docking technology screening the effective ingredients for the laxative effect of Aster tataricus,on this basis,further to explore the possibility of Aster tataricu as health care products development.Specific research contents are as follows:(1)To elucidate the laxative effect of Aster tataricus and explore its possible mechanism of action.In vivo,loperamide hydrochloride was used to establish mice constipation model,and the effects of ATE on intestinal charcoal transit,residual amount and water content of colonic feces,content of neurotransmitters in blood and colonic tissue sections of mice were observed.In vitro,stripped intestinal segments were suspended in the constant temperature perfusion system of isolated tissues to simulate the environment of isolated tissues in animals and humans.After equilibration,the effects of ATE on spontaneous contractile tension of intestinal segments and the contractile tension after agonist pretreatment were observed.In vivo results showed that ATE could significantly increase the small intestinal charcoal transit,reduce the amount of residual feces in colon,and increase its water content.In addition,ATE could effectively relieve colonic pathological damage caused by loperamide as well,and increase the contents of 5-HT and AchE in serum,indicating that ATE may play a laxative role by regulating the content of neurotransmitters.In vitro results showed that ATE had a certain influence on the spontaneous contractile tension and amplitude of duodenum,and could significantly inhibit the contractile tension induced by a single concentration of external agonists KCl and Ach,and gradually recovered to near the basal tension value.At the same time,after pretreatment with ATE or blockers,it was found that the inhibitory effect of ATE on Ach and CaCl2 was similar to that of atropine,an M receptor blocker,and verapamil,a Ca2+ channel blocker,by accumulatively increasing the concentrations of external agonists,respectively.We infered that the mechanisms of action of ATE on intestinal diastolic activity is related to antagonizing the binding of Ach to M receptor and inhibiting Ca2+influx.(2)By using UHPLC-Q-TOF-MS/MS and corresponding data analysis strategies,the chemical components of the ATE and the intestinal contents were qualitatively analyzed.First of all,the best analytical and detection conditions were established to obtain high quality mass spectrometry data.Secondly,based on the analysis of the whole components of Aster tataricus in the previous stage,the Agilent Mass Hunter Qualitative Analysis B.07.00 software was used to rapidly identify the chemical components in the ATE and the components entering the intestinal tract in this study.Finally,the presence of the compound was confirmed based on the parent ion and fragment ion information in the primary and secondary mass spectrometry data,combined with reference substance and the characteristic fragment ions information aided identification,32 chemical constituents were identified in the ATE,including 14 peptides,8 organic acids,4 flavonoids,3 terpenoids and 3 coumarins,and there were 28 chemical constituents in the ATE that got into the small intestine.(3)Molecular docking technology was used to screen the active ingredients in Aster tataricus.Molecular docking,as a powerful tool for screening drug targets and pharmacodynamic components,has been widely used in the research and development of drugs in the pharmaceutical field.In this study,SYBYL-X 2.0 software was firstly used to calculate the Total Score values of 32 compounds binding to M2 receptor,M3 receptor and PKC protein,and 10 common active ingredients with Total Score values greater than 5 were screened out,respectively Astin J,Asterin A,Asterin F,Chlorogenic acid,3,4-Dicaffeoylquinic acid,3,5Dicaffeoylquinic acid,4,5-Dicaffeoylquinic acid,Kaempferol,Quercetin,Luteolin;Then,compounds with a Total Score values greater than 5 were selected to be plotted by SYBYL-X 2.0 software to obtain the three-dimensional binding pattern diagram of receptor-ligand and hydrogen bond interaction diagram formed between ligand and amino acid residues.Finally,Discovery Studio 4.0 software was used for related drawing and analysis,and all the interaction figures of the formation of two-dimensional ligands and amino acid residues could be obtained.(4)Based on the previous studies on the moistening intestines of Aster tataricus and the practical application of the university-level innovation and entrepreneurship project"moistening intestines tea of Aster tataricus",this study further investigated the curative effect of Aster tataricus combined with Radish seed in the treatment of constipation,in order to provide a certain theoretical basis for the development of Aster tataricus as a health care product.According to the test method of defecation function in the standard of health care products,Aster tataricus combined with Radish seed was used to treat mice with constipation caused by loperamide,and its effect on intestinal charcoal transit and fecal excretion of mice were observed in this study.The experimental results showed that the water extract of Aster tataricusRadish seed could significantly promote the small intestinal charcoal transit rate,increase the amount of defecation and the fecal water content of mice.Meanwhile,it also could effectively improve the abnormality of colon tissue caused by loperamide.To sum up,this study comprehensively elucidated the efficacy of Aster tataricus for defecation through in vivo and in vitro experiments,and clarified the mechanisms of action of Aster tataricus in treating constipation by regulating the content of neurotransmitters,blocking the binding of Ach and M receptor,and antagonizing Ca2+influx.Ten ingredients,such as Astin J,Asterin A,Asterin F and Chlorogenic acid,were screened out by UHPLC-Q-TOF-MS/MS and molecular docking technology as the effective ingredients for the laxative effect of Aster tataricus.At the same time,this study found that Aster tataricus combined with Radish seed have a significant laxative effect.This study can not only provide scientific basis for the development of Aster tataricus as a health care product,but also provide ideas for the research on the pharmacological substance basis of other Chinese herbs,and actively explore the development of the modernization of Chinese herbal compound preparation,which has high scientific and application value. |
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