Research On The Material Basis And Mechanism For Anti-ulcerative Colitis Of Polygonum Divaricatum L.

[Research background and purpose]Ulcerative colitis(UC)is one of the most common and difficult to cure digestive diseases,and it was defined as one of the world's refractory diseases by the World Health Organization.Clinically,glucocorticoids,immunosuppressive agents,non-steroidal drugs and other western medicines are mainly used to treat the disease.However,due to the long-term application of the above-mentioned western medicines,there are serious side effects such as body weight gain,osteoporosis and hypertension.So far,there is still lack of a safe,effective and preventable drug.In recent years,traditional Chinese medicine has attracted more and more attention in the treatment of UC because of its unique advantages of safety,low toxicity and effectiveness,and has shown broad application prospects.Polygonum divaricatum L.is a herb of Polygonum.It is used as a dry whole herb.It has the functions of warming the kidney,eliminating the accumulation of food,clearing away heat and stopping bleeding,and stopping diarrhea.It treats intestinal tingling and has less frequent stools,stool with pus,mixed with mucus,tenesmus,diarrhea,stool thin yellow green,etc.in North China,Northeast folk and clinical P.divaricatum L.is often used to treat enteritis,diarrhea,dysentery and other diseases.In order to develop a new effective drug from traditional Chinese medicine for the treatment of UC,this article takes the whole grass of P.divaricatum L.which has a therapeutic effect in the folk as the research object,and intends to carry out research from the following four aspects:?.Establishment of a rat model of ulcerative colitis(UC);?.P.divaricatum L.Pharmacodynamic study of anti-UC;?.P.divaricatum L.Study on the chemical constituents of the effective parts of anti-UC;?.P.divaricatum L.Screening and molecular mechanism of in vitro anti-inflammatory activity of chemical components of anti-UC effective parts;It is expected that the above-mentioned research will clarify the material basis and mechanism of action of P.divaricatum L.against ulcerative colitis,and provide new theories and new developments for the development of multi-component and multi-target anti-UC drugs with clear,well-defined and effective curative effects.Ideas.[Research methods and results] 1 Establishment of a rat model of ulcerative colitis(UC)A rat model of ulcerative colitis was induced by a mixture of 2,4,6-trinitrobenzenesulfonic acid(TNBS)/ethanol.Different doses of TNBS were mixed with different concentrations and different doses of ethanol to establish the model.Observed and scored the normal living conditions,disease activity index,gross morphology of colonic mucosa and colon histopathology in normal control group and model group A,B,C and D rats.Thus,a reasonable TNBS dose,ethanol concentration and dose were determined to establish a UC model.The experimental results showed that the model of UC rats was successfully induced by 50 mg/kg TNBS and 0.25 ml of 50% ethanol mixture,and the mortality of rats was controlled to ensure the stability and Repeatability of the experiment.2 Pharmacodynamic study of P.divaricatum L.on anti-ulcerative colitis(1)Study on the pharmacodynamic effects of total extract of P.divaricatum L.to anti-ulcerative colitis.To study the therapeutic effect of total extracts of P.divaricatum L.on UC model rats,and to clarify its mechanism of action.The results showed that the total extract of P.divaricatum L.had a certain therapeutic effect on TNBS/ethanol-induced UC model rats,and its therapeutic effect was positively correlated with the dose administered.With the increase of the dose,the therapeutic effect was more significant..At the same time,P.divaricatum L.has increased the activity of SOD in colon tissue of rats with UC,reduced the content of MDA and the activity of MPO,revealing that it inhibits the occurrence of cellular lipid peroxidation and reduces the probability of infiltration by inflammatory cells.Ultimately improve inflammation of the colon.(2)Study on the effective parts of P.divaricatum L.anti-UC model rats.P.divaricatum L.coarse powder was extracted by water,45% ethanol,95% ethanol,and the extract was concentrated under reduced pressure to obtain extract.The extract was degreased with water and suspended in petroleum ether,and then washed with a gradient of pure water,35% ethanol-water,70% ethanol-water and 95% ethanol-water on AB-8 macroporous adsorption resin to obtain a water elution site,35% ethanol elution site,70% ethanol elution site and 95% ethanol elution site.The efficacy of the anti-UC model rats was studied on the four different concentrations of ethanol eluting sites.The experimental results show that the low and high dose groups of 35% ethanol elution site and the low and high dose groups of water can improve the general living conditions of UC model rats,reduce DAI,CMDI,HS scores,the MDA content and MPO activity in colon tissue were decreased,SOD activity in colon tissue was increased,and the gene expression of TLR4 and NF-kBp65 in colon tissues was effectively inhibited,and the contents of NO,IL-1?and TNF-? in serum of UC rats were significantly down-regulated.Among them,the 35% ethanol elution site dose group showed the best effect and better anti-inflammatory effect.3 Study on the chemical constituents of the active part of anti-UC of P.divaricatum L.31 compounds were isolated and purified from the effective parts of P.divaricatum L.anti-UC model by using modern separation analysis techniques such as polyamide column chromatography,reversed phase silica gel(ODS-C18)column chromatography and preparative high performance liquid chromatography.The compound was identified by modern spectral analysis techniques such as UV,IR,NMR and MS to obtain 25 monomeric compounds,mainly 10 flavonoids: quercetin(1),quercitrin(2),hyperin(3),rutin(4),kaempferol(5),isoquercitrin(6),myricetin(7),avicularin(8),astragalin(9),iIsorhamnetin(10);12 phenolic acids: epicatechin(11),epicatechin gallate(12),epigallocatechin gallate(13),catechin(14);ethyl gallate(15),resveratrol(16),gallic acid(17),protocatechuic acid(18),ferulic acid(19),succinic acid(20),p-hydroxybenzoic acid(21),caffeic acid(22);daucosterol(23);piceid(24);syringin(25).The 1-25 compounds identified above were all obtained from P.divaricatum L.for the first time.4 Screening and molecular mechanism of anti-inflammatory activity of the chemical constituents of the active part of anti-UC of P.divaricatum L in vitro.In vitro inflammatory model was established by lipopolysaccharide(LPS)stimulation of RAW264.7 macrophages,and 25 monomeric compounds isolated from the effective fraction of P.divaricatum L.anti-UC were screened for in vitro anti-inflammatory activity.ELISA was used to detect the contents of IL-1? and TNF-? in cell supernatants by four active components of compound 7 myricetin,compound 12 epicatechin gallate,compound 15 gallic acid ethyl ester and compound 24 resveratrol.The effect of 4 active ingredients on the expression of iNOS,TLR4 and p-NF-?Bp65 protein in cells was detected by Western blot.The experimental results showed that 10 anti-inflammatory active ingredients were selected from the effective parts,which were 4 flavonoids,5 phenolic acids and 1 stilbene compound.The four active ingredients can reduce the levels of inflammatory factors IL-1? and TNF-? in the cell supernatant in a dose-dependent manner,inhibiting the expression of iNOS,TLR4 and p-NF-kBp65 signaling in cells,and exert anti-inflammatory effects.[Conclusion] 1.Elucidation of the material basis of P.divaricatum L against UC: P.divaricatum L total extract has a significant therapeutic effect on UC model rats,and its 35% ethanol elution site is an effective site for anti-UC,from the effective site.25 monomer compounds were isolated and identified,and 4 flavonoids,5 polyphenols and 1 stilbene were selected as active ingredients for the treatment of UC,which laid a solid foundation for finally elucidating the material basis of P.divaricatum L against UC.basis.2.Preliminary analysis of the mechanism of action of P.divaricatum L against UC: P.divaricatum L anti-UC mainly inhibits the activation of NF-kB signaling pathway by inhibiting the expression of TLR4/iNOS gene or protein,thereby down-regulating the release of related inflammatory factor NO?IL-1? and TNF-?,ultimately achieve an anti-inflammatory effect.