Study On The Purgative Mechanism Of Quercetin And The Extraction Of Quercetin In Pine Needles

Backgroud and objective:Slow transit constipation (STC) is the most common chronic constipation one is colonictransit intestinal motility dysfunction caused by weakened intestinal contents transmitted slow intractable constipation, mainly for the number of bowel movements, and it is intended to reduceweaken or even disappear, the majority of patients with abdominal distension, loss of appetite andother symptoms. At present, studies STC conditioning and intervention for these factors, but theresults are not satisfactory. So explore the pathogenesis of STC, which in turn taken due totreatment become a research hotspot.However, the pathogenesis is unclear STC. In recent years, studies on the mechanism of STChas a new direction. Studies have found that intestinal epithelial function plays an important rolein the process of STC occurrence and development. First, the normal intestinal epithelium is themost important part of the composition of the intestinal barrier function. Once the intestinal barrieris damaged, there will be bowel dysfunction. Secondly, the intestinal mucosal epithelium canregulate hormone secretion liquid, thus affecting the intestinal peristalsis. Meanwhile, STCaquaporin (AQPs) increased expression in colon mucosa increased intestinal absorption ofmoisture contents, the stool becomes hard, dry and difficult to discharge. In addition,Lactobacillus, Bifidobacterium and other competitively inhibit the intestinal mucosa of the gutbacteria, can secrete acid to provide70%of the energy of the intestinal epithelium, provide theimpetus for bowel function. Therefore, maintaining the integrity of the intestinal mucosa andprotect intestinal epithelial function for the treatment of STC have a negligible effect.The antioxidant, anti-inflammatory effects of drugs and natural products can effectivelyprevent the intestinal mucosa by free radicals, inflammatory mediators, cytokines damage,protect the integrity of the intestinal mucosa, thereby promoting recovery STC bowel function.Because of the need for prevention and treatment of STC long intervention, so the choice oftreatment on drugs tend to use a wider range of food safety of natural products. Therefore, toexplore a long-term effective and non-toxic side effects of natural products is important.Quercetin (Quercetin) is the main component of flavonoids, the wide variety of sources, hasantioxidant, anti-inflammatory and anti-infection and anti-tumor activity of a variety of biologicaleffects. Modern pharmacological tests showed quercetin on cardiovascular disease, diabetes,cancer and other chronic diseases have a positive role in prevention and treatment, while thestudy also confirmed that quercetin nontoxic to humans, non-teratogenic and carcinogenic,long-term food security high. So as to protect the intestinal mucosa quercetin, promote therecovery of bowel function, relieve chronic constipation transport raw materials.This study intends to investigate constipation laxative effect in mice with chronic transportproperties of quercetin and quercetin protect the intestinal mucosa research, mitigationmechanisms STC, as well as pine needles in the extraction and separation of quercetin for foodadjuvant treatment of constipation and the development of functional foods laxative provide atheoretical basis.Method:1Establish a mouse model of chronic transit constipation1.1Use of compound diphenoxylate tablets suspension fed mice to establish the model of chronictransit constipation1.2Evaluation of defecation in miceEach mouse was observed and recorded the first row of black stools time, within6h black stoolgrains and observe black stools and weight traits.1.3Production of mouse colon tissue biopsy, observed changes in colonic biopsy pathology inmice. 2Evaluation of quercetin purge function2.1observe,6h grains and weight within the bowel, intestinal contents of grains and weightEffect of quercetin on the first row of each group of mice melena time to evaluate the laxativeeffect of quercetin.2.2Determination of serum malondialdehyde (MDA) content.2.3Production of mouse colon tissue biopsy, colon pathology observed in each group of micesliced change.Effect of quercetin on intestinal floraDetermination of E. coli, Enterococcus, Lactobacillus and Bifidobacterium content coloniccontents and faeces of mice3.1plate count method.3.2Determination of lactic acid in serum.3.3Determination of the pH of the contents of the colon of mice3.4Determination of murine colonic TNF-α, IL-6expression, EGF mRNA of the4needles in optimizing the extraction and separation of quercetin4.1Optimization pine extract rutinUsing response surface method of central composite design to optimize the extraction processparameters rutin pine needles, using aluminum nitrate-extract content of liquid sodium nitritecolorimetric detection of rutin.4.2Preparation of crude quercetinThe extraction process according to the optimized, rutin extract was obtained, then concentratedto no alcohol taste, adjust the pH of concentrated hydrochloric acid was added, the crystalsprecipitated home freezer overnight, centrifuged, the supernatant. The precipitate is placed in adrying oven at60℃about12hours, the crude quercetin.4.3Preparation of quercetin fineTake crude quercetin, plus2%H2SO4solution is heated to a boil direct fire, bright yellowprecipitate precipitation. Cooled, centrifuged, the supernatant, the precipitate washed with waterto neutral. Precipitation drying, fine quercetin.4.4Qualitative Analysis4.4.1ferric chloride test for detection of phenolic compounds in the samples.4.4.2hydrochloride-flavonoids magnesium assay samples.4.4.3Molish carbohydrate reaction assay samples.4.5High Performance Liquid Chromatography (HPLC) analysis of the quantitativeUsing the HPLC method for coarse and fine Quercetin Quercetin two rutin and quercetin samplesfor qualitative and quantitative analysis.4.5methodological studyThrough repeated experiments, the experimental precision, stability experiments testingequipment, samples and experimental methods.Result:Establish a mouse model of chronic transit constipation1.1Evaluation of defecation in miceDiphenoxylate can significantly prolong the first row of black stools available, reducing theoverall weight and defecation in mice6h grains (P <0.05), in line with the characteristics of STC.1.3Production of mouse colon tissue biopsy, observed changes in colonic biopsy pathology inmice. STC villi can lead to colon of mice reduced wall thinning, glandular lumen narrowing, glandular atrophy.Evaluation of quercetin purge function2.1quercetin can significantly shorten the first row of black stools with mice, the increase in thetotal stool weight and grains (P <0.05) Description quercetin have a laxative effect in mice6h.2.2quercetin can significantly reduce serum malondialdehyde (MDA) levels (P <0.05).2.3Each group of mice colon tissue pathology, pine needles more normal dose colon and colonvilli, digestive gland cells and compared with model group increased.Effect of quercetin on intestinal flora3.1Comparison with constipation group,100mg/kg and400mg/kg fed quercetin can significantlyincrease the number of colonies of mouse intestinal lactobacilli (P <0.05), can significantly reducethe number of colonies (P <0.05) enterococci.3.2Effect of quercetin on intestinal flora.Gavage100-400mg/kg quercetin can significantly increase serum levels of lactic acid (P<0.05).3.3mouse colon contents PH value of each measurementEach dose group could significantly reduce the pH of the colon contents (P <0.05).3.4Determination of murine colonic TNF-α, IL-6expression, EGF mRNA of theEach dose group can significantly reduce the expression of the colon of mRNA of TNF-α (P<0.05), could significantly reduce the dosage of1,3,4colon mRNA expression of IL-6(P <0.05),4dose groups the expression of mRNA can significantly improve the EGF (P <0.05).4needles in optimizing the extraction and separation of quercetin4.1Optimization pine extract rutinRutin pine needles get optimum extraction conditions were: ethanol concentration of30%,solid-liquid ratio of1:50(g: ml), extraction temperature is65℃, extraction time was120min.Pine needle extract rutin forecast rate is10.28%.4.2Qualitative Analysis4.2.1ferric chloride test for detection of phenolic compounds in the samples.After the extract rutin, quercetin and refined crude quercetin ethanol solution of ferricchloride solution was added, the solution becomes blue and green can be observed, indicatingthat the three caught contain phenolic compounds.4.2.2hydrochloride-flavonoids magnesium assay samples.After the extract rutin, quercetin and refined crude quercetin magnesium and concentratedhydrochloric acid was added, compared to the blank tube, three were caught showing orange-redto purple. Showed three caught contain flavonoids.4.2.3Molish carbohydrate reaction assay samples.Between the liquid extract and rutin concentrated sulfuric acid is formed perinone, thereaction is positive, carbohydrate-containing materials. Coarse and fine Quercetin Quercetinsolution was not observed between liquid purple ring, the reaction was negative, does not containcarbohydrates.4.3High Performance Liquid Chromatography (HPLC) analysis of qualitative and quantitative4.3.1Qualitative AnalysisRutin standard retention time2.65-3.05min, quercetin standard retention time7.42-7.82min.Crude rutin and quercetin solution containing quercetin, quercetin fine solution containingquercetin, rutin free. 4.3.2Quantitative AnalysisCrude solution containing quercetin and rutin concentration of1.48×10-3mg/mL andquercetin1.94×10-3, finishing quercetin solution containing quercetin3.96×10-3, after acidhydrolysis, quercetin the concentration increased.4.4methodological studyThrough repeated experiments RSD0.35%,1.42%RSD precision experiments, the stability ofthe experimental RSD was0.86%.Conclusion:1Quercetin has a laxative effect intestines, relieve chronic transit constipation symptoms.2Quercetin can activate beneficial to reduce harmful bacteria, reduce cytokines on intestinaldamage, protect the intestinal mucosa.3Pine needles are rich in quercetin, rutin extraction rate increases, then the acid solution, can bemore quercetin.