Studies On The Bioactive Components And Action Mechanism Of Jujubae Fructus Reinforcing Spleen And Stomach Effects

This dissertation included four chapters.Chapter one,Literature reviewA detailed review was performed to the main active ingredients and pharmacological activity of Jujubae Fructus.The research progress of pathological characteristics and animal model construction of TCM spleen deficiency syndrome were also summarized.The aim of this review was to provide ideas and reference for studies on the action mechanism of Jujubae Fructus reinforcing spleen and stomach effects,and to provide basis for the utilization and development of Jujubae Fructus.Chapter two,Study on the action mechanism of Jujubae Fructus reinforcing spleen and stomach effects based on intestinal microbiota and metabolomics interrelationSection 1,Study on the action mechanism of Jujubae Fructus reinforcing spleen and stomach effects based on the spleen deficiency animal model caused by bitter-cold drug1.Spleen deficiency rat model was established by giving Sennae Folium extract intragastrically.During the time of model establishment,animals in treatment groups were orally administered with Jujubae Fructus.After 10 days of administration,the blood,urine,spleen and small intestine tissues of rats were collected for peripheral blood routine determination,biochemical indicators measurement and pathological analysis to evaluate the protective effect of Jujubae Fructus on spleen deficiency rats caused by Sennae Folium.The results showed that the weight increasing rate,serum amylase,D-xylose and gastrin levels were significantly decreased,and the spleen index,serum cholinesterase and somatostatin levels were significantly increased when the model had been established.The peripheral blood routine indicators of model group were significantly changed,and the small intestine injury were also found.After the administration of Jujubae Fructus,the indicators were all improved.This indicated that Jujubae Fructus might be effective in reinforcing spleen and stomach by regulating the digestive system,immune system and energy metabolism2.The metabolic changes associated with spleen deficiency and the intervention of Jujubae Fructus on spleen deficiency rats were analyzed based on metabolomics.31 potential endogenous metabolites associated with spleen deficiency were identified(22 in plasmas,9 in urine).After oral administration of Jujubae Fructus,27 endogenous markers turned back to normal level at some extent,which were involved in retinol metabolism,pentose and glucuronate interconversions,nicotinate and nicotinamide metabolism,ether lipid metabolism,selenoamino acid metabolism.The results showed that Jujubae Fructus could improve spleen deficiency rats,and revealed the possible action mechanism of Jujubae Fructus reinforcing spleen and stomach effect from metabolomics aspect3.The 16S rDNA sequencing method was used to study the structure and diversity of intestinal bacteria in rats with spleen deficiency caused by Sennae Folium,and to explore the regulation of Jujubae Fructus on the intestinal bacteria of rats with spleen deficiency.The result of LEfSe analysis showed that intestinal flora of spleen deficiency rats had changed from phylum units to the genus units when compared with the control group.31 differential genera were found.After oral administration of Jujubae Fructus,the levels of Allobaculum,Turicibacter,Corynebacterium,Proteus,Aerococcus,Enterococcus,Epulopiscium,Morganella,Collinsella,Brevibacillus were significantly decreased.The results indicated that Jujubae Fructus might achieve therapeutic effect by improving the disorder of intestinal flora in spleen deficiency rats4.To comprehensively analyze the relations among plasma metabolites(31 identified potential biomarkers),and gut microbiota(the differential flora at phylum and genus levels),a correlation matrix was generated by calculating the Pearson’s correlation coefficient.And the interaction diagram between intestinal microbiota and host metabolites under pathological conditions of spleen deficiency was constructed.The results showed that Aerococcus was positively correlated with niacinamide,17a-Hydroxypregnenolone,and vitamin A.Jujubae Fructus can significantly decrease the relative abundance of Aerococcus.It is speculated that the increasement of Aerococcus was caused by diarrhea of spleen deficiency,and then the increasement promoted the pathological process of spleen deficiency by affecting the retinol metabolism,nicotinate and nicotinamide metabolism.Aerococcus may be a candidate genus for predicting and treating spleen deficiency and related metabolic disorders.Section 2,Study on the bioactive components and action mechanism of Jujubae Fructus reinforcing spleen and stomach effects based on the inflammatory bowel disease animal model1.Inflammatory bowel disease rat model was established by feeding 5%dextran sulfate sodium.After orally administered of Jujubae Fructus and its chemical components(triterpenic acid,oligosaccharide,polysaccharide),the DAI value,contents of inflammation-related factors IL-6,IL-10 and TNF-a in colon tissue were significantly decreased.And the peripheral blood routine indicators of model group were significantly improved.Jujubae Fructus and its chemical components also reduced colon intestine injury.The intervetion effects of each administration group on indicators related to inflammatory bowel disease were not completely consistent.This indicated that the different chemical components of Jujubae Fructus had different regulation effects on rats with inflammatory bowel disease.It is speculated that Jujubae Fructus improved inflammatory bowel disease by the combined action of its chemical components.2.The metabolic changes associated with inflammatory bowel disease and the intervention of Jujubae Fructus and its chemical components were analyzed based on metabolomics.43 potential endogenous metabolites associated with inflammatory bowel disease were identified(13 in plasmas,30 in urine),which were involved in nicotinate and nicotinamide metabolism,pantothenate and CoA biosynthesis,arachidonic acid metabolism,pentose and glucuronate interconversions and et al.After oral administration of Jujubae Fructus and its chemical components,some endogenous metabolites turned back to normal level at some extent.However,the action trends of each chemical component were not completely consistent,and the high dose of Jujubae Fructus regulated the largest number of endogenous metabolites.Arachidonic acid metabolism pathway was verified by western blotting.It was found that Jujubae Fructus and its chemical components could significantly inhibit the expression of PLA2,COX2,5-LOX proteins in the pathway.The results showed that Jujubae Fructus could improve inflammatory bowel disease rats,and revealed the possible action mechanism of Jujubae Fructus reinforcing spleen and stomach effect from metabolomics aspect.It is speculated that the bioactive components of Jujubae Fructus improving inflammatory bowel disease were the combined action of its chemical components.3.The 16S rDNA sequencing method was used to study the effect of Jujubae Fructus and its chemical components on the diversity of intestinal bacteria in inflammatory bowel disease.The study found that the overall structure of intestinal flora in the cecal part of inflammatory bowel disease rats didn’t change significantly,and the disorder begain to appear in the colon part.27 differential genera were both found from colonic contents and fecal,with about 50%overlapping.After oral administration of Jujubae Fructus and its chemical components,the results showed that the high-dose Jujubae Fructus and the triterpenic acid component had a good regulation effect on the intestinal flora of the colon part.Jujubae Fructus and its chemical components could improve the structure of unbalanced fecal flora at different extent.4.To comprehensively analyze the relations among plasma metabolites(43 identified potential biomarkers),and gut microbiota(the differential flora at phylum and genus levels),a correlation matrix was generated by calculating the Pearson’s correlation coefficient.And the interaction diagram between intestinal microbiota and host metabolites under pathological conditions of inflammatory bowel disease was constructed.The results showed that Rikenella was positively correlated with 4,6-dihydroxyquinoline,urocanic acid,and nicotinic acid mononucleotide.It is speculated that the decrease of Rikenella was caused by inflammatory bowel disease,and then the decrease promoted the pathological process of inflammatory bowel disease by affecting the intestinal barrier function,histidine metabolism,nicotinate and nicotinamide metabolism.Rikenella may be a candidate genus for predicting and treating inflammatory bowel disease and related metabolic disordersChapter three,Detoxification mechanism of Jujubae Fructus and Crotonis Semen Pulveratum based on the gastrointestinal system in mice1.Based on normal mice,the effect of Jujubae Fructus(JF)on the gastrointestinal toxicity and diuretic effect of Crotonis Semen Pulveratum(CT)was investigated by urine output measurement,biochemical indicators determination,and small intestinal tissue pathological analysis.The results showed that JF slowed down the rapid diuretic effect of CT,and significantly increased serum interleukin-2(IL-2),interleukin-6(IL-6),gastrin(GAS),somatostatin(SS).JF also reduced small intestine injury caused by CT.The results suggest that JF exhibited a tendency to reduce the toxicity of CT in the aspects of serum immune index,intestinal movement,and intestinal damage.In addition,the JF could also slow down the rapid diuretic effect of CT,behaving a tendency to reduce the clinical effect of CT.2.Based on normal mice,the 16S rDNA sequencing method was used to study the changes of intestinal bacteria when CT used alone and combined with JF.The results showed that JF improved the disorder of intestinal flora caused by CT.Low dose CT combined with JF significantly decreased the relative abundance of Sphingomonas and Oscillospira.The level of Bilophila was decreased after the combined application of high dose CT and JF.The results suggest that JF exhibited a tendency to reduce the toxicity of CT in the aspect of intestinal microflora structure.Chapter four,Pharmacokinetic of multiple triterpenic acids from Jujubae Fructus extract in vivo1.An ultra-performance liquid chromatography coupled with mass spectrometry(UPLC-MS/MS)method was established and applied for the simultaneous determination of seven triterpenic acids in normal and immunosuppressed rat plasma samples.The established method was validated and found to be specific,accurate,and precise for the seven triterpenic acids.The results showed that the AUCo-t and AUCo-∞of epiceanothic acid in immunosuppressed rats were significantly higher than those in normal rats,and the CLz/F of epiceanothic acid in immunosuppressed rats were significantly decreased.In contrast,the AUC0-t,AUC0～∞,and Cmax of alphitolic acid in model rats showed relatively lower values compared to those in normal rats.CLz/F of alphitolic acid was markedly higher in model rats than in normal rats.This research might provide an insight for the clinical usage of triterpenic acids from Jujubae Fructus.2.An established UPLC-MS/MS method was applied for the simultaneous quantitation of seven triterpenic acids in plasma samples of normal and acute liver injury rats induced by CCl4The results showed that there were significant differences(p<0.05)in the pharmacokinetic parameters of seven triterpenic acids between model and normal groups.The AUCo-t and AUCo-∞ of epiceanothic acid and pomonic acid in model rats were significantly higher than those in normal rats,and the CLz/F of them were significantly decreased.In contrast,the above parameters for alphitolic acid,betulinic acid and betulonic acid exhibited the quite different trend.This pharmacokinetic research might provide useful information for the clinical usage of triterpenic acids from Jujubae Fructus.