Study On Antioxidant Effect Of Total Flavonoids In Ziziphi Spinosae Folium Based On Intestinal Flora

Rationales: Ziziphi Spinosae Folium(LZJS),the leaf of Ziziphus jujuba Mill.Var.spinosa(Bunge)Hu ex H.F.Chou(ZJS),which are rich in resources and are wasted due to the lack of effective utilization.In Compendium of Materia Medica,an ancient Chinese pharmacopeia,LZJS has been used as a traditional medicine to heal sores and relieve pain.The pharmacological evaluation has shown that LZJS has potential hypnotic,antifungal,and hepatoprotective activities Moreover,a variety of nutrients,including carbohydrates,minerals,vitamins,and amino acids,are available in the LZJS.The study on the effects of sleep improvement and anti-oxidation on Ziziphi Spinosae Folium is one of the hot spots in the field of research.Our previous study found that the total flavonoids content in LZJS could be as high as 8.04%,and the extraction and purification process was optimized to obtain the total flavonoids content in LZJS(purity 41%,yield 10%).Characterization of total flavonoids showed that the flavonoids in LZJS were mostly flavonols(quercetin,Kaempferol)as aglycone and had high antioxidant activity.LZJS products are widely available in drinks and tea.They are usually taken orally,and LZJS flavonoids(LZJSF)are metabolized by human intestinal bacteria.However,the conjugation to glycoside has limited the absorption of the flavonoid glycosides within the small intestine.And the flavonoid glycoside is hydrolyzed by intestinal bacteria to release the aglycone which can then be absorbed in the colon.Therefore,this study proposed “Does flavonols of LZJS produce antioxidant active products under the metabolic transformation of human intestinal flora? Does it have antioxidant effect in vivo? What is the mechanism? ” Research hypothesis.Establish a rapid,accurate and efficient method for the determination of components in LZJSF.The chemical composition and antioxidant activity of LZJSF before and after intestinal bacterial transformation were compared to clarify the antioxidant activity products of LZJSF.Exploring the antioxidant mechanism of LZJSF through animal models is necessary to fully develop and utilize LZJSF resources.Objective:The chemical composition and antioxidant activity of LZJSF were compared before and after the transformation of intestinal bacteria in order to clarify the antioxidant activity products of LZJSF,With D-gal induced oxidative damage in rats as the research object,the multi-pathway and multi-target action mechanism of LZJSF for improving D-gal induced oxidative damage in rats was illustrated from the perspectives of classical pharmacodynamics,metabolomics and intestinal flora.Methods:1.A method of simultaneous determination of 6 components in LZJSF by LC-MS/MS based on MRM-IDA-EPI mode was established.2.This study evaluated the chemical constituents and compared the antioxidant activity between LZJSF and fermented LZJSF in the human intestinal bacteria.Furthermore,the metabolic processes of the LZJSF,as well as the changes in the main metabolites during fermentation at different time points,were determined.Moreover,three chemical methods and PC12 cells in vitro were used to evaluate the antioxidant capacity of LZJSF and FLZJSF.3.Rat model of oxidative injury was established by intramuscular injection of D-gal.Morris water maze was used to evaluate the effects of LZJSF on learning and memory of D-gal-induced oxidative injury in rats.Furthermore,the effects of LZJSF on antioxidant indexes such as Malondialdehyde in serum and brain tissue of D-gal induced oxidative injury were also investigated.1H-NMR metabolomics technology combined with multivariate statistical method was used to analyze the change rules and metabolic pathways of endogenous metabolites in serum of D-gal-induced oxidative damage rats,and the regulatory effects of LZJSF on endogenous small molecules and metabolic pathways were investigated.Finally,the effects of LZJSF on D-gal-induced oxidative damage to intestinal flora in rats were investigated from the perspective of intestinal flora.Results:1.Built a solution based on the pattern of MRM-IDA-EPI and determination of 6 kinds of ingredients in LZJSF,the method of the detection sensitivity is high,on the basis of accurate quantitative MRM,further joint EPI scans under the condition of low concentration generates secondary ion full scan,qualitative quantitative analysis at the same time,to prevent false positive peak interference judgment.The average contents of quercetin-3-O-Rob,quercetin-3-O-Rut、quercetin-3-O-β-D-Gal,quercetin-3-O-β-D-Glu,quercetin-3-Rha,quercetin were 10.4,8.3,0.1,0.3,0.2,0.00 5%,respectively.2.The in vitro faecal incubation system was successfully established.The composition determination before and after transformation showed that the glycosides of LZJSF were hydrolyzed to glycosides by intestinal bacteria,and the glycosides were then hydrolyzed to small phenolic acids.The content of the glycoside compounds quercetin-3-O-Rut,quercetin-3-O-Rob,quercetin-3-O-β-D-Glu,quercetin-3-O-β-D-Gal,quercetin-3-Rha,kaempferol-3-O-Rut in samples transformed for 0 h,1 h and 2 h were high,the content of glycogen compounds Quercetin,Taxifolin and Kaempferol in 4 and 6 h after transformation was high,The converted 24 h and 48 h samples mainly contained 3,4-dihydroxyphenylacetic acid,3-hydroxyphenylacetic acid,4-hydroxyphenylacetic acid,m-hydroxyphenylpropionic acid,3-phenylpropionic acid,4-hydroxybenzoic acid,p-hydroxyphenylpropanoic acid.The chemical activity determination of the samples before and after transformation showed that the scavenging capacity of DPPH· and O2-was 4,6 h>0,1,2 h>24,48,The same trend was also found in H2O2 induced PC12 cell damage,and the content of MDA and ROS was 24 h>0 h>4 and 6 h.3.The intervention effect of LZJSF on D-gal rats was lower than that of Control rats in terms of activity distance,activity time,number of times across the original platform and swimming speed,indicating the success of the model.The behavioral,serum and brain biochemical indexes of LZJSF were compared,and the results showed that high dose of LZJSF could effectively eliminate the excess free radicals in the body,repair the oxidative stress damage of the organism,and improve the learning and memory of the oxidative damaged rats.(2)in the serum metabolome study of D-gal rats by LZJSF,the VIP>1 value of the OPLS-DA model and the P value of t-test were used to find that 12 metabolites in the D-gal group were significantly changed,while 11 metabolites,such as Valine,Isoleucine,Leucine and Acetic acid,were callback in the high-dose group after administration.Met PA results show that high-dose LZJSF mainly plays a role by regulating the metabolism of taurine and taurine,the metabolism of glycine serine and threonine,and the metabolic pathway of pyruvate.(3)Effects of LZJSF on intestinal flora of D-gal rats.Alpha diversity analysis found that low and high doses of LZJSF restored Alpha diversity in the bacterial flora,LZJSF through change the abundance of bacteria in the levels of Phylum(Firmicutes,Bacteroidetes,Firmicutes/Bacteroidetes),family(Lactobacillus,Bacteroidales-S24-7)and genus(Unclassified S24-7,Lctobacillus)played a regulatory role in the intestinal flora of oxidized rats.(4)The Pearson correlation analysis found that the differential metabolites associated with oxidative damage were positively or negatively correlated with the regulation of intestinal flora.Conclusion: In this paper,a method for the determination of 6 chemical components based on MRM-IDA-EPI mode was established.The method has good repeatability and high accuracy,and can be used for the determination and quality control of 6 chemical components in total flavonoids of Ziziphi Spinosae Folium.Eighteen prototypic components and seven metabolites of total flavones of Ziziphi Spinosae Folium were identified after intestinal flora transformation,and the transformation law of glycoside compounds with quercetin and kaempferol as parent nucleus was clarified.4 h and 6 h were the key time points for the metabolic transformation of total flavonoids,and the content of glycosides decreased significantly while the glycosides increased significantly.The antioxidant activity was strongest at both chemical and cellular levels.Total flavonoids in Ziziphi Spinosae Folium could improve the memory damage and oxidative stress induced by D-galactose in rats.Total flavonoids in Ziziphi Spinosae Folium play an antioxidant role by regulating(1)the metabolism of taurine and taurine(2)the metabolism of glycine,serine and threonine(3)the metabolism of pyruvate.The total flavones in Ziziphi Spinosae Folium can regulate the intestinal flora of oxidation-damaged rats by affecting the abundance of bacteria at the levels of phylum,family and genus.Differential metabolites associated with oxidative damage were positively or negatively correlated with the regulation of intestinal flora.