Interactions Between Kudingcha Dicaffeoylquinic Acids And Intestinal Microbiota

Kudingcha is mainly made from the plants in genus of Aquifoliaceae Ilex,I.kudingcha C J.Tseng and 1.latifolia Thunb.It is an important alternative tea beverage in China,and it is of diverse biological activities with a long history of consumption.Kudingcha is rich in saponins,polysaccharides and polyphenols,and the main components of polyphenol are caffeoylquinc acids(CQAs).Kudingcha CQAs consist of mono-caffeoylquinc acids(mono-CQAs)and dicaffeoylquinc acids(diCQAs).Compared to mono-CQAs,there are fewer studies about diCQAs,and Kudingcha possesses a high mount of diCQAs.Therefore,Kudingcha diCQAs were selected as the subject in the present study.Dietary polyphenols have reputations of antioxidant,anti-inflammatory,lipid-lowering and anti-obesity,and catch much attention of researches.Due to their chemical properties such as high molecular weight and multi-hydroxyl groups,however,polyphenols have low bioavailability.Dietary polyphenols persist in upper digestive tract,and most of them reach the colon in their intact forms,whereas they interact with the considerable intestinal microorganisms inhabiting there.The intestinal microflora is of enormous metabolizing potential,and it is also closely associated with the health of host.The bilateral effects would happen between the dietary phenolics and the intestinal microbiota.The polyphenols could affect the composition and metabolic pathways of the microflora,while the intestinal microbiota would convert the phenolics to the metabolites and improve their bioavailability.Conclusively,dietary polyphenols may exert their bioactivities through the microbiota.Ilex Kudingcha wass utilized as the raw material in the subject,and three diCQA compounds were isolated and purified.On the basis of characterization of their conversions in the digestive tract,the interactions between Kudingcha diCQAs and intestinal microbiota were investigated.The study has important significance in demonstrating the biological activities and their function mechanisms of Kudingcha diCQAs.The main research contents and results are presented as follows:(1)Isolation and Purification of Three DiCQA Compounds from KudingchaIn this chapter,high-speed counter current chromatography(HSCCC)coupled with column chromatography was employed to purify Kudingcha diCQAs.Kudingcha was extracted with hot water,and the infusion was freeze dried to afford Kudingcha crude extract;the crude extract was introduced to HP-20 macroporous resin chromatography,and the 70%ethanol effluent fraction was collected as the Kudingcha diCQAs after the column eluted with water;3,4-diCQA and a mixture of 3,5-and 4,5-diCQAs were obtained by HSCCC with the solution system containing n-hexane-acetate ethyl-methanol-0.1%hydrochloric acid(HCl);3,5-and 4,5-diCQAs were acquired by Toyopearl HW-40S gel chromatography finally.The purities of the obtained 3,4-,3,5-and 4,5-diCQAs 94.8%,84.6%and 87.3%,respectively.(2)Simulated Digestions of Kudingcha DiCQAs in vitro In this chapter,simulated digestions were used to investigate the hydrolysis and conversions of Kudingcha diCQAs in oral cavity,stomach,small intestine and colon.In addition,model of Caco-2 cell line was utilized to study the anti-inflammatory effects of diCQAs and their intestinal metabolites.The results showed that Kudingcha diCQAs would persist in artificial saliva,gastric and pancreatic solutions,and they would not be hydrolyzed by the digestive enzymes or the brush border esterase of epithelial cell mimicked by Caco-2 cells;in anaerobic fermentation with fecal slurry,simulation of intestinal microbial metabolism,diCQAs could be hydrolyzed to mono-CQAs and free caffeic acid(CA),which could be further converted to(DHCA),and the degree of hydrolysis was associated with the sugar and microbial composition in the culture medium.The three Kudingcha diCQA compounds and their intestinal metabolites,mono-CQAs,CA and DHCA,could all inhibit the IL-8 level expressed by lipopolysaccharides(LPS)-induced Caco-2 cells.(3)Effects of Kudingcha DiCQAs on Intestinal Microbiota in vitroIn the present chapter,anaerobic fermentation model and high throughput sequencing technology were applied to investigate the effects of Kudingcha diCQAs on intestinal microflora in various carbohydrate conditions.The data exhibited that addition of diCQAs increased the diversity of the microbiota;diCQAs had more distinct effects on intestinal microbiota than different carbohydrates;diCQAs increased the relative abundance of the genera of Bacteroides,Escherichia/Shigella,Bifidobacterium,Parasutterella,Alistipes,Romboutsia,Oscillibacter,Veillonella,Butyricimonas,Phascolarctobacterium,Lachnospiracea incertae sedis,Gemmiger,Streptococcus,Clostridium sensu stricto and Haemophilus;the abundances of Ruminococcus,Anaerostipes,Dialister,Megasphaera,Megamonas and Prevotella were reduced;diCQAs also had effects on pH and production of short-chain fatty acids,and acetate and lactate contents were improved with reduced levels of propanoic and butyric acids.In general,Kudingcha diCQAs have modulating potential on intestinal microecology.(4)Primary Studies on an Intestine-Derived Kudingcha DiCQAs Hydrolyzing Bacterial Strain and EnzymeIn this chapter,a strain of bacteria with the ability to hydrolyze Kudingcha diCQAs was isolated from human feces,and it was characterized and named as Lactobacillus sp.M-B.Lactobacillus sp.M-B cultured on glucose,it could not catalyze diCQAs;Lactobacillus sp.M-B could hydrolyze 3,4-diCQA and slight amount of 4,5-diCQA,and it was hard to catalyze 3,5-diCQA.Lactobacillus sp.M-B was able to express a bacterial diCQAs-hydrolyzing endoenzyme,and the target enzyme was purified by ammonium sulfate precipitation,DEAE-Sepharose Fast Flow anion exchange and Sephadex G-100 molecular exclusion chromalography in turn.The diCQAs hydrolyzing enzyme was of a molecular weight of about 50 kDa.It could hydrolyze the caffeoyl group on different positions of diCQAs to generate corresponding mono-CQAs,and it could also catalyze mono-CQAs to the end product of CA.The optimal enzymic reaction conditions were 37℃ and pH 6.5.Magnesium ion(Mg2+)could activate the diCQAs-catalyzing enzyme,and it might be the coenzyme;manganese ion(Mn2+)and ethylenediaminetetraacetic acid(EDTA)could inactivate the enzyme activity,especially EDTA whose inhibition could reach 90%.(5)Modulating Effects of Kudingcha DiCQAs on Lipid Metabolism,Inflammation and Intestinal Microbiota in vivoIn this chapter,C57BL/6J mouse model was employed to investigate the effects of Kudingcha diCQAs on lipid metabolism,inflammation and gut microflora of high-fat diet fed mice.The data showed that Kudingcha diCQAs decreased the serum levels of total cholesterol(TC)and low density lipoprotein-cholesterol(LDL-C);hepatic expressions of synthesis and accumulation of fat related genes,including FAS,SREBP-1c,PPARγ and LXRa,at mRNA level were reduced,and transcriptions of lipid catabolism related genes(CPT-1 and PPARa)were increased indicating the anti-obesity effect of Kudingcha diCQAs;Serum cytokines and endotoxin LPS concentrations and transcript expressions of liver cytokines all were decreased indicating their anti-inflammatory activities.Kudingcha diCQAs could affect the intestinal microbial diversity and overall structure.Kudingcha diCQAs had no impact on the relative abundance of Firmicutes,Bacteroidetes or their ratio,but diCQAs could increase the abundance of genera of Akkermansia and Bifidobacterium in high-fat diet fed mice and decrease Bilophila and Olsenella levels.In addition,the shift in intestinal microecology was closely associated with host lipid metabolism.Conclusively,Kudingcha diCQAs could reduce host lipid accumulation and inflammation level via modulating intestinal microbial composition,especially the abundance of Akkermansia,but the accurate mechanism still need further investigation.