Ameliorative Effect Of Small Peptides And Phenolics From Common Bean(Phaseolus Vulga L.) Milk And Yogurt On Inflammation And The Underlying Mechanisms

Inflammatory response is the common pathogenesis shared by the high fatal chronic diseases such as cardiovascular disease,diabetes and cancer.Intestine suffers high inflammatory risk due to the long-term exposure to the exogenous agents.The hazardous substances,once ingested into guts,can cause oxidative stress response as well as the subsequent nonspecific immune responses which could lead to the intestinal barrier damage,the increasing permeability of gut,and even invasion of the harmful substances to the gut tissues,thus,induced chronic intestinal inflammation.Phenolics and small peptides derived from food play a role in the prevention of intestinal inflammation.Common beans are known as a healthy food since its high richness in proteins and phenolic compounds and desirable in vitro antioxidant activity.The worldwide production of common beans is high,however,the utilization of common beans is limited because of its singular products in the market and hardto-cook property especially in the developed countries.In this study,two varieties of common beans(Navy bean and light red kidney bean)were developed into bean milk and yogurts based on their features of high richness in proteins and phenolic compounds.Gradually screening of the bioactive compounds was performed with in vitro digestion,super-filters,ion exchange column and antioxidant and anti-inflammatory trials on cell model.Phenolics and peptides were verified as the antioxidant and anti-inflammatory compounds in the common bean milk and yogurts.A co-culture cell model consisted of intestinal epithelial cells(Caco-2)and vascular endothelial cells(EA.hy926)was established.The transepithelial ability to cross the epithelial cell monolayer,and the antioxidant and anti-inflammatory effect of the phenolics and peptides in common bean milk and yogurts were measured in the Caco-2 mono-culture and Caco-2/EA.hy926 co-culture cell models.This study firstly identified three small anti-inflammatory peptides that could be transported across the intestinal epithelial cell monolayer and quantitated the transport ratio of them.Besides,a high-fat diet/LPS co-stimulation mice model was also established to study the protective effect of the small MW GI digestates of lightred kidney bean milk and yogurt on gut and systemic inflammation.The main findings in this study are listed below:1.Antioxidant and anti-inflammatory compounds were identified as peptides and phenolics in common bean milk and yogurts.After verified that the small MW fractions of common bean milk and yogurt GI digestates showed significantly stronger antioxidant and anti-inflammatory effect compared to the large MW fractions,the small MW fractions were isolated to phenolic fractions and peptide fractions by ion exchange columns.11 phenolic compounds were identified in the phenolic fractions which were mainly feruloyl ester derivatives.In addition,three peptides e.g.?-glutamyl-S-methylcysteine,?-glutamyl-leucine and Xle-Val-Xle were identified in the peptide fractions.Fermentation enhanced both phenolic and peptide contents in yogurt samples compared with their corresponding milk samples.As a result,yogurt samples showed higher cellular antioxidant activity and anti-inflammatory activity than the milk samples.2.Common bean milk and yogurts derived peptides showed anti-inflammatory effect in Caco-2 cells.The common bean milk and yogurts derived peptides significantly inhibited the gene expression of TNF-?,IL-1?,IL-8,and IL-6 induced by TNF-? in Caco-2 cells.The synthesis standards of three peptides ?-glutamyl-Smethylcysteine,?-glutamyl-leucine and Leu-Leu-Val also showed anti-inflammatory effect by inhibiting IL-8 secretion induced by TNF-? in Caco-2 cells.Standards of the three peptides showed similar anti-inflammatory effects at the high-(4 m M)and middoses(2 mM),while ?-glutamyl-S-methylcysteine showed weaker effect at the low dose(1 mM)compared with the other two peptides.NPS-2143,an agonist to the CaSR pathway,blocked the anti-inflammatory effect of the common bean milk and yogurts derived peptides significantly(P<0.05)which suggested that the antiinflammatory effect of the small peptides in Caco-2 cells may depend on CaSR pathway,meanwhile,the two ?-glutamyl peptides can be served as potential chelating agents to CaSR.3.Common bean milk and yogurts derived peptides were able to be transported to the basolateral side of the Caco-2 monolayer.In the Transwell model,three peptides in common bean milk and yogurts were able to be transported across theepithelial cell monolayer.Leu-Leu-Val showed the highest transport ratio among the three peptides,while ?-glutamyl-S-methylcysteine showed the highest concentration in the basolateral side because of its high amount in the peptide samples.By using the specific competitive agonist,Gly-Sar,to block the function of transporter PepT1,transportation of Leu-Leu-Val was completely being blocked while this blocking effect on the transportation of the two ?-glutamyl peptides was not significant(P>0.05).These results suggested that transportation of Leu-Leu-Val completely depends on PepT1,however,there may be other mechanisms of the transportation of the two ?-glutamyl peptides.4.Common bean milk and yogurts derived peptides showed anti-inflammatory effect in C2BBe1/EA.hy926 co-culture cell model.The transepithelial peptides of common bean milk and yogurts significantly inhibited the secretion of IL-8 and the gene expression of ICAM-1,VCAM-1,IL-8,IL-1? and IL-6 induced by TNF-? in EA.hy926 cells significantly(P<0.05).Western blot analysis indicated that common bean small peptides exert anti-inflammatory functions by blocking NF-?B and MAPK signal pathways.The anti-inflammatory effect of common bean derived peptides was significantly compromised by Gly-Sar,which acted as the competitive agonist of PepT1(P<0.05).This phenomenon suggested that the anti-inflammatory effect of bean peptides was dependent on the intestinal uptake.5.Common bean milk and yogurts derived phenolics could be metabolized and transported by C2BBe1 cells.Common bean derived phenolics were first hydrolyzed to free ferulic acid by esterase secreted by the intestinal epithelial cells,and then transported to the basolateral side by the form of free aglycone.The feruloyl ester derivatives were completely metabolized in the apical side after 24 h.Therefore,common bean derived phenolics were suggested to be rapidly metabolized and transported by the C2BBe1 cell monolayer.6.Common bean milk and yogurts derived phenolics could protect cells from inflammation responses induced by oxidative stress.In Caco-2 cells,the common bean derived phenolics significantly inhibit the secretion of IL-8 induced by t-BOOH.In C2BBe1/EA.hy926 co-culture cell model,after being metabolized and transferred,the transepithelial phenolics were able to inhibit p38 MAPK signal pathway in theEA.hy926 cells,thus suppressed gene expressions of the pro-inflammatory mediators e.g.ICAM-1,VCAM-1,IL-8,TNF-?,IL-6 and COX-2 after 8-h-stimulation with oxLDL.After 24 h oxLDL stimulation,common bean derived phenolics could still inhibit the secretion of IL-8 significantly among the four groups(P<0.05).7.GI digestates(< 10 kDa)of common bean milk and yogurts had an ameliorating effect on obesity and insulin resistance in mice.High-fat diet(NC)and high fat diet/LPS co-stimulation(PC)caused an increase in body weight gain rate,liver weight,white adipose tissue weight and brown adipose tissue weight,moreover,an elevation in triglyceride,cholesterol and low-density lipoprotein in plasma.Lowgrade insulin-resistance was also observed in these groups.In addition,hypertrophy of adipocytes,macrophage-infiltration and lower adiponectin synthesis were observed in white adipose tissues of NC and PC mice.Whereas no significant differences were observed in common bean derived milk and yogurt on the amelioration of all these metabolic disturbances mentioned above in co-stimulated mice(P>0.05).These effects may due to the anti-inflammatory effect of peptides and phenolics in common bean milk and yogurts.8.GI digestates(< 10 kDa)of common bean milk and yogurts showed protective effects on enteritis and systemic inflammation.High fat diet(NC)and high fat diet/LPS co-stimulation(PC)significantly increased the intestinal permeability,as well as endotoxin,MCP-1 and TNF-? concentrations in plasma.In addition,gene expressions of the proteins related to the integrity of intestinal barrier e.g.JAM-A,Ocld,Cld1,ZO-1,Muc2,and IgA in colon were decreased in NC and PC mice,meanwhile,three microbial sensing gene TLR4,NOD2 and Reg3 g were increased.MCP-1,TNF-?,IL-1?,IL-17 A and IL-6 which are referred as pro-inflammatory mediators were increased in NC and PC mice,while the anti-inflammatory cytokine IL-10 was decreased.There was no significant difference between NC and PC in most parameters,nevertheless,PC tend to show severer inflammation degree than NC.Common bean derived milk and yogurts attenuate inflammation in all these parameters.