Research On The Mechanism Of Metabolic Transport And Immunomodulation Of Different Dried Thinned Peach Polyphenols

Thinned peach is a part of young fruits artificially removed in the 4th to 5th weeks after peach blossom,in order to obtain high-quality peach fruit and sustained high yield.Thinned peach is a plant resource with high added value that can be utilized,which contains more diverse phytochemicals compared with its ripe fruit,especially polyphenols.There are approximately1.25 million tons of thinned young peach in China every year.Generally,drying treatment is a necessary procedure before processing and utilization of thinned peach,to prevent putrefaction during transportation and storage and improve the extraction efficiency of active components.However,different drying methods inevitably change the polyphenol composition and further affect its biological activity.In this study,five treatments of freeze drying（FD）,natural drying（ND）,hot air drying at 50°C（HAD50）,70°C（HAD70）and 90°C（HAD90）were adopted for thinned peach to study their polyphenol compositions,antioxidant activities,bioassessibility,metabolic transport and immunomodulatory mechanisms.The specific research results were as follows:（1）The physicochemical properties,polyphenols components and in vitro antioxidant activities of different varieties of thinned peach and nectarine were comprehensively analyzed.Compared to the ripe fruits,the soluble solids content（SSC）and p H values of the thinned peach and nectarine were significantly lower,while the firmness and titratable acid（TA）were higher（p<0.05）.Glucose and fructose accounted for 74.04%～81.37%of total soluble sugars in the thinned fruits,while sucrose accounted for 59.57%～77.88%of total soluble sugars in the ripe fruits.Malic acid,quinic acid,aspartic acid and glutamic acid were the main organic acids and amino acids in the thinned fruits,which were higher than those in the ripe fruits.The total polyphenols content（TPC）,total flavonoids content（TFC）and antioxidant activities of the thinned fruits were 8.07,6.16 and 3.95～11.95 times higher than those of the ripe peach and nectarine,respectively.The main polyphenols in thinned peach and nectarine were neochlorogenic acid,catechin and chlorogenic acid,accounting for 88.80%～95.45%of the total polyphenols,which is more than 10%higher than that in ripe fruits（70.18%～84.56%）.In addition,multivariate analysis results indicated that TPC,TFC,neochlorogenic acid,catechin,chlorogenic acid,3-p-coumaroylquinic acid,1-caffeoylquinic acid,kaempferol-3-o-rutinoside,myricetin,and sucrose were the characteristic variables and important markers of thinned fruits.Therefore,polyphenols in thinned peach were the key research object of this subject.（2）The compositions,contents and in vitro antioxidant activities of polyphenols in the thinned peach treated with five drying methods（FD,ND,HAD50,HAD70 and HAD90）were studied,and the interaction of three main polyphenols（neochlorogenic acid,chlorogenic acid and catechin）in the process of enzymatic oxidation was analyzed.The results showed that the polyphenols in thinned peach would undergo different degrees of oxidative changes,including isomerization,oxidation polymerization and degradation reactions.There was a competitive oxidative inhibitory effect between caffeoylquinic acids（chlorogenic acid and neochlorogenic acid）and catechin during the drying process of thinned peach.In the antioxidant activities of thinned peach,the contribution of catechin was higher than neochlorogenic acid and chlorogenic acid,and were significantly affected by polyphenol oxidation.Moreover,the antioxidant activities of neochlorogenic acid,catechin and chlorogenic acid were higher than their enzymatic oxidized products.Overall,different drying treatments significantly changed the compositions,contents and antioxidant activities of thinned peach polyphenols.（3）The digestive stability,bioaccessibility and metabolic properties of the different dried thinned peach polyphenols were studied using in vitro simulated gastrointestinal digestion and Caco-2 cells models.The results indicated that different drying treatments markedly affected the stability and bioaccessibility of thinned peach polyphenols in simulated gastrointestinal digestion and metabolic transport in Caco-2 cells.Thereinto,the HAD50 thinned peach exhibited the highest bioaccessibility of 91.19%and well absorption behavour(P_{app AP-BL}=13.40×10^-6 cm/s and P_{app BL-AP}=13.24×10^-6 cm/s)in Caco-2 cells.Furthermore,the transepithelial transport of the dried thinned peach polyphenols was affected by P-glycoprotein（P-gp）-mediated efflux and organic anion transporting polypeptide（OATP）-mediated influx effects,there was a synergistic effect of neochlorogenic acid,chlorogenic acid and catechin in transepithelial transport.In addition,12 metabolites were detected and characterized in Caco-2cells after the polyphenols were absorbed by cells.Overall,compared with FD and ND,HAD50thinned peach polyphenols exhibited higher bioaccessibility and transport property during in vitro digestion and metabolic transport.（4）The potential immunomodulatory activities and mechanisms of different dried thinned peach polyphenols on macrophages RAW264.7 were explored.The results indicated that the dried thinned peach polyphenols could exert potential immunomodulatory effects through the NF-κB and Nrf2 signaling pathways in RAW264.7 cells.Specifically,the thinned peach polyphenols effectively altered the nuclear translocation of NF-κB and Nrf2,as well as the downstream cytokines interleukin-1β（IL-1β）,interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）and interferon-γ（IFN-γ）production,superoxide dismutase（SOD）activity and reactive oxygen species（ROS）levels,exerted potential immunomodulatory activity.Thereinto,the effects of ND and HAD in NF-κB pathway were weaker than that of FD,and the HAD50thinned peach polyphenols could activate more effectively Nrf2 nuclear translocation than FD.In addition,50μg/mL of thinned peach polyphenols enhanced cell signaling pathways associated with stress response,while 800μg/mL restrained the development of inflammation in macrophages.These results indicated that different drying treatments notably impacted the immune responses of thinned peach in RAW264.7 macrophages,the differences are associated with the drying process and the incubation concentration.