Study On The Factors Affecting Bioavailability Of Avenanthramides And Construction Of Nano Co-encapsulated Particle

Avenanthramides are the most important polyphenols in oats,which have strong antioxidant,anti-inflammatory and anti-itching activities.However,the bioavailability of avenanthramides is very low,which limits its application in the field of health food to a great extent.Therefore,it is of great significance to clarify the reason and mechanism of low bioavailability of avenanthramides and to establish appropriate methods to improve their bioavailability.This paper starts with the establishment of in vitro digestion/Caco-2 cell model,comprehensively analyzes the factors and mechanisms that affect the bioavailability of avenanthramides by exploring the metabolic mechanism of avenanthramides in the process of digestion and absorption,and puts forward a new delivery strategy:constructing a co-entrapment system based on nanoparticles and designed synergies.The results of this research will not only improve the bioavailability of avenanthramides,but also provide innovative ideas and scientific methods for improving the bioavailability of other polyphenols.Analysis of avenanthramides and phenolic acid content of Chinese oat varieties and their bioavailability.The results showed that N-?3',4'-dihydroxy?-?E?-cinnamoyl-5-hydroxyanthranilic acid?2c?,N-?4'-hydroxy?-?E?-cinnamoyl-5-hydroxyanthranilic acid?2p?and N-?4'-hydroxy-3'-methoxy?-?E?-cinnamoyl-5-hydroxyanthranilic acid?2f?were the main avenanthramides in 9 oat varieties collected by the author,and there were significant differences?p<0.05?in avenanthramide contents?0-146.94?g/g DW?and phenolic acid content?35.65-143.52?g/g DW?in different oat varieties,among which Longyan 3 had the highest avenanthramide contents?146.94?g/g DW?,and Zhaotong had the highest phenolic acid content?143.52?g/g DW?.The content of avenanthramides in oat bran was significantly higher than that in corresponding oat grains?p<0.05?.Then the antioxidant activities of 9 oat varieties and 4 oat brans were determined by chemical?ORAC?and cell model?CAA?respectively,and the correlation analysis was also carried out.The results showed that Bayan 1 had the highest ORAC values?21.18?mol TE/g DW?,Zhaotong showed the strongest cellular antioxidant activity?33.38?mol QE/100g DW?,and the correlation coefficient?R²?between avenanthramide contents and CAA values?R²=0.20?was much lower than that between phenolic acid contents and CAA values?R²=0.74?.At the same time,there was a great correlation between avenanthramide contents and ORAC values?R²=0.81?,but there was no correlation between phenolic acid contents and ORAC values?R²=0.17?.The bioavailability of avenanthramides and phenolic acids was studied by in vitro digestion/Caco-2 cell model.the results showed that the bioaccessibility of 2c,2f and 2p was 16.14%,61.99%and 74.45%,respectively,and the bioaccessibility of phenolic acids was between 24.72%and 88.57%.After being absorbed by Caco-2 cell,all phenolic compounds except gallic acid were detected on the basolateral?BL?side.The intestinal uptake rates and bioavailability of avenanthramides?2c,2f and 2p?were?0.93%-3.10%?and?0.15%-2.31%?,respectively,which were much lower than intestinal uptake rates?7.42%-27.68%?and bioavailability?5.43%-22.01%?of phenolic acids.In addition,before absorption,the ORAC values of avenanthramides 2c,2f and 2p?4.17-7.02?mol TE/?mol?were significantly higher than those of ferulic acid?2.46?mol TE/?mol?and caffeic acid?3.21?mol TE/?mol?,but the antioxidant activity of the sample solution on the BL side after absorption was opposite to that before absorption?p<0.05?.Study on the factors and mechanism of limiting the bioavailability of avenanthramides.The stability and metabolic mechanism of avenanthramides during digestion and absorption were further revealed:avenanthramide 2c,2f and 2p were stable in gastric acid?pH2?,but decreased due to the combination of pepsin and trypsin during digestion.Moreover,bile salts had no significant effect on the contents of avenanthramides?p<0.05?.In addition,2f and 2p were stable in small intestinal pH environment?pH 7.5,no enzyme,no bile salt?.However,2c may undergo non-enzymatic oxidation and dehydrogenation dimerization in the small intestinal pH environment,forming quinones and homodimers?MW:628?,resulting in a loss rate of more than 80%in intestinal digestion.In the absorption process,monoamine oxidase?MAO?in phase I metabolic enzymes is involved in the oxidation of 2c,2f,2p;Carboxylesterase?CES?is involved in the hydrolysis of 2c and 2f;The results of quantitative analysis showed that phase I reaction was the main metabolic pathway of avenanthramides in the absorption process.In addition,2c,2f and 2p were affected by P-gp and MRP2 efflux in Caco-2 absorption model.Effect of food substrate on bioavailability of avenanthramides.The effects of skimmed milk powder,milk protein and different types of starch on the bioavailability of avenanthramides were studied.The results showed that after mixing with whey protein,casein and skim milk powder,the bioaccessibility of avenanthramide 2c was increased by 3.6 times,1.5 times and 1.2 times,respectively,the uptake rates of avenanthramide 2c was increased by1.4 times,2.4 times and 1.8 times,respectively,and the bioavailability of avenanthramide 2c was increased by 4.1 times,2.7 times and 1.7 times,respectively.Moreover,the bioavailability of avenanthramide 2f and 2p decreased significantly when mixed with casein and skimmed milk powder?p<0.05?.In addition,after mixing with ungelatinized and gelatinized amylose,amylopectin and common starch,the bioaccessibility of avenanthramide 2c increased slightly?1.4-1.9 times?,but the uptake rates decreased in different degrees.Finally,the bioavailability of avenanthramide 2c did not increase significantly?p<0.05?,while the bioaccessibility,intestinal absorptivity and bioavailability of avenanthramide 2f and 2p decreased significantly?p<0.05?.The effect of flavonoids on the absorption rate of avenanthramides was further studied.The results showed that apigenin and naringin had no significant effect on the apparent permeability coefficient(P_apppp value)of avenanthramide 2f and 2p?p<0.05?,but the P_app value of avenanthramide 2c increased from 0.65×10^-66 cm/s to 2.03×10^-6 cm/s and 1.78×10^-6 cm/s,respectively.When quercetin was mixed with avenanthramides,the P_app values of 2c,2f and 2p were significantly higher than those of the control group?p<0.05?.Particularly,the intestinal absorption rate of avenanthramide 2c increased by 4.3 times after mixing with quercetin,and the addition of quercetin inhibited the methylation of avenanthramides 2c during absorption.Construction of avenanthramide 2c nano co-encapsulated particles and bioavailability analysis.Firstly,the optimum preparation method of nano co-encapsulated particles were studied:quercetin and avenanthramide 2c were dissolved at pH 12 and pH 7,respectively during the acidification process?p H 12-pH 6?of sodium caseinate and whey protein solution.The encapsulation efficiency and storage stability of quercetin and avenanthramide 2c in sodium caseinate were significantly higher than those in whey protein?p<0.05?.Moreover,the encapsulation efficiency of quercetin and avenanthramide 2c in sodium caseinate was 94.3%and 80.6%,respectively,when the concentration of quercetin was 0.25 mg/mL and avenanthramide 2c was 0.5 mg/mL.The nanoparticles remained stable during 21 days of storage.Furthermore,the formation mechanism of sodium caseinate-quercetin-avenanthramide2c nanoparticles was further revealed:the deprotonation of quercetin made it negatively charged to become solubilized at pH12,while sodium caseinate disintegrated and quercetin combined with sodium caseinate statically.When the pH was decreased to 7,the loose protein micelles were repolymerized into particles,and the protonation of quercetin was wrapped in the protein particles.Furthermore,avenanthramide 2c combined with sodium caseinate statically.When the solution pH is adjusted from 7 to 6,avenanthramide 2c was embedded in the protein particles,and further reduces the particle size.Finally,nano co-encapsulated particles increased the bioaccessibility and uptake rates of avenanthramide 2c by 2.2 times and6.1 times,respectively,and finally increased the bioavailability of avenanthramide 2c by 13times.