The Antioxidant Activity Of Pomegranate Peel Polyphenols And Its Enzymatic Hydrolysates And Their Protective Effects On Oxidatively Stressed Mice

Polyphenols are a class of ubiquitous secondary metabolites in plants.They attracted a lot of attention from researchers because of their potent antioxidant activities.Pomegranate is a kind of fruit rich in polyphenols,of which the polyphenols are mostly in the unedible peel.In our country pomegranate production is huge and the pomegranate peel polyphenols are abundant in resource.The antioxidant ability of pomegranate peel polyphenols is high,and its antioxidant ability was reported to be the highest among about 1,000 plants.Therefore,it is promising for pomegranate peel polyphenols in the application in animals as antioxidants.However,there exists a lot of high molecular ellagitatins in pomegranate peel polyphenols,which could not be absorbed by animal and therefore restrictes its in vivo antioxidant activity.Moreover,the high molecular ellagitatins can also precipitate protein and metals,which affects the nutrients absorption.Aimed at the in vivo antioxidant activity problem of pomegranate peel polyphenols,this paper first investigated the in vitro and in vivo antioxidant activities of the representative compounds in pomegranate peel polyphenols,and then establish the optimum enzymolysis conditions of pomegranate peel polyphenols with tannase.Taking the pomegranate peel polyphenols and the enzymolysis pomegranate peel polyphenols as study objects,this paper then analyzed their chemical compositions,in vitro antioxidant and protein precipitation abilities,in vivo metabolites and antioxidant abilities,and finally study their oxidative injury repairing effects in oxidatively stressed animal model.1 Assessment of in vitro and in vivo antioxidant activities of three representative polyphenols with different molecular weights in pomegranate peelThe aim of this test was to evaluate punicalagin(PG)—the major compound in pomegranate peel polyphenols,and its two hydrolysates in different hydrolysis degrees—punicalin(PL)and ellagic acid(EA)for their antioxidant activity in vitro and oxidative injury repair effects in vivo.Taken water soluble vitamin E,Trolox,as a reference agent,we tested the DPPH· sacavenging activity,superoxide anion(O2·-)scavenging activity,Ferric reducing antioxidant power(FRAP),and lipid peroxidation inhibition(LPO)of the two kinds of polyphenols.Results showed that the trends of in vitro antioxidant activities were EA>PG>PL>Trolox in scavenging DPPH·,PG>PL>EA?Trolox in scavenging O2·-,EA>PG?PL>Trolox in FRAP,and Trolox>PG>EA>PL in LPO inhibition.EA treatment increased the ADG,T-AOC in plasma,liver and intestine,SOD activity in liver and intestine,GSH-Px activity in intestine,and intestinal villus height to crypt depth ratio(P<0.05),and decreased the MDA content in plasma,liver and intestine,and the mRNA expressions of pro-inflammatory factor TNF-a,IL-6 and IFN-y(P<0.05).PL increased the intestinal SOD activity and GSH-Px activity(P<0.05),and decreased the intestinal MDA content,mRNA expressions of TNF-a and IL-6(P<0.05).PG increased the intestinal SOD activity and GSH-Px activity(P<0.05),and decreased the intestinal MDA content and IL-6 mRNA expressions(P<0.05).Conclution:EA,PL and PG all had powerful in vitro antioxidant capacities,and they had different antioxidant advantages in acting against different kinds of radicals;EA was more effective than PL and PG in protecting against oxidative injury in vivo.This indicated that the degradation and conversion of pomegranate peel polyphenols could help to improve its in vivo antioxidant acitiviy.2 Optimization of the enzymolysis and purification of pomegranate peel polyphenolsThe aim of this test was to reduce the content of high molecular polyphenols such as PG,increase the content of small molecular polyphenols such as EA by tannase hydrolyzation,and establish the optimum conditions of hydrolyzation and purification of the polyphenols.Using high performance liquid chromatography(HPLC)as the detection method,EA increasing rate and PG degradation rate as enzymolysis degree indices,we conducted the enzymolysis test on pomegranate peel polyphenols with tannase.Using the single factor design and orthogonal combination design,we optimized the enzymolysis indices,and established the macroporous resin purification parameters of enzymolysis products by using the methods of static adsorption and dynamic adsorption.Results show that the optimum reaction conditions for the enzymolysis were pH5.2,45 ?,enzyme substrate ratio 30 mL/g,16 h.Static adsorption and adsorption experiments showed that D101 macroporous resin could absorb the maximum polyphenol,the best elution was 95%ethanol,the best loading volume was 300 mL,and the best elution volume was 120 mL.Under the optimum condition of macroporous resin purification,the primary pomegranate peel polyphenols(PP)was obtained,the yield of which was 71.39%,the total polyphenol content of which was 81.50%,the PG content of which was 43.64%,and the EA content of which was 4.85%.Under the optimal enzymatic hydrolysis condition and the optimal macroporous resin purification condition,the enzymolysis pomegranate peel polyphenols(EP)was obtained,the yield of which was 54.09%,the total polyphenol content of which was 80.40%,the PG content of which was 0%,and the EA content of which was 45.73%.3 Analysis of the compositon compounds and the physicochemical property of pomegranate peel polyphenols and its enzymatic hydrolysatesTaking the pomegranate peel polyphenols(PP)and the enzymolysis pomegranate peel polyphenols(EP)as study objects,this part analyzed their chemical compositions using liquid chromatography-mass spectrometry technology(LC-MS/MS)as the testing means,and then investigated their in vitro antioxidant,protein and metal ions precipitation abilities.Results showed that seven compounds in PP were identified,which were(1)T=15.31,m/z=1083.0618,punicalgin a;(2)T=15.88,m/z=783.0700,pedunculagin;(3)T=17.38,m/z=1083.0614,punicalagin b;(4)T=20.92,m/z=757.0900);(5)T=22.27,m/z=633.0737,corilagin;(6)T=30.41,m/z=300.9996,ellagic acid;(7)T=36.60,m/z=401.0879,cetraric acid.Four compounds in EP were identified,which were:(1)T=23.14,m/z(291.0152),brevifolin carboxylic acid;(2)T=25.68,m/z=600.9895,gallagic acid;(3)T=26.20,m/z=247.0258,brevifolin;(4)T=30.33,m/z=300.9995,ellagic acid.In vitro antioxidant assays showed that the DPPH· scavenging ability,O2·-scavenging ability and FRAP of PP were better than that of Trolox,while LPO inhibition ability was lower than that of Trolox.For EP,its DPPH· scavenging ability was close to that of Trolox,O2·-scavenging ability was lower than Trolox at low concentrations,but higher than Trolox at high concentrations,FRAP was between that of PP and Trolox,and LPO ability was lower than Trolox.Under the condition of pH2 and pH7,the precipitation to bovine serum albumin,Cu2+,and Zn2+ of EP were lower than that of PP.In conclution,after the enzymatic degradation,the types and molecular weight of pomegranate peel polyphenols reduced,and their in vitro antioxidant ability reduced to the similar level with commonly used antioxidant Trolox.The protein and metal ion precipitation ability were reduced as well.4 The identification of the serum metabolites of pomegranate peel polyphenols and its enzymatic hydrolysates in mice and their effects on redox status in normal miceThe in vivo metabolites of the primary pomegranate peel polyphenols(PP)and enzymolysis pomegranate peel polyphenols(EP)were identified with LC-MS/MS.Then the effects of PP and EP on redox status in normal mice and the possible mechanism were investigated.Results showed that,four phenolic metabolites were detected in the serum of mice orally taken PP or EP 400 mg/kg bw after 0.5h,which were urolitins A,4-vinylphenyl hydrogen sulfate,and two unknown compouds.Among that,4-vinylphenyl hydrogen sulfate was specific in EP metabolites,and urolitins A was specific in PP metabolites.EP consumption decreased the protein carbonyl carbon level in serum(P<0.05),and increased the GSH level in liver(P<0.05),while PP consumption only decreased the GSH level in liver(P<0.05).Both PP and EP significantly increased the expression of antioxidant genes Keap1,Nfe212,Hmox1,Nqo1,and Gclc in liver(P<0.05),and that in EP group were significantly higher than that in PP group.Other parameters were not significantly affected(P>0.05).In conclution,EP consumption increased the metabolites in the blood of normal mice,decreased the oxidation level,raised the GSH content,and upregulated the mRNA expressens of genes in Keapl-Nrf2 genetic pathway in a larger extent as compared with the PP.5 The oxidative injury repair effects of pomegranate peel polyphenols and its enzymatic hydrolysates in oxidatively stressed miceThe oxidative injury repairing effects of the primary pomegranate peel polyphenols(PP)and enzymolysis pomegranate peel polyphenols(EP)in oxidized fish oil(OFO)induced oxidatively stressed animal model was studied.Results showed that:compared with fresh fish oil(FFO)group,in OFO group the average daily gain(ADG)was decreased(P<0.05),peroxidized products(MDA and NO)in serum increased(P<0.05),activities of duodenal trypsin and lipase were significantly reduced(P<0.05),the morphological of jejunum villi was seriously destroyed,anti-oxidation pathway gene(Keapl,Nfe212,Gclc,and Hmox1)expression in jejunum was significantly increased(P<0.05),a variety of damage repair factor(Cldnl,Ocln,Oggl,Tfam,and Tjpl)gene expression were decreased significantly(P<0.05).Compared with OFO group,10 mg/kg bw doses of PP and EP significantly improved ADG to the same level with FFO group(P<0.05),significantly increased the jejunum antioxidant pathways genes(Nfe212,Nqol,Gclc and Hmoxl)expression,and increased the gene expression of damage repair factors(Cldnl,Ocln and Tfam)(P<0.05);Serum nitric oxide(NO)in 50 mg/kg bw dose of OFO+PP and 30 and 50 mg/kg bw dose of OFO+ EP were significantly lower than OFO group(P<0.05).The serum MDA content of 30 mg/kg bw dose of OFO+EP was decreased(P>0.05).The duodenal amylase activity of 50 mg/kg bw dose of OFO+ PP was decreased significantly(P<0.05).Other parameters did not show significant difference(P>0.05).The jejunum epithelial lymphocyte positive rate in 10 and 50 mg/kg bw doses of OFO+PP,and 10,30,50 mg/kg bw doses of OFO+EP were significantly lower than OFO group(P<0.05).The jejunum epithelial goblet cells positive rate in 50 mg/kg bw dose of OFO+ EP was significant increased(P<0.05).In conclution,EP consumption reduced the MDA and NO content in serum,specifically improved the jejunal secretion function,and did not affect the activity of duodenal digestive enzymes.PP and EP up regulated the gene expressions of antioxidant pathways and damage repair factors in vivo in different degrees,and the regulation effects of EP group were higher than that of PP group.To sum up,this study investigated the antioxidant activity in vitro and in vivo of three representative polyphenol compounds in pomegranate peel,and raised the viewpoint that small molecular polyphenols may exhibit more active antioxidant activity than the large molecular polypheonls.Base on this viewpoint,we hydrolyzed the pomegranate peel polyphenols by tannase,optimized the hydrolyzation and purification conditions,and obtained the small molecular enzymatic hydrolysate,of which ellagic acid was the main component.This enzymatic hydrolysate had lower anti-nutrient action,higher antioxidant activity in vivo,and higher intestinal injury repairing activity compared to the original polyphenols.This study provided new ideas for the exploration of new feed additives with anti-oxidative stress effects.