The Form And Distribution Of Polyphenols In Walnuts And Their Correlationship With Antioxidant Activities

This study focused on forms,distributions and antioxidant activities of phenolics in different walnut（kernel and skin）cultivars based on the analysis method for determination of existence forms of phenolics.More components and key antioxidant phenolics were further explored in walnut.The results are as follows:Through the optimization of the extraction and determination methods of polyphenols,the separation and quantification of three forms of phenolics（soluble free phenolics,soluble esterified phenolics and insoluble bound phenolics）were achieved.Free phenolics were the most important form（51.1%-68.1%）.Phenolic acids（including hydroxybenzoic acid and hydroxycinnamic acid）accounted for 80%of the total phenolic compounds,followed by flavanols（6.4-12.5%）>flavonols（1.3-4.8%）>flavones（0.2-1%）.Ellagic acid was the most important phenolic compounds with mean content of 119.71μg/g.In terms of solvent efficiency,acetone was better at extracting soluble phenolics（free and esterified phenolics）,while methanol is better at extracting insoluble bound phenolics.For different types of phenolics,methanol had higher extraction efficiency for hydroxybenzoic acids,hydroxycinnamic acids and flavonols in free phenolics,while acetone had higher extraction efficiency for hydroxybenzoic acids,hydroxycinnamic acids and flavanols in esterified phenolics.The differences of hydroxybenzoic acids and flavanols in bound forms were 57.6%and 82.4%,respectively,while there were no significant difference with other types of phenolics.Based on the analyzed methods of three forms of phenolics,the distribution and content of phenolics in kernel and skin of 12 walnut cultivars were determined.The contents of free,esterified and bound phenolics were 9.24-23.86 mg/g,2.31-8.40 mg/g and 1.70-3.46 mg/g,respectively;the contents of freel,esterified and bound phenolics were 120.24-257.92 mg/g,39.02-82.48 mg/g and 21.15-44.85 mg/g,respectively.The content of free phenolics（67.10%in kernel and 66.92%in skin）was highest,followed by esterified and bound phenolics.Phenolic acids and flavanols are the most important types of phenolics with contents of over80%.Ellagic acid,gallic acid and catechins were the main phenolic compounds in kernel and skin.Especially,the content of ellagic acid reached 45.65%（kernel）and 51.56%（skin）,respectively.In addition,kaempferol,quercetin-7-O-β-D-glucoside and dihydroquercetin were found only in the free phenolics in kernel,while p-coumaric acid was found only in the bound phenolics in skin.According to the results of phenolic contents in 12 walnut cultivars,single factor analysis of variance showed that there were significant differences with the phenolic contents of kernel and skin among different cultivars（p<0.05）.The average coefficient of variation was 16.97%for kernel and 16.97%for skin.For different types of phenolics,the main differences among different cultivars in kernel were phenolic acids,flavanols and flavonols（p<0.05）,and the coefficients of variation of phenolic acids（bound）and flavanols（esterified）were up to 125.74%and 55.91%,respectively.The main differences among different cultivars in skin were phenolic acids,flavanols,flavonols and flavones（p<0.05）.The coefficients of variation of flavonols（bound）and flavones（free）were up to 72.34%and 109.49%,respectively.There were 28 and25 kinds of detected phenolic compounds in the kernel and skin,respectively.There were significant differences among cultivars with most phenolic compounds,especially for phenolic acids.The biggest difference was up to 185.60%in kernel and 159.15%in skin,respectively.DPPH free radical scavenging experiment in vitro was employed to determine antioxidant activity of phenolic extracts from 12 walnut cultivars.The results showed that the DPPH free radical scavenging ability of free phenolics in kernel and skin(IC₅₀:13.90-21.60μg/m L;6.28-8.10μg/m L)was significantly stronger than those of esterified phenolics(IC_50:27.97-41.88μg/m L;19.49-29.94μg/m L)and bound phenolics(IC₅₀:58.14-102.62μg/m L;38.50-57.07μg/m L).There was a significant positive linear correlation between the content of total phenolics and antioxidant activity（p<0.05）,and the mean correlation coefficient was as high as 0.85.Ellagic acid was the most important antioxidant component in walnut skin based on the method of dot value,and its contribution of 83.81%（free）,81.87%（esterified）and53.06%（bound）,respectively.In walnut kernel,ellagic acid was also the most important antioxidant component in free phenolics with contribution of 80.81%.In esterified phenolics,ellagic acid and gallic acid were the most important antioxidant components with contribution of 25%and 25.7%respectively.The main antioxidant phenolic compounds in bound phenolics were ellagic acid,gallic acid,ferulic acid,erucic acid,syringic acid and protocatechuic acid.