Study On Antioxidant Properties Of Winter Vine-prunings And Their Acitve Phenolic Constituents

Winter vine-prunings, the main solid wastes derived from grape planting, are not paidenough attention because of lack of understanding and the related research. Currently, thedisposal methods for the overwhelming majority of vine-prunings, such as in situ burning orburial, easily cause environmental pollutions, thus, proper managements of these wastesbecome an urgent need for the sound development of modern grape industy. In thisdissertation, domestic yields and basic physicochemical characteristics of vine-prunings werefirstly investigated. Afterwards, the antioxidant property of vine-prunings from10commoncultivars （Kyoho, Red Globe, Thompson Seedless, Victoria Blanc, Hongmeigui, CabernetSauvignon, Cabernet Franc, Chardonnay, Merlot and Pinot Noir） were systematically studiedvia establishment and optimal screening of antioxidant methods, analysis of polyphenolcontent and profile, activities of phenolic monomers and their structure-activity relationships,contributions of monomers to the total antioxidant activity and interactions among them. Thestudy aims to provide theoretical support to the future exploration and utilization of thesepruning wastes. Main conclusions can be drawn as follows:（1） The annual yield of winter vine-prunings per tree ranged from1.33to3.21kg （fresh）,with an average of2.35kg, yields per vine of table grapes and pergola trellis were higher thanwine cultivars and standard trellis, respectively. The domestic output of vine-prunings reacheda value of3770kt based on the current cultivation scale, among8major grape producingregions, Huanghegudao and Ganning had the highest and lowest yield per vine, respectively,while Xinjiang and Dongbei owned the highest and lowest total output, respectively. Vine-prunings showed acidic pH and abundant organic matter and K, Ca, P, Mg, Fe, Mn, etc., andhigher contents of dry matter, total organic carbon, dietary fibre and Mn compared with grapepomace.（2） Titantnium salts spectrophotometry （TSS） assay, a simple, convenient and accurateantioxidant method, was firstly established for the determination of H₂O₂-scavenging activityof antioxidants. Optimized parameters: hydrogen peroxide:10mM; phosphate buffer:60mM （pH7.4）; titanic sulfate:50mM; sulphuric aicd:1.2M; reaction temperature:37°C; reactiontime:30min; final volume:5.1mL; detection wavelength:404nm. The sensitivity of TSSassay was23times higher than the traditional method; besides, the precision, repeatability andaccuracy of TSS were also superior to the traditional one.（3） Vine-prunings extracts （VPE） exhibited various degrees of antioxidant activities in19test syetems, belonging to four broad categories of antioxidant mechanisms （radical-scavenging, antilipidperoxidation, metal chelation, and reducing power）, and all theseactivities were in dose-dependent manners in respective concentration ranges tested. Varietaldifferences in antioxidant activities were abserved in all VPE, some of which were significant（P <0.05）; moreover, varietal differences were influenced by antioxidant models. The averageantioxidant activity of ten VPE was lower than Trolox （positive control） in scavenging DPPH,ABTS⁺and NO, higher than Trolox in scavenging HOCl, ONOO^-, O₂^-, OH and H₂O₂,comparable with Trolox in scavenging ROO, one-third of β-carotene in scavenging1O₂^-, one-half of TBHQ in combating peroxidation of phosphatidylcholine, one-seventh of EDTA inchelating Fe2+. Moreover, all VPE presented distinct reducing properties.Highly significant and positive relations （P <0.01） were observed between the methodsused for determining same parameters, or with same purposes, while diverse correlationshipswere found between those with different purposes. Abiding by the principle ‘not only shouldwe preferentially adopt the methods with biological relevance, but also we should guaranteethe test comphrehensiveness and avoid the repetition of assays with same mechanism,meanwhile, both the efficiency and cost should be considered’, nineteen antioxidant assayswere compared and evaluated, and11different methods, including ABTS （scavengingABTS⁺）, PMS/NADH （O₂^-）, TBA （OH）, TSS （H₂O₂）, Crocin/ABAP （ROO）, RNO （1O₂^-）,TNB （HOCl）, EB （ONOO^-）, TBARS （antilipidperoxidation）, Ferrozine （chelation） andCUPRAC （reducing power）, were chosen as the methodology for the future use.（4） A novel HPLC-UV method for the simultaneous determination of12phenoliccompounds from vine-prunings at their maximal absorption wavelengths （MAW） viawavelength switching was proposed. Optimized chromatographic conditions: VP-ODS C18column （250mm×4.6mm,5μm）; mobile phase:3%acetic acid （v/v） and acetonitrile;gradient elution; wavelength mode: detection wavelengths were switched to thecorresponding MAW0.05min before the signal response of each analyte, then switched backto400nm0.05min after the end of the signal; flow rate:0.8mL/min; column temperature:30°C. The newly proposed method significantly improved the sensitivities of several phenolicsin comparison with the constant wavelength （280nm） detection commonly used. （5） Vine-prunings were rich in polyphenol contents, and there were differences amongcultivars. The highest contents of total phenolics, flavonoids, flavanols and hydroxycinnamicacids were found in cv. Cabernet Sauvignon, while the highest total proanthocyanidins wasfrom cv. Pinot Noir. Twelve phenolic monomers （trans-resveratrol, catechin, epicatechin,rutin, morin, quercetin, gallic, protocatechuic, vanillic, caffeic, syringic and p-coumaric acid）were dectected in all cultivars. trans-Resveratrol, catechin and epicatechin were the threemost abundant monomers occuring in vine-prunings, representing33%,25%and25%of thesum quantified by HPLC. Vine-prunings possessed high levels of trans-resveratrol, rangingfrom138to291mg/100g dry weight, and were expected to be the biggest potential source ofnatural trans-resveratrol when comparing with the current ones.Polyphenol levels demonstated a close relationship with antioxidant activities, and theroles of different constitutes in antioxidant effects of vine-prunings were not exactly the same.A significant and positive correaltion was found between total phenolics and all antioxidantparameters excepting Fe2+-chelation. Total flavonoids, flavanols and proanthocyanidins werecorrelated significantly positively with some antiradical parameters as well as reducing powerand antilipidperoxidation, while total hydroxycinnamic acids was only correlated stronglywith O₂^-and HOCl. In terms of monomers, both catechin and epicatechin were found tocorrelate significantly positively with most antioxidant parameters except ONOO^-. The nextmost relevant was trans-resveratrol, which showed significant positive correlation with ROO,1O₂^-, HOCl and antilipidperoxidation.（6） The antioxidant activity and structure-activity relationship of phenolics were affectedby test methods, which exerted a greater influence on simple phenolic acids thanpolyphenolics. In Crocin/ABAP assay, the activity of phenolic acids with pyrogallol structure> catechol structure> single phenolic hydroxyl group; methoxylation of hydroxyl groupweakened the activity, while the increase of methoxy groups enhanced the activity; under thesame aromatic substitution, the activity of phenolic acids with propenoic side chain（hydroxycinnamic acids） were stronger than those with carboxylic group （hydroxybenzoicaicds）. In ABTS assay, the effects of methoxylation and methoxy group number on allphenolic acids except gallic acid, and hydroxyl group number on hydroxycinnamic acids,were opposite to those observed in Crocin/ABAP assay. Flavonoid compounds presented verysimilar structure-activity relationships in both Crocin/ABAP and ABTS. The catecholstructure in the B-ring, the3-hydroxyl group in the C-ring, and the C2-C3double bond inconjugation with the4-oxo group in the C-ring were key features for flavonoids to maintainhigh antioxidant activities. （7） The sum of theoretical contributions of12individual phenolics can explain19.7⁵0.8%and34.1⁵7.9%of total antioxidant activities measured by Crocin/ABAP andABTS assays, respectively. trans-Resveratrol, catechin and epicatechin were the threemonomers giving the highest contributions in all vine-prunings （n=10）.Different phenolics （protocatechuic acid, catechin, epicatechin, p-coumaric acid andtrans-resveratrol） occurring in vine-prunings present in equimolar mixtures can interact, andtheir concrete interaction types depended on phenolic species and test methods. In mostcombinations of Crocin/ABAP assay, protocatechuic acid and epicatechin exerted synergisticeffects on the antioxidant activity, while p-coumaric acid presented antagonistic effects;meanwhile, both catechin and trans-resveratrol exhibited additive effects. In ABTS assay, theeffects of trans-resveratrol, catechin and epicatechin were generally synergistic, whileprotocatechuic and p-coumaric acids were mainly antagonistic or additive. It should bestressed that the combination trans-resveratrol/catechin/epicatechin demonstrated obvioussynergistic effects in both assays, in combination with the relationships between these threemonomers and antioxidant activities as well as their contributions to the total activity, we canconclude that trans-resveratrol, catechin, and epicatechin were the predominating activeconstituents of vine-prunings.