Vis 4, 121-128 2015) Characterization of seed proanthocyanidins of thirty-two red and white hybrid grape varieties De Manoa! RScucui L, Moun, Thai M. Ds Ros, A. Pant 'M-Gasen M. Gs R, Canna? aus Visova, red Rc Fuse >is or a > pioneer Ste Apne Une Sa Pao ad. Summary ‘Grape seed extracts are known for ter beneficial ‘ardiovascalar dents de the fee radial acaveng {fo improve the organoleptic characterises of wines, ‘forthe carton of mast and wines: PAs need ‘ntratt of 2 hybrid ad three 1 vere grape var ‘ties were characterized by MALDI-TOF mas spec {womery. Signals of 18 compounds were Mente as IMT, (nteNa ana [MeK adc of Beye and ‘type PAs formed from catechinepictehia sun pt camer and with allyson degree 7. The beraverage molecular weight (M, ofthe samples, {parameter correlated withthe mleclar weight of palmer and the polypenolc content of extract were so determined. Profs ofthe hybrid grape varieties tnere compared with hove of tree nnfere samples dled an references, "Ter 108-0 showed high on tt antiaidant polypenols and Seyve Vilard 12390" higher content of higher ogomers. These to grape Key words: proanbacynisis hybsd eps sets, MALDI-TOF: plypenots mass pectomety. troduction ‘The study of wap exacts fom sever aris of vines is becoming inseasing)y important, due to their ‘we inte food murcees,parmaceticaland cols. fn indies fn grapes, condensed eis» alo ale rsntocjaniine (PAs) -are present high eves batik {heh and sents (Downe of 2003, Mas ta 2007) ‘These compounds ply an inportant oetoogcl le a they are responsible fr soe enolic properties of ‘wine, purely teres and asingency. Grape se ‘tonne are tee sometime ed staves f m= rove te ogtolepi characteris of produce (asa ea 2012) Moreover, igholeasr weigh aap seed ‘toning re ammonia in cenology fr he clare thon of matand wins ey should ot change he ole ‘ory popriso colar of product and should aero ‘val chica speictns, sah simu eel of {eal Aavancs (10mg eatechin equlvaleat of eeno= Jogial a, OV 200) ‘Grape ed exacts se alo known fo have bees ‘in effecs on ath and on earovarclr dae, be ‘ng many sib tthe oad ro aoa ‘Rivity aod fee adalacevenging properties of PAS (Geass 200, Yan apd Tensbo 200). These ioe Tope! pops of PAs. ad of polyenes in geen ave edo aresed nrest inthe recovery of rod ‘sin te eologeal industry assed and sin exacts a ‘ed as deury supplements ad food preservatives i ‘heruraceteal inst Prope ae flan oligomers and polymers with soc connie (*pcuehin and )epieatehin Ut ned lneravani bonds at CHR andor CCS Peston (Biype compounds) In A-type compounds one ‘raare C227 and C2-C5 ether bod re preset (Fi 1) {Mtns af 1990, Rsaoo ox Sita oa 198, Suse ‘or Burson and Sane 2000, PAS of rape stds are femplex mires of oligomer and polymers composed ty (peaehin (repesechin end (epee -O- tala, wit oe galoy! group etd To the beteey fhe ing and the presence of B-ype and Asype bond {Bicaoo or Save a af 1991, Patino a. 1984, Pass a 2007) Prodiphinii, comamg ()ealoatecn tnd C)epiplloesachin unit fave been fund In rape ‘Sng, pulp nd ems (Sou ea 1996, 200, Mave at 2007, ‘The degree of poymezaton (DP) of PAS in gape seals varies widely andi generally lower han in grape ‘hin (Scogr ta 1996). Sever authors epoca {gape sod PAs conespond to lgomers: PAS wih DP up {0°11 were ened In seed extat profile eerined, ty "Mars Asie Laer Depton lniaon Tie of Flight” a pecromery (MALDI-TOF) (Keisatn ea 2000, and spoT by MALDI sn FTICR ass peo try (Vane Chien 200, Roesnaaci eta 2012) PAS ‘wih DP upto 8 were deed by peronning hl {nd ge permeation cheratoprapy (Pau a 1934, Gorepantrce Dr Fs, Cn err in psn as l'on pri, Cen Rice prt ola VT, Vase XVI Agee 01S Compan (70) aly “39.08.6079 ltnecre samenFig Sue foals an res PAS) np seed Loss ot 1999) Moreover, in he electspray mass spectra obaed by detinfson he sigtals of [M3 As ions corespondng to up to 28 unis, were observed (Bavssac eat 200), In gerd by mass pecrometc tecige # earce detsion of phe oer weigh As is ofen observed pinply duet on suppression phenomena ised 1 the high abundance af lower mo. Fela seis Davos a 2003, Puree ea 2008, Movaci ff 2010, Meus and Fico 2012) "The deg of polymerization ane galolaton ats the “notre” properties eased by he meacton As with salary leoptein heihenee perception ‘of astngeey has ben observed wih incr ‘res i the degree of polymerization silly, an i ‘Steed depe of galoylatin favours eighened percep. ‘om ofcourse (VON a. 2003). In the lt cet, = number of French-Ameria hy ea grape varies were pce, ih tes oF in ‘platessa toda, Caen tht cutars have ‘een il sed beau in several ours eg in he European Cammunip) they ae ot teude ine st ‘of varies teed for producing wine (CE Regulation no ‘277008, ad in my cases ony conserved in ge plas colecions. These hybrids ron charles by ‘Sigh ape roan pr hee ae deo he ei ‘nce to sates, witout sing pss. Moreover they ‘Be fin duracrzed by peculiar nines of chemical ‘Secondary menboles wih pet the Fyne vane fies Fo example stuly of the anoryani poles af wengroneo thes re rapes showed tat most of them ‘a quaiaively an quantitvely very ch in meno ‘Sos and dipcoside compous (De Rosso al 2012) 2 say of eae sed taylor showed tht some ‘Of these varies (eg Bact I fave pele conten af ‘lool, highansarrsed wiayerise compound (De ‘Maat ea 2012 Inthe present work. PAS pote of ‘hi. hybrid grapes inating both rel and white ae ‘ebes was sided MALDLTOF and MALDI-TOF TOF nals were permed wbichave eenproved beet ‘eee in tan des REE ef 208 Manz fa 2010) PAs pole of sees ofthese varies was aver sid bere an ete highpolyphenoe conten sme fen could be intereing fer profoctionofaced exacts er the fod oenologia and narcetcs nd. Three ‘vinfra sles, ‘Araen’ Chardonnay snake Snuigns, were sud ag reine compare it lye! potenti wth he yd saps Methods Samples and reagents: Tay.io hybed yap sanpls (21 ted and I) wht) and tee Fn For gape samples ramen, Charony” a ‘Cube ne Shogo?) wee collected in 2010 a il shwlog- fal pene fom te fv plans of each variety pest inthe CRA-VIT (icuhie Revere Cae) Grapevine Germpla Colleton (Sues, Trevi tly al Imei ze. The Ist of samples sted reported in Tab 1, Methanol, heate and Folin Coca reagent vere parcnsed fo Fuk Sipe Ali (a, aly Proanthocyanidins extraction: Sexdsof 20 bees were manly separated fom skins an pls, eid paper see wig an gra with i togen ty weg a snr The sop we efit wth eae ao 110 wh) by hepa the sample under si ‘since wha methanol/water sltion (130 vi) 8 ‘aio I-10 (ws (XU ta 2010) The ston was kept de steing neni centage 2683 for 10 mn {15 °Cand ead on paper fie.‘Seo prouthoeyaidns of iy two red and white bed pe varies 3 ti gripe vies side: rmnbeneege molec weit Re pape wy Wie pe ae be i ast m R Bison a R boutesoveans i cian) mt R cmcews in © Semin is Seva ioen ie Ri Syeeun inser is & SSeeviuediaa00 ee R Soeewanae ee R soevimiaess tan W Gateeis2 be ine vt Tapper nee etic epg I es i i ts us * nema rity Clog pts! (Chacon RAC caecoe nce of conn Total Polypheno! index: Tl polyhe oli inde of exact was determined by Flies ‘ethed (DI STEFANO apd CRAVERO 1991). A volume {1 mL of methanol exact was 41! died with ‘tlc and wanted in 20 mi ask Five ilies of Auld wae, | mofo Ciocaea reagent aod $m ‘of sou carbonate slution (10% wi) were ade 0 ‘hetample nde rescon war cain 1s Atte fend of econ the aberance Of sluen a700 mwas ‘measured wings Cary S0 Sos UV-Vis Specrophstter (ran, Milan tay). Tat pole index was cleltd ty te equation (1) (Opeth mpg) = 186 Ay (vi) (v2) (1) hn umber fon fhe si le V, vole of eae ed (01) Vg ttl volume of pe sed ext, wight of ape. MALDI-TOF mars spectrometry: An yee wereperfores sing MALDI-TOF. TOF UlraeX- ‘rome (Bruker Datos, Bremen, Gereany) instrument. cupped wit | ir sata ser 355m) and ‘pert n refer pone on mode. The sen {a onions employed wo ansae meu species i the m= ange 4503800 were: Lm Soe I: 25.00 KV; fen Source 2: 2230 4V, Leng: 7.704, Pulsed on exeac= tow 60s, Reflector: 2648 RV, Reflector 2: 1348 AV. ‘The mate solution was prepared by doling 10 me of eysaline 2digdroxy enzoie ai (DH in | - ‘sf metanaeter sition (030), The extra tine were ced fen tines wih a metal water 6 tin 70:30 v) Five wo tis solution wee mined wits thesame volume of mate soln nd! Hof tere Ing more was deposed direly one ties sel ‘Spe hole and allowed to ry Before gout in ‘he mass spectrometer Extra mas eae (Pepi Ctration Standard) was based on monosoopic valeot of [MoH of Bradyinin Angiotensin, Anglotesin 1, (1839, Somatwtain 38 a mie 79799916, 1046 520, 12866883, 1347736, 16125290, 20930868, 2465 990 and 3187471, respecvels TOF/TOF experiments were erfomed using tbe LIFT device withthe following exe ‘men conitons ISI: 78, 182-675RVELiN: 18 V5, in 3.7 4; Refletor 25 RV; Delay tne 7 Statistical analysisi, Relative nests of the sins were refed f the highest signal and sib Jest angular tnsfrmaion node to nome ts isbn. was performed sing the equation (2) yoann) @ ‘whee sth Valeo he anton variable, asin ‘hese fnction 2) he sured root fhe ro ponton (Goues and Gow 984). To select he vrs Still significa fr aeetton beeen Vin fea ad fyb samples One-way Analysis of Variance (ANOVA) with the Lest Signin Diflecce (Fisher test) and 95 % significant eve, was applied (Canin Pugs ea 2008), Siatcl antec were performed sing MINITAB Stasi! Software Ine. QUIS), Reese 14 fr Winows Sate Calige,Penneyanis). Results nt Discusion Characterization of proanthocyani- ins: The fist sep of ths roy valved the det eatin ofthe best MALDI condor nore oot the best epee ad he beet sigan a, eros maces were sted uch sap a, sno-tiydrnpeinamic ue. 2Saydraytenoie ned (DEB) diana an the te spectra wer aoe ing _DHB, witha reproduc oie abundance of bo 9. 97% measured onthe main peaks ofthe spect) and 2 sigubso-mose value i te range of 7-8, Une ch ono the Spectra characters by te pres of shunant caonsed species Ne’ K°) nd, in some ate, ‘of prtonated moleclr specs Mexsrement wer 0 famed in both ear ndseften ade our nt et povided esata which ied fom hse devi by Mena eel (2010, showing thatthe refs mode ‘else signa ae eprodabe dt Postive MALDI-TOF pear of pape seed exacts show decreasing peak itenies with neening mv tes (Fig espe mater) According fo a ‘hele, peas conespoeding [AN ond MK dives of PAS ae foun, while [MPH were observed ony in oe cases Ta, 2) Kune a 200, Wes al 2007) MALDI én may revealed sever ses lt molecular species ergnang fom Atype and ype tumin. Dept positive MALDI-TOF analy did mt roc the sgalsf izmers ihe han: 4000, te ‘pect provied much aforation wich lowed de Featon ofthe signals of 14 pute PA. The presence ‘of 5 pttive bare pus Ta. 2) «for example the lon at m= 181 = maybe dc fo pentane det or sdanlyied (26) eramerNa adduct shouldbe noted {hat the dierene of 288 Daobuevd for many ial F De Manon eal co aon of father etchinpicteshin mono ‘vie oat oF 132 Da may be oe presence ot aie {eid moieties inthe molecule site (Fig ? of spe etl material). Moreover, worth notes on wa wo gle aid molecules give eqn mass ofthe ato of a gallctechinplga ect tn ‘hemlet (+304 Da) (ther eigen series ve bse tte nd hie ‘males ae lowe tan ht of ype es parla rks at-2 and 6 De fave bon abarved. These sees fan be sctited othe presence of PAs wth ae o sone ‘ype bonds in the sample. For example, nthe case of IMO tiers, a clster wih peaks eferng 2 and ¢ Da as been observed and theca be expe bythe ree exc oftwo Bype bonds inst BED rf one BPS bond alone Ape Bnd oe at m= 15) a of t0 A ‘ypetond (on atm 863). Tegal n= 1189, et ‘ed [MH teramer Aspe, ind th presen oF ‘hye ype bo althogh tistheretelly potable beens ofstechiarace: Looking a te oksned eral Show in Ta. 2, his analyte approached tothe en ‘fein ofboth A- and ype tains vp to ndesamers id depres of allylation (DG) raging frm 0107 1n fener DP and DG ofthe PAs denied se similar fo thee previously reported in Vv gape sed exacts (Keown ea 200, Havas oa 03). “wo ther signa tm 56 and S89 were detect ‘ot posly duet [MMI species of dyéred Baype fu Acype dimers, especie, a demi by Motte ‘Foca (2012), TOPTOF exprnerts were cated ‘ut on more abundant A- and Bye olgmes a care ‘eae he roel src. AS a eal, the TOF? TOF mass spect of nem mi 1481 (B99 sigue) and 68 mer with am A-type and 2B bong) re report in Fig 3 of supplemental mati. As ean be ‘enn inthe case of ean at a8 fgnent oda to the loses of earechntpetecin mois (248 Dare eldetectble together with oases of 17a gic acd) nd 152 Da, orgmting om tearangment of the iy Aropyran heterocycle. When eA) rd reset the mace the TOF/TOF mas speinen becomes more ampli Inthe cae of i atm 86 te aly de Serbed less of 268 Ds and 152 Ds atl dts, butnefagmens, sich sth tw 68s preset oe ‘ating fom the fos fa rad species whose mols lar mala i C0, (81 Da. This eu be exlained by rearagzmets of poate specie doco Hy the ‘Asypebood which ene sti hindee ofthe moe cule Ineensing the polyertion degre the TORTOF Ins peta fhe A type PAs eco a) complied te loses of 152 Da and 268 Ds at sil deere bat fragment ions crgeatng fom rearanement ae very shunt ands ery ict asin srt Be, ‘The presece of A-type PAs in sape ree priclry ‘neesting base, snc near, these ous have been proposed as ctive components ich {nha ie aderence of col ture epi cl sure hus preventing bate cloasaior and sbeeqent inson (Foo eo 200), A-type PAS hve only Been found in few plats and fits ol. 2003, 2Seed rontocyaniins af thy. edad white hybi ape varies ns Samisopuv yp and Ape proses ein MALDI-TOF spec of ames sued: lie ten “pen penay BER SoM z e wine 26 11931208 118, 119 1208, 1207 ed bs Ba fata ask ‘eiremer 1G 1305, ine as “13 1325, 1327 1348, 1 = ‘er lw ves ey wn is ‘ome 1 iso Wearan”atae ‘met tation ae reer Werte ter em far 10 tenses anne fesone 20 veneer es rae 30 Chins” tains = racist aus 6 tomes sons oan toneie iene Bam mre Baa yasmans imese Sova Baas a ioe ss Sono Saw Bena one 20 amare Baa Srenroe Mentos Boni Senet Secor ere Stoners same can Sam aanane Sner¢ arias “hn Sms Serie eae =a Sham naw Sime mn Siar eer sar oo Sons Pa a Sore sane Sere si Sime 20 ‘hee sods (Ya 2010) pasa cinnamon Gu era (Pasos ea 2007), PAs with ono wo intelvanid 3003), Mashing ou ning, the presence o alloyed type bonds wee aso iene in ark tem of Pea ‘nsypevnain pape sess acct em demonaed_ aon (Baio. 1995126 Seeds PA profile of the grape varie ties: In sone samples, the Botzman taped curve of IMALDI pet was nrupe at ow mv vasa the highest ines mas observed fr the sgl atm 589 (ram, Ter 100-31, Taree Bran 624, Cabernet Sauvignon!) and w/z 577 (Clio Code 28, Sete 1057 "Seyve Wilard 2-369 and'25-39, "Ter 1086, "Tea 9746) Feo samples th most bd on wa tm 119) comaponding fo mer 2G sna InSibel 8357 ar Sey Vln 12-357 a mz 905 ct responding to diner 20 sodium ads nthe case of the scsson une enifestion a ‘inion the MALDI ‘pecram was chrcteized by high chemical Bakgrnd hd it was more complica than the oer samples. tsar te sigs of Atype PAs were fe ihe tan [Binge ones Monoter, oer oo Sess of tors sting fhm 89 and 711 (559 152) respective ad diag the eal atm BA, 1135, 1433, 171,199 nd 2287 anda 99,1287, 1863, 2151, 2639 aod respstvely, wereaberved (Fig 4 of spplemenal me Fal) These te ses dle om puaive PAs A-type By 8 Da(Moes wd Fucus 2012) “onl polyetl index of exacts wa eterna nd sata re reported Tab | This inde pate ee {ul fora comparin of polyphenols content of he sn. Pes. The ests ged fom iim of 200 meg” [bape toa maxima of 500 mpeg rape (Ter 1088), ‘wile those fund vine sampler are betwee 150 TO mek wae. To campue the sampler, ako the sumberaverage molecular weight (M4) was elle by {Sing th ols testy of MALDI spt scoring qusion OF Men E vty: ° wher / she abit ters of exch ain decid (Neue of a 200, Mae ef a 2007) andr reported in “ab. 1. The average M, fr bye rapes was 197597, hie iat of tetera same 13764 18 These dua mach thse the Mere fr soasmersal tran facto of rape and pple (Me a 2007) nd gh Tight ht he main peaks in MAL spect comesond to ‘lige Total uyphenol index end Mz of hardow ‘ay exact lover thane dtp etre for {he sme variety afer pholuinolss (1400 mek 30D mpg (Mone eat 2007) ba i baby do {ote diferent nla methods wed ad samples ‘eas obverve tat the ihe the dere of poner ‘Stn te lowers on pea nent Ma a ihe an in ur Sere Vale 130, alten tively In Seye Vila 12390, very hah pope ‘coment was ofan (1 ga gripe). Sever ses ‘Showed a ori onlin between nn chin eth ‘Sn eranolotic aratrts the ihe te MV he Tower the biterest andthe sworger he asrngency (Vou er a 2003) Mr di ofthese te vais show Bihar ‘ones of high tani, as consequence hei ex ‘aes are are interesting for oeoloies es, ‘Some lb pes ware cher of Aye PA ihe spect snore in prc, Bd ele Seyve 4828, Coolere 28, Sebel 10878, Seyve Vilars F Dy Maso 12.306, Seye Vilad 25-367, Seyve Vill 23-399, ‘Gai 26549, GE84-2-9 cava Brant 624, Sibel 110 a Seve Villard 12-375 showed PA with wo tive Ape Hon up 0 names and a rfl paral similar to caer (Fig. 2) (Pera eal 2012). The high coment of Ahpe Pas, characterize by acral a tides tivity (HOME ef af 2005), ry ter ‘cease he ners fr euacetcal wes these grapes. “Te pe be of inl nT. 2 wae eed 10 109 by sing the menses of [MoH MON an [s40K) species a those of PAs wha eat one Ape od. Raber redicon of the varales was shined by perloming one-way ANOVA ad Fisher’ tat ing ‘he meat vale of the Ryd saps Tomas the mean ‘efor yon arabes ih a high Fst so Pile 0.08 wee cossered inion in iereat ng been ytd an singer varieties Fen PAS ‘suing more characteris a ibid apes, in pric lar A-type [MNal diner egal (or (MOK timer, ‘vtpe INONaY” dimer debated, and Buype (MN) carer teagan 1.3), Conctasions In spite of the presnce inthe matics of som and possum sls, which my aft sini, MALDI-TOF analysis has provided arp and ompee aractrition of ape sel exacts allowed the idemieaton 35 Bape und 95 A‘ype pcathoyantin, respectively ‘A hig amount of anonidat polyphenols aks the act ptt interesting for aes testa Ais, an high MW gape sed tins are used forthe ‘lnifiaton of must abd wins Seed PAs of xe yb {pape were sce fr he fist ne, atm of them Pretent sources of rata compounds potentially very in (esting for he ty In pr, "Ter! 108-6 nd fxrnt showed igh coment of atin polyno Despite MALDI-TOF slowed ot Pas jun ecames he comparison with oe ifr any bo grape septs evidenced tha Seve Vilar 12-590 rated by «relevant presence of higher oligomers Ie firs permed by ee permenon cromaton ‘spy wl confirm reevant reece lo of PAs with MW ‘a 100 (Kinyo a 203) th sed exact of is ape vey can tea nesting sure of oeologiel famine Asa consequence, hex ais wil show lo Ineesngofemlepc properties thy cm he et for rauctono rape ues sn epost a sures SE PAS fr indstal purposes. Acknowledgements “Ths as elo iin he VITENERGY! Pr, wit eng ran alan Min Ape, a and Fore Poles dston S009 The ace a nls RGVFAO Pre de Ian My of Apa Feat and Fry Paley, eh cnt oman ‘GECRAVIT gmp cio,Seed probes of havo red and whit yi pe vars by Vtitllds CLLESEDE ili Il COLL pth CEE EL ig 2 Pacenage of inp and Bye Ph een) vine sample sed (men ab) Sey Vir 2-397 6) See 1478 cambery enc (Pa eal 202) 5 PEEL osnaeenasyy saauaSaaEEE pupa nsaaad or iit hin cient ins oes, ibe te ey Pe. asi Taio ao0r ‘eta os [M&As ere 8.30 oc MEK} penne 16 Inet ie a0 MR rane Seer MRT ner 816 B36 ReferencesSimi things ea oy cre “ISA a3. oman mar sn ie ‘in ea em rey ne ‘Sthay panna em Dae Fagg ote Be on NDA pe per ‘Swen ey imam gee {E ptphe nisnpa ocn aes Gin A.A: Pend KA, “asta Sn neva apse ‘scent Sah nee ca ent ae Fn BH ulstennin bee Lam igi oem ‘Soap anni a ee St: Gnas of partyin pe sn eon oy ip Ce Ss GL M2088, Ape aio pwns reg a Ses eno Kena A oA 298: Ai fp by Ai mim veep ro ‘mm "Sb" Mrs een tpt malngctoicm aoe 1 Suman nen pe panaoganion Seige cpt Fen "or Anemone meena font ‘cot poe "Fc 207 Ominous oe Pw nea rn om pope i erent sgn coach Our Enpernin | gr Fn 84 TET oon We Orns Bs Ome CB "ere Loan R018 MALOLTOE NS mh in aman Par ame Al 8188372 aie ean BS URC ESI yf ian ‘hee el nem mrs tom» Be F DeMaria ul un Yo ter Ong N21 “pti on cao ema Fn ESSN ea dconnn ie em ey iran tn 12 ea mele nd nus COE. ae PC § Do MR ME Te MCs Meas SO Boe pd set Ci ‘eepobmas wens hp ecb ‘con ti dp ou mui eine ope Fromm 0s Scan fone aaa Te rater ttre hain SF ORT ee ‘eimaie ‘roantocyaidins em grape skins. noche 4, ‘Chorals ligula 1) pecanthcoald charac by mam Seeceteors eee mR "a Crs, V; Wares, E 2; 208; The moudelpopenis of iexeaaae ieee Compren of genie min ern rage Sa ieee retain ccm di Food PE eee rune Dangtotnmce ine og et ‘yatta RE ie Heap cl pape se ‘Reco Sponr $2014