Evolution of Catechins and Oligomeric Procyanidins during Grape Maturation of Castelão Francês and Touriga Francesa

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1 230 Jorão et l. Evolution of Ctehins n Oligomeri Proyniins uring Grpe Mturtion of Cstelão Frnês n Tourig Frnes António M. Jorão, 1,2 Jorge M. Riro--Silv, 1 * n Olg Lureno 1 Quntittive hnges in oligomeri proyniins [imeri proyniins (B1, B2, B3, B4), glloylte imeri (B1-3-0-gllte, B2-3-0-gllte, n B gllte), trimeri proyniins (C1, T2)] n monomers [(+)-tehin n (-)-epitehin] in sees, stems, n skins of mturing Cstelão Frnês n Tourig Frnes (Vitis vinifer L.) grpes were mesure using reverse-phse high-perfomne liqui hromtogrphy (HPLC). For skins, only imeri proyniins (B1, B3, n B4) were etete uring mturtion. The level of oligomeri proyniins from sees, stems, n skins in oth grpe ultivrs stuie erese uring ripening, minly in the first stges of mturtion. (+)-Ctehin n (-)-epitehin from sees in oth grpe vrieties inrese uring the first stges of ripening, followe y strong erese n stiliztion. Proyniins B4 n B2 were the most unnt iniviul oligomeri proyniins from sees in Cstelão Frnês n Tourig Frnes, respetively, uring mturtion, while in the se of stems n skins, B1 ws the prinipl oligomeri proyniin in oth grpes ultivrs stuie. Key wors: Grpes, proyniins, tehins, HPLC, mturtion, sees, stems, skins The flvnol ompouns known s pronthoyniins ffet the olor, tste stringeny, stility, n ging hrteristis of wines [3,12,17,20,23,25,41,43]. Severl uthors hve reently reporte tht grpe n wine proyniins ply positive role in humn helth, ting s ntioxints [16,25,40], therogeni protetors [15,22], oxygen free ril svengers [28], ntihypertensives [2], ntirinogeni gents [19], ntivirls [39], n ntimiroils [4]. These phenoli ompouns re present in the soli prts of the grpe (stems, skins, n prtiulrly sees) n exist in ifferent forms (imers, trimers, tetrmers, n polymers). In the se of polymers, Czohnsk et l. [5] reporte tht polymeri pronthoyniins onsist exlusively of repeting flvn-3-ol units. The mjority of these polymers present monomers with preominntly 2.3-is stereohemistry, with the sme onfigurtion s (-)-epitehin [6,37]. Sun et l. [38] stuie the trnsfer of tehins n ll pronthoyniins from soli prts of the grpe luster into wine, n the results suggeste tht grpe sees n stems, together with the skins, re importnt soures of wine pronthoyniins. In generl, B1 is the most unnt oligomeri proyniin in stems n skins, while B2 is present in the highest onentrtion in sees [1,7,9,11,13,29,30,31]. The glloylte proyniins re present in onsierly lower onentrtions thn the nonglloylte proyniins [11,30]. However, B1 is 1 Universie Téni e Liso, Instituto Superior e Agronomi, Lortório Ferreir Lp (Setor e Enologi), Lison, Portugl; 2 Present ress: Esol Superior Agrári e Viseu, Cmpus Politénio, 3500 Repeses-Viseu, Portugl. *Corresponing uthor [Emil: jrirosil@is.utl.pt] Aknowlegments: The uthors thnk José Mri Fonse Sus S.A., Azeitão, Portugl, for grpes n for support. Mnusript sumitte Mrh 2000; revise Otoer 2000 Copyright 2001 y the Amerin Soiety for Enology n Vitiulture. All rights reserve. the most unnt oligomeri form in re n white erries [1,7,29,30,31]. The ontent of proyniins in grpes epens on the grpevine vriety n the yer [1,8,9,10,11,18,24,34]. In the stuies ville, some ifferenes hve een foun etween the flvnol omposition of the skins n the sees uring grpe mturtion, ut few stuies hve fouse on stems. In erries, Dumzert et l. [8] reporte n erly inrese n then erese in the (+)-tehin ontent of ripening Muz grpes grown in Frne. In 1992, Fernnez e Simon [10] reporte tht in Ceniel grpes, the vritions in the onentrtion of these monomeri phenoli omponents present in erries uring the mturtion perio exhiite two stges: n inrese roun verison, followe y erese roun mturity. Czohnsk et l. [5] reporte tht the mjor omponents (+)-tehin n (-)-epitehin, present in the erries of Sieel (seletion not reporte) n Gmy noir grpes, unerwent omprtively little flutution uring mturtion. However, Romeyer et l. [34] stuie mturtion of the erries of four Vitis vinifer vrieties (Grenhe, Ugni ln, Crignn, n Meo) n reporte tht monomeri flvn- 3-ols isplye mximum t verison n tht proyniins B1, B2, n B4 exhiite n inrese ontinuously uring mturtion. A similr evolution in erries ws reporte y Lee n Jworski [18]. The results otine y Czohnsk et l. [5] showe tht proyniins B1, B2, n B gllte exhiite highest onentrtions in the erly stges of evelopment, followe y erese in the lst stges of mturtion. These stuies reporte the quntifition of (+)-tehin n (-)-epitehin n some imeri proyniins, prtiulrly in entire erries n sees, uring grpe ripening. It ws thus of gret interest to evlute other imeri n trimeri proyniin ontents uring grpe mturtion, onsiering them seprtely in sees, stems, n skins. The present work, whih is prt of 230

2 Evolution of Ctehins n Oligomeri Proyniins 231 lrger stuy of the ehvior of phenoli ompouns uring mturtion of grpes [13,14], esries the evolution of the mjor smll flvnol moleules uring the ripening perio in two importnt grpe ultivrs grown in Portugl in orer to explin the oligomeri flvnol evolution uring grpe mturtion. Mterils n Methos Grpes. Tourig Frnes n Cstelão Frnês (Vitis vinifer L.) grpes (200 erry smples) were otine in 1995 from vineyr lote in Plmel (south of Lison). Berries were frozen t -18 C until proessing. The smpling perio strte t verison (5 July for Tourig Frnes n 12 July for Cstelão Frnês). Physiohemil nlysis of grpes. Tourig Frnes n Cstelão Frnês grpes were nlyze t tehnil mturity for weight of 200 erries, estimte loholi egree, ph, titrtle iity, totl phenols, totl nthoynins, olor ensity, olor hue, mli i, n trtri i following the proeures of OIV [21]. All etermintions exept phenols, nthoynins, n olor were one on the must. The other mesurements were one on n extrt otine from mertion of the rushe grpes t 25 C for 24 hr in ph 3.7 uffer. Flvnol extrtion. The sees, skins, n stems were seprte mnully from the lusters (pproximtely 6, 20, n 40 g, respetively) from eh ultivr. After this seprtion, eh frtion ws immerse immeitely in 100 ml of methnol n store t -24 C for 24 hr uner N 2. At the sme time, 5 mg of sori i ws e to voi oxition. Eh frtion ws then extrte following the proeure of Bourzeix et l. [1]: 100 ml of 80% methnol ws followe y 100 ml of 50% methnol eh for 4 hr n istille wter ws e to the smple n ple t -24 C for 15 hr. After this perio the resiue ws extrte with 75% etone for 1 hr. It ws foun tht the extrtion performe with gittion extrte more tehins n proyniins thn tht without gittion. Thus, the extrtion ws rrie out with mnul gittion throughout the y (over perio of 8 hr) for pproximtely 2 min/hr (totl time, ± 16 min ily). For eh smple, pproximtely 5 ml of the extrt ws purifie n frtionte on polymie olumn (Mherey-Ngel, Duren, Germny) s esrie y Riro Silv et l. [26]. Elution ws rrie out with 80 ml of wter juste to ph 7 to eliminte phenoli is. After this proeure, the elution ws first rrie out with 50 ml of ethyl ette (30% v:v) to elute tehins, followe y 50 ml of etone (75% v:v) to elute proyniins. The tehins n proyniins frtions were psse through 0.45 µm memrne filter n 50 µl liquots were injete into the HPLC olumn. HPLC nlysis. The equipment use for nlytil HPLC ws Konik 500B system (Konik Instruments, Spin) with 7161-LC Rheoyne vlve. Detetion ws me t 280 nm with Konik UV-Vis etetor moel 206 PHD, ouple to Konikron t tretment system. The olumn ( mm, with prtile size of 5 µm) ws reverse-phse C 18 Superspher 100 (Merk, Drmstt), protete y gur olumn of the sme mteril. A moifition of the metho [26] ws use for the HPLC seprtion of iniviul tehins n proyniins. The moile phses for monomeri flvnols were (A) eti i/ iistille wter (2.5:97.5, v:v) n (B) etronitrile/a (80:20, v:v). The liner grient ws with 93% of A n 7% of B for 26.1 min, followe y 88% of A n 22% of B for 90 se. After this the olumn ws wshe with methnol/wter (50:50, v:v) for 15 min. The flow rte use ws 0.9 ml/min. The moile phses for proyniins were (A) eti i/ iistille wter (10:90, v:v) n (B) iistille wter. The liner grient ws run with 10% of A n 90% of B to 70% of A n 30% of B for 45 minutes, followe y nother liner step of 90% of A n 10% of B for 25 min, fter whih it remine onstnt (isorti) for 12 min. The flow rte ws 1mL/min. Between injetions, the olumn ws wshe with methnol/ wter (50:50, v:v) for 15 min. All nlyses were one in uplite. Flvnol ientifition. The hemil strutures of ll tehins n oligomeri proyniins otine n quntifie in this stuy were onfirme using previously esrie proeures [27,33]. Dt nlysis. During grpe mturtion (for eh omponent of eh ultivr), n nlysis of vrine n omprison of tretment mens (Dunn s multiple rnge test, 5% level) ws performe using the SttGrphis progrm (Mnugistis, Rokville, MD, USA). Results n Disussion Berry weight n omposition. Totl nthoynins, erry weight, n estimte loholi egree tene to e muh higher in Cstelão Frnês thn in Tourig Frnes grpes (Tle 1), while the vlues of totl phenols, olor ensity, n olor hue were muh higher in Tourig Frnes thn in Cstelão Frnês grpes (exept the vlues of totl nthoynins). The vlues of ph, titrtle iity, mli i, n trtri i seeme to e similr for the two grpe vrieties t mturtion. Tle 1 Physiohemil omposition of Tourig Frnes (TF) n Cstelão Frnês (CF) grpe vrieties t tehnologil mturity (6 Sept. 1995), Plmel, Portugl. TF CF Berry weight (g) (verge) Estimte loholi egree (% v/v) Titrtle iity (g/l tr..) ph Totl nthoynins (mg/g erry) Color ensity (s.u.) Color hue (s.u.) Totl phenols (o.. 100) Mli i (g/l) Trtri i (g/l)

3 232 Jorão et l. Ctehins n oligomeri proyniins in sees. At the eginning of mturtion, the proyniin ontents in Cstelão Frnês sees were generlly higher thn in Tourig Frnes sees (Tle 2). During grpe see mturtion, the flvnol onentrtion generlly exhiite rpi erese in the first 22 ys, followe y grul erese in the lst stges. These results re in or with other uthors [5, 7,8,34]. However, some proyniins (B4, B2, T2, n B2-3-0-gllte) exhiite smll inrese uring the lst 30 ys of mturtion, prtiulrly in Cstelão Frnês sees. For Tourig Frnes, the evolution of (+)-tehin n (-)-epitehin from grpe sees ws hrterize y rpi inrese, followe y erese in the lst stges of mturtion (Tle 2). This evolution onfirms stuies with other vrieties [10,34]. For Cstelão Frnês onsistent erese in these two ompouns uring mturtion ws oserve, exept for epitehin, whih showe slight inrese etween the first n seon smpling tes. Severl stuies with other grpe ultivrs t ommeril mturity showe tht (+)-tehin n (-)-epitehin were present in higher onentrtions thn ny of the iniviul proyniins present in sees [1,9,35]. We otine similr results with Tourig Frnes n Cstelão Frnês. At tehnil mturity, for Tourig Frnes the vlues of (+)-tehin n (-)-epitehin were 3.30 n 2.30 mg/g of see, respetively, n for Cstelão Frnês, the onentrtions of (+)-tehin n (-)-epitehin were 2.11 n 4.55 mg/g of see, respetively. However, the lower onentrtions of monomers oserve in Tourig Frnes sees t the erliest smpling te is n unusul result, perhps ttriute to rpi oligomeriztion, whih woul eplete the monomers. Further experiments re neessry to explin the phenomen. Tle 2 Distriution (mg/g see) of flvnols in sees of Tourig Frnes (TF) n Cstelão Frnês (CF) grpes uring mturtion, Plmel, Portugl, July 12 July 26 July 16 August 6 Septemer TF CF TF CF TF CF TF CF B A B C C 0.08 B A B C D 2.02 B A B C D 0.52 B A B C D 2.64 B1-3-0-gllte 1.31 A n 1.17 B C D 0.17 B2-3-0-gllte 1.26 A B C D 0.26 B gllte n 1.79 n C D 0.03 C A B C D 0.45 Trimer A B C D 0.93 (+)-Ctehin 0.60 A B C D 2.11 (-)-Epitehin 0.74 A B C D 4.55 Verison te. Eh vlue represents the men from uplite smples. Sttistil signifine of the ifferenes etween smpling tes for eh omponent of eh ultivr one y Dunn s multiple rnge test. Mens within eh line (pitl letter for TF n lowerse for CF) followe y the sme letter re not signifintly ifferent (p < 0.05). n: not etete. Most stuies hve foun tht B2 n B4 re the mjor proyniins foun in sees [1,7,11,29,30,31]. The present stuy suggests tht t mturity, B4 ws the most unnt proyniin (2.64 mg/g of see) in Cstelão Frnês (Tle 2). In Tourig Frnes the min proyniin t mturity ws B2 (1.35 mg/g see), followe y B1-3-0-gllte (0.81 mg/g see; Tle 2). On the other hn, B gllte ws present in sees of oth ultivrs in the lowest onentrtion. Riro Silv et l. [26,27] reporte tht glloylte imeri proyniins re generlly the lest unnt flvonols in the ifferent prts of the grpe luster. Oligomeri proyniins in skins. For oth vrieties, the evolution of oligomeri proyniins in skins ws quite similr to those proyniins etete in sees (Tle 3). Thus, the onentrtion of these ompouns in oth ultivrs generlly exhiite rpi erese in the first 32 ys of mturtion, fol- Tle 3 Distriution (mg/g skin) of proyniins in skins of Tourig Frnes (TF) n Cstelão Frnês (CF) grpes uring mturtion, Plmel, Portugl, July 12 July 26 July 16 August 6 Septemer TF CF TF CF TF CF TF CF B A A B B 0.08 B A A 0.01 n n n n B4 n n 0.01 B n 0.01 B n 0.02 B n Trimer A B B B 0.02 Verison te. Eh vlue represents the men from uplite smples. Sttistil signifine of the ifferenes etween smpling tes for eh omponent of eh ultivr one y Dunn s multiple rnge test. Mens within eh line (pitl letter for TF n lowerse for CF) followe y the sme letter re not signifintly ifferent (p < 0.05). n: not etete.

4 Evolution of Ctehins n Oligomeri Proyniins 233 lowe y grul erese, exept with B1 n the trimeri proyniin T2, where smll inrese in lst 30 ys of mturtion ws oserve. During the smpling perio, proyniin B4 oul not e etete in Cstelão Frnês skin extrts (Tle 3). The proyniin B3 ws etete in oth ultivrs only in the eginning of the mturtion proess. For Cstelão Frnês, the proyniin B1 ws the most unnt ompoun uring the entire mturtion perio, while for Tourig Frnes, the trimeri proyniin T2 ws the most unnt ompoun etete (from 0.08 to 0.06 mg/g of skin). Ctehins n oligomeri proyniins in stems. Chnges of flvnol monomers n oligomeri proyniins uring mturtion were oserve in oth vrieties (Tle 4). Cstelão Frnês isplye generl erese for ll oligomeri proyniins uring grpe mturtion. However, Tourig Frnes isplye more omplex evolution. Ctehin, T2, B1, C1, B gllte, n B2-3-0-gllte showe ontinul inrese uring mturtion, while B3 erese. Proyniins B2, B4, n B1-3-0-gllte onentrtions peke on 16 August. Thus, for the first 32 ys of mturtion, the vlues of these proyniins (B2, B4, n B1-3-0-gllte) exhiite rpi inrese in stems (from unetete to 0.90 mg/g, 0.26 to 1.04 mg/g, n to 1.62 mg/g, respetively) followe y strong erese (from 0.90 mg/g to 0.27 mg/g, 1.04 to 0.13 mg/g, n 1.62 to 0.28 mg/g, respetively). (+)-Ctehin gretly erese uring mturtion in oth vrieties. (-)-Epitehin in Tourig Frnes erese in the eginning of the smpling perio followe y n inrese (Tle 4). (-)-Epitehin in Cstelão Frnês ontinue to inrese throughout the smpling perio. Tle 4 Distriution (mg/g stem) of flvnols in stems of Tourig Frnes (TF) n Cstelão Frnês (CF) grpes uring mturtion, Plmel, Portugl, July 12 July 16 August 6 Septemer TF CF TF CF TF CF B A B C 1.93 B2 n B C 0.10 B A B C 0.04 B A B C 0.14 B1-3-0-gllte 0.03 A B C 0.12 B2-3-0-gllte 0.44 A B C 0.09 B gllte n B B 0.22 C A B C 1.52 Trimer A A C 8.93 (+)-Ctehin A B C 0.07 (-)-Epitehin 8.31 A B C 1.13 Verison te. Eh vlue represents the men from uplite smples. Sttistil signifine of the ifferenes etween smpling tes for eh omponent of eh ultivr one y Dunn s multiple rnge test. Mens within eh line (pitl letter for TF n lowerse for CF) followe y the sme letter re not signifintly ifferent (p < 0.05). n: not etete. For oth vrieties, B1 ws the min oligomeri proyniin in stems uring mturtion, n t tehnil mturity, it ws 6.5 n 1.93 mg/g for Tourig Frnes n Cstelão Frnês, respetively. At tehnil mturity the most unnt proyniin in oth vrieties ws the trimeri proyniin T2 (7.28 n 8.93 mg/g stem, respetively), whih onfirms other stuies [30,31,32]. The teneny for higher onentrtions of flvnols to our in the erly stges of erry evelopment is pprently typil n suggests tht these polyphenols re tively metolize throughout the growing seson. Aoring to Singleton n Esu [36], these re synthesize fter verison similrly to nthoynins, sugrs, n other grpe omponents. However, Vlero et l. [42] suggeste tht these ompouns erese uring grpe mturtion s onsequene of inrese weight of erries. Conlusions Oligomeri proyniins from sees, stems, n skins in oth Vitis vinifer L. ultivrs erese uring grpe mturtion, primrily in the first stges. However, the vlues foun for Cstelão Frnês were in generl higher thn those reporte y Riro Silv et l. [31], whih proly inites the importne of the vintge n/or the terroir. For exmple, uring 1995, the temperture in the Plmel region (south of Lison) ws on verge hotter thn norml in Portugl (in August the mximum verge temperture ws 32 C). The glloylte imeri proyniins (B1-3-0-gllte, B gllte, n B gllte) onstitute the smllest group of proyniins in sees n stems uring grpe mturtion. At tehnil mturity, Cstelão Frnês h the highest onentrtion of proyniins in sees, while Tourig Frnes grpes h the highest onentrtion in stems. Grpe sees ontine the most flvnols n so represent the est soure of oligomeri pronthoyniins for ommeril reovery. Literture Cite 1. Bourzeix, M., D. Weyln, n N. Herei. Étue es téhines et es proyniols e l grppe e risin, u vin et utres érivés e l vigne. Bull. OIV : (1986). 2. Cheng, J.T. Antihypertensitive priniples from the leves of Melstom nium. Plnt Me. 59: (1993). 3. Cheynier, V., n J. M. Riro Silv. Oxition of grpe proyniins in moel solutions ontining trnsffeoyltrtti n polyphenoloxise. J. Agri. Foo Chem. 39: (1991). 4. Chung, K.T., T.Y. Wong, L.I. Wel, n Y.W. Hung. Implitions of foo tnnins for humn helth. In Proeeings of Polyphenols Communitions 96, Boreux, Frne, pp (1996). 5. Czohnsk, Z., L.Y. Foo, n L.J. Porter. Compositionl hnges in lower moleulr weight flvns uring grpe mturtion. Phytohemistry 18: (1979). 6. Czohnsk, Z., L.Y. Foo, R.H. Newmn, n L.J. Porter. Polymeri pronthoyniins. Stereohemistry,

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