Total polyphenolic compounds contents (TPC), total antioxidant activities (TAA) and HPLC determination of individual polyphenolic compounds in selected Moravian and Austrian wines

1. In oduc ion In ecen yea s, many pape s ha e paid a en ion o he bioac i e compounds, pa icula ly o he an ioxidan ac i i y o polyphenolic compounds in ood and be e ages, due o hei posi i e e ec on he human body. As consume s ha e become mo e conscious o he heal h bene i s o phenolic compounds and hei an ioxidan ac i i ies ia he con en ional media, he be e age indus ies ha e ecognized new ma ke ing oppo uni ies o hei p oduc s. The e o e, he phenolic compounds and hei an ioxidan capaci y in oods and be e ages become an impo an quali y pa ame e , especially in niche ma ke s conce ned wi h heal h bene i s. Wine is a widely consumed be e age in he wo ld, wi h housands o yea s o adi ion. I is an excellen sou ce o a ious classes o polyphenols. The phenolic compounds a e esponsible o he senso y cha ac e is ics, pa icula ly colo , as ingency, bi e ness and a oma [1,2]. The phenolic compounds in ed wine exhibi a boa d spec um o bene icial pha macological p ope ies, belie ed o be ela ed o hei an ioxida i e p ope ies. An i-a he ogenic, an i- umou , an i-ulce , and an i-in lamma o y ac i i ies ha e all been demons a ed by he consump ion o ed wine and ed wine phenolic compounds [3-8]. As one o he winemaking p ocedu es, he phenolic compounds o he wine g ape a e one o he mos impo an aspec s ha de e mine wine quali y. A la ge numbe o published pape s ha e ocused on he essen ial con ibu ions o phenolic compounds p o iles o wine quali y and senso y p ope ies [1,2]. The phenolic p o iles in wine depend on he phenolic compounds p esen in he g apes, he ex ac ion pa ame e s, winemaking echnologies as well as e men a ion empe a u e, yeas s ain, p ocessing enzymes, cap managemen , and alcohol concen a ion [9-11]. On he o he hand, he phenolic compounds o g apes a e a ec ed by many ac o s such as ag o Cen al Eu opean Jou nal o Chemis y * E-mail: [email p o ec ed] 1Depa men o Biochemis y and Food Analysis, Tomas Ba a Uni e si y in Zlín, CZ-762 72 Zlín, Czech Republic 2Ins i u e o Chemis y, Depa men o Analy ical Chemis y, Masa yk Uni e si y in B no, CZ-62500 B no, Czech Republic Badam se seg Soyollkham1, Pa el Valášek1, Mi osla Fiše a1, Vlas imil Fic1, Vlas imil Kubáň1,2, Ignác Hoza1 To al polyphenolic compounds con en s (TPC), o al an ioxidan ac i i ies (TAA) and HPLC de e mina ion o indi idual polyphenolic compounds in selec ed Mo a ian and Aus ian wines Resea ch A icle Abs ac : © Ve si a Sp. z o.o. Recei ed 9 No embe 2010; Accep ed 23 Ma ch 2011 Keywo ds: Phenolic compounds • Wine • To al an ioxidan ac i i y • To al phenolic con en • HPLC/UV-VIS Wine samples (G üne Vel line (GV) and Zweigel (ZW) om ou di e en geog aphical egions o Aus ia and Czech Republic) we e analyzed o de e mine hei o al phenolic con en (TPC) by applying he Folin-Ciocal eau me hod, o al an ioxidan ac i i y (TAA) by FRAP ( e ic educing an ioxidan powe ) and DPPH (1,1-diphenyl-2-pic yl-hyd azyl) assays, and o iden i y and quan i y ele en phenolic compounds using a HPLC/UV-VIS me hod. Cen . Eu . J. Chem. • 9(4) • 2011 • 677-687 DOI: 10.2478/s11532-011-0045-3 677 To al polyphenolic compounds con en s (TPC), o al an ioxidan ac i i ies (TAA) and HPLC de e mina ion o indi idual polyphenolic compounds in selec ed Mo a ian and Aus ian wines echnical p ocesses, gene ic a ia ion, ma u i y, clima ic and geog aphical condi ions [12-14]. O he ac o s ha in luence he ex en o phenolic ex ac ion a e he molecula weigh , size and ype o phenolic molecules, he su ace a ea, he concen a ion g adien , o he empe a u e ea men s including g ape and mus eezing and he mo- ini ica ion, and ac o s ha a ec cell pe meabili y, such as pec oly ic enzyme selec ion [15]. Also, he en i onmen al condi ion ( empe a u e, annual p ecipi a ion le els, al i ude and geochemical cha ac e is ics) can a ec he g apes ma u a ion and consequen ly he concen a ion o hei phenolic compounds. Many pape s dealing wi h phenolic compounds o wine and g apes and hei o al an ioxidan capaci y ha e been published. Howe e , li le a en ion has been paid o compa ing he phenolic compounds o wine g apes om di e en o igins in Mo a ian wine, as well as compa ing he phenolic con en s and an ioxidan ac i i ies o phenolic compounds. Fla onoids, phenolic acids, la onols and ans- es e a ol and o he g oups o compounds could be key agen s o he an ioxidan ac ion on he human me abolism pa hway, he eason why we a e quali ying he wines om a nu i ional poin o iew. This s udy de e mines he o al con en o phenolics, iden i ies and quan i ies indi idual phenolic compounds and de e mines he o al an ioxidan ac i i y in wine samples collec ed om ou di e en geog aphical egions o Aus ia and Czech Republic ( wo wine ies in Aus ia – Poysdo and G oss ieden hal; and wo wine ies in he Czech Republic - Velké Bílo ice and Velké Hos ě ádky - Bošo ice). The s udy assesses he in luence o di e en geog aphical condi ions on he phenolic composi ion and e alua es he ela ionship be ween an ioxidan po en ial and he phenolic con en o Mo a ian and Aus ian wine. 2. Expe imen al P ocedu e 2.1. Ins umen a ion A single beam lash scan diode a ay spec opho ome e co e ing 330-800 nm Bioch om Lib a S6 (Bioch om L d, Camb idge, UK) was used o spec opho ome ic assays. The Ul iMa e® 3000 HPLC sys em consis ed o Ul iMa e 3000 RS pump, Ul iMa e 3000 RS au osample , Ul iMa e 3000 RS column compa men and Ul iMa e 3000 RS diode a ay de ec o (Dionex Co., Sunny ale, CA, USA). Ch oma og aphic sepa a ion was ca ied ou on Supelcosil LC-18-DB column (250×4.6 mm, 5 µm, Supelco, USA) a 30°C by g adien elu ion wi h a mobile phase con aining sol en A (5% / aqueous ace oni ile (ACN) acidi ied wi h 0.35 mL i luo oace ic anhyd ide (TFAA) and sol en B (50% / aqueous ace oni ile acidi ied wi h 0.25 mL TFAA). Run ime was 30 min and he low a e was 1 mL min-1. 2.2. Chemicals Folin-Ciocal eau eagen , gallic acid, 2,4,6- is-(-2- py idyl)-s- iazine (TPTZ) and 2,2-diphenyl-1-pic yl- hyd azyl (DPPH) we e ob ained om Sigma-Ald ich (S einheim, Ge many). A s anda d solu ion o DPPH c = 0.20 mol L-1 was p epa ed in me hanol. A wo king DPPH solu ion was p epa ed a c = 100 µmol L-1 con aining an ace a e bu e o pH 4.3 wi h a a io o 1:2 (DPPH:bu e ). Tannin was ob ained om Me ck KGaA (Da ms ad , Ge many). Phenolic e e ence s anda ds including gallic acid, ca echin, anillic acid, ca eic acid, p-couma ic acid, e ulic acid, sinapic acid, u in, cinnamic acid, que ce in and es e a ol we e pu chased om Ald ich (Zwijnd ech , Belgium). Lab-Scan ace oni ile (ACN) was ob ained om POCH A.S. (Gliwice, Poland). O he chemicals and eagen s o eagen g ade pu i y we e pu chased om Pen a, Ch udim and/o Lachema, B no, (bo h Czech Republic). All solu ions we e p epa ed wi h deionised (DI) wa e (Aquaosmo ic, Tišno , Czech Republic). 2.3. Sampling A o al o 32 wine samples including 16 whi e and 16 ed wines (all wines om he 2009 in age) we e collec ed be o e bo ling in Vinopol L d. (Velké Bílo ice, Czech Republic). Wines we e made om G üne Vel line (GV) and Zweigel (ZW) wine g apes g own in ou di e en geog aphical egions o Aus ia and Czech Republic; wo wine ies in Aus ia (Poysdo and G oss ieden hal) and wo wine ies in he Czech Republic (Velké Bílo ice and Velké Hos ě ádky - Bošo ice). Fou locali ies in mu ual dis ances (mo e han 40 km) we e selec ed o sampling o minimize he isks posed by wea he condi ions. The Aus ian ineya ds o he wine ies we e loca ed in wo dis ic s. Poysdo is loca ed in Aus ia’s la ges wine-g owing a ea called “Wein ie el” (16.650 ha). G oss ieden hal is embedded in he wine-g owing a ea called “Wag am” (2.800 ha). In he Czech Republic, he ineya ds a e loca ed in he Mo a ian sub-dis ic called “Velkopa lo ická“ (5.200 ha). G apes we e ha es ed in ou epe i ion uni s (see Fig. 1) om each ineya d; wine samples we e made by each epe i ion g ape samples. Table 1 shows a lis o analyzed wine samples. The clima ic condi ions o he wo wine-g owing a eas in Aus ia show nonsigni ican di e ences. Poysdo (225 m abo e sea le el) and G oss ieden hal (277 m abo e sea le el) a e a ec ed by he Pannonian clima e ha is cha ac e ized by ho d y summe s and 678 B. Soyollkham e al. cold win e s. The annual a e age empe a u e is 9.1°C and he annual p ecipi a ion is 480 mm. The geology o he egion shows ha Poysdo (embedded in he Vienna basin) and G oss ieden hal (embedded in he Molasse basin) is cha ac e ized by loess and loess- clay. The pedology o he ineya ds di e s be ween he wo wine-g owing a eas; in Poysdo he soil ype is a i gin soil and in G oss ieden hal i is a b own soil. In he Czech Republic, he ineya ds in Velké Bílo ice (185 m abo e sea le el) and Velké Hos ě ádky - Bošo ice (215 m abo e sea le el) a e a ec ed by he con inen al clima e wi h an occasional in luence o A lan ic clima e. The annual a e age empe a u e is 9.4°C and he annual p ecipi a ion is 510 mm. The geology o he wine- g owing a ea is cha ac e ized by calca eous clay, ma l and sands one. Vi gin soil cha ac e izes he soil ype o he ineya ds . 2.4. Sampling s a egy and wine making p ocedu es Sample p ocedu es we e pe o med in acco dance wi h he ollowing c i e ia in di e en geog aphical egions o he expe imen al ields: i) 400 g ape ines pe expe imen al ield, ii) 4 epe i ion uni s, iii) c op age 6 – 25 yea s, i ) dis ance be ween ows 2 – 2.5 m, ) applica ion o ull mechaniza ion and iennial dep h-loosene , i) b eak ou o g ape ines 8 – 10 pe in age. Acco ding o a s anda d winemaking p ocedu e, he e is no wood con ac in he expe imen al cella o Vinopol. A e c ushing 100 kg o g apes, diammonium phospha e, SO2 and Saccha omyces ce e isiae s ain we e added o each wine . The g ape skin was sepa a ed om he juice using s anda d p essing p ocedu e. P essed juice was added o he 50-L glass bo les and e men ed by s anda d p ocedu e a 8°C, and he cap was punched down wo imes pe day. Wines we e cold- s abilized o se e al weeks a 8°C and il e ed using ce amic il e s. 2.5. Me hods 2.5.1. Folin-Ciocal eau me hod The TPC was de e mined acco ding o he Folin- Ciocal eau me hod [16]. B ie ly, a 0.025 mL sample was mixed wi h 1 mL o he 10- old dilu ed Folin-Ciocal eau eagen and allowed o s and o 3 min. Then 5 mL o 200 g L-1 sodium ca bona e (Na2CO3) was added, and a inal olume was made up o 50 mL wi h DI wa e . Each sample was measu ed spec opho ome ically a 765 nm a e 30 min o s anding agains he blank. Fi e-poin calib a ion was s ic ly linea (R2>0.9999) in he concen a ion ange 0 - 250 mg L-1 wi h annin as he s anda d. The eg ession equa ion was A = 0.0018 c + 0.0028. The de e mined alues we e exp essed as annin equi alen s (TE, mg L-1). All samples we e analyzed as iplica es. S anda ds and samples gleaned highly epea able esul s. 2.5.2. DPPH adical sca enging ac i i y [17,18] A mix u e o an undilu ed sample (0.1 mL) wi h a 10 mL wo king DPPH solu ion was measu ed immedia ely a 515 nm agains a me hanol blank (AC(0) ). The mix u e was hen incuba ed a oom empe a u e and in he da k o 30 minu es and was again measu ed spec opho ome ically a 515 nm (AA( )). The gallic acid (GA) calib a ion cu e was plo ed as a unc ion o he pe cen age o he DPPH adical sca enging ac i i y. The measu emen was compa ed o he s anda d calib a ion cu e, and he ee adical sca enging ac i i ies we e exp essed as mic omoles o gallic acid equi alen s (GAE) pe millili e o sample (µmol mL-1). The calib a ion cu e was s ic ly linea (A = 855.59, c = 16.015, R2 = 0.9980, whe e A is abso bance alue, c is he concen a ion o gallic acid in s anda d solu ions) in he concen a ion in e al 0.02 – 0.08 µmol mL-1 gallic acid. The µmol mL-1 inhibi ion o he DPPH adical caused by a wine sample we e de e mined acco ding o he ollowing o mula: (AC(0) – AA( ))/AC(0) × 100, whe e AC(0) is he abso bance o he sample a = 0 min and AA( ) is he abso bance o sample a = 30 min). All samples we e analyzed as iplica es. Table 1. Lis o analysed wine samples Sample Code* Type o wine Vineya ds (dis ic ) Wine ies SGV Whi e Velkopa lo ická Velké Bílo ice SZW Red PGV Whi e Velkopa lo ická Velké Hos ě ádky - Bošo ice PZW Red OGV Whi e Wein ie el Poysdo OZW Red BGV Whi e Wag am G oss ieden hal BZW Red * GV – G uene Vel line , ZW – Zweigel , a ia ions 1 – 4 acc. Fig. 1 we e collec ed Figu e 1. Plan o he expe imen al ields o sampling om indi idual expe imen al ineya ds in Czech Republic (S, P) and Aus ia (O, B). 679 To al polyphenolic compounds con en s (TPC), o al an ioxidan ac i i ies (TAA) and HPLC de e mina ion o indi idual polyphenolic compounds in selec ed Mo a ian and Aus ian wines 2.5.3. Fe ic ion educing an ioxidan powe (FRAP) The educing ac i i y o he samples was de e mined by he FRAP me hod [19]. A 0.1 µmol L-1 s anda d solu ion o gallic acid (GA) was p epa ed in H2O. The oxidan in he FRAP assay was p epa ed by mixing 5 mL o 10 mmol L-1 2,4,6- ipy idyl-s- iazine (TPTZ) in wa e , 50 mL o ace a e bu e pH 3.6, and 5 mL o FeCl3•H2O (20 mmol L-1). A sample (25 µL) was added o he 4 mL eagen . Abso bance was measu ed spec opho ome ically a 593 nm (A0min). Then he sample solu ion was allowed o s and a oom empe a u e and in he da k o 10 min and measu ed again a 593 nm (A10min). The di e ence o abso bances (ΔA = A10min - A0min) o he eac ion mix u e was calcula ed and ela ed o ΔA o a Fe(II) s anda d solu ion. The di e ence in abso bance ΔA was linea ly p opo ional o he concen a ion o he an ioxidan and indica ed inc eased educing powe . The measu emen was compa ed o a calib a ion cu e o he p epa ed gallic acid solu ion, and hen inal esul s we e exp essed as mic omoles o gallic acid equi alen s (GAE) pe millili e o he sample (µmol mL-1). The calib a ion cu e was s ic ly linea (A = 1.0800 c + 0.0072, R2 = 0.9999, whe e A is he abso bance alue, c is he concen a ion o gallic acid in s anda d solu ions) in he concen a ion in e al 0.02 – 0.1 µmol mL-1 gallic acid. All samples we e analyzed as iplica es. 2.5.4. HPLC analysis o phenolic composi ion The indi idual phenolic compounds we e quan i ied using a HPLC me hod [20] using g adien elu ion wi h he mobile phase con aining sol en A (5% / aqueous ACN acidi ied wi h 0.35 mL i luo oace ic anhyd ide (TFAA) and sol en B (50% / aqueous ACN acidi ied wi h 0.25 mL TFAA). The UV de ec o was se a 205, 210, 275 and 375 nm. The wine sample was il e ed using 0.45 µm po e size Nylon memb ane il e 13 mm (FFNN1345-100, G onus, SMI labHu L d., Maisemo e, Glouces e shi e, UK) using il e de ices (Millipo e, Bed o d, MA, USA) be o e injec ing. The injec ion olume was 20 µL. Indi idual phenolic compounds we e iden i ied by compa ing e en ion imes and UV spec a o he co esponding s anda d compounds. Da a we e quan i ied using he co esponding calib a ion cu es o he indi idual s anda d compound. 3. Resul s and Discussion In his s udy, a o al o 32 wine samples including 16 whi e and 16 ed wines, which we e made om he G üne Vel line and Zweigel g ape a ie ies, we e selec ed o de e mine he o al phenolic con en s (TPC) and he o al an ioxidan ac i i y (TAA). G üne Vel line is a a ie y o whi e wine g ape g own p ima ily in Aus ia and in he Czech Republic. Zweigel is a ed wine g ape a ie y ha is he mos widely g own in Aus ia oday. 3.1. To al phenolic con en s The di e en a ia ions o ed and whi e wine samples we e es ed o TPC in ou se s o analyses. The TPC a ied om 218 o 328 mg L-1, a e aging 263 mg L-1, o he ou whi e wine samples SGV and om 1182 o 1232 mg L-1, a e aging 1216 mg L-1, o he ou ed wine samples SZW om Velké Bílo ice. The o al phenolic con en s anged om 268 o 283 mg L-1, a e aging 274 mg L-1 o PGV samples and om 564 o 729 mg L-1, a e aging 651 mg L-1 o ed wine samples PZW om Velké Hos ě ádky - Bošo ice. Samples PGV-3 and PZW-3 showed a high con en o o al phenolics; he same as SGV-3 and SZW-3. I can be assumed ha he high eading o he phenolic con en s o he g ape samples depends on he loca ion o he ineya d whe e he g apes g ew, hei shel e om he wind, in ensi y o sunligh adia ion as well as shaded o non shaded clus e s and o he ac o s. In he nex se o analyses, wine samples om Aus ian egions we e es ed ( ou ed wines and ou whi e wines). The TPC a ied om 260 o 304 mg L-1, a e aging 275 mg L-1, o he whi e wine samples OGV and om 824 o 878 mg L-1, a e aging 846 mg L-1, o he ed wine samples OZW collec ed in Poysdo . The co esponding alues om 117 o 210 mg L-1, a e aging 179 mg L-1, in whi e wine BGV, and om 1068 o 1184 mg L-1, a e aging 1122 mg L-1 TE, in ed wine BZW om G oss ieden hal we e ob ained. In his se , he highes TPC o he ed wines was ound in OZW-4 and o he whi e wines was ound in OGV-3 samples. Figs. 2 and 3 show (see also Table 2) he TPC in whi e and ed wine samples. The mean alues o he o al phenolic con en s in whi e wine samples we e de e mined in he in e al om 263 o 275 mg L-1 TE, excep o he BGV (179 mg L-1 TE). Ou alues o whi e wines we e abou 50% highe compa ed o he esul s published by Simone i e al. [21] and app oxima ed he alues published by Komes e al. [22], Sánche - Mo eno e al. [23], Heinonen e al. [24], Jewell [25] and S e ana o e al. [26]. The TPC o eigh whi e wines om Sou h Mo a ia we e de e mined by S a il e al. [27] and alues o TPC we e app oxima ely 40-60 % lowe han ou esul s. Fig. 2 shows he an ioxidan ac i i y o ou di e en a ia ions g own in ou di e en ineya ds, a o al o 16 whi e wine samples. As shown in his igu e, he highes TPC we e ound o wine samples O (275 mg L-1) g own in Wein ie el, Aus ia and P (274 mg L-1) g own in Velké Hos ě ádky - Bošice, Czech Republic. 680 B. Soyollkham e al. The a e age alues o TPC in ed wine samples anged om 651 (PZW) o 1216 (SZW) mg L-1 TE. These esul s we e in he ange o he p e iously summa ized [27-29] da a (824-4059 mg L-1) by C ozie e al. [28], excep o he PZW samples (564-729 mg L-1). Mo eo e , TPC in ed wine we e app oxima ely 40-75% lowe compa ed o he esul s published by selec ed au ho s [21-28,30,31]. The TPC o 16 ed wine samples we e compa ed wi h each o he . The esul s a e shown in Fig. 3. The highes TPC we e de e mined o ed wine samples S (mean alue o ou a ia ions, 1216 mg L-1) g own in Velkopa lo ická, he Czech Republic and o ed wine samples B (mean alue o ou a ia ions, 1122 mg L-1) g own in Wag am, Aus ia. Howe e , he ed wine samples showed a low con en o o al phenolics o wine samples O which we e g own in Wein ie el, Aus ia. Gene ally, he o al phenolic con en s o whi e wine samples we e obse ed a ela i ely high le els (117- 328 mg L-1), bu hey we e s ill a leas 5- imes lowe han he ed wines (564-1216 mg L-1). In he whi e wine samples, he lowes TCP (117 mg L-1) was de e mined in wine samples B g own in Wag am, bu in con adic ion, o ed wine samples he high con en was ound in wine samples B g own in he same egion. These esul s indica ed ha he geog aphical o igin, a e age annual empe a u e, annual le els o p ecipi a ion and pedology s ongly in luenced he wine quali y and quan i y. 3.2. An ioxidan ac i i y o wines The o al an ioxidan ac i i y alues o he wine samples, using he DPPH and FRAP me hods, a e gi en in Figs. 4 and 5. Wine samples showed high TAA, mainly hose wi h high le els o o al phenolic con en s. Acco ding o he FRAP alues de e mined in whi e wines, he TAA can be quali ied in dec easing o de (GAE mmol L-1): OGV-3 (0.50) ~ OGV-4 (0.50) > PGV-3 (0.43) ~ SGV 3 (0.43). These esul s indica ed ha OGV and SGV ha e a highe an ioxidan ac i i y han PGV and BGV whi e wines. The highes an ioxidan ac i i y was ound in BGV-2 and BGV-1 ed wines (3.8-3.5 mmol L-1, i.e., 7 o 11- imes highe han ha in whi e wines). The lowes alue, less han hal o he highes alue, was ound in PZW-4 ed wine (1.4 mmol L-1). The a e age an ioxidan ac i i y alues in each ineya d de e mined by he FRAP me hod we e 0.43 (SGV), 0.4 (PGV), 0.49 (OGV) and 0.38 (BGV) in whi e wines, mo eo e , 2.9 (SGV), 1.53 (PGV), 1.73 (OGV) and 3.46 (BGV) mmol L-1 in ed wines, espec i ely. Fu he mo e, acco ding o he DPPH a e age alues de e mined in whi e wines, he o al an ioxidan ac i i y can be quali ied in his dec easing o de (GAE mmol L-1): PGV (0.35) > OGV (0.32) > BGV-3 (0.3) > SGV (0.29). These esul s we e in disag eemen wi h he alues o he highes an ioxidan ac i i y in whi e wines which we e de e mined by FRAP me hod. Howe e , esul s o he highes an ioxidan ac i i y in ed wine was ound in BGV-2 ed wine (2.01 mmol L-1, i.e., 5- o 8- imes highe han ha in whi e wines). Also,, he lowes alue, less han hal o he highes alue, was ound in he PZW and OZW ed wine samples (0.67 and 0.68 mmol L-1). These esul s we e in ag eemen wi h he alues de e mined by he DPPH me hod. The mean alues o he an ioxidan ac i i ies in each ineya d de e mined by he DPPH me hod we e 0.43 (SGV), 0.4 (PGV), 0.49 (OGV) and 0.38 (BGV) mmol L-1 in whi e wines, mo eo e , 2.9 (SGV), 1.53 (PGV), 1.73 (OGV) and 3.46 (BGV) mmol L-1 in ed wines, espec i ely. I was di icul o compa e ou alues o he TAA wi h he li e a u e da a. The majo i y o au ho s ha e used a ious me hods such as inhibi ion o lipid oxida ion, he ORAC me hod, he ABTS me hod, he DPPH me hod wi h he e alua ion o EC50 ( he sample concen a ion necessa y o educe he emaining DPPH by 50%). On he o he hand, i is possible o pa ially e alua e, some o he alues de e mined by he FRAP, DPPH me hods using he T olox s anda d, e.g. S a il e al. [27]. Fo he DPPH me hod, alues we e de e mined Figu e 3. To al phenolic con en s in 16 ed wines om indi idual expe imen al ineya ds in Czech Republic (S, P) and Aus ia (O, B). SDs alues see Table 2. Figu e 2. To al phenolic con en s in 16 whi e wine samples om indi idual expe imen al ineya ds in Czech Republic (S, P) and Aus ia (O, B). SDs alues see Table 2. 681 To al polyphenolic compounds con en s (TPC), o al an ioxidan ac i i ies (TAA) and HPLC de e mina ion o indi idual polyphenolic compounds in selec ed Mo a ian and Aus ian wines Table 2. Phenolic compounds Sample No Whi e wines Red wines SGV PGV OGV BGV SZW PZW OZW BZW 15.70 ± 0.00 5.79 ± 0.01 5.53 ± 0.01 5.71 ± 0.02 14.5 ± 0.03 8.93 ± 0.01 14.2 ± 0.02 16.8 ± 0.08 25.66 ± 0.00 5.67 ± 0.31 6.08 ± 0.01 6.01 ± 0.02 14.7 ± 0.01 9.46 ± 0.02 12.9 ± 0.02 17.4 ± 0.04 Gallic acid 35.71 ± 0.00 5.80 ± 0.05 5.83 ± 0.01 5.44 ± 0.02 14.2 ± 0.05 9.65 ± 0.08 14.4 ± 0.02 16.8 ± 0.04 45.04 ± 0.31 5.71 ± 0.01 6.19 ±0.01 5.36 ± 1.00 14.2 ± 0.05 9.23 ± 0.01 14.1 ± 0.02 16.9 ± 0.02 A e age 5.52 ± 0.32 5.74 ± 0.06 5.90 ± 0.29 5.63 ± 0.29 14.4 ± 0.23 9.31 ± 0.31 13.9 ± 0.68 16.9 ± 0.30 15.01 ± 0.03 9.90 ± 0.0 4.63 ± 0.01 6.46 ± 0.07 23.5 ± 14.85 23.5 ± 0.03 25.6 ± 0.04 20.2 ± 0.28 28.08 ± 0.02 10.57 ± 0.01 4.21 ± 0.01 8.38 ± 0.05 38.7 ± 0.01 13.8 ± 0.02 25.0 ± 0.01 22.8 ± 0.05 Ca echin 310.11 ± 0.07 11.44 ± 0.76 6.21 ± 2.23 7.00 ± 0.03 29.7 ± 0.46 24.9 ± 0.47 23.9 ± 0.04 25.1±0.82 46.77 ± 0.02 10.69 ± 0.09 6.79 ± 0.03 6.63 ± 0.04 29.7 ± 0.46 24.1 ± 0.02 24.8 ± 0.03 24.7 ± 0.04 A e age 7.49 ± 2.15 10.65 ± 0.63 5.46 ± 1.23 7.11 ± 0.87 30.4 ± 6.24 21.5 ± 5.21 24.8 ± 0.70 23.2 ± 2.24 11.02 ± 0.02 - - 1.02 ± 0.02 3.11 ± 0.02 - 1.68 ± 0.01 1.14 ± 0.01 20.93 ± 0.01 - 0.99 ± 0.03 0.75 ± 0.0 2.71 ± 0.03 1.25 ± 0.02 1.62 ± 0. 0 1.11 ± 0.0 Vanillic acid 30.72 ± 0.00 - 1.19 ± 0.01 - 2.36 ± 0.00 1.28 ± 0.04 1.68 ± 0.02 1.45±0.02 40.81 ± 0.04 - - 0.88 ± 0.0 2.36 ± 0.00 1.37 ± 0.01 1.64 ± 0.02 1.09 ± 0.01 A e age 0.87 ± 0.13 - 1.09 ± 0.14 0.88 ± 0.13 2.63 ± 0.35 1.3 ± 0.06 1.65 ± 0.03 1.19 ± 0.16 1ND 0.14 ± 0.01 0.13 ± 0.01 ND 2.81 ± 0.21 1.40 ± 0.01 2.35 ± 0.01 0.56 ± 0.0 2ND - 0.19 ± 0.02 0.60 ± 0.02 2.64 ± 0.01 0.75 ± 0.01 1.53 ± 0.01 0.49 ± 0.0 Ca eic acid* 3- 2.10 ± 0.39 3.58 ± 0.0 0.61 ± 0.01 2.34 ± 0.17 1.68 ± 0.01 2.28 ± 0.01 0.52±0.004 40.05 ± 0.00 - 0.23 ± 0.01 0.34 ± 0.01 2.34 ± 0.17 10.4 ± 0.33 2.13 ± 0.01 4.08 ± 0.01 A e age - - 1.03 ± 1.69 0.51 ± 0.15 2.53 ± 0.23 3.6 ± 4.59 2.07 ± 0.37 1.41 ± 1.77 12.26 ± 0.00 - 2.31 ± 0.13 ND 4.05 ± 0.00 2.40 ± 0.01 2.92 ± 0.02 ND 22.31 ± 0.00 - - ND 4.41 ± 0.00 2.40 ± 0.01 2.91 ± 0.01 ND Fe ulic acid 3- - - ND 4.04 ± 0.08 2.42 ± 0.01 2.92 ± 0.15 ND 42.27 ± 0.00 - - ND 4.04 ± 0.08 2.42 ± 0.01 2.86 ± 0.01 ND A e age 2.28 ± 0.02 - . - 4.13 ± 0.18 2.41 ± 0.01 2.90 ± 0.02 ND 12.56 ± 0.01 2.71 ± 0.01 2.58 ± 0.02 2.71 ± 0.08 7.09 ± 0.06 3.65 ± 0.01 5.23 ± 0.05 4.25 ± 0.05 22.48 ± 0.00 2.52 ± 0.01 2.50 ± 0.02 2.62 ± 0.01 9.23 ± 0.04 5.22 ± 0.01 5.28 ± 0.03 5.58 ± 0.01 Sinapic acid 32.47 ± 0.02 2.59 ± 0.01 2.49 ± 0.14 2.74 ± 0.01 3.94 ± 0.02 3.03 ± 0.00 4.92 ± 0.03 4.18±0.10 42.50 ± 0.00 2.56 ± 0.04 2.63 ± 0.01 7.40 ± 1.20 2.76 ± 0.01 5.36 ± 0.01 4.19 ± 0.05 A e age 2.50 ± 0.04 2.61 ± 0.01 2.53 ± 0.04 2.67 ± 0.05 6.91 ± 0.19 3.66 ± 1.10 5.19 ± 0.19 4.55 ± 0.68 13.43 ± 0.01 3.29 ± 0.01 3.56 ± 0.01 3.37 ± 0.00 3.95 ± 0.01 10.4 ± 0.08 6.93 ± 0.01 5.34 ± 0.01 23.44 ± 0.01 3.32 ± 0.01 3.46 ± 0.02 3.45 ± 0.01 5.12 ± 0.00 9.49 ± 0.09 3.83 ± 0.01 5.82 ± 0.01 Ru in 33.64 ± 0.01 3.32 ± 0.01 3.41 ± 0.17 3.41 ± 0.04 3.94 ± 0.02 7.19 ± 0.09 3.56 ± 0.01 6.11±0.02 43.40 ± 0.00 3.31 ± 0.01 3.50 ± 0.01 3.49 ± 0.01 3.95 ± 0.02 8.84 ± 0.04 4.51 ± 1.61 7.25 ± 0.05 A e age 3.47 ± 0.01 3.31 ± 0.01 3.48 ± 0.06 3.43 ± 0.05 4.24 ± 0.58 8.97 ± 1.35 4.51 ± 1.61 6.13 ± 0.81 1- ND 0.93 ± 0.02 0.86 ± 0.01 1.19 ± 0.00 0.84 ± 0.01 1.00 ± 0.01 1.42 ± 0.01 2- ND ND 0.56 ± 0.48 0.86 ± 0.00 0.85 ± 0.00 0.96 ± 0.02 1.38 ± 0.03 Res e a ol 30.96 ± 0.01 - 0.93 ± 0.01 - 0.97 ± 0.14 - 1.05 ± 0.002 1.29±0.01 40.84 ± 0.01 - 0.88 ± 0.03 - 0.97 ± 0.14 - 0.98 ± 0.01 1.32 ± 0.0 A e age 0.90 ± 0.08 - 0.91 ± 0.02 0.71 ± 0.21 0.99 ± 0.13 0.84 ± 0 0.99 ± 0.03 1.35 ± 0.05 1- ND ND 4.90 ± 0.17 ND ND 2- ND ND 4.57 ± 0.09 ND ND Cinnamic acid 3- - ND 4.66 ± 0.02 ND ND 4- ND ND 4.18 ± 0.57 - ND A e age - - - 4.57 ± 0.29 - ND ND 15.47 ± 0.04 5.43 ± 0.51 5.14 ± 0.32 5.71 ± 0.02 3.63 ± 0.01 3.58 ± 0.00 3.57 ± 0.03 3.65 ± 0.01 25.20 ± 0.14 5.13 ± 0.28 5.01 ± 0.18 6.01 ± 0.02 3.59 ± 0.00 3.57 ± 0.02 3.62 ± 0.02 3.67 ± 0.01 Que ce in** 35.02 ± 0.16 9.39 ± 0.61 4.92 ± 0.17 5.44 ± 0.02 2.14 ± 0.00 2.04 ± 0.00 3.56 ± 0.001 3.63±0.003 45.05 ± 0.01 4.63 ± 0.01 4.69 ± 0.09 5.36 ± 1.00 2.14 ± 0.01 3.57 ± 0.00 3.59 ± 0.004 3.69 ± 0.0 A e age 5.18 ± 0.21 6.14 ± 2.18 4.94 ± 0.18 5.63 ± 0.29 2.87 ± 0.84 3.19 ± 0.76 3.58 ± 0.02 3.66 ± 0.02 *ca eic acid – de ec ed a 275 nm, **que ce in - de ec ed a 375 nm, p-couma ic acid was no de ec ed in any sample, ND – no de ec ed Con en o phenolic compounds in wine samples de e mined by HPLC in G uene Vel line (GV) and Zweigel ebe (ZW) o ou ineya ds (P, S, O B) in Czech Republic (P, S) and Aus ia (O, B) collec ed om 4 di e en sampling loca ions in each ineya d measu ed in iplica es. 682 B. Soyollkham e al. wi h in e als app oxima ely 0.6-2.9 mmol L-1 o whi e wines and alues wi h in e al app oxima ely 3-9 mmol L-1 (TE) o ed wines. These alues we e abou 3-15 imes highe han ou alues, and, o he FRAP me hod, ou alues we e lowe by 30-90% in ed wines, and 50-80% in whi e wines, espec i ely [27]. Bee e al. [32] p esen ed he alues o DPPH in he ange om 0.8-1.06 mmol L-1 TE o whi e wines. Ou alues we e lowe han one hal compa ed o hese alues. The di e ences could be caused by he di e en s anda d used in he expe imen s. 3.3. Indi idual phenolic con en The ollowing compounds we e iden i ied in he wine samples (see Fig. 6): he phenolic acids (i.e., gallic acid, anillic acid, ca eic acid, p-couma ic acid, e ulic acid, sinapic acid and cinnamic acid), ca echin, es e a ol, que ce in and u in. Con en s o he indi idual phenolics in wines om ou di e en wine-p oducing sub- egions o he Czech Republic and Aus ia a e epo ed in Table 2. Figu e 4. To al an ioxidan ac i i ies in whi e wine samples de e mined by he DPPH and FRAP me hods (GAE mmol L-1, SDs see Table 2) Figu e 5. To al an ioxidan ac i i ies in ed wine samples de e mined by he DPPH and FRAP me hods (GAE mmol L-1, SDs see Table 2) 683 To al polyphenolic compounds con en s (TPC), o al an ioxidan ac i i ies (TAA) and HPLC de e mina ion o indi idual polyphenolic compounds in selec ed Mo a ian and Aus ian wines The esul s ob ained con i med a a ia ion in he phenolic con en s among wines es ed due o hei di e en geog aphical o igin. A compa ison o hese esul s wi h li e a u e alues was di icul because o he sample p epa a ion and because he ch oma og aphic condi ions signi ican ly di e ed. Mo eo e , he con en da a o indi idual phenolics we e limi ed o a ew compounds and samples in his s udy. Howe e , he ob ained esul s we e in ag eemen wi h he alues epo ed in li e a u e. Gallic acid was he mos abundan phenolic compound (mean 5.69 mg L-1) in whi e wines; he highes le el (6.19 mg L-1) was ound in OGV-4 sample om he Wein ie el ineya d, while he lowes amoun (5.04 mg L-1) o gallic acid was ound in SGV-4 sample om he Velkopa lo ická ineya d. Resul s we e compa ed o he p e iously published da a by Malo aná e al. [33], Ras ija e al. [16], and Komes e al. [22], wi hin he concen a ion ange o gallic acid ( om 5.16 o 28.3 mg L-1) de e mined in samples om he Cana y Islands, (0.7-8.4 mg L-1) ound in samples om C oa ia and 2.63 mg L-1 om Zago je, espec i ely. Gallic acid (mean 13.1 mg L-1) in ed wine was 3- o 5- imes and 5 imes lowe han he esul s published o Tu kish and I alian wines, espec i ely [34,35]. Ca echin, wi h a mean concen a ion o 7.6 mg L-1, was he second mos abundan phenolics in whi e wines and wi h 24.5 mg L-1 also in ed wines; his was om 3- o 10- imes highe han he C oa ian wines esul s (mean 2.86 mg L-1) and simila o (mean 25.1 mg L-1) he esul s o Tu kish ed wines, espec i ely [34,35]. The highes amoun o anillic acid was ound (in a e age 2.64 mg L-1) o SZW ed wine and he lowes was (a e age alue 0.87 mg L-1) o SGV (whi e wine). High alues o anillic acid, anging om 4.66 o 5.22 mg L-1, we e de ec ed in some ed wines om Tu kish egions [34]. Mo eo e , low alues o his acid, anging om 0.05 o 0.28 mg L-1, we e ound in Spanish wines [36]. Ca eic acid anged om 0.01 o 10.4 mg L-1 in whi e and ed wines. These esul s we e simila o esul s in Spain wines (4.09 mg L-1) and I alian ed wines ( anged om 2.5 o 17.9 mg L-1), espec i ely [35,36]. p-Couma ic acid and cinnamic acids we e de ec ed excep ionally in some samples in much lowe amoun s, bu i was di icul o quan i y hei exac concen a ions. The a e age alues o e ulic acid anged om 2.28 o 2.31 mg L-1 in whi e wines and 2.41 - 4.13 mg L-1 in ed ones. These esul s aligned wi h he esul s by Komes e al. [22], ( anged om 1.88 o 3.2 mg L-1). Mean concen a ions o sinapic acid we e 2.55 mg L-1 in whi e wines and 5.07 mg L-1 in ed wines, espec i ely. Amoun s o u in and que ce in anged om 3.29 o 10.4 mg L-1 and om 2.04 o 9.39 mg L-1 in whi e and ed wines, espec i ely. Ou esul s we e in ag eemen wi h alues ob ained by Malo aná e al. [33] and Ras ija e al. [16]. Res e a ol, a compound wi h mul iple heal h bene i s, was ound in all wine samples, excep o PGV. Amoun s we e compa able wi h he concen a ion anges ound in he li e a u e [16,37]. The o al phenolic con en signi ican ly co ela ed wi h he an ioxidan ac i i y and con en s o indi idual phenolic compounds in he wines in es iga ed (see Fig. 7 as an example) i he spec um o he phenolics ( hei ela i e abundance) and ma ix o a sample we e simila . Fo ins ance, he o al con en s o Figu e 6. HPLC-UV-VIS DAD ch oma og am o mix u e o polyphenols s anda ds a 210 nm, Supelcosil LC-18-DB column, 30°C, g adien elu ion: sol en A (95% ( / ) ace oni ile acidi ied wi h 0.35 mL TFAA) and sol en B (50% ( / ) aqueous ace oni ile acidi ied wi h 0.25 mL TFAA), injec ion olume 10 µL, low a e o 1 mL min-1. 684 B. Soyollkham e al. phenolic compounds and he an ioxidan ac i i y (DPPH me hod) alues o S a ia ions g own in Velkopa lo ická, P a ia ions g own in Velké Hos ě ádky - Bošo ice, O a ia ions g own in Wein ie el and B a ia ions g own in Wag am showed a good co ela ion. These esul s ag ee wi h he ela ionship be ween he o al phenolic con en and he o al an ioxidan po en ial alues o wines (R2 = 0.9876) by Minussi e al. [30]. Mo eo e , in SGV and SZW, he o al an ioxidan ac i i ies o wines in es iga ed co ela ed well wi h gallic acid (R2 = 0.95), ca echin (R2 = 0.81), anillic acid (R2 = 0.97), e ulic acid (R2 = 0.94), sinapic acid (R2 = 0.73), u in (R2 = 0.56), que ce in (R2 = 0.62) and es e a ol (R2 = 0.39), espec i ely. In PGV and PZW, he o al an ioxidan ac i i ies o wines in es iga ed well co ela ed wi h gallic acid (R2 = 0.98), ca echin (R2 = 0.74), sinapic acid (R2 = 0.37), u in (R2 = 0.93) and que ce in (R2 = 0.35), espec i ely. In OGV and OZW, he o al an ioxidan ac i i ies o wines in es iga ed co ela ed wi h gallic acid (R2 = 0.97), ca echin (R2 = 0.98), anillic acid (R2 = 0.99), ca eic acid (R2 = 0.44), u in (R2 = 0.19), que ce in (R2 = 0.88) and es e a ol (R2 = 0.22), espec i ely. In BGV and BZW, he o al an ioxidan ac i i ies o in es iga ed wines co ela ed wi h gallic acid (R2 = 0.95), ca echin (R2 = 0.91), anillic acid (R2 = 0.37), sinapic acid (R2 = 0.91), u in (R2 = 0.92), que ce in (R2 = 0.74) and es e a ol (R2 = 0.82), espec i ely. Ou esul s we e in ag eemen wi h some in es iga ions p esen ed in he li e a u e. Acco ding o F ankel e al. [38], he ela i e an ioxidan ac i i y o 20 selec ed Cali o nian wines co ela ed wi h o al con en s o phenolics in wines (R2 = 0.94), and concen a ion o gallic acid (R2 = 0.92), ca echin (R2 = 0.75), que ce in (R2 = 0.68), ca eic acid (R2 = 0.63) and u in (R2 = 0.50). Sánchez-Mo eno e al. [39] obse ed ha he ee adical-sca enging ac i i y o gallic acid was he highes ; annic acid, ca eic [acid, que ce in and u in ac i i ies we e in e media e and e ulic acid and es e a ol we e he lowes . 4. Conclusions The highes TPC alues we e ound o ed wine samples (1230 mg L-1) g own in Velkopa lo ická, Czech Repuiblic. The lowes TPC alue (564 mg L-1) was ound o ed wine samples g own in Wein ie el (app oxima ely hal o he alue), Aus ia.The TPC alues o whi e wine samples we e ela i ely high bu se e al imes lowe han compa ed o ed wines. The lowes TPC alue was ound in he whi e wine g own in Wag am in con as o he ed wine samples om he same egion. The highes TAA by he FRAP me hod was ound in ed wines (3.5 - 3.8 mmol L-1, i.e., 7 o 11- imes highe han ha in whi e wines) while he lowes TAA alue (1.41 mmol L-1), lowe han hal o he highes alue, was ound in a ed wine g own in Velké Hos ě ádky - Bošo ice. The highes TAA by applying he DPPH me hod was ound in a ed wine (2.0 mmol L-1, i.e., s ill 5- o 8- imes highe han ha ound in whi e wines). Gallic acid was he mos abundan phenolic compound (mean 5.7 mg L-1) in whi e wines; he highes le el (6.2 mg L-1) was ound in a whi e wine sample om Wein ie el, while he lowes amoun (5.04 mg L-1) o gallic acid was ound in a sample om he Velkopa lo ická egion. Ca echin, wi h a mean concen a ion o 7.6 mg L-1, was he second mos abundan phenolics in whi e wines, and wi h 24.5 mg L-1, also in ed wines. The highes concen a ion o anillic acid was ound (in a e age 2.6 mg L-1) o ed wines and he lowes was (a e age alue 0.9 mg L-1) o SGV whi e wines. The ca eic acid concen a ion anged om 0.01 o 10.4 mg L-1 in whi e and ed wines. p-couma ic acid and cinnamic acids we e de ec ed in some samples in much lowe amoun s bu i was no possible o exac ly quan i y hei concen a ions. The a e age concen a ions o e ulic acid and sinapic acid we e 2.3 mg L-1 and 2.6 mg L-1 in whi e wines and 2.4 - 4.1 mg L-1 and 5.1 mg L-1 in ed wines, espec i ely. Amoun s o u in and que ce in anged om 3.3 o 10.4 mg L-1 and om 2.0 o 9.4 mg L-1 in whi e and ed wines, espec i ely. Acco ding o he esul s, i was concluded ha i) he o al con en o phenolics and o al an ioxidan ac i i y/ o al an ioxidan capaci y and he concen a ion o phenolic compounds could be a indica o o possible iden i ica ion o wines’ geog aphical o igin, ii) he o al con en s o phenolic compounds signi ican ly co ela ed wi h he an ioxidan ac i i y and he con en s o indi idual phenolic compounds i he spec um o phenolics ( ela i e abundance) and he ma ix we e simila , iii) he gallic acid was he mos abundan compound; annic acid, ca eic acid, que ce in and u in ac i i ies we e in e media e; and he e ulic acid and es e a ol showed he lowes in luence on he ee adical-sca enging ac i i y. Figu e 7. Rela ionship be ween he o al polyphenolic compounds con en s (TPC) and an ioxidan ac i i y (TAA) by DPPH me hod alues o SGV and SZW (as an example). (♦) whi e wine (●) ed wine 685