Phenolic Contents and Compositions in Skins of Red Wine Grape Cultivars among Various Genetic Backgrounds and Originations

In order to analyze and compare the phenolic characteristics of red wine grapes with diverse genetic backgrounds, skin phenolics among 21 different cultivars belonging to Vitis vinifera L., East Asian and North American Vitis species and hybrids, as well as 2 varieties of muscadine grapes were estimated by HPLC-MS/MS. There were 45 anthocyanins, 28 flavonols, 8 flavan-3-ols, 9 cinnamic acids, 5 benzoic acids, 5 ellagic acids and 2 stilbenes detected in all the samples. Total contents of each phenolic type varied significantly among the different grape cultivars investigated. There was also a large variability in the phenolic compositions of different grape groups. The differences in anthocyanin composition were obvious between V. vinifera and non-V. vinifera grapes and also between the grapes originating from Eurasia and North America. Quercetin-3-glucuronide and quercetin-3-glucoside were marker flavonol compounds for Euvitis grape skins. Flavan-3-ol monomers were dominant in the skins of muscadine and non-V. amurensis East Asian grapes, whereas polymers were more common in V. vinifera and North American grapes. The muscadine grapes were very rich in flavonols, flavan-3-ols and ellagic acids. Via principal component analysis, these grape cultivars were clustered into three groups according to their characteristic phenolic content and composition.


Introduction
The grape (Vitis L.), which has a long history of cultivation and utilization, is one of the most important commercial fruit crops worldwide. There are more than 70 grape species and a large number of grape cultivars growing all over the world [1]. Vitis L. is divided into two subgenera: Euvitis Planch. and Muscadinia Planch. Based on geographical distributions, there are three groups of Euvitis species, including V. vinifera, East Asian and North American Vitis species. Muscadinia Planch., which refers mainly to V. rotundifolia Michx., is originated in the southeastern United States. This grape is also called muscadines and is genetically distinct from Euvitis species [2].
With more than 30 grape species being reported, China is the most important original center of East Asian Vitis species [3]. Vitis vinifera, the predominant Vitis species distributed and cultivated worldwide today, is believed to have been introduced to China more than 2000 years ago. At present, wine grape varieties cultivated in China mostly belong to V. vinifera plus a small percentage of Oriental species and hybrids. Recently, more American grapes and French hybrids for wine were introduced to China and received a great deal of attention due to their strong disease and pest resistance and stress tolerance [1].
Phytochemicals in grapes are mostly phenolic compounds. According to their molecular structure, the phenolic compounds are divided into four classes: one phenolic ring (cinnamic acids and benzoic acids), two phenolic rings (stilbenes), three rings (anthocyanins, flavonols and flavan-3-ols) and complex ring (ellagic acids) [4]. Anthocyanins, flavonols and flavan-3-ols, which have the nuclear molecular structure of C 6 -C 3 -C 6 , are also called flavonoid compounds. The remaining compounds are termed non-flavonoids. Grape skins contain abundant, widely varied phenolics. These phenolics play an important role in the sensory properties and nutrition of berries and wines. Many studies demonstrated that these phenolics could reduce the incidence of serious chronic diseases such as cancer and cardiovascular diseases, due to their antioxidant abilities [4][5][6].
Liquid chromatography-tandem mass spectrometry (HPLC-MS) technology has become a popular tool for the efficient identification of phenolic compounds. As a result, a lasrge amount of phenolic compounds have been identified and quantified in the grape berries and wines from V. vinifera [7][8][9], muscadines [10][11][12] and some hybrids [13,14]. However, few reports have reported phenolic content and coposition in different grape species/cultivars originated or cultivated in China, especially non-anthocyanin compounds. Thus the purpose of the present study was to perform an extensive analysis of phenolic compositions in the skins of a selected group of red wine grape cultivars with very diverse genetic backgrounds (Table 1). This information will be very useful for evaluating the winemaking and nutritional potential of these different grapes.
With regard to monoglucoside and diglucosides anthocyanins, there was a distinct separation among the different species. The V. vinifera grapes only contained the monoglucoside anthocyanins, but V. rotundifolia grapes only had diglucosides anthocyanins. Among the East Asian species and V. amurensis × V. vinifera hybrids, diglucosides anthocyanins were dominant in most grapes (accounting for 86.81% of TAs on average) except V.quinquangularis "Mao" and the hybrids "Zuohongyi" and "Hasang". While monoglucoside anthocyanins were the main anthocyanins in North American grapes and Euro-American hybrids (accounting for 74.76% of TAs on average), except V.aestivalis "Black Spanish" and the hybrid "St. Croix".
Among acylated anthocyanins, the p-coumaroyl derivatives were the most abundant ones with a high propotion (21.61% of TAs on average) in most samples with the exception of V. amurensis and V. rotundifolia grapes, in which no acylated anthocyanins were detected. The percentage of acetyl derivatives was high in the two V. vinifera grapes, but very low in non-V. amurensis East Asian grapes, North American grapes, and Euro-Asian and Euro-American hybrids.
Colored anthocyanins play an important role in the quality of red grapes and wines, which make the anthocyanin profile a major index for classifying grape cultivars and wine. In anthocyanin biosynthesis, there are three branches from naringenin: 4'-substituted, 3',4'-substituted and 3',4',5'-substituted anthocyanins. The latter two have an absolute advantage among grape anthocyanins, but their proportion of TAs largely depends on the expression level of flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Meanwhile the expression levels of methyltransferase, glucosyltransferase and acetyltransferase directly influence the degree of methylation, glucosylation and acylation [15]. As a result, the grapes with different genotypes displayed diverse anthocyanin compositions. Liang et al. (2008) concluded malvidin derivatives were the most abundant anthocyanins in the majority of germplasms, and monoglucoside derivatives were in V. vinifera and both mono-and di-glucoside derivatives in other Vitis germplasms. Their data were based on the analysis of the anthocyanin composition in the skins of 110 Euvitis grape cultivars [16]. For muscadine grapes, 3,5-diglucosides of delphinidin, cyanidin and petunidin accounted for approximately 90% of TAs [17].
In the present study, we found some interesting trends worth investigating in further research in the molecular mechanism differences of anthocyanin biosynthesis in red wine grapes with various genetic backgrounds and originations. i) Methylated and 3',4',5'-substituted anthocyanins were dominant in the skins of V. vinifera and East Asian grapes native to Eurasia, the proportion of which was significantly higher than in North American and muscadine grapes native to American. ii) Mono-glucosides were the only anthocyanin type in European grape skins and dominant in North American grape skins, while di-glucoside anthocyanins were the only type in muscadines and dominant in East Asian grapes.
iii) The proportion of acetyl anthocyanins was significantly higher in V. vinifera grapes than in non-V. vinifera grapes.
Kaempferol derivatives were the second most important flavonols in the skins of North American grapes and Euro-American hybrids, accounting for 24.04% and 10.19% of TFOs, respectively. However, myricetin derivatives were the second most abundant ones in East Asian grapes, Euro-Asian hybrids and muscadine grapes, accounting for 10.39%, 15.54% and 13.34% of TFOs, respectively. The proportions of other flavonol aglycone derivatives were low in most samples except the two V. vinifera grapes, in which isorhamnetin, dihydroquercetin, syringetin and kaempferol derivatives accounted for 18.49%, 14.69%, 14.36% and 12.54% of TFOs, respectively.
Flavonols are related to bitterness, and act as copigments of anthocyanins in the wines [21]. Many previous studies investigated flavonol composition in V. vinifera grape skins [22][23][24]. Our research was the first to show that quercetin-3-glucuronide and quercetin-3-glucoside were the characteristic flavonols in the skins of Euvitis red grapes. These compounds were not detected in muscadine samples. The large amount of diverse flavonols and dihydroflavonols was an outstanding characteristic of flavonol profiles for the skins of muscadine grapes, which might enhance their antioxidant capacity and make their taste different.

Flavan-3-ol Profiles
Flavan-3-ols mainly contribute to the structure of wines. Flavanol polymers (condensed tannin) play a particularly important role in the astringency of wines. They may react with anthocyanins through an intermolecular copigmentation process, leading to the definition and stability of color in red wines [25]. There were significant differences in total content and chemical compositions of flavan-3-ols (TFAs) among various grape cultivars ( Table 2). Among American cultivars and Euro-American hybrids, V. labrusca "Niagara Rosada" had the highest TFA content in all the tested samples (1243.67 µg CE/g DW), while V. labrusca "Catawba", V.aestivalis "Black Spanish", and the French hybrid "Marechal Foch" had less than 20 µg catechin equivalence (CE) /g DW. The two muscadines also possessed high TFAs (451.48µg CE/g DW for "Noble" and 350.62 µg CE/g DW for "Alachua"). A relatively low TFA contents was found in most East Asian cultivars, and none were detected in most V. amurensis cultivars and its hybrids except "Hasang" and "Changbaijiu" (562.73 and 381.80 µg CE/g DW).
There were 8 flavan-3-ol compounds detected in all, including 4 monomers (gallocatechin, epigallocatechin, catechin, epicatechin), 3 dimers and 1 trimer (Table 4). In the skins of the V. vinifera grapes, North American grapes and Euro-American hybrids, the flavan-3-ols were mainly comprised of polymers, accounting for 70%-100% of TFAs, except in the French hybrid "Marechal Foch". However, monomers were the chief flavan-3-ol type in the muscadines and non-V. amurensis East Asian species. The most abundant monomers were found in V. rotundifolia "Noble" and "Alachua" (338.20 and 265.63 µg CE/g DW, respectively). Among all the flavan-3-ols, the procyanidin dimer was the most important compound in the non-muscadine grapes. The high levels of TFA in the skins of "Niagara Rosada", "Hasang" and "Changbaijiu" were correlated to their extremely high contents of procyanidin dimmer levels. For monomers, catechin and epicatechin were the most common, but their levels were still low in most non-muscadine grapes.

Non-Flavonoid Phenolic Profiles
The total cinnamic acids (TCA) contents in the tested samples ranged from trace amounts to 230 µg caffeic acid equivalence (CAE)/g DW ( Table 2). The three cultivars with the highest levels of TCA were V. xunyangensis "Mi", the V. amurensis × V. vinifera hybrid "Zuoyouhong" and V. dividii "Black pearl" (229.37, 219.42 and 216.19 µg CAE/g DW, respectively). In addition, V. quinquangularis "Mao", V. labrusca "Fredonia" and V. amurensis "Changbaijiu" also had a high TCA content (174.13, 154.96 and 109.73 µg CAE/g DW, respectively). However, only trace cinnamic acids were detected in V. vinifera "Merlot" and "Cabernet Sauvignon". Nine different cinnamic acids were found in the skins among the cultivars investigated, with chlorgenic acid, hexose ester of ferulic acid, and fertaric acid were more common than others. Fertaric acid was the most abundant cinnamic acids in the East Asia group, accounting for 49.87% of TCAs (Table 5).        Five benzoic acids were also identified, and hexose ester of vanillic acid was the most common, followed by hexose ester of chlorgenic acid (Table 5). In general, low benzoic acids were detected in all the cultivars (Table 2). For example, the highest total benzoic acid content (TBC) was only 27.85 µg gallic acid equivalence (GAE)/g DW, which was found in V. ficifolia "Sangye", whereas the lowest content was found in V. labrusca "Catawba" (0.58 µg GAE/g DW).

Principle Component Analysis
Twenty-two evaluation parameters in all the cultivars investigated (4'-substituent, 3',4'-substituent, 3',4',5'-substituent, methylated, non-methylated, monoglucoside, diglucosides, acetyl, caffeoyl, coumaroyl and total anthocyanins; quercetin, myricetin, kaempferol derivatives and total dihydroflavonols and flavonols; monomeric, polymeric and total flavon-3-ols; total cinnamic acids, total benzoic acids, total ellagic acids and total stilbenes) were subjected to Principle Component Analysis (PCA) in order to separate these grapes according to their skin phenolic characteristics. The first three principal components (PCs) possessed relatively high percentages of total variance. PC1 described 27.94% of the variance, which had high contributions from 4'-substituted anthocyanins, quercetin derivatives, myricetin derivatives, total flavonols, and monomeric and total flavon-3-ols, as well as total ellagic acids. PC2 described 25.32% of the variance, and correlated with 3',4'-substituted, 3',4',5'-substituted, methylated, non-methylated, diglucosides, total anthocyanins and total benzoic acids. PC3 accounted for 12.50% of the variance and was mostly described by kaempferol derivatives, and polymeric and total flavon-3-ols. Figure 1 shows the distribution of the 23 grapes in the three-dimensional space of PC1, PC2 and PC3 and the two-dimensional space of PC1 and PC3. The score plots of these samples revealed three distinct groups. Group A, located in the positive axis of PC3, included all the V. Vinifera, North American grape cultivars, Euro-American hybrids, and the V. amurensis × V. vinifera hybrid "Hasang". This group was linked by their relatively high contents of kaempferol derivatives or polymeric or total flavon-3-ols in the skins. Group B, in the negative PC3 axis, was comprised of all the East Asian grapes and Euro-Asian hybrids except "Hasang". This group was matched through no or low content of kaempferol derivatives or polymeric or total flavon-3-ols in their skins. The two mascadine grapes comprised group C, located in the significantly higher position of the PC1 axis. This group was characterized by the presence of pg-3,5-diglucosides and ellagic acids, and by high contents of monomeric flavan-3-ols and flavonols.

Materials
Berries of the five V. amurensis grape cultivars, three V. amurensis × V. vinifera hybrids, four American cultivars and three Euro-American hybrids were collected from the experimental vineyard of the grape germplasm repository of China Agricultural University in Beijing. The two V. vinifera grape cultivars were from the nearby vineyard of Ji county in Tianjing. The berries of the other four East Asian species were collected from the experimental vineyard of the grape germplasm repository of Henan Academy of Agricultural Science in Zhengzhou. The berries of the two muscadine grape cultivars were collected from the experimental vineyard of Guangxi Academy of Agricultural Science in Nanning (Table 1).
All the grape berries were harvested at technological ripeness upon ripening in 2009, determined based on the former years ripening dates and as judged from seed color change to dark brown without senescence of berry tissue. Two 100-berry batches were sampled from at least 50 cluster selections at similar positions of 6 whole vine selections. Each group of berries was considered as one replication resulting in two replications for every grape cultivar or hybrid.
The fresh samples were placed in refrigerated bags and taken to the laboratory. Then the skins were separated manually from the berries and immediately freeze-dried (LGJ-12, Songyuan Huaxing Corporation, Beijing, China). Dried specimens were ground thoroughly with a stainless-steel grinder (FW-135, Taister Corporation, Tianjin, China), and stored in vacuum-packaged polyethylene pouches at −20 °C for subsequent analysis.
for providing grape berries for this study: Jun Wang at China Agricultural University, Chong-Huai Liu at Zhengzhou Fruit Research Institute, and Yu Huang at Guangxi Agricultural Academy.