molecules

Article Variability in the Content of Trans-Resveratrol, Trans-ε-Viniferin and R2-Viniferin in Grape Cane of Seven Vitis vinifera L. Varieties during a Three-Year Study

Jan Tˇríska 1,*, NadˇeždaVrchotová 1, Josef Balík 2, Ivo Soural 2 and Radek Sotoláˇr 2 1 Global Change Research Institute, Academy of Sciences of the Czech Republic, v. v. i., Lipová 9, Ceskˇ é Budˇejovice37005, Czech Republic; [email protected] 2 Faculty of Horticulture, Mendel University in Brno, Valtická 337, Lednice 69144, Czech Republic; [email protected] (J.B.); [email protected] (I.S.); [email protected] (R.S.) * Correspondence: [email protected]; Tel.: +420-723-059-668

Academic Editor: Isabel C. F. R. Ferreira Received: 21 April 2017; Accepted: 31 May 2017; Published: 3 June 2017

Abstract: Grape canes are a waste product from viticulture that show potential as an industrially extractable source of stilbenes, which are valuable for medical and other purposes. In this work, grape canes collected in three consecutive years (2014–2016) at six different places in South Moravia, Czech Republic were extracted, and the contents of trans-resveratrol, trans-ε-viniferin, and r2-viniferin were determined by high-performance liquid chromatography. The study included three blue grape varieties of Vitis vinifera L. (Cabernet Moravia, Blaufränkisch, and Piwi variety Laurot) and four white grape varieties (Chardonnay, Green Veltliner, Piwi variety Hibernal, and Piwi variety Malverina). From the viewpoint of producing extracts with high stilbenes content, the Hibernal variety is clearly the best. The mean amounts of the stilbenes for this variety at all localities and for all three years were 4.99 g/kg for trans-resveratrol, 3.24 g/kg for trans-ε-viniferin, and 1.73 g/kg for r2-viniferin. The influence of vintage, locality, and variety on the amounts of stilbenes was studied using PCA analysis. In contrast to expectations, there was no strong impact of locality on stilbenes content. The differences were varietal for most varieties, regardless of the area of cultivation. Laurot and Hibernal varieties did differ significantly in that respect, however, as they exhibited clear dependence on location.

Keywords: stilbenes; Vitis vinifera L.; grape cane; Moravian wine region

1. Introduction In recent years, increasing attention has been focused on waste products generated in agriculture and in the food industry because such materials can be a source of valuable substances. One group of such substances is the stilbenes. These can be derived from grape canes (a waste material from viticulture), which are very rich in trans-resveratrol and trans-ε-viniferin. Considering the large amounts of stilbenes found in grape canes (3.45 g of trans-resveratrol per kg dry weight (d.w.) of cane and 1.3 g of trans-ε-viniferin per kg d.w. of cane) and their market values, it is estimated [1] that it would be possible to obtain several thousand US dollars per hectare through the commercial use of these compounds. Zhang et al. [2] analysed 165 samples of canes from seven major wine regions in China using high-performance liquid chromatography, and prepared a very thorough economic calculation for obtaining trans-resveratrol from waste canes. Regarding extraction, Rayne et al. [1] recommended a mixture of alcohol and water (70–80% alcohol) for the quantitative extraction of trans-resveratrol and trans-ε-viniferin. Karacabey et al. [3] extracted from milled canes of the Pinot Noir variety using water alone and water with alcohol (7.4%, 15%, and 25%) in a pressure apparatus at

Molecules 2017, 22, 928; doi:10.3390/molecules22060928 www.mdpi.com/journal/molecules Molecules 2017, 22, 928 2 of 10 temperatures of 85, 95, 105, 120, 140, and 160 ◦C. They found that the extraction yields were reduced at temperatures above 95 ◦C for both trans-resveratrol and trans-ε-viniferin. The maximum yield of trans-ε-viniferin amounted to 1.65 g/kg of lyophilized canes. It is difficult to compare the published data about the content of trans-resveratrol and trans-ε-viniferin in canes because various authors have studied different white and red varieties, including resistant and sensitive ones, and prepared samples of canes under different drying and storage conditions. Thus, the content of trans-resveratrol and trans-ε-viniferin varies in the range of 0.038–1.53 g/kg for trans-resveratrol and white varieties, 0.046–0.967 g/kg for trans-resveratrol and blue varieties, 0.62–1.92 g/kg for trans-ε-viniferin and white varieties, and 0.43–2.30 g/kg for trans-ε-viniferin and blue varieties [4]. Soural et al. [5] found as much as 6.03 g/kg of trans-resveratrol in the Cabernet Moravia variety. Pawlus et al. [6] studied the profile of nine stilbenoids in 16 grape varieties (Vitis amurensis, Vitis arizonica, Vitis berlandieri, Vitis betulifolia, Vitis cinerea, Vitis x champini, Vitis x doaniana, Vitis labrusca, Vitis candicans [syn. Vitis mustangensis], Vitis riparia, Vitis rupestris, Vitis vinifera, Muscadinia rotundifolia, Vitis vinifera x Muscadinia rotundifolia). Those authors isolated 15 stilbenoids, confirmed their structures by mass spectrometry and nuclear magnetic resonance spectrometry, and found that the greatest yield of trans-ε-viniferin was 5.74 g/kg in variety Vitis riparia Pull. Vergara et al. [7] studied the distribution of trans-resveratrol, trans-piceatannol, and trans-ε-viniferin in Chilean Pinot Noir and Gewürztraminer and found the highest concentration of trans-ε-viniferin of 0.87 g/kg in canes of Pinot Noir. They also found in this variety that—in contrast to other varieties—it is advantageous to keep the canes after trimming in the vineyard for a period of two months, during which the content of the trans-ε-viniferin practically doubled. Guerrero et al. [8] described the cane stilbene composition of 22 grape cultivars. The authors chopped samples into small pieces, dried them at 40 ◦C, and performed the extraction using an acetone/H2O mixture at room temperature. The most abundant stilbene in all cultivars was trans-ε-viniferin, which reached concentrations ranging from 0.90 g/kg d.w. in Palomino fino to 2.81 g/kg d.w. in Gewürztraminer. Those authors also used principal component analysis (PCA) to better understand the differences between cultivars. The great interest in trans-resveratrol in the past and in trans-ε-viniferin today is due to their biological properties. Certain of these properties are very promising from the viewpoint of human medicine. For example, trans-ε-viniferin glucoside is an attractive new candidate for protecting brain cells, and could therefore be useful in treating Alzheimer’s disease [9]. This probably relates to the finding that trans-(−)-ε-viniferin activity increases the activity of mitochondrial sirtuin 3 and protects cells in a model of Huntington’s disease, which is virtually untreatable [10]. Trans-ε-viniferin is active not only as a preventive agent against cancer, but also as a substance having direct cytotoxicity against selected cancer cell lines [11]. The results in a study published by Basri et al. [12] are very promising, and show that a combination of trans-ε-viniferin and vancomycin is active against methicillin-resistant Staphylococcus aureus—a bacterium responsible for difficult infections in humans and animals. Stilbenes from canes have also been studied as natural fungicides. Methanolic and ethanolic crude extracts from grape canes of Pinot Noir, Gamaret, and Divico varieties have exhibited significant antifungal activity against three major fungal pathogens affecting grapevines: Plasmopara viticola, Erysiphe necator, and Botrytis cinerea [13]. Stilbene complex mixtures from Vitis vinifera L. wastes (cane, wood, and root) have recently been used as a cheap source of bioactive compounds for the development of natural fungicides against, for example, Plasmopara viticola [14]. Because all the aforementioned and future studies have required and will require a starting material such as trans-ε-viniferin in far greater amounts, a successful search for sources of these substances and a description of their distribution in different grapevine varieties with regards to sampling procedure and storage conditions is highly desirable and promising. As is apparent from the overview presented above, it is necessary to thoroughly study the distribution of stilbenes in canes in relation not only to the variety and sampling points, but also to the variability in stilbenes content through time. Our work was therefore focused on studying and Molecules 2017, 22, 928 3 of 10

Moravia, Czech Republic during a three-year study. To our knowledge, this is the first such comprehensivecomparing stilbenes study contents of its kind. of seven varieties of grape canes in six different places in South Moravia, Czech Republic during a three-year study. To our knowledge, this is the first such comprehensive 2.study Results of its kind.

2. ResultsSeven varieties of Vitis vinifera L. from the Moravian wine region were included in the experiment, encompassing three blue grape varieties: Cabernet Moravia (CM; sampling points: 3, 5, 6), BlaufränkischSeven varieties (Bl; sampling of Vitis viniferapoints: 3,L. 5, from6), and the Laurot Moravian (La; sampling wine region points: were 1, 2, 6); included and four inwhite the grapeexperiment, varieties: encompassing Chardonnay three (Ch; blue sampling grape varieties: points: 3, Cabernet 5, 6), Green Moravia Veltliner (CM; (GV; sampling sampling points: points: 3, 5, 6),3, 5,Blaufränkisch 6), Hibernal (Hi; (Bl; sampling points: 1, 3, 4, 5, 6), 6), and and Malverina Laurot (La; (Ma; sampling sampling points: points: 1, 2,1, 6);4, 6). and The four varieties white andgrape six varieties: sampling Chardonnay sites are described (Ch; sampling in the points:Materials 3, 5,and 6), Methods. Green Veltliner In our (GV; study sampling during points:2014–2016, 3, 5, we6), Hibernalfocused (Hi;on determining sampling points: three 1, 4,do 6),minant and Malverinastilbenes (Ma;in grape sampling canes: points: trans-resveratrol, 1, 4, 6). The varietiestrans-ε- viniferin,and six sampling and r2-viniferin. sites are described in the Materials and Methods. In our study during 2014–2016, we ε focusedLow on values determining of trans-resveratrol three dominant were stilbenesfound in inthe grape Blaufränkisch canes: trans (Bl)-resveratrol, variety at alltrans three- -viniferin,sampling pointsand r2-viniferin. and during all three observed years. The lowest values were recorded in 2014, and the differenceLow valueswas statistically of trans-resveratrol significant were as compared found in the to Blaufränkisch2015 and 2016 (Bl) (Figure variety 1). atIt allis interesting three sampling that pointsthe contents and during of the all two three other observed stilbenes— years.trans The-ε lowest-viniferin values and were r2-viniferin—in recorded in 2014, the Bl and variety the difference did not wasreach statistically such high significantvalues (Figures as compared 2 and 3), to 2015but were and 2016relatively (Figure close1). It to is the interesting values thatof the the Malverina contents ε variety.of the two other stilbenes—trans- -viniferin and r2-viniferin—in the Bl variety did not reach such high valuesHigh (Figures values2 and of all3), butthree were stilbenes relatively were close foun tod thein the values Hibernal of the variety. Malverina The variety. amounts of trans-ε- viniferinHigh in valuesLaurot ofat allall three sampling stilbenes sites were in found 2015 were in the comparable Hibernal variety.to the values The found amounts in the of ε Hibernaltrans- -viniferin variety. in The Laurot cont atents all of three r2-viniferin sampling and sites trans in 2015-resveratrol were comparable in Laurot torecorded the values at Sampling found in Sitethe Hibernal2 were many variety. times The higher contents than of those r2-viniferin at Sampli andngtrans Sites-resveratrol 1 and 6. A in similar Laurot effect recorded was at also Sampling found inSite the 2 wereLaurot many variety times for higher the contents than those of attrans Sampling-resveratrol. Sites 1 Large and 6. differences A similar effect in the was contents also found of r2- in viniferinthe Laurot at varietyvarious for sampling the contents sites ofweretrans also-resveratrol. found in the Large Cabernet differences Moravia in the variety, contents and of a r2-viniferin significant differenceat various samplingwas also determined sites were also between found years. in the Cabernet Moravia variety, and a significant difference was alsoRegarding determined r2-viniferin, between it years.is interesting that in 2014, the lowest value (statistically significant, p < 0.05) Regardingwas always r2-viniferin, recorded at itSampling is interesting Site 6 thatwhen in comparing 2014, the lowest the same value variety. (statistically With the significant, exception ofp < the 0.05) Hibernal was always variety, recorded the lowest at Sampling value Site among 6 when different comparing varieties the same in 20 variety.16 was With also the recorded exception at Samplingof the Hibernal Site 6. Findings variety, thein the lowest year value2015 were among very different diverse. varieties in 2016 was also recorded at SamplingThe highest Site 6. Findingsamount inof thetrans year-resveratrol 2015 were in very the Cabernet diverse. Moravia variety was recorded at the samplingThe highestsites in 2014. amount of trans-resveratrol in the Cabernet Moravia variety was recorded at the sampling sites in 2014.

trans-resveratrol

7000 2016 6000 2015

5000 2014

4000

3000 mg/kg

2000

1000

0 Bl -5 Bl -3 Bl -6 La -La 2 -La 1 -La 6 Hi -4 Hi -1 Hi -6 GV -3GV -6GV Ch -5 Ch -3 Ch -6 Ch GV -GV 5 Ma - 4 - Ma 1 - Ma 6 - Ma CM -5 CM -3 CM -6

Figure 1. Contents of trans-resveratrol in grape canes during three years (contents in mg/kg dry weight).Figure 1. Example Contents explanation of trans-resveratrol of abbreviations: in grape CM-5—“CM” canes during isthree thevariety, years (contents “5” is the in sampling mg/kg dry site (seeweight). Materials Example and explanation Methods and of Table abbreviations:1). Bl: Blaufränkisch; CM-5—“CM” Ch: is Chardonnay; the variety, CM:“5” is Cabernet the sampling Moravia; site (seeGV: GreenMaterials Veltliner; and Methods Hi: Hibernal; and Table Ma: 1). Malverina; Bl: Blaufr La:änkisch; Laurot. Ch: Chardonnay; CM: Cabernet Moravia; GV: Green Veltliner; Hi: Hibernal; Ma: Malverina; La: Laurot.

Molecules 2017, 22, 928 4 of 10

MoleculesMolecules 20172017,, 2222,, 928 928 44 of of 10 10 trans-epsilon-viniferin

trans-epsilon-viniferin 5000 2016 4500 2015 5000 2016 4000 2014 45003500 2015 40003000 2014 35002500 3000 mg/kg 2000 25001500 mg/kg 20001000 1500500 10000 500 Bl - 5 Bl - 3 Bl - 6 La - 2 La - 1 La - 6 Hi - 4 Hi - 1 Hi - 6 Ch - 5 Ch - 3 Ch - 6 Ch Ma - 4 Ma - 1 Ma - 6 Ma -GV 5 -GV 3 -GV 6 0 CM - 5 CM - 3 CM - 6

Bl - 5 Bl - 3 Bl - 6 La - 2 La - 1 La - 6 Hi - 4 Hi - 1 Hi - 6 Ch - 5 Ch - 3 Ch - 6 Ch Ma - 4 Ma - 1 Ma - 6 Ma -GV 5 -GV 3 -GV 6 CM - 5 CM - 3 CM - 6 Figure 2. Contents of trans-ε-viniferin in grape canes during three years. Example explanation of abbreviations:Figure 2. Contents CM-5—“CM” of trans-ε is-viniferin the variety, in grape “5” is canes the sampling during threesite (see years. Materials Example and explanation Methods and of FigureTableabbreviations: 1). 2. Contents CM-5—“CM” of trans-ε-viniferin is the variety, in grape “5”is canes the sampling during th siteree (see years. Materials Example and explanation Methods and of abbreviations:Table1). CM-5—“CM” is the variety, “5” is the sampling site (see Materials and Methods and Table 1). r2-viniferin

r2-viniferin 2016 3000 2015 2016 2500 2014 3000 2015 2000 2500 2014 1500

mg/kg 2000 1000 1500 mg/kg 500 1000 0 500 Bl -5 Bl -3 Bl -6 La - 2 La - 1 La - 6 Hi - 4 Hi - 1 Hi - 6 GV -3GV -6GV Ch - 5 Ch - 3 Ch - 6 Ch Ma - 4 Ma - 1 Ma - 6 Ma GV -GV 5 0 CM - 5 CM - 3 CM - 6

Bl -5 Bl -3 Bl -6 La - 2 La - 1 La - 6 Hi - 4 Hi - 1 Hi - 6 GV -3GV -6GV Ch - 5 Ch - 3 Ch - 6 Ch Ma - 4 Ma - 1 Ma - 6 Ma GV -GV 5 Figure 3. CM - 5 ContentsCM - 3 CM - 6 of r2-viniferin in grape canes during three years. Example explanation of Figure 3. Contents of r2-viniferin in grape canes during three years. Example explanation of abbreviations: CM-5—“CM” is the variety, “5” is the sampling site (see Materials and Methods abbreviations: CM-5—“CM” is the variety, “5” is the sampling site (see Materials and Methods and and Table1). FigureTable 1). 3. Contents of r2-viniferin in grape canes during three years. Example explanation of abbreviations: CM-5—“CM” is the variety, “5” is the sampling site (see Materials and Methods and TableBecause 1). the the amounts amounts of oftranstrans-resveratrol-resveratrol are approximately are approximately twice twicehigher higher than those than of those trans- ofε- viniferintrans-ε-viniferin and the andamounts the amounts of r2-viniferin of r2-viniferin are smalle arest, smallest,the sums theof all sums monitored of all monitored stilbenes (Figure stilbenes 4) essentially(FigureBecause4) essentially reflect the amounts the reflect amounts of the trans amounts of-resveratrol trans of-resveratrol.trans are-resveratrol. approximately The highest The twice highest total higher content total contentthan of thosestilbenes of stilbenes of trans in the-ε in- viniferinCabernetthe Cabernet and Moravia Moraviathe amounts variety variety atof all r2-viniferin at sampling all sampling sitesare smalle sites occu occurredrredst, the in 2014.sums in 2014. Forof all the For monitored Laurot the Laurot variety, stilbenes variety, Sampling (Figure Sampling Site 4) essentially2Site varied 2 varied considerably reflect considerably the fromamounts from sites sites of1 andtrans 1 and 6.-resveratrol. For 6. For othe otherr varieties, The varieties, highest with with totalsome some content exceptions, exceptions, of stilbenes the the differences differences in the Cabernetbetween locationsMoravia varietyand years at allwere sampling small. sites occurred in 2014. For the Laurot variety, Sampling Site 2 varied considerably from sites 1 and 6. For other varieties, with some exceptions, the differences between locations and years were small.

Molecules 2017, 22, 928 5 of 10

Molecules 2017, 22, 928 5 of 10 Molecules 2017, 22, 928 Sum of stilbenes 5 of 10

14000 Sum of stilbenes 2016

12000 2015 14000 2016 2014 1000012000 2015 2014 800010000

6000 8000 mg/kg 6000

4000mg/kg

2000 4000 2000 0 0 Bl - 5 Bl - 3 Bl - 6 La - 2 La - 1 La - 6 - 4 Hi - 1 Hi - 6 Hi Ch - - 5 Ch - 3 Ch - 6 Ch Ma - 4 - Ma 1 - Ma 6 - Ma -GV 5 -GV 3 -GV 6 CM 5 - CM 3 - CM 6 - Bl - 5 Bl - 3 Bl - 6 La - 2 La - 1 La - 6 - 4 Hi - 1 Hi - 6 Hi Ch - - 5 Ch - 3 Ch - 6 Ch GV -GV 5 -GV 3 -GV 6 Ma - 4 - Ma 1 - Ma 6 - Ma CM 5 - CM 3 - CM 6 -

FigureFigure 4. TotalTotal content content of of stilbenes stilbenes in in grape canes during three years.years. Ex Exampleample explanation explanation of of Figure 4. Total content of stilbenes in grape canes during three years. Example explanation of abbreviations: CM-5—“CM” is the variety, “5” is the sampling site (see Materials and Methods and abbreviations:abbreviations: CM-5—“CM” CM-5—“CM” is theis the variety, variety, “5” “5” is is the the samplingsampling site site (see (see Materials Materials and and Methods Methods and and Table1). Table Table1). 1).

3.3. Discussion Discussion3. Discussion FiguresFiguresFigures 1–1–44 show 1–4show theshow the amounts the amounts amounts of stilbenes of of stilbenesstilbenes in the analysedinin the analysedanalysed varieties. varieties. Tovarieties. illustrate To Toillustrate the illustrate relationships the the relationshipsbetweenrelationships varieties, between between locations, varieties, varieties, and locations, years, locations, we and performed and ye years,ars, principalwe performedperformed component princi principal analysispal component component (PCA) analysis usinganalysis the (PCA)Statistica(PCA) using 12 using program.the Statistica the Statistica The 12 results program.12 program. are presented The The results results in are Figuresare presented5–8. in in Figures Figures 5–8. 5–8.

Figure 5. Principal component analysis for 2014. (A) projection of varieties and localities into Figurecomponent 5. Principal plane; component(B) projection analysis of component for 2014. weights (A of) stilbenes. projection of varieties and localities into component plane; (B) projection of component weights of stilbenes. FigureIn 5.2014 Principal (Figure component5), the Cabernet analysis Moravia for 2014.variety (A differed) projection from ofother varieties varieties and and localities also differed into componentby sampling plane; site (CM-3(B) projection vs CM-5 of andcomponent CM-6), weightsas did Laurot of stilbenes. (La-1 vs La-2 and La-6). Hibernal also Indiffered 2014 (Figurefrom other5), thevarieties. Cabernet Moravia variety differed from other varieties and also differed by samplingIn 2014The site (Figure (CM-3Laurot 5),variety vs CM-5the Cabernetdiffered and CM-6), again Moravia even as did varietyin Laurot2015, differedan (La-1d differences vs from La-2 other andin the La-6). varieties contents Hibernal ofand stilbenes also also differed differed in byfrom samplingLaurot other varieties.depended site (CM-3 on locationvs CM-5 (La-1 and vs CM-6), La-2 and as La-6). did Hi-6Laurot differed (La-1 from vs La-2the other and Hibernal La-6). Hibernal sites. The also differedTheother from Laurot varieties other variety exhibited varieties. differed practically again no evendependence in 2015, on andthe area differences of cultivation in the (i.e., contents sampling of site), stilbenes but in LaurotThea dependence depended Laurot variety on variety location differed was (La-1 obvious again vs even La-2in the andin case 2015, La-6). of Chardonnay and Hi-6 differences differed (3, 5, 6)in from vs the Cabernet thecontents other Moravia of Hibernal stilbenes (3, 5, sites. in Laurot depended on location (La-1 vs La-2 and La-6). Hi-6 differed from the other Hibernal sites. The The other varieties exhibited practically no dependence on the area of cultivation (i.e., sampling site), otherbut a varieties dependence exhibited on variety practically was obvious no dependence in the case on of the Chardonnay area of cultivation (3, 5, 6) vs (i.e., Cabernet sampling Moravia site), (3,but 5, a6) dependence vs Blaufränkisch on variety (3, 5, was 6). The obvious variance in the in case values of forChardonnay Green Veltliner (3, 5, 6) and vs MalverinaCabernet Moravia varieties (3, was 5, slightly larger.

Molecules 2017, 22, 928 6 of 10 Molecules 2017, 22, 928 6 of 10 6) vs Blaufränkisch (3, 5, 6). The variance in values for Green Veltliner and Malverina varieties was slightly6) vs Blaufränkischlarger. (3, 5, 6). The variance in values for Green Veltliner and Malverina varieties was Molecules 2017, 22, 928 6 of 10 slightly larger.

FigureFigure 6. Principal 6. Principal component component analysis analysis for 2015. for 2015. ( A) projection (A) projection of varieties of varieties and andlocalities localities into into componentFigurecomponent 6. plane; Principal plane; (B) projection (Bcomponent) projection of componenanalysis of component fort weights 2015. weights of(A stilbenes.) ofprojection stilbenes. of varieties and localities into component plane; (B) projection of component weights of stilbenes.

FigureFigure 7. Principal 7. Principal component component analysis analysis for 2016. for 2016. (A) projection (A) projection of varieties of varieties and andlocalities localities into into component plane; (B) projection of component weights of stilbenes. componentFigure 7. plane; Principal (B) projection component of componentanalysis for weights 2016. of(A stilbenes.) projection of varieties and localities into component plane; (B) projection of component weights of stilbenes. In 2016,In 2016, the theHibernal Hibernal variety variety differed differed considerab considerablyly from from the others, the others, and andSampling Sampling Sites Sites 6 and 6 and4 4 for Hibernalfor HibernalIn 2016, differed the differed Hibernal from from Sampling variety Sampling differedSite Site1. 1.considerably from the others, and Sampling Sites 6 and 4 forThe HibernalThe present present differed results results from indicate indicate Sampling that that the Site the difference 1. difference is is varietal varietal for for most most varieties, varieties, regardless ofof thethe area areaof of cultivation cultivationThe present (sampling (sampling results indicate sites).sites). LaurotLaurot that the andand difference Hibernal is varieties varietal differ for most significantly, significantly, varieties, as regardless in their cases of the we wearea foundfound of cultivationa a clear dependence (sampling on sites). location Laurot (sampling and Hibernal sites). varieties differ significantly, as in their cases weThe found Theabove a above clear results resultsdependence are complemented are complemented on location by (sampling PCA by PCA includ including sites).ing all three all three years years (Figure (Figure 8). For8). For the thesake sake of of clarity,clarity, Thethe thevariousabove various results sampling sampling are complementedsites sites are arenot notindicated, by indicated, PCA but includ butonlying only the all thevarieties three varieties years according (Figure according to 8). colour. toFor colour. the In sake the In of the caseclarity,case of the of the the varieties various varieties Malverina,sampling Malverina, sites Green Green are notVeltliner, Veltliner, indicated, andand but Blaufränkisch,Blaufränkisch, only the varieties the the respective respective according points pointsto colour. are are all locatedInall the locatedcaseclose ofclose together, the together, varieties indicating indicating Malverina, that thethat Green value the value ofVeltliner, stilbenes of stilbenes and in the Blaufränkisch, respectivein the respective canes the does canesrespective not does depend notpoints depend on locationare all on locatedlocation(sampling close (sampling sites) together, or sites) year. indicating or In year. the case Inthat the of the Laurot,case value of Laurot, onof thestilbenes otheron the in hand, other the respective thehand, points the points arecanes spaced doesare spaced farnot apart depend far and onare location located (sampling in three quadrants. sites) or year. Furthermore, In the case of it isLaurot, obvious on thatthe other the Hibernal hand, the variety points differs are spaced from far the other varieties. The spacing of the Chardonnay points is also interesting.

Molecules 2017, 22, 928 7 of 10

apart and are located in three quadrants. Furthermore, it is obvious that the Hibernal variety differs Moleculesfrom the2017 other, 22, 928varieties. The spacing of the Chardonnay points is also interesting. 7 of 10

Figure 8. Principal component analysis for all three years. (A) projection of varieties and localities into componentFigure 8. Principal plane; ( Bcomponent) projection analys of componentis for all three weights years. of ( stilbenes.A) projection of varieties and localities into component plane; (B) projection of component weights of stilbenes.

TheThe issueissue ofof thethe diversitydiversity ofof thethe distributiondistribution ofof stilbenesstilbenes obtainedobtained fromfrom grapevinegrapevine canescanes isis ratherrather complex.complex. AsAs wewe havehave alreadyalready pointedpointed outout inin thethe article,article, thethe processesprocesses themselvesthemselves (different(different storagestorage andand dryingdrying times)times) affectaffect thethe contentscontents ofof thesethese substances.substances. OurOur previousprevious articlearticle [[5]5] studiedstudied extractionextraction methodsmethods forfor CabernetCabernet Moravia,Moravia, whichwhich affectedaffected yields.yields. BecauseBecause wewe havehave designeddesigned ourour samplingsampling pointspoints soso thatthat atat samplingsampling pointpoint No.No. 66 (Znojmo—Oblekovice)(Znojmo—Oblekovice) wewe havehave allall sevenseven varieties,varieties, wewe addedadded datadata forfor totaltotal rainfallsrainfalls andand sumsum ofof effectiveeffective temperaturestemperatures onon thisthis localitylocality forfor 2014,2014, 2015,2015, andand 20162016 (see(see TableTable2 2 MaterialsMaterials andand Methods).Methods). FromFrom thethe studiedstudied parameters,parameters, rainfallrainfall resultedresulted higherhigher inin 2014.2014. DueDue toto thethe stressstress conditionsconditions whichwhich induceinduce stilbenestilbene biosynthesisbiosynthesis [[15]15] (especially(especially transtrans-resveratrol),-resveratrol), thethe higherhigher rainfallrainfall inin 20142014 mightmight havehave increased the fungal pressure. This means that plants might have been stressed, and in this way the content of trans-resveratrol increased. It is obvious from Figure1 that at sampling point No. 6 varieties Molecules 2017, 22, 928 8 of 10

CM, La, Ma, and Ch responded in the expected way, while varieties Hi, Bl, and GV responded in the opposite way. Although the territory of the Moravian wine region is relatively small, we can still say that the contents of stilbenes in some varieties of vines was highly dependent on the year and location (sampling sites), but the effect was not found to be the same for all varieties. The reason may be that the varieties are not grown evenly across South Moravia. For all investigated varieties, the dominant stilbenes were trans-resveratrol, trans-ε-viniferin, and r2-viniferin. To our knowledge, such an extensive set of data from a three-year study about the content of stilbenes in canes of different varieties of Vitis vinifera L. at various locations has not yet been published in the literature.

4. Materials and Methods

4.1. Varieties and Localities Seven varieties of Vitis vinifera L. canes from the Moravian wine region were included in the experiment—three blue grape varieties: Vitis vinifera L. variety Cabernet Moravia (CM), Vitis vinifera variety Blaufränkisch (Bl), and Piwi variety Laurot (La); and four white grape varieties: Vitis vinifera L. variety Chardonnay (Ch), Vitis vinifera L. variety Green Veltliner (GV), Piwi variety Hibernal (Hi), and Piwi variety Malverina (Ma). The six sampling sites (1–6) are described in Table1. Type of middle vine Rhine-Hesse training was used in all varieties. The canes from seven varieties were sampled at the end of February each year (2014, 2015, 2016).

Table 1. Sampling sites of grape canes.

Sampling Site Wine Area and Varieties Wine Sub-Region Lednice 1 Mikulovská (GPS: 48◦47024.000 N, 16◦47049.2” E) Hi, La, Ma Hlohovec 2 Mikulovská (GPS: 48◦47007.400 N, 16◦46044.200 E) La Velké Bílovice 3 Velkopavlovická (GPS: 48◦51054.000 N, 16◦53024.000 E) Bl, CM, GV, Ch Rakvice 4 Velkopavlovická (GPS: 48◦51046.500 N, 16◦48000.300 E) Hi, Ma Kostice 5 Slovácká (GPS: 48◦44028.800 N, 16◦58028.500 E) Bl, CM, GV, Ch Znojmo-Oblekovice 6 Znojemská (GPS: 48◦49026.400 N, 16◦05030.800 E) Bl, CM, GV, Hi, Ch, La, Ma

Table 2. Wine Area: Znojmo—Oblekovice (sampling site 6).

Year 2016 2015 2014 Rainfall total (mm) 428 359 545 Sum of effective temperatures (◦C) 1450 1470 1337

4.2. Sample Preparation Collected canes were dried in darkness for three months at room temperature. They were then ground using a laboratory grinder. Powdered grape canes were extracted for 165 min at 50 ◦C as described in our previous publication [5].

4.3. Chromatography Separation The samples were separated using an HP 1050 high-performance liquid chromatography instrument (Hewlett–Packard, Palo Alto, CA, USA) on a 3 µm, 150 mm × 2 mm, Luna C18(2) column Molecules 2017, 22, 928 9 of 10

(Phenomenex, Torrance, CA, USA) with water-acetonitrile-o-phosphoric acid mobile phase. We used a G1315B diode array detector (DAD, Agilent, Prague, Czech Republic) and G1321A fluorescence detector (FLD, Agilent, Prague, Czech Republic). We followed the method detailed in our previous publication [5]. The content of trans-resveratrol was calculated according to a calibration curve for trans-resveratrol. The content of trans-ε-viniferin was calculated according to the calibration curve for trans-ε-viniferin, the content of r2-viniferin was calculated according to the calibration curve for trans-ε-viniferin in and the obtained value was multiplied by the ratio of MW of r2-viniferin/MW of trans-ε-viniferin. Data were recorded as mg/kg dry weight. The method was validated in terms of limits of detection and of linearity [16]. Trans-resveratrol (LOD 0.056 µg/mL, LOQ 0.187 µg/mL), trans-ε-viniferin (LOD 0.158 µg/mL, LOQ 0.525 µg/mL).

4.4. Standards and Solvents Standards were purchased from the following sources: trans-resveratrol (purity > 99%) from Sigma-Aldrich (Prague, Czech Republic), trans-epsilon viniferin from Sigma-Aldrich (Prague, Czech Republic), methanol, and acetonitrile from Merck (Prague, Czech Republic, LiChrosolv, gradient grade for LC), and ortho-phosphoric acid p.a. from Fluka (Prague, Czech Republic).

5. Conclusions The measurements of trans-resveratrol, trans-ε-viniferin, and r2-viniferin in studied cane extracts from six different places in South Moravia and from seven varieties of Vitis vinifera L. did not show the expected strong impact of locality. The largest difference was found in the variety Piwi Laurot at Sampling Site 2 for trans-resveratrol and r2-viniferin. A significant difference was also observed in the Cabernet Moravia variety at Sampling Sites 3 and 6 for r2-viniferin. From the viewpoint of producing extracts with high stilbenes content, the Hibernal variety is clearly the best. The mean amounts of the aforementioned stilbenes for this variety at all localities and for all three years was 4.99 g/kg for trans-resveratrol, 3.24 g/kg for trans-ε-viniferin, and 1.73 g/kg for r2-viniferin.

Acknowledgments: Financial support for this research was provided by Project No. LD14038 of the Ministry of Education, Youth and Sports of the Czech Republic. Supported was also provided by the Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program I (NPU I), grant no. LO1415. We also wish to thank V. Peˇrina,E. Tománková, T. Neˇcas,and V. Rucki for providing plant materials. Author Contributions: Jan Tˇríska, NadˇeždaVrchotová, Josef Balík, Ivo Soural and Radek Sotoláˇrdesigned the research and wrote the paper. NadˇeždaVrchotová and Ivo Soural performed sample measurements and data evaluation. All authors analysed the data and read and approved the final manuscript. Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Not available.

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