Free Radical Scavenging Activity and Anthocyanin Profile of Cabernet Sauvignon Wines from the Balkan Region

The present study is focused on anthocyanin derivatives characterizing the antioxidant activity of Cabernet Sauvignon wines produced from different vineyard regions in the Balkans. These bioactive compounds were quantified with a high performance liquid chromatography (HPLC)-diode array detection (DAD) method. The antiradical activity was estimated by the ability of the wine to scavenge the stable 2,2`-diphenyl-1-picrylhydrazyl free radical (DPPH•). The results show that the total anthocyanin content varied from 205.88 to 1940.28 mg/L, depending on agroclimatic factors and the enological practices of the corresponding vineyard region. The most prominent antocyanin in all investigated Cabernet Sauvignon wines was malvidin-3-O-monoglucoside, which accounted for 50.57% of total content, followed by its acetyl derivatives, 15.45%, and p-coumaryl derivatives 5.66%. The relationship between the anthocyanin derivatives and free radical scavenging activity is discussed. A high correlation between total anthocyanin content and DPPH· scavenging ability of tested wines was confirmed (r2 = 0.9619). The significant correlations were obtained between antiradical activity and the sum of 3-monoglucoside (r2 = 0.95594), the sum of 3-acetyl-3-glucoside (r2 = 0.9728) and the sum of p-coumaryl-3-glucoside (r2 = 0.8873) of wine samples. It can be concluded that, the anthocyanin composition can be used as biochemical marker for the authenticity of red grape cultivar and their corresponding single-cultivar wine.

Cabernet Sauvignon is the world`s most widely recognized red wine grape variety. From France, the grape spread across Europe, New World and to South Africa. Despite its importance in the world of wine, the grape is a relatively new variety, being the product of a chance crossing between Cabernet Franc and Sauvignon Blanc sometime during the 17 th century in southwestern France.
The Balkan region has some good modern vineyards, 80 per cent of which are planted with red wine grapes, including Cabernet Sauvignon and Merlot, as well as the indigenous grapes. Considering the economic importance of winemaking in the Balkan countries, the aim of this work was to evaluate the free radical scavenging activity of eighth Cabernet Sauvignon wines produced from different agronomical and winemaking regions, and their correlation with the total and the real content of the main anthocyanin derivatives.

The content and the distribution of anthocyanins in Cabernet Sauvignon wine samples
Anthocyanin composition is an important quality parameter for red grapes because of the significance of these compounds in determining color of the resulting wines [14]. All investigated Cabernet Sauvignon wines produced from different vineyards in the Balkan region are listed in Table 1. The red wines from the Balkan region were found to contain significantly higher amounts of anthocyanins [10,12,24]. The changes in content of anthocyanin composition and radical scavenging activity in investigated Cabernet Sauvignon wine samples are presented in  [17][18][19][20]. Light exposure to grape clusters and berry size are two of the most important [1]. The results obtained confirm a variation in the anthocyanin composition of wine samples tested. The range of the data obtained is in agreement with the available literature [15][16][17][18][19][20].
Monomeric anthocyanins are the most labile phenolic compounds in wine, typically decreasing at a rate of about 50% per year [14,25]. Anthocyanin extraction and stability are affected by winery production practices. Monomeric anthocyanins usually decline during fermentation and maceration, but the process may continue throughout the life of a wine. Wine characteristics such as pH, SO 2 , and acetaldehyde influence these processes and anthocyanin interactions with other phenolic compounds. Stability of anthocyanins can be enhanced through so called copigmentation. Copigmentation plays a crucial role in wine ageing and maturation. Acylated anthocyanins containing two or more aromatic acyl groups may affect the color through a mechanism called intramolecular copigmentation [19,20].
Anthocyanins also interact with other flavonoids and related compounds to produce an increase in color intensity and a shift in the wavelength of maximum absorbance toward higher wavelengths. Such a phenomenon is called intermolecular copigmentation, which can take place in acidic, neutral and even slightly alkaline aqueous solution. Polymeric anthocyanin content of the Cabernet sauvignon wine samples from the 2007 and 2008 vintage seasons (Table 2), using the pH-differential method [21] was determined from 45.50 to 69.77%. As wines age, a greater proportion of their anthocyanin content is polymerized, then color density varied from 6.76 to 11.56 (mean 7.16) and hue from 0.62 to 1.12 (mean 0.96) absorbance units.

HPLC analysis of Cabernet Sauvignon wine samples
Anthocyanins profile of grape and wine, determined by the relative proportions of the different anthocyanins, are characteristic for each grape variety and wine, corresponding. Moreover, concentrations of different compounds can vary significantly within grape cultivars according to environmental conditions and winemaking practices [1,20,29].
The findings showed that malvidin-3-O-monoglucoside was the most abundant anthocyanin in all wines that had a prevalence of tri-hydroxylated anthocyanins. By contrast, cyanidin was the least abundant anthocyanin pigment, as demonstrated for a number of other wines [12][13][14][15][16][17][18]26]. A similar profile has been reported for Syrah [18,22], Hellenic native grape varieties [12], and Corsica red wines [13], but different patterns were observed for Cabernet Franc and Pinot Noir wines from British Columbia [3]. Another point worth mentioning is that the order of abundances based on average value of distribution for each anthocyanin was the following: Mvgl > Mvgl-ac > Ptgl > Pngl > Dpgl > Mvgl-p-coum > Cygl The Cabernet Sauvignon wines have a percentage of methoxylated (peonidin, petunidin and malvidin, sum 62.54%) higher than non-methoxylated (cyanidin and delphinidin, sum 6.12%) anthocyanin forms. With regard to the acylation of the glycosyl group, the distribution of non-acylated anthocyanins, mean 66.03% (from 55.72 to 74.53%) were the most abundant fraction in all wine samples. The mean amount of contribution of acetylated anthocyanins was 22.30% (from 17.74 to 25.58%), followed by coumarylated 9.25% (from 2.84 to 14.03%) in all wines. According to the wine sample results, it is obviously that the content of anthocyanin constituents of single-cultivar wines coming from different viticulture regions and wine producers were different, which may be related to the thickness of the grape skin, the climate that the grape grows in, the degree of ripeness of the grape, the application of different vinification techniques and the wine's ages [1,2,29].

The correlation between the radical scavenging activity and anthocyanin derivatives of the Cabernet Sauvignon wine samples
The antioxidant activity of the wine samples was estimated by the ability of the sample to scavenge the stable DPPH• free radical [10,16,23,24]. The decrease in absorbance at 517 nm is taken as a measure of the extent of radical-scavenging. All wines show a higher DPPH• radical scavenging activity (Table 2) in the range from 70.03 to 83.53% (mean 73.76%). Anthocyanins are considered very good antioxidant agents, their high activity being attributed to their peculiar structure, namely the oxonium ion in the C ring [24]. But the number of sugar residues at the 3-position, the oxidation state of the C ring [25], the hydroxylation and methylation pattern, as well as the acylation by phenolic acids [16] are considered crucial factors for the expression of antioxidant effects.
The percentage of DPPH radical scavenging activity against the content of the total anthocyanins, the sum of 3-monoglucoside, the sum of 3-acetyl-3-glucoside and the sum of p-coumaryl-3-glucoside of wine samples are plotted in Figures 2 to 5.  The strong correlation between total anthocyanins and DPPH · scavenging ability (r 2 = 0.9619) of tested wines was confirmed. The significant correlations were obtained between antiradical activity and the sum of 3-monoglucoside (r 2 = 0.95594), the sum of 3-acetyl-3-glucoside (r 2 = 0.9728) and the sum of p-coumaryl-3-glucoside (r 2 = 0.8873) of wine samples.

Wine samples
Eight selected Cabernet Sauvignon wines with definite geographical origin, from the 2007 and 2008 vintage seasons, produced from different agronomical and winemaking regions in the Balkans, were analyzed. All of them are listed in Table 1.

Determination of indices for anthocyanin pigment degradation, polymeric color, browning, and hue
Indices for anthocyanin degradation of the wine can be derived using the pH-differential method described by Giusti and Wrolstad [21]. The absorbance at 420 nm of the disulphide treated sample serves as an index for browning. The color density of the control sample and the polymer color of the disulphide bleached sample wine are calculated as follows: The value of hue is calculated as follows: The ratio between polymeration color and color density is used to determine the percentage of the color that is contributed by polymerized material: Polymeric color (%) = (polymeric color/color density) × 100

Free radical scavenging activity
The free radical scavenging activity of the wine samples was analyzed by using the 2,2′-diphenyl-1picrylhydrazyl (DPPH • ) assay [10,23,24] for antioxidant activity which is based on measurement of the loss of DPPH • color by change of absorbance at 517 nm caused by the reaction of DPPH • with tested wine sample. The reaction was monitored by a UV/VIS spectrophotometer. The diluted wine sample (wine was diluted with water, 1:10 v/v) and fresh 1x10 -4 M DPPH • metabolic solution were putted into a cuvette at the room temperature. After 20 min incubation period at room temperature, the absorbance was read against a blank at 517 nm. Scavenging capacity of DPPH • in percent (%) of each wine sample was calculated from the decrease of absorbance according to the relationship: Radical scavenging activity (%) = (1 -A sample -A blank /A control ) × 100 where A control is the absorbance of control reaction (3x10 -4 M DPPH• solution), A blank is the absorbance of dilution wine sample and A sample is the absorbance of the dilution wine sample with same concentration of DPPH• solution. Three analytical replicates were carried out on each sample wine.

High performance liquid chromatography (HPLC-DAD) analysis
Anthocyanins were analyzed by direct injection of the samples, previously filtered through a 0.45 µm pore size membrane filter, in an Agilent Technologies 1200 chromatographic system equipped with a Agilent photodiode array detector (DAD) 1200 with RFID tracking technology for flow cells and UV lamp, an automatic injector, and a ChemStation software. The column was termostated at 30 o C. After injecting 5 µL of wine sample, separation was performed in an Agilent-Eclipse XDB C-18 4.6 × 150 mm coumn. The HPLC grade solvents used were formic acid/water as solvent A and acetonitrile/formic acid/water as solvent B. The elution profile was as follows: from 0 to 28 min, 0.0% B, from 28 to 35 min, 25% B, from 35 to 40 min, 50% B, from 40 to 45 min, 80% B, and for last 10 min again 0% B. The detection wavelength was 520 nm. The different anthocyanin compounds were identified by comparing their retention times and spectral characteristics with data given in the literature [18,20,27,28]. Quantitation was made by means of a calibration curve obtained by injecting standard solutions of malvidin-3-monoglucoside chloride with different concentrations. The range of the linear calibration curve (r 2 was 0.9991). Results were expressed as mg/L sample.

Statistical analyses
Three analytical replicates were carried out on each sample wine. Measurements were averaged, and results are given as mean ± standard deviation (SD). Correlation between the anthocyanin content and antiradical efficiency was established using regression analysis at a 95% significance level (P ≤ 0.001).

Conclusions
From the results obtained, we can conclude that the amount of anthocyanins is important for understanding of antioxidant potency of red wines. The mechanism by which anthocyanins are absorbed and metabolized in the body is currently unclear. The high anthocyanin content in the investigated Cabernet Sauvignon wines contributes to its increased radical scavenging activity. It can be concluded that, the anthocyanin composition can be used as biochemical marker for the authenticity of red grape cultivar and their corresponding single-cultivar wine.