ANTIOXIDANT PROPERTIES, TOTAL PHENOLIC AND TOTAL FLAVONOID CONTENT OF THE SLOVAK WHITE WINES – WELSCHRIESLING AND CHARDONNAY

The biologically active compounds in wines, especially phenolics, are responsible for reduced risk of developing chronic diseses (cardiovascular diseases, cancer, diabetes, Alzheimer disease, etc.), due to their antioxidant activities. Twenty six Slovak white wines, produced from different geographical origins, were examined in this study. The antioxidant activity, total phenolic and flavonoid contents of two types monovarietal wines – Welschriesling and Chardonnay were evaluated. All three mentioned parameters were determined by UV-VIS absorption spectrometry. The results showed that both types of Slovak white wines were high in polyphenols (average content was 303.2 mg GAE.L in Welschriesling, resp. 355.6 mg GAE.L in Chardonnay) and flavonoids (average content was 51.9 mg CE.L in Welschriesling, resp. 60.1 mg CE.L in Chardonnay), as well as a high antioxidant activity (average value was 35.0% inhibition of DPPH in Welschriesling, resp. 43.3% inhibition of DPPH in Chardonnay), comparable to the wines produced in other regions in the world. Among the white wines, Chardonnay had higher content of total polyphenols, as well as flavonoids and higher values of antioxidant activity. Our results confirmed very strong linear correlations between all three analysed parameters (TPC, TFC and AA): TPC and TFC (r = 0.818), AA and TPC (r = 0.699), resp. TFC and AA (r = 0.693).


INTRODUCTION
The terms antioxidant and free radical are popular expressions used by nutritionists, scientists and general public. Free radicals are chemical species, compounds and/or elements with one or two unpaired electrons in their outer layer, which can be created in a multiple ways. They can be exogenic (e.g. ultraviolet radiation, xenobiotics and infections) or endogenic (Andzi Barhé and Feuya Tchouya, 2014). A lack of antioxidant or an overproduction of free radicals can lead to an imbalance between the oxidant and antioxidant system ( Wine is an alcoholic fermented beverage with considerable amounts of phytochemicals (Gresele et al., 2011). Wine is very rich source of polyphenols, such as catechins, epicatechins, quercetin, rutin, myricetin, anthocyanins, fenolics acids (gallic acid, caffeic acid, p-coumaric acid, etc.), trans-resveratrol and many others polyphenols and compounds. Many of these componds have been reported to have multiple biological activities, including cardioprotective, anti-carciogenic, antiviral and antibacterial properties ( and anti-diabetic (Marfak, 2003) properties. Currently, chemoprevention is being used in medicine as a new strategy to prevent cancers. Natural phytochemicals, including wine and/or grape polyphenols, appear to be very promising substances to block, reverse, retard or prevent the process of carcinogenesis (Russo, 2007).
The total amount of polyphenols in wines has been estimated to range from 200 to 6000 mg.L -1 (Quideau et al., 2011). The highly variable level of phenolic compounds in wine is due to differences in grape variety and source as well as processing. Wine polyphenols have been reported to be bioavailable in several studies (Nardini et al., 2009;Vitaglione et al., 2005). These compounds are directly related to the quality of wines, so phenolic analysis can be used as an effective tool in characterizing defferent wines. Many factors can influence the phenolic composition if wines, including grape variety and the technology applied (Mulero et al., 2011).
The purpose of this study was to determine and evaluate chosen properties (the content of total polyphenols, content of total flavonoids and antioxidant activity) and their mutual correlations in white wines -Welschriesling (typical Central European variety) and Chardonnay (most famous world variety), originated from 3 most important Slovak vineyard areas.

Samples
Analysed, bottled, white, especially dry wines Welschriesling (WR), resp. Chardonnay (CH) and their characteristics are mentioned in Table 1 and 2. Wine samples with origin in various Slovak vineyard areas (VA) were purchased in retail network, to provide that analysed samples of wine would have the same properties as wines that are consumed by common consumers (properties of wine affected by various factors, such as period and conditions of storage or distribution of wine).

Antioxidant activity determination
Antioxidant activity (AA) was assessed by method of Brand-Williams et al. (1995) using of DPPH (2,2diphenyl-1-picrylhydrazyl) radical. This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen-donating antioxidant due to the formation of the nonradical form DPPH-H (Chanda and Dave, 2009). This transformation results in a color change from purple to yellow, which is measured spectrophotometrically. Briefly, 0.1 mL of white wine was added to 3.9 mL of a 6x10 -5 mol.L -1 solution of DPPH in methanol. A control sample containing the same volume of water instead of extract was used to measure the maximum DPPH absorbance. After the reaction had been allowed to take place in the dark for 10 min, the absorbance at 515.6 nm was recorded to determine the concentration of remaining DPPH. The percentage inhibition of the initial concentration of DPPH radical was calculated as: % inhibition = [(ADPPH -Awine)/ADPPH] x 100. The results were also expressed as Trolox equivalent antioxidant capacity using calibration curve method.

Determination of total polyphenol content
Total polyphenol content (TPC) was determined by modified method of Singleton and Rossi (1965). 0.1 mL of wine sample was pipetted into 50 mL volumetric flask and diluted with 5 mL of distilled water. To diluted mixture 2.5 mL Folin-Ciocalteau reagent was added and after 3 minutes 7.5 mL of 20% solution of Na 2 CO 3 was added. Then the sample was filled with distilled water to volume 50 mL and after mixing left at the laboratory temperature for 2 hours. By the same procedure the blank and calibration solutions of gallic acid were prepared. Absorbance of samples solutions was measured against blank at 765 nm. The content of total polyphenols (TP) in wines was calculated as amount of gallic acid equivalent (GAE) in mg per 1 litre of wine.

Determination total flavonoid content
Total flavonoid content (TFC) was assessed by aluminium chloride method (Chang et al., 2002). 1 mL of wine sample and 4 mL of deionised water were added to a 10 mL volumetric flask. 5 min after adding 0.3.mL of 5% sodium nitrite, 0.3 mL of 10% aluminium chloride was added. 2 mL of sodium hydroxide with cincentration 1 mol.L -1 was added to the reaction mixture after 6 min incubation. The final volume was immediately made up to 10 mL with deionised water. The absorbance of the solution was measured at 510 nm against blank solution. The content of total flavonoids (TF) in wine samples was calculated as amount of catechin equivalent (CE) in mg per 1 litre of wine.

Statistical analysis
Statistical analysis were performed using the software Statistica 6.0 (StatSoft) and the results were evaluated by analysis of variance ANOVA.

RESULTS AND DISCUSSION
All studied parametersthe content of total polyphenols, the content of total flavonoids and antioxidant activity of the slovak wines Welschriesling, resp. Chardonnay are described in Table 3 and Table 4.   According to the average value of TPC an order for wines -Chardonnay could be as following: wines from South Slovak VA >wines from Nitra VA >wines from Little Carpathian VA. Gained results also did not exert statistically significant differences (at significance level p = 0.05) between TPC in wines -Chardonnay made in various vineyard areas in Slovakia.
Total On the basis of value of AA for wines -Chardonnay an order could be as following: wines from South Slovak VA >wines from Nitra VA >wines from Little Carpathian VA. Gained results also did not exert statistically significant differences (at significance level P = 0.05) between values of AA in wines -Chardonnay made in various vineyard areas in Slovakia.
The results showed that both types of Slovak white wines were high in polyphenols and flavonoids, as well as a high antioxidant activity, comparable to the wines produced in other regions in the world. Similar conclusions were  In order to investigate the mutual relations between analyzed parameters, the linear regressions were obtained. The statistical evaluation of the obtained results confirmed very highly significant correlations at significance level p <0.001 between all 3 studied parameters: TPC and TFC (r = 0.818), AA and TPC (r = 0.699), resp. TFC and AA (r = 0.693). This was consistent with many other articles in the literature, which also reported a very high degree of correlation between the total phenols, total flavonoids and antioxidant properties of wines (

CONCLUSION
The phenolic, resp. flavonoid content and antioxidant properties of white wines (Welschriesling and Chardonnay) made in 3 most important Slovak vineyard areas was evaluated in the present study. All 3 studied parameters of analysed Slovak winestotal polyphenol content, total flavonoid content and antioxidant activity are comparable to the wines produced in other regions in the world. Slovak white wines -Welschriesling and Chardonnay have high content of healthy useful phenolic compounds and high antioxidant activity. The results didn´t showed statistically significant differences for all 3 studied parameters (except AA in Welschriesling) in wines made in different vineyard areas in Slovakia. On the basis of statistical evaluation of our results we can state that statistically very highly significant correlations were demonstrated between all 3 parameters (TPC, TFC and AA).