Comparison of Total Phenolic Content and Total Antioxidant Activity in Local Red Wines Determined by Spectrophotometric Methods

In this study, the antioxidant activity of local red wines was determined by four different analytical methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2'-azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS∙+) radical cation decolourisation, ferric reducing/antioxidants power (FRAP) and cupric ion reducing capacity in the presence of neocuproine (CUPRAC) for the evaluation of reducing power (PR). They were correlated with the total phenolic content (TPC) determined with the Folin-Ciocalteu’s reagent using gallic acid as a standard. The total polyphenol concentration was found to vary from 2599.90 to 4846.57 mg/L gallic acid equivalents (GAE). The total antioxidant activity determined by DPPH, ABTS, FRAP and CUPRAC methods was found to vary from 7.49 15.93 mmol/L, 12.02 24.73 mmol/L, 12.65 27.68 mmol/L and 13.19 31.07 mmol/L, respectively. The total phenolic contents of red wine samples exhibited a good correlation (p < 0.01) with antioxidant properties.


Introduction
Antioxidants are substances that, at low concentrations, prevent or retard the oxidation of easily oxidisable biomolecules such as lipids, proteins or DNA.They counteract free radicals and thus prevent oxidative damage [1] [2].There are two basic categories of antioxidants, namely, synthetic and natural.The interest of natural antioxidants has increased in recent years because of their presumed safety and potential nutritional and therapeutical effects [3].Epidemiological studies indicated that a higher level of natural antioxidants (vitamins, carotenoids, phenolics) in human everyday diet can protect against cardiovascular diseases, cataract, cancer and aging-related disorders [4].Among natural antioxidants, red wine has attracted particular interest due to a high content of biologically active compounds [3].Phenolic compounds are one of the most important quality parameters of wines, and they contribute to organoleptic characteristics such as color, astringency, and bitterness [5].These compounds are also active in biochemical processes and have nutraceutical effects on human health, including antimicrobial, anticarcinogenic, and antioxidant properties [6].Consequently, numerous papers have focused on the determination of antioxidant capacities of wines and correlation with their polyphenol content [5]- [12].
Several in vitro methods have been developed to measure antioxidant capacities of food, beverages and biological samples.The most commonly used antioxidant capacity assays were 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) assay [13], 2,2-azino-di-(3-ethylbenzothialozine-sulphonic acid) (ABTS) assay [14], ferric ion reducing antioxidant power (FRAP) assay [15] [16], cupric ion reducing capability (CUPRAC) assay [17] and oxygen radical absorbance capacity (ORAC) assay [18] [19].These methods differ from each other in terms of their assay principles and reaction conditions.Single assay will not accurately reflect all antioxidants.Therefore it is recommended that at least two or all of these assays be combined to provide a full profile of antioxidant capacity of foodstuff [20].
The main objective of this study was to determine the polyphenolic content and antioxidant capacity of local wine samples and to compare the antioxidant capacity of these samples applying four most widely used spectrophotometric methods: DPPH, ABTS, FRAP, CUPRAC and to estimate correlation of antioxidant capacities with total phenolics.

Wine Samples
8 red wine samples were purchased from local stores in Elazığ and Malatya (Table 1).Samples were opened, protected against sunlight and stored at 4˚C.Analyses were carried out within a few days.Each wine was analyzed 5 times.

Equipment
Absorbance measurements were performed on a UV mini 1240 UV-Visible spectrophotometer (Shimadzu, Japan).

Determination of Total Phenolic Content
Spectrophotometric determination of the TPC was carried out with Folin-Ciocalteu method as adapted for wine analysis [21] using gallic acid as the standard.This method is based on the reduction of a phosphotungstenphophomolybdate complex by phenolics to blue reaction products.For the preparation of calibration curve, 0.1 mL aliquots of 50, 100, 150, 200, 250 and 300 mg/L aqueous gallic acid solutions or 0.1 mL 20-fold dilution samples of red wines (diluted with 13% (v, v) ethanol) were introduced into a test tube and then 2 mL of 2% sodium carbonate was added.After incubation for 2 min, 0.1 mL of Folin-Ciocalteu's reagent (diluted with water 1:1, v/v) was added.After a further 30 min the absorbance was measured at 750 nm using a spectrophotometer.
Results were expressed as mg gallic acid equivalents per litre of wine (mg GAE/L).

ABTS• + Radical-Scavenging Assay
ABTS assay was based on a previously published method with some modifications [14], using Trolox (6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid), a water-soluble analog of vitamin E as standard.ABTS• + radical cation was produced by mixing 7 mM ABTS solution and 2.46 mM potassium persulfate aqueous solution.This stock solution was kept in the dark at room temperature for 12 -16 h for incubation.The ABTS• + solution was then diluted with ethanol (approximately 1:90, v/v) to obtain an an absorbance of 0.7 ± 0.02 at 734 nm.After 100 µL diluted sample or Trolox standard solution was added to 2.4 mL ABTS• + solution, absorbance was measured at exactly 6 min.Results were expressed as Trolox-equivalent antioxidant capacity (TEAC) values (mM).The standard curve was linear between 0 -500 µM Trolox.

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay was performed according to previously described method with some modifications [15].The FRAP reagent was prepared freshly.The stock solution included 25 mL acetate buffer (300 mM, pH 3.6), 2.5 mL 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl and 2.5 ml of 20 mM FeCl 3 •6H 2 O.The mixture was warmed at 37˚C before using.150 µL diluted sample or Trolox standard solution was added to 2.85 mL FRAP solution.The absorbance was measured at 593 nm after 30 min.Aqueous solutions of Trolox concentration were between 0 and 300 µM for calibration.

Cupric Reducing Antioxidant Capacity (CUPRAC)
Cupric reducing antioxidant capacity assay was carried out according to the method of Apak et al. [17]. 1 mL each of 10 mmM Cu(II), 7.5 mM neocuprine, 1 M ammonium acetate buffer (pH 7) solutions and 0.6 mL water were mixed in a test tube.0.5 mL of diluted wine or Trolox standard solutions were added to the initial mixture.
The absorbance was measured at 450 nm after 30 minutes.Trolox standard solutions were prepared at a concentration range from 40 to 400 µM.

Statistical Analysis
Data is expressed as means ± SD of five paralel measurements.Correlation analysis was performed by Spearman rho coefficient.p < 0.01 was regarded as statistically significant.

Total Phenolic Content and Total Antioxidant Capacity
The total phenolic content (TPC) and total antioxidant capacity content (TAC) of the local wine samples are presented in Table 2. TAC was measured by four different assays DPPH, ABTS, FRAP and CUPRAC using mmol Trolox equivalents per litre of wine (mmol TE/L) to express the results.The TPC of the wine samples were expressed mg of gallic acid equivalents per liter of wine (mg GAE/L).
TPC values of local wines ranged from 2599.90 to 4846.57mg GAE/L.The total phenolic contents determined by the Folin-Ciocalteu method for wines from other countries are as follow: M. S. Fernandez-Pachon et al. 1313 -2389 mg GAE/L for Spanish red wines [23].Kallithraka [35] (Table 4).
Different authors have reported very different Trolox equivalents values.This is expected since experimental conditions of DPPH, ABTS, FRAP and CUPRAC methods were different from each other in many details.Significant differences in antioxidant activity existed not only experimental conditions but also variety of red wines due to different origins and winemaking procedures.

Correlation between Antioxidant Capacity and Total Polyphenols
Correlation analysis was used to explore the relationships amongst the different antioxidant parameters measured for all wine samples (Table 5).The total phenolic contents of red wine samples exhibited a good correlation (p < 0.01) with antioxidant properties.Amongst the methods the strong correlation between DPPH and Folin    lin method was 0.864; CUPRAC and Folin method was 0.848.Amongst the methods used for quantifying antioxidant activities, the correlation between FRAP and CUPRAC; FRAP and ABTS were 0.886 and 0.870, respectively.These results are in agreement with other reports in the literature [5] [6] [8]- [12] [30] [32].

Conclusions
Since the above methods differ from each other in terms of assay principles and reaction conditions, one single method is not enough to show all the antioxidants.Therefore this study compared four most widely used spectrophotometric methods.A good correlation between the antioxidant activities determined by DPPH, ABTS, FRAP and CUPRAC methods versus total phenol content (Folin Method) was observed for the red wines in this

Table 1 .
The wine samples tested.

Table 2 .
Total antioxidant capacity and total phenolic content of wine samples.

Table 3 .
Comparison of present study and published values of total phenolic content in red wines (mg gallic acid equivalents/L).

Table 5 .
Spearman rho correlation coefficients (R) among the different methods.