Polyphenolic Composition and Antioxidant Activities of Grape Seed Extract

Transcription

1 International Journal of Food Properties ISSN: (Print) (Online) Journal homepage: Polyphenolic Composition and Antioxidant Activities of Grape Seed Extract Anamarija I. Mandic, Sonja M. Đilas, Gordana S. Ćetković, Jasna M. Čanadanović-Brunet & Vesna T. Tumbas To cite this article: Anamarija I. Mandic, Sonja M. Đilas, Gordana S. Ćetković, Jasna M. Čanadanović-Brunet & Vesna T. Tumbas (2008) Polyphenolic Composition and Antioxidant Activities of Grape Seed Extract, International Journal of Food Properties, 11:4, , DOI: / To link to this article: Copyright Taylor and Francis Group, LLC Published online: 17 Nov Submit your article to this journal Article views: 1167 Citing articles: 55 View citing articles Full Terms & Conditions of access and use can be found at

2 International Journal of Food Properties, 11: , 2008 Copyright Taylor & Francis Group, LLC ISSN: print / online DOI: / POLYPHENOLIC COMPOSITION AND ANTIOXIDANT ACTIVITIES OF GRAPE SEED EXTRACT Anamarija I. Mandic, Sonja M. Ðilas, Gordana S. 1etkovic, Jasna M. Canadanovic-Brunet, and Vesna T. Tumbas University of Novi Sad, Faculty of Technology, Novi Sad, Serbia Grape seed extracts (GSEs,) obtained from Italian and Rhine Rieslings, were examined for polyphenolic composition and antioxidant activities using HPLC and ESR spectrometry. The seed extraction was carried out with ethyl acetate and ethanol. The contents of polyphenols, flavan-3-ols and antioxidant activities were found to be higher in ethyl acetate than in ethanolic extracts. IC 50 values were mg/ml and mg/ml for the stable DPPH radical in ethanolic and ethyl acetate extracts, respectively. The values for the shortlived OH radical were mg/ml and mg/ml, in the given order. The significant correlations between the antioxidant activities of GSEs and polyphenols were established (P < 0.05). Owing to their antioxidant activities, the cultivars could be used as a source to produce a GSE. Keywords: Grape seed extract, Flavan-3-ols, HPLC, Antioxidant activity, ESR spectrometry, White grape variety. INTRODUCTION Polyphenolic compounds are very effective to counteract the oxidative stress and have a protective and beneficial role for human body. [1,2] As GSE contains a lot of polyphenols, it is considered as a superior natural antioxidant and is used as a health promoting product. The use of synthetic antioxidants in the food has been under scrutiny for toxicological reasons, and therefore the interest in the natural antioxidants has steadily been increasing. [2,3,4] The antioxidant and radical scavenging activities of a large number of polyphenolic compounds isolated from plants have been studied. [5 9] The monomeric phenolic compounds [(+)-catechin, ( )-epicatechin, and ( )- epicatechin-3-o-gallate], dimeric, trimeric, and tetrameric procyanidins are the most interesting constituents of grape seeds. The procyanidins are bioconstituents of white and red grapes, but the most concentrated procyanidin amounts are thought to be in the seeds of red grapes. The research of the composition of polyphenolic compounds has mainly been focused to the red grape varieties in most papers, while only few studies reported on the white grape varieties. [10 14] Received 15 January 2007; accepted 20 July Address correspondence to Anamarija I. Mandic, University of Novi Sad, Faculty of Technology, Bul. Cara Lazara, Novi Sad , Serbia. anam@technol.ns.ac.yu 713

3 714 MANDIC ET AL. The aim of this work was to determine the polyphenolic composition and antioxidant activities of GSEs obtained by two different extraction procedures. Two white grape varieties, Italian and Rhine Rieslings, harvested in two seasons in Serbia were used. The antioxidant activities for scavenging the stable DPPH and short-lived OH free radicals were determined using the contemporary technique, ESR spectrometry. The correlation between the composition and the antioxidant activities were calculated. The obtained data indicate that the GSEs antioxidant activities might be of interest as they contained a lot of functional polyphenols. MATERIAL AND METHODS The samples (about 50 kg of pomace) were collected at the Navip winery in the years of 2003 and The grapes used for processing were harvested at optimum technological maturity, as judged by indices of sugar and acid contents, established by the laboratory in the Navip winery. The average sugar contents in grapes in the years of 2003 and 2004 were 17.0 and 15.8%, and the acid contents 6.4 and 7.0%, respectively. After pressing, the pomace of two Vitis vinifera cultivars, Italian and Rhine Rieslings, was sampled for experiments. The seeds were separated from other pomace components (skin and stems), dried at room temperature, and stored at 24 C prior to extraction. Chemicals All solvents used for the extraction and spectrophotometric determination were analytic grade. Methanol and ethanol were purchased from Lachema (Neratovice, Czech Republic) and ethyl acetate from Kemika (Zagreb, Croatia). Acetonitrile and methanol, LiChrosolv, gradient grade for chromatography and vanillin were obtained from Merck (Darmstadt, Germany). 2, 2-Diphenyl-1-picrylhydrazil stable radical (DPPH radical), 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO), gallic acids, (+)-catechin, ( )-epicatechin, and FeCl 2 4H 2 O were purchased from Sigma-Aldrich Co. (St. Louis, MO). Folin-Ciocalteau reagent was obtained from Fluka (St. Gallen, Switzerland) and Trolox from Aldrich (Milw aukee, WI). Extraction Procedures Two different extraction procedures were used to prepare the GSEs. In each of them, 100 g of grape seeds was extracted with 400 ml of the solvent in a sealed bottle at room temperature for 48 h, with occasional mixing. Extraction with ethanol (procedure I). Forty ml of water and 360 ml of ethanol were added to 100 g of whole grape seeds. The obtained extract was separated by filtration through a filter paper and the solvent was removed by evaporation under the reduced pressure, at maximum temperature 40 C. The yields of the obtained GSEs were calculated. The following abbreviations for the obtained GSEs according to the procedure I were introduced: I-1-i and I-2-i for GSEs for the Italian Riesling harvested in years of 2003 and 2004, respectively; and I-1-r and I-2-r for GSEs for the Rhine Riesling harvested in years of 2003 and 2004, respectively. Extraction with ethyl acetate (procedure II). The extraction with 90% ethyl acetate was carried out according to the method described by Pekic et al. [15] The following abbreviations for the obtained GSEs according to the procedure II were introduced: II-1-i and

4 COMPOSITION AND ANTIOXIDANT ACTIVITIES OF GRAPE SEED EXTRACTS 715 II-2-i for GSEs for the Italian Riesling harvested in years of 2003 and 2004, respectively; and II-1-r and II-2-r for GSEs for the Rhine Riesling harvested in the years of 2003 and 2004, respectively. All the obtained extracts were stored at 24 C until analysed. Spectrophotometric Determination of Total Soluble Polyphenols and Flavan-3-ols in Extracts The amount of the total soluble polyphenols in the extracts was determined spectrophotometrically according to the Folin-Ciocalteau method. [16] Gallic acid was employed as a calibration standard and the results were expressed as gallic acid equivalents in grams per 1 kg of dry seed weight. The amount of total flavan-3-ols was assayed colorimetrically by the vanillin method using (+)-catechin as a standard. [17,18] The values were expressed as catechin equivalents in grams per 1 kg of dry seed weight. All analyses were performed in triplicate and the results were averaged. HPLC Analysis of GSEs HPLC analyses were conducted with Hewlett-Packard Liquid Chromatograph HP 1090 equipped with Diode Array Detector (DAD). A reversed-phase column (Zorbax SB-C18, 5 μm, mm i.d.), protected by guard column (Zorbax SB-C18, 5 μm, mm i.d., Agilent, USA) was used throughout this research. The detection was performed at 277 nm and the absorption spectra of the compounds were recorded between 210 and 400 nm. The solvent gradient was formed by varying the proportion of the solvent A (1% acetic acid in water, v/v) to the solvent B (acetonitrile). [19] The solvent linear gradient elution programme was as follows: 0 20 min, 95 87% A; min, 87% A; min, 87 78% A; min, 78 10% A; min, 10% A. The column was equilibrated to the initial conditions, 95% A, 10 min. The flow rate was set at ml/min. The column was operated at room temperature (22 C). The sample injection volume was 10 μl, and the injection was performed manually. The GSE was dissolved in 10% (v/v) methanol in water and the obtained concentration was 1.0 mg/ml. All solutions were filtered through 0.45 μm pore size nylon filters (Rotilabo Spritzenfilter 13 mm, Roth, Karlsruhe, Germany) before injecting them into the HPLC system. Phenolic components in a sample extract were identified by matching the retention time and their spectral characteristics against those of the standards. The purity of the peaks was determined to ensure the identification. The external standard method was a technique used for quantification. For each component, [gallic acid, (+)-catechin, and ( )-epicatechin] a stock solution was made from the commercial standards which was dissolved in 10% (v/v) methanol in water and the obtained concentration was 1.0 mg/ml. The diluted stock solutions were used for calibration. The final concentrations were in the range of mg/ml. The peak areas from the chromatograms were plotted against the known concentrations of the standards. The equations generated via linear regression were used to establish the concentrations of the phenolic compounds in the extracts. When reference compounds were not available, the calibration of (+)-catechin was used. The mean values with the standard deviations (S.D.) were reported. Antioxidant Activity The antioxidant activities of the GSEs were determined as the measure of radical scavenging using DPPH and OH radicals, measured by the electron spin resonance (ESR)

5 716 MANDIC ET AL. spectrometer. The spectrophotometric method was used to determine the DPPH radical. The value of the inhibitory concentration (IC 50 ), defined as the amount of an antioxidant necessary to decrease the initial free radical concentration by 50%, was calculated for each of the obtained GSEs. ESR spectrometry DPPH radical assay. The blank probe was obtained by mixing 400 μl 0.4 mmol/l methanolic solution of DPPH radical and 200 μl of methanol. A volume of μl of 1% methanolic solution of the investigated extract was added to (200-x) μl of methanol and 400 μl of 0.4 mmol/l methanolic solution of DPPH radical. The range of the investigated extract concentrations was mg/ml. The mixture was then stirred for 2 min and transferred to a quartz flat cell ER-160FC. The ESR spectra were recorded on the ESR spectrometer Bruker 300E (Rheinstetten, Germany) under the following conditions: field modulation 100 khz, modulation amplitude: G, receiver gain, , time constant ms, conversion time ms, centre field G, sweep width G, x-band frequency 9.64 GHz, power 20 mw, and temperature 23 C. [20] The antioxidant activity (AA) value of each extract was defined using the following Eq. (1): AA (%) = 100 ( h0 h ) / h0, (1) where h 0 and h x are the height of the second peak in the ESR spectrum of DPPH radical of the blank and the probe, respectively. The value of the calculated IC 50 was expressed as mg GSE/mL of DPPH radical for each extract. Hydroxyl radical assay. It is known that the hydroxyl radicals are the major active oxygen species causing the lipid oxidation. [21] The Fenton reaction (Fe 2+ + H 2 O 2 Fe 3+ + OH + OH) was used as a source of hydroxyl radicals to test the reactions between the hydroxyl radicals and the extracts. Using the spin trap, such as DMPO, it is possible to convert reactive hydroxyl radicals into stable nitroxide radicals (DMPO-OH adducts) with spectral hyperfine splittings that reflect the nature and structure of these radicals. The hydroxyl radicals were obtained by the Fenton reaction in the system: 0.4 ml, c(dmpo) = 0.3 mmol/l; 0.4 ml, c(h 2 O 2 ) = 10 mmol/l; 0.4 ml, c(fe 2+ ) = 10 mmol/l (blank). The influence of the extracts on the formation and stabilization of hydroxyl radicals was investigated by adding the extracts to the Fenton reaction system in the range of concentrations of mg/ml. The ESR spectra were recorded after 5 min, with the following spectrometer settings: field modulation 100 khz, modulation amplitude G, receiver gain, , time constant ms, conversion time ms, centre field G, sweep width G, x-band frequency 9.64 GHz, power 20 mw, temperature 23 C (20). The AA value of each extract was defined using the following Eq. (2): where h 0 and h x are the height of the second peak in the ESR spectrum of DMPO-OH spin adduct of the blank and the probe, respectively. The values of calculated IC 50 were expressed as mg GSE/mL of OH radical for each extract. The magnetic field scanning was calibrated using Fremy s salt (peroxylamine disulphonate). The splitting constants were calculated from the computer-generated second derivatives of the spectra after optimizing the signal-to-noise ratios and were verified by computer simulations. x AA (%) = 100 ( h0 h ) / h0, (2) x

6 COMPOSITION AND ANTIOXIDANT ACTIVITIES OF GRAPE SEED EXTRACTS 717 Spectrophotometric assay for DPPH. In a test tube containing 3 ml of methanolic GSE solution, prepared at five different concentrations ranged between 0.1 and 3.5 μg/ml, 1 ml of c(dpph radical) = 90 μmol/l (dissolved in 95% methanol in water, v/v) was added (sample). The blank was prepared by adding 1 ml of 95% methanol, instead of DPPH radical solution, to 3 ml of GSE solution at each concentration. The reaction mixture was allowed to stay in the dark at room temperature for 60 minutes. The absorbance was read at 515 nm using 95% methanolic solution as a reference solution. [22] The percentage of the remaining DPPH radical (%DPPHŽ rem ) was calculated for each concentration of the GSE from the obtained absorbance for the sample (A s ) and the blank (A 0 ) using the following Eq. (3): % DPPHrem = ( As / A0 ) 100. (3) The values of the calculated IC 50 were expressed as mg GSE/mg of DPPH radical for each extract. Statistics All determinations were made in the triplicate and the values were averaged and reported along with the standard deviation (± Standard Deviation). The differences between the results for the tested grape varieties, the years of sampling and the extraction procedures were analysed using multifactor analysis of variance. The mean values were compared by the Duncan s multiple range test. Pearson correlation coefficients between all the obtained antioxidant activities and the content of polyphenols in GSEs were determined with the significance level of P < RESULTS AND DISCUSSION Proanthocyanidins from the grape seeds are extracted with the methanol, ethanol, acetone and ethyl acetate, or with their mixtures; for analytical and preparative purposes. [23] Proanthocyanidins are well soluble in ethyl acetate and this solvent possesses significant selectivity towards the extraction of the flavan-3-ols of lower molecular masses. [15] The choice of extractants used in this examination was made according to their low toxicity and therefore the ethanol (procedure I) and ethyl acetate (procedure II) were selected. The yields of the GSEs from the seeds of two cultivars harvested in two years, using ethanol and ethyl acetate for the extraction, expressed in grams of GSE per kg of dry seeds, are presented in Table 1. It is well known that the genetic potential of the individual species for the polyphenol biosynthesis and maturation stage may affect polyphenol content in seeds, along with the variations from season to season. [24 26] As it was expected, due to the referred causes, the differences in the GSE yields between the years of harvesting and the used extraction methods were observed. The yields of the extracts obtained with ethyl acetate for the Italian Riesling (II-1-i and II-2-i) were higher than the yields ( g/kg of dry seeds) reported by Pekic et al. [15] for the same grape variety. These differences could be attributed to the variations due to the seasons within the grape variety. The yields of the obtained GSEs ranged from 4.40 to 15.6 g/kg of dry seeds. The average yield, when ethyl acetate was used, was 5.41 g/kg and when ethanol was applied

7 718 MANDIC ET AL. Table 1 Yields of GSEs of investigated white grape varieties, expressed in grams of GSE per kg of dry seeds. Cultivar Year of harvesting Extraction procedure Sample Yield (g/kg of dry seeds) Italian Riesling 2003 Ethanol (I) I-1-i 15.6 ± 0.78 Ethyl acetate (II) II-1-i 6.03 ± Ethanol (I) I-2-i 15.0 ± 0.47 Ethyl acetate (II) II-2-i 5.94 ± 1.13 Rhine Riesling 2003 Ethanol (I) I-1-r 14.8 ± 3.26 Ethyl acetate (II) II-1-r 5.25 ± Ethanol (I) I-2-r 14.7 ± 0.01 Ethyl acetate (II) II-2-r 4.43 ± 0.26 for the extraction it was 15.0 g/kg of dry seeds. The yields of ethyl acetate extracts were approximately three times lower than those of ethanol extracts, as it could be seen in Table 1. Total Soluble Polyphenols and Flavan-3-ol Determination in GSEs The results of spectrofotometric determination of total soluble polyphenols and flavan-3-ols contents in GSEs are shown in Table 2. The Folin-Ciocalteou method is based on the reducing power of the phenolic hydroxyl groups. This method is not very specific as it detects all phenols with varying sensitivities. In this paper, the total soluble polyphenols of the obtained GSEs are expressed as gallic acid equivalents. The contents of the total soluble polyphenols in GSEs varied from 3.59 to 11.7 g/kg of dry seeds. Comparing the yields obtained from the methanolic extracts, g/kg of dry seeds, reported by Revilla et al. [14] for the white grape varieties, the yields were similar or higher than those obtained in this research. The GSEs prepared according to the procedure I contained %, and according to the procedure II, % (w/w GSE) polyphenols. As it was mentioned above, the yields of ethyl acetate extracts (II) were lower than those of the ethanolic extracts (I) because ethyl acetate is more selective. The content of the total soluble polyphenols was higher in ethyl acetate extracts (II) than in ethanolic ones (I). In order to determine the flavan-3-ols content in the extracts, the vanillin assay was performed and the (+)-catechin was used as a standard for calibration. The content of flavan-3-ols in GSEs varied from 3.70 to 11.5 g/kg of dry seeds and again it was higher for the extracts obtained according to the procedure II. The results for the tested GSEs Table 2 Contents of total soluble polyphenols, flavan-3-ols, and total phenolics determined by HPLC in GSEs of investigated white grape varieties (g/kg of dry seed). Sample Total soluble polyphenols Flavan-3-ols Total phenolics (HPLC) I-1-i 11.8 ± ± ± 0.08 II-1-i 4.92 ± ± ± 0.21 I-2-i 11.3 ± ± ± 0.20 II-2-i 4.83 ± ± ± 0.16 I-1-r 11.2 ± ± ± 0.04 II-1-r 4.22 ± ± ± 0.18 I-2-r 10.9 ± ± ± 0.40 II-2-r 3.59 ± ± ± 0.09

8 COMPOSITION AND ANTIOXIDANT ACTIVITIES OF GRAPE SEED EXTRACTS 719 indicated that the extracts obtained according to the procedure I contained %, and according to the procedure II % (w/w GSE) flavan-3-ols. The correlation analysis showed that the content of the total soluble polyphenols was significantly correlated with flavan-3-ols content in GSEs (r = 1, P < 0.05). HPLC Analysis of Extracts A typical HPLC chromatogram of GSE is shown in Figure 1. The separation of the phenolic compounds in GSE was achieved within 60 min. Under the described chromatographic conditions, the components were eluted in the following order: gallic acid, procyanidin-b 3, procyanidin-b 1, (+)-catechin, procyanidin- B 4, procyanidin-b 2, ( )-epicatechin, procyanidin-c 1, dimer gallate, and procyanidin-b 5. The content of the total phenolic compounds determined by HPLC in GSEs ranged between 2.15 and 5.61 g/kg of dry grape seeds (Table 2). The distribution (%) of gallic acid, procyanidin-b 3, procyanidin-b 1, (+)-catechin, procyanidin-b 4, procyanidin-b 2, ( )- epicatechin, procyanidin-c 1, dimer gallate, and procyanidin-b 5 in GSEs by HPLC is given in Tables 3 and 4. (+)-Catechin was predominant in all investigated GSEs. In previously reported data for the white varieties by Bakkalba4i et al., [10] the content of ( )-epicatechin (38 64%) was higher in most cultivars than the content of (+)-catechin (36 61%). In the extracts, reported by Revilla et al., [14] (+)-catechin (31 61%) was more abundant than ( )-epicatechin (8.6 40%) in the majority of the investigated varieties. Fuleki & Ricardo da Silva [27] found that the ( )-epicatechin (21 43%) had been the most abundant compound in most white grape cultivars followed by (+)-catechin (14 27%). The obtained amounts of (+)-catechin (38 53%) and ( )-epicatechin (20 36%) in this research were similar to the findings of the authors listed above. Figure 1 HPLC chromatogram of a GSE II-1-r, recorded at 277 nm. Peak assignment (t R, min): (1). gallic acid (9.3); (2). procyanidin-b 3 (20.6); (3). procyanidin-b 1 (22.4); (4). (+)-catechin (25.0); (5). procyanidin-b 4 (27.5); (6). procyanidin-b 2 (29.1); (7). ( )-epicatechin (33.4); (8). procyanidin-c 1 (39.5); (9). dimer gallate (42.1); and (10). procyanidin-b 5 (49.9).

9 720 MANDIC ET AL. Table 3 Distribution (%) of phenolic compounds in GSEs in Italian Riesling, white grape cultivar. Phenolic compund I-1-i II-1-i I-2-i II-2-i Gallic acid 4.54 ± ± ± ± 0.27 Procyanidin-B 3 ND 1.56 ± 0.43 ND 1.51 ± 0.53 Procyanidin-B ± ± ± ± 0.45 (±)-Catechin 44.5 ± ± ± ± 1.77 Procyanidin-B ± ± ± ± 0.39 Procyanidin-B ± ± ± ± 0.24 ( )-Epicatechin 36.0 ± ± ± ± 1.49 Procyanidin-C ± ± ± ± 0.46 Dimer gallate 3.13 ± ± ± ± 0.40 Procyanidin-B ± ± ± ± 0.53 ND: not detected. Table 4 Distribution (%) of phenolic compounds in GSEs of Rhine Riesling, white grape cultivar. Phenolic compund I-1-r II-1-r I-2-r II-2-r Gallic acid 6.10 ± ± ± ± 0.15 Procyanidin-B ± ± 0.91 ND 1.58 ± 0.57 Procyanidin-B ± ± ± ± 0.22 (+)-Catechin 45.0 ± ± ± ± 2.39 Procyanidin-B ± ± ± ± 0.43 Procyanidin-B ± ± ± ± 0.44 ( )-Epicatechin 23.3 ± ± ± ± 0.81 Procyanidin-C ± ± ± ± 0.40 Dimer gallate 1.84 ± ± 0.74 ND 4.23 ± 0.39 Procyanidin-B ± ± ± ± 0.58 ND: not detected. The amount of the total soluble polyphenols in the extracts determined by the Folin- Ciocalteau spectrophotometric method was on average 1.5 to 2.5 times higher than the values obtained by HPLC (Table 2). The content of phenolic compounds in the ethyl acetate extracts reported by Guendez et al. [24] was on average three times lower determined by HPLC than the one obtained by the Folin-Ciocalteau methodology. Those results are in agreement with the results obtained in this research. The total phenolic content determined by HPLC was significantly correlated with the values determined by the Folin-Ciocalteau methodology (r = 0.90, P < 0.05) and was also significantly correlated with the content of flavan-3-ols (r = 0.90, P < 0.05). Antioxidant Activity DPPH radical scavenging activity of extracts. The relative intensity of the ESR spectroscopy signal is directly related to the concentration of free radicals. In this research, the stable DPPH radical was used to investigate the antioxidant activities of GSEs. The ESR spectrum of the stable DPPH free radical in the presence of 0.03 mg/ml of GSE (II-2-r) is shown in Figure 2.

10 COMPOSITION AND ANTIOXIDANT ACTIVITIES OF GRAPE SEED EXTRACTS 721 Figure 2 ESR spectrum of DPPH radical in the presence of 0.03 mg/ml of GSE sample II-2-r. Figure 3 Antioxidant activities of different concentrations of GSEs on DPPH radicals. (a) Extracts prepared in pocedure I; and b) extracts prepared in pocedure II. Bars represent the S.D. The antioxidant activity of GSEs at different concentrations on the DPPH radical is presented in Figure 3. The DPPH radical scavenging activity of extracts was examined within the range of the mass concentrations from 0.02 to 0.15 mg/ml. Generally, better results were obtained for the extracts prepared according to the procedure II than with the extracts prepared according to the procedure I. For the extracts prepared using the procedure I IC 50 was ( ± mg/ml), while it was much lower ( ± mg/ml) for the extracts obtained in the procedure II. The difference in the antioxidant activity between the procedures I and II was significant (P < 0.01). It was found that the antioxidant activity increased while increasing the concentration of all extracts. The GSEs influenced the DPPH radical depending on the type of the extraction procedure and the concentration of the extracts. Hydroxyl radical scavenging activity of extracts. The antioxidant activity of the GSEs was investigated on the capacity of the extracts to scavenge hydroxyl radicals. The ESR spectrum of DMPO-OH spin adduct in the Fenton reaction system in the presence of mg/ml of GSE II-1-r, is shown in Figure 4.

11 722 MANDIC ET AL. Figure 4 ESR spectrum of DMPO-OH spin adduct in the presence of mg/ml of II-1-r. The influence of the different concentrations of the GSEs on the formation of hydroxyl radicals generated in the Fenton reaction is presented in Figure 5. The addition of the extracts to the reaction system ( mg/ml) inhibited the ESR signal intensity of DMPO-OH spin adduct. The addition of 0.25 mg/ml of the extracts prepared according to the procedure II scavenged 100% of hydroxyl radicals (Figure 5).The extracts prepared in the procedure I scavenged from 54 to 82% of hydroxyl radicals at the same concentration. Based on the calculated IC 50 values, the following order of the antioxidant activities were established: II ( ± mg/ml) > I ( ± mg/ml). The difference in antioxidant activities between the procedures I and II was significant (P < 0.01). It was also evidenced that the antioxidant activity increased while increasing the concentration of all extracts. In order to evaluate the results obtained for free radical scavenging activities of the GSEs, IC 50 values were determined for Trolox which is often used as a referent antioxidant. The IC 50 values for Trolox were and mg/ml for DPPH and OH radicals, Figure 5 Scavenging effect of different concentrations of GSEs on DMPO-OH spin adduct. (a) Extracts prepared in procedure I; and b) extracts prepared in procedure II. Bars represent S.D.

12 COMPOSITION AND ANTIOXIDANT ACTIVITIES OF GRAPE SEED EXTRACTS 723 respectively. Summarizing the results for the DPPH and OH radical scavenging activities of the GSEs, it could be concluded that all the examined extracts showed good free radical scavenging activities. There is negligible data available in the literature for the similar ESR conditions which could be used to compare the obtained results with. However, Calliste et al. [28,29] used the similar conditions to determine scavenging activities in a grape marc extract. Yamaguchi et al. [30] examined the scavenging effects of the GSE on the OH radical formed in an H 2 O 2 /NaOH/DMSO system. Spectrophotometric Assay for DPPH Radical The values of IC 50 obtained for the DPPH radical, determined spectrofotometrically, are shown in Figure 6. The values varied between ( mg sample/mg DPPH radical). The following order of antioxidant activity, according to the calculated IC 50 values, expressed as mg sample/mg DPPHŽ, was established: II ( mg sample/mg DPPH radical) > I ( mg sample/mg DPPH radical ). The values of IC 50 for the samples obtained in the procedure II are in the same range as the values obtained in the work of Bakkalba4i et al. [10] for the GSEs of the white grape varieties using acetone for the extraction ( mg sample/mg DPPH radical). Comparison of GSEs Antioxidant Activities with Different Extraction Procedures Analysis of variance, using F-test, showed that there were no significant differences in the antioxidant activities among the tested grape varieties and the years of harvesting, but the differences between the applied extraction procedures were highly significant. The differences, using the Duncan s multiple range test for the antioxidant activities expressed as IC 50, were determined for the GSEs obtained in the extraction procedures I and II, and the results are shown in Table 5. The antioxidant activities of the GSEs, prepared in the procedure I, were significantly lower from the activities of the GSEs obtained in the procedure II (P < 0.01) for both free radicals. Figure 6 Values of IC 50 (mg sample/mg DPPH radical) of investigated GSEs on DPPH radical obtained by spectrophotometric method.

13 724 MANDIC ET AL. Table 5 Values of IC 50 for DPPH and OH for applied extraction procedures. ESR determination Spectrophotometer Extraction Procedure IC 50 (mg/ml DPPH ) IC 50 (mg/ml OH ) IC 50 (mg/mg DPPH ) I a a a II b b b Numbers following the different letters (a, b) in the column show the significant differences using the Duncan s multiple range test, at P < GSEs Antioxidant Activities and Phenolic Compounds Correlations The relations between the pairs of the GSEs analytical parameters were determined by the correlation analysis. The results are presented in Table 6. The significant correlation (r = 0.95) was established between the antioxidant activities of the GSEs on the DPPH and OH radicals determined by ESR spectrometry. The significant correlation between the values determined by the spectrophotometric method for the DPPH radical activity and antioxidant activity of the DPPH (r = 0.73) and OH radicals (r = 0.83) by ESR spectrometry was established. The total phenolic content found by HPLC was significantly correlated with the values determined by the Folin-Ciocalteau methodology (r = 0.90) and with the content of flavan-3-ols (r = 0.90) as well. Finally, considering all the obtained results, the tested GSEs possess substantial free radical scavenging activities. These extracts showed the scavenging activities of both stable DPPH and short-lived OH radicals. CONCLUSION The high flavan-3-ols contents and the GSEs antioxidant activities of the white grape varieties, Italian and Rhine Rieslings, grown in Serbia, were determined in this article. It was detected that one kg of grape seeds could yield 5.41 or 15.0 g of GSE on average, depending on the used extraction methods. The contents of the total soluble polyphenols and flavan-3-ols were higher in ethyl acetate than in ethanolic extracts. The GSEs antioxidant activities on DPPH and OH radicals showed that the antioxidant activities were significantly higher in the ethyl acetate extracts than in the ethanolic Table 6 Correlation coefficients and their significance level for analytical parameters in GSEs. Parameter IC 50 (mg/ml OH ) - ESR IC 50 (mg/mg DPPH ) spectrophoto meter Total soluble polyphenols Flavan-3-ols Total phenolics (HPLC) IC 50 (mg/ml DPPH ) ESR 0.95* 0.73* 0.93* 0.92* 0.96* IC 50 (mg/ml OH ) - ESR 0.83* 0.96* 0.95* 0.94* IC 50 (mg/mg DPPH )-spectrophotometer 0.72* 0.70* 0.73* Total Soluble Polyphenols 1.00* 0.90* Flavan-3-ols 0.90* *Values statistically significant (P<0.05).

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