Polyphenols Content of Some Table Grape Varieties (V. vinifera) and Raisins on Their Basis

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1 Oxidation Communications 34, No 2, (2011) Analytical investigations in biological systems Polyphenols Content of Some Table Grape Varieties (V. vinifera) and on Their Basis N. Gougoulias a, N. Mashev b * a Department of Plant Production, Technological Educational Institute of Larissa, Larissa, Greece ngougoulias@teilar.gr b Agricultural University, 12 D.Mendeleev Blvd., 4000 Plovdiv, Bulgaria nimash@abv.bg ABSTRACT The fresh grape and raisins of the seedless table varieties Sultanina, Corinth noir, Flame seedless and Kishmish Turkmenski (V. Vinifera L.) were investigated for the content of total phenols (ТР), nonflavonoid phenols (NFP), flavonoid phenols (FP), total flavanols (F-3-ols), the antiradical activity (DPPH ) and ferric reducing antioxidant power (FRAP). The content of TP in the grape varied from 754 to 1437 mg/kg, NTP from 263 to 575 mg/kg, FP from 491 to 862 mg/kg GAE, F-3-ols from 171 to 225 mg/kg CE, and in the raisin from 2510 to 4840 mg/kg, from 856 to 1817 mg/kg, from 1654 to 3032 mg/kg, and from 919 to 1471 mg/kg, for the corresponding grape varieties, respectively. The fresh grape and raisins of the Corinth noir variety displayed the highest antiradical (DPPH ) and FRAP activity, while the white grape variety Sultanina manifested the lowest. The variations in phenol and phenol fractions composition and the antioxidant activity of raisins are correlated with the process of grape drying. Keywords: grape, raisins, polyphenols, DPPH activity, FRAP activity. AIMS AND BACKGROUND Many laboratory, clinical and epidemiological studies demonstrate that the fruit- and vegetable-rich food reduces the risk of degenerative diseases such as cardiovascular and cancer. A number of investigations have evidenced that besides nutritive and mineral ingredients they contain a lot of vitamins as vitamin С, Е (tokopherols), ß-caroten (carotenoids) and particularly various phenol compounds (monophenols and polyphenols) which possess strong antioxidant properties 1 4. More than 8000 types of * For correspondence. 405

2 phenol compounds have been detected in plants, which in addition to their functions have the significant capacity to scavenge and neutralise the free oxygen radicals in the cells of aerobic organisms 5,6. The free radicals are strong oxidisers, which damage the basic biological molecules (proteins, lipids, nucleic acids), destroy the proceeding of the normal biochemical process in the cells and in the human organism induce the occurrence of many diseases 7. Grape and its products wine, juices, raisins, jams, bottled fruits, etc. being rich source of phenol antioxidants, are intensively studied as foods or supplement foods for different diets and diseases The obtaining of raisins from grape is accompanied by changes in the phenol compounds composition leading to the formation of new chemical structures which show strong antioxidant effects as well In addition, the process of grape transformation in raisins is also accompanied by the proceeding of the so-called the Мaillard reactions resulting in the formation of Amadori compounds with antioxidant action 14. This is of significance for the comparison of polyphenols and antioxidant capacity of grape and its raisins in view of evaluating the nutritive and healthy properties of these products. The purpose of the study included: (1) determination of the amount of total phenols and the major polyphenol fractions in some seedless table grape varieties and the raisins obtained from them, and (2) assessment of the antiradical and ferric reducing antioxidant power of fresh grape and raisins. EXPERIMENTAL Grape and raisins. of the seedleass grape varieties Sultanina, Corinth noir, Flame seedless and Kishmish Turkmenski of Vitis vinifera L. was investigated. The grape of Sultanina and Corinth noir varieties was grown in the region of the village Vrina, Ahios Olimpicus, Peloponess Greece and that of the Flame seedless and Kishmish Turkmenski from the region of the village Bresnik, at the experimental files of the Viticulture Department of the Agriculture University Plovdiv, Bulgaria. The grape from the two regions was collected in complete technological maturity. The raisins from all grape varieties were prepared in Bulgaria, following a classical procedure including sun-drying and dipping in boiling water for 6 10 s. The drying was carried out to 14% reduction in the water content. Extract preparation. The extracts were obtained after twofold treatment of 5 g sample with 60% methanol after 1-hour storage at dark and room temperature. The combined extracts after centrifugation were brought to 50 ml with aqueous methanol and used for further chemical analysis 15,16. Determination of total polyphenols content. Total polyphenols (TP) contents were determined with the Folin-Ciocalteu reagent according to the method of Singleton and Rossi 17 and were expressed as gallic acid equivalent (GAE). 406

3 Determination of phenol fractions. The non-flavonoid phenols (NFP) were determined with the F.-C. reagent after the removal of flavonoid phenols (FP) with formaldehyde according to the method of Kramling 18. FP content was determined as a difference between the content of TP and NFP, and were expressed as gallic acid equivalent (GAE). The total flavanols (F-3-ols, catechins and procyanidins) were assayed using p- dimethylaminocinnamaldehyde (p-dmaca) reagent after the method of Li et al. 19 and were presented as catechin equivalent (CE). Determination of antioxidant activity. The antiradical activity of the methanol extracts was determined according to the method of Brand-Williams et al. 20 using the stable free radical 2,2 -diphenyl-1-picrylhydrazyl (DPPH). The activity was evaluated in µmol DPPH/g sample as well as Trolox equivalent (synthetic vitamin E). The ferric reducing antioxidant power (FRAP) was evaluated according to the method of Benzie et al. 21 and was expressed as µmol FRAP reagent/g grape. The activity was also presented as ascorbic acid equivalent (AAE) in mg% per fresh matter. The results are means of two years and the chemical analyses were the mean of three parallel samples. The statistic treatment of the results was performed according to well-accepted methods using a program making possible the calculation of t of the Student test at a confidence level p<0.05. RESULTS AND DISCUSSION The content of ТР in the fresh grape of the investigated table varieties varies from 754 to 1437 mg/kg fresh matter and from 3016 to 5748 mg/kg dry matter (Table 1). The grape of Corinth noir is distinguished by the highest TP content, and the Sultainina variety by the lowest. Table 1. Total phenols content (ТР, GAE) No Variety mg/kg fresh matter mg/kg dry matter 1 Sultanina 754 ± Corinth noir 1437 ± Flames seedless 1104 ± Kishmish Turkmenski 1343 ± Sultanina 2510 ± Corinth noir 4840 ± Flames seedless 3845 ± Kishmish Turkmenski 4518 ± The decrease of the water content in the raisins to 14% increases the relative amount of polyphenols. Thus, the TP in the grape varieties under study varies from 407

4 2510 mg/kg for Sultanina to 4840 mg/kg for Corinth noir. The raisins of the Kishmish Turkmenski are richer in total phenols than that of the Flame seedless, respectively 4518 and 3845 mg/kg. Related to the amount of dry matter, the content of TP in the raisins is reduced as compared to that of the fresh grape. According to some studies, the decrease in TP content during raisins obtaining is much stronger and can reach 40% depending on the variety type and the applied technology 14. The raisins from the investigated by us grape varieties and obtained by sun-drying and dipping in boiling water for 6 7 s have a minimum loss of TP which is statistically significant only in the case of Kishmish Turkmenski (p<0.05). It was of interest to examine the distribution of the separate polyphenol fractions in the fresh grape and its raisins, after thermal treatment. We have focused our studies on 3 phenol fractions, i.e. nonflavonoid (NFP), flavonoid (FP) and total flavanols (F-3-ols ). The amount of NFP in the fresh grape and raisins is demonstrated in Table 2. It is seen that NFP in the grape varies from 263 to 575 mg/kg, and in the raisins from 856 tо 2837 mg/kg fresh matter. A similar tendency as in the case of total phenols is observed: the fresh grape and raisins of the white variety Sultanina contains lower amount of NFP in comparison with the red grape varieties and the highest is found for the Corinth noir variety. The comparison of the content of NFP with respect to the total phenols in the fresh matter of the grape and its raisins shows that they represent about 33 40% от ТР. The amount of NTP in the dry matter of the raisins is reduced on average by 9% (р< 0.05), while for the Corinth noir variety it is 14% in comparison with the dry matter of the grape. These results demonstrate that the thermal treatment of the grape induces increase in the amount of the phenol nonflavonoid fraction. Some authors 11,13 reported that the NFP fraction consists mainly of gallic, hydroxybenzoic, coumaric, caffeic, ferulic, cinnamic and other acids, and their derivatives. These compounds affect not only the grape and raisins taste but they also manifest strong antioxidant properties. Table 2. Nonflavonoid phenols content (NFP) No Variety mg/kg fresh matter mg/ kg dry matter 1 Sultanina 263 ± Corinth noir 575 ± Flames seedless 354 ± Kishmish Turkmenski 484 ± Sultanina 856 ± Corinth noir 1817 ± Flames seedless 1387 ± Kishmish Turkmenski 1523 ±

5 Total flavanoids were determined as the difference between the TP and NFP, after removal of the flavanoids through precipitation and filtration using a formaldehyde solution according to the method of Singleton et al. 17 The results on FP content of the fresh grape and its raisins are shown in Table 3. The flavonoid phenols are ranging within 491 and 962 mg/kg in the grape, and 1025 and 2417 mg/kg in the raisins. The fresh grape and the raisins of the white variety Sultanina are not so rich in flavonoid phenols (491 and mg/kg) in comparison with the red varieties, particularly with the grape and raisins of Corinth noir, which amount to 962 and 2417 mg/kg, respectively. It has been established that the ratio of flavonoid phenols to total phenols of 65 70% in the fresh grape of all varieties is reduced to 35 40% in the raisins. The fraction of flavonoid phenols includes variouis subgroups of phenol compounds differing in structure and properties. The fraction of flavons, flavonols, flavanones, flavanols and anthocyanins 22 is of utmost significance for the grape and grape products raisins, juices, wines, jams, etc. Although flavanols are within the flavonoid composition, we have investigated them separately using the specific method of Li et al. 19, applying p-dimethylaminocinnamic aldehyde (p-dmaca) as a proper reagent for these compounds. Here one should mention some important for the grape and raisins polyphenols that determine the taste, flavour and antioxidant properties as catechine, epictachine, proanthocyanidins (tannin compounds) and other biologically active compounds with unknown origin 11. The content of total flavanols (flavan-3-ols) in the fresh grape and raisins under study are presented in Table 4. The data demonstrate that depending on the grape variety the content of F-3-ols in the fresh grape ranges from 141 to 225 mg/kg СЕ, and in the raisins from 819 to 1471 mg/kg. The content of the flavanols fraction in the grape varies from 25 to 30% of flavanoids, while in the raisins it increases and varies from 41 to 50%. The amount of the flavanols in the raisins dry matter ranges from 28 to 47%, which is higher than that in the dry matter of the corresponding grape varieties. Table 3. Flavonoid phenols content (FP) No Variety mg/kg fresh matter mg/kg dry matter 1 Sultanina 491 ± Corinth noir 862 ± Flames seedless 720 ± Kishmish Turkmenski 859 ± Sultanina 1654 ± Corinth noir 3023 ± Flames seedless 2454 ± Kishmish Turkmenski 2995 ±

6 Table 4. Total flavanols content ( F-3- ols, СЕ) No Variety mg/kg fresh matter mg/kg dry matter 1 Sultanina 171 ± Corinth noir 225 ± Flames seedless 203 ± Kishmish Turkmenski 218 ± Sultanina 819 ± Corinth noir 1471 ± Flames seedless 1004 ± Kishmish Turkmenski 1259 ± Our results indicate that the processes of sun drying of the grape induce the occurrence of processes increasing the oxidation-reduction potential of the phenol compounds in the raisins, which react selectively with the Folin-Ciocalteu reagent. In addition to the increase in the content of total phenols and other phenol fractions as a result of the reduction of water content in the raisins, some changes occur in their quality connected with the oxidative-reductive enzymatic and non-enzymatic catalysis of the reactions. In their studies on the individual phenol compounds in the fresh grape and raisins, some researchers established increased amounts of vanillic, protocatechuic, cоumaric, gallic, ferulic, caffeic acids, coutaric, as well as in quercetin and kaempherol glycosides 11,12. According to Vinson et al. 14, Sanz et al. 23, the fruits drying is accompanied by the proceeding of the Maillard reactions, and hydroxymethylfurfuraldehyde (NMF) is obtained from the products of Amidori. The latter does not react with the Folin-Ciocalteu reagent and does not affect the content of phenol compounds. We have determined the in vitro antioxidant activity of the fresh grape and raisins using 2 methods: applying the free stable radical DPPH and the ferric reducing antioxidant power with FRAP reagent. The results on the antiradical activity of the fresh grape and raisins are presented in Table 5. The fresh grape and raisins of the Corinth noir variety display the highest antiradical activity, respectively 1.46 and 7.81 µmol DPPH/g, and the fresh grape and raisins of the Sultanina variety the lowest, 0.73 and 5.39 µmol DPPH/g, respectively. The activity of the red grape varieties presented as vitamin E equivalent (Trolox) ranges between 0.34 and 0.67 mм ТЕАС, and for the raisins of the same varieties between 2.50 and 2.91 mм ТЕАС. Taking into consideration the content of dry matter in the fresh grape as 22 25%, and 85% in the raisins, it should be expected that the activity expressed as TEAC will increase both for the raisins of the white grape variety and of the red ones. 410

7 Table 5. Antiradical (DPPH) activity No Variety μmols DPPH/g mm Trolox/kg (ТЕАС) 1 Sultanina 0.73 ± Corinth noir 1.46 ± Flames seedless ± Kishmish Turkmenski 1.28 ± Sultanina 5.39 ± Corinth noir 7.81 ± Flames seedless 6.86 ± Kishmish Turkmenski 7.14 ± The results for the ferric reducing antioxidant power of the fresh grape and raisins estimated by the FRAP reagent are presented in Table 6. It varies between 2.36 and 4.07 mmol FRAP/g for the grape berries and between and µmol FRAP/g for the raisins. We observed again a higher antioxidant activity of the fresh grape and raisins for the red varieties in comparison with the white variety Sultanina. The fresh grape and raisins of the Corinth noir variety have the highest antioxidant activity. It is known that the fresh grape contains relatively low amount of vitamin C about 10 mg%. Although its content is reduced in the raisins as a result of grape drying, the ferric reducing antioxidant power of the phenol compounds expressed as vitamin C (ascorbic acid) equivalent is high both in the fresh grape and particularly in the raisins, which make them valuable and healthy food with high antioxidant properties and suitable ingredient in other nutritive products and diets. Table 6. Ferric reducing antioxidant power (FRAP) No Variety μmols FRAP/g mм AAE/kg 1 Sultanina 2.36 ± Corinth noir 4.07 ± Flames seedless ± Kishmish Turkmenski ± Sultanina ± Corinth noir ± Flames seedless ± Kishmish Turkmenski ± In literature there are scarce data on the fractional polyphenol composition and the antioxidant activity of fresh grape and raisins. Zhao and Hall 24, Karadeniz et al. 11 have established that the methanol extracts of raisins also increase 5 6 times the antioxidant activity in comparison to the fresh grape. The studies of Greek authors 411

8 reveal that the polyphenol content, antioxidant activity and individual composition of the raisins are in close relation to the grape variety type, the technology of their producing and the region of grape cultivation 12,13. The results for the antiradical activity of the fresh grape and raisins of the investigated table varieties and estimated by the use of the stable free radical 1,1-diphenyl, 2-picrylhydrazyl (DPPH), varied from 0.73 µmol DPPH /g to 7.81 µmol DPPH /g fresh matter, depending on the variety and processing technology (Table 5). This activity, expressed as Trolox equivalent (synthetic vitamin E), and depending on the grape variety, changes from 0.23 to 0.67 mm ТЕАС for the grape, and from 1.68 to 2.91 mm ТЕАС for the raisins. The fresh grape and raisins of the Corinth noir variety manifest the highest antiradical activity among the other red varieties under study and the white grape variety Sultanina has the lowest activity. These relations are also supported by the results of the assay of the antioxidant potential of the fresh grape and raisins with the FRAP method, where the activity is determined in dependence on the oxidative-reductive potential of the system, and the transformation of the non-coloured Fe 3+ - complex in the coloured Fe 2+ -complex 21. The ferric reducing antioxidant power of the phenol compounds in the grape of the investigated varieties ranges between 2.36 and 4.07 µmol FRAP/g, and in the raisins between and µmol FRAP/g fresh matter (Table 6). The presentation of this activity as ascorbic acid (vitamin C) equivalent gives the following data: it ranges from 2.36 to 4.07 mm AAE/kg in the fresh grape, and from to mm AAE/kg fresh matter in the raisins. And again we should outline the higher antioxidant activity of the grape and raisins of the Corinth noir variety of the red varieties and the lowest activity of the grape and raisins of the white grape variety Sultanina. Our data support the findings of some authors who established a positive correlation between the antioxidant activity of the grape and the content of total phenols 8,10,25,26. However, it should be noted that while the ratio of the average antiradical activity (DPPH) of the fresh matter of the grape and raisins is 1:5.71, the average ferric reducing antioxidant power (FRAP) is 1: Calculated per a dry matter of the grape and raisins these ratios become 1:1.68 and 1:3.26, respectively, i.e. the ferric reducing antioxidant power of the raisins is two time higher than that assayed by DPPH radical. These results give us ground to conclude that some of the chemical compounds in the raisins, which appeared during the grape drying, do not exert antiradical activity with respect to DPPH, but have ferric reducing antioxidant power. Most likely these are different products of Amadori, formed during the Maillard reactions at the interaction between free amino acids and sugars, interactions between tannins (oligomeric forms of proanthocyanidins, polymers) and proteins, etc. 11,23 The comparison of the antioxidant activity of the grape and other fruits with that of their dehydrated products shows that the phenol compounds are superior antioxidants to the antioxidant vitamins С, Е and ß-carotene (Vinson). The raisins extracts from the Sultanina and Corinth noir varieties display high antioxidant efficiency not only to free radicals but also to LDL oxidation, total GSH decrease, cytotoxicity, etc. 12,13 Our 412

9 data show that only the traditional grape varieties Sultanina and Corinth noir, which are used for the production of raisins in Greece, but also some table seedless grape varieties with high-yield and good nutritive qualities such as Flame seedless and Kishmish Turkmenski can be successfully used for this purpose. CONCLUSIONS The raisins from the table seedless varieties Sultanina, Corinth noir, Flame seedless and Kishmish Turkmenski contain polyphenol and phenol fractions characteristic for the grape used for their production. The Corinth noir, one of the grape varieties for raisins producing in Greece, is distinguished by the highest content of ТР, NTP, FP, F-3-ols, as well as by the highest antiradical (DPPH ) and ferric reducing antioxidant power (FRAP). The sun-dried raisins from the grape varieties Sultanina, Corinth noir, Flame seedless and Kishmish Turkmenski are nutritive products containing compounds with high antioxidant efficiency and can be successfully recommended for their including in different healthy diets. REFERENCES 1. E. Vasilopoulou, K. Georga, M. B. Joergencen, A. Naska, A. Trihopoulou: The Antioxidant Properties of Greek Foods and Flavonoid Content of Mediterranean Menu. Curr. Med. Chem. and Metab. Agents, 5, 35 (2005). 2. C. Rice- Evans: Screening of Phenolics and Flavonoids for Antioxidant Activity. In: Antioxidant Food Suplements in Human Health (Eds L. Pcker, M. Hiramatsu, T. Yoshikawa). Acad. Press, F. Vegilli, C. Scaccini, L. Pasker, G. Rimbach: Cardiovascular Disease and Nutrition Phenolics. In: Anioxidants in Food (Eds J. Pokporny, N. Yanishlieva, M. Gordon). CRC, I. T. Johonson: Antioxidants and Antitumour Properties. In: Antioxidants in Food (Eds J. Pokporny, N. Yanishlieva, M. Gordon). CRC, F. Shahidi: Natural Antioxidants: Chemistry, Health Effects and Applications (Ed. F. Shahidi). AOCS Press, Champaign Illinois, 1997, L. BRAVO: Polyphenols: Chemistry, Dietary Sources, Metabolism and Nutritional Significance. Nutrition Reviews, 56 (11), 317 (1998). 7. B. Halliwell, J. M. Gutteridge: Free Radicals in Biology and Medicine. 2nd ed. Clarendon Press, Oxford, 1989, J. Kanner, E. Frankel, R. Granit, B. German, J. E. Kinsella: Natural Antioxidants in Grapes and Wines. J. Agric. Food Chem., 42, 64 (1994). 9. M. Bourzeix, D. Weyland, N. Heredia: A Study of Catechins and Procyanidins of Grape Clusters, the Wine and Other By-products of the Wine. Bulletin de L OIV, 59, 1171 (1998). 10. A. ARNOUS, D. P. MAKRIS, P. KEFALAS: Effect of Principal Polyphenolic Components in Relations to Antioxidant Characteristics of Aged Red Wines. J. Agric. Food Chem., 49 (12), 5736 (2001). 11. F. Karadeniz, R. W. Durst, R. E. Wrolstad: Polyphenolic Composition of. J. Agric. Food Chem., 46, 5343 (2000). 12. A. Chiou, V. T. Karathanos, A. Mylona, F. N. Salta, F. Preventi, N. K. Andrikopoulos: Currants (Vitis vinifera L.) Content of Simple Phenolics and Antioxidant Activity. Food Chemistry, 102, 516 ( 2007 ). 413

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