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Antioxidant activity of six varieties of grapes from the city of Pyatigorsk harvest 2013.

INTRODUCTION

Grapes are among the oldest plants cultivated by the humanity, and rank first among all fruit crops. Grape varieties that are now grown in the vineyards around the world--are the result of wine-growing that began 60 million years ago. Numerous ice ages and climate changes led to the disappearance of some varieties and the appearance of some new ones. The grapevine is a wood creeping plant--a liana. In the wild, it rises to a height of 20 meters or more, as it tends to be higher than the trees that shade it. This suggests that the grapevine is a photophilous plant. In the shade, it grows poorly and gives low yields: the vines gradually get thinner, ripen poorly, are more damaged by frost and eventually die [1].

Grapes are distinguished from other crops by a variety of useful properties. Grapes are a nutritious, dietary and therapeutic product. One kilogram of fresh grapes provides about 30% of calories of the daily human diet. Grapes contain 14-30% of sugar, a significant amount of organic acids (tartaric, malic, citric, etc.), which increase appetite and improve digestion and prevent the formation of kidney stones [1]. Grapes and grape juice are used as therapeutic agents for feeding retarded children, for the rehabilitation of the patients after cardiac and gastro-intestinal diseases, polyarthritis, after surgeries. Muscatel grape varieties contain antibiotic aromatics. Pectins contained in the skin of dark-coloured grapes, bind radioactive metals to insoluble salts and thus remove them from the body [2]. Due to the presence of biologically active substances, grapes help to treat anemia and nervous system disorders. Grapes are a source of a whole vitamin complex: A, Bj, B2, B6, B7, C, K, and P. Grapes contain many microelements, including manganese, zinc, rubidium, fluorine, vanadium, iodine, titanium, cobalt, etc. Dark-coloured grapes contain very useful tannins and colorants, having bactericidal and antiradiation action. According to nutritionists, the annual consumption rate of grapes per person must be 18 kg [3].

Wine-growing is a large sector of agriculture and is aimed at cultivating high yields of table and wine grapes [4]. Wine grapes are distinguished by the indicators of acidity and sugar content, which determine the type of produced wine. They are rated in terms of mechanical characteristics and chemical composition, which in turn depend on the conditions of grape cultivation and its biological characteristics. Canteens and technical grape varieties, can be cultivated in almost all areas where winegrowing is possible, which affects the quality of the crop. Therefore, when grown in different climatic and soil conditions the same wine grape varieties can be used for a different industrial purpose [5].

The aim of our research was to study the chemical composition and antioxidant power of six table grape collected on the territory of the Russian Federation in the city of Pyatigorsk: Risus, Saperavi, Levokumsky, Gurzufsky pink, Rkatsiteli, Moldova [6].

Risus--technical grape varieties. Ripens in the end of September--beginning of October. Production period of 150 days. The bushes medium vigor. Flower bi-sexual. Bunch cylindrical, winged, heavy, middle weight 220, Berry middle, round, greenish-white. Skin firm, elastic. The pulp is juicy. The berry seed 2-3 medium size. Productivity is high (150-200 kg/ha) and is stable with grape juice saccharinity 20-22 % and acidity 9-11 g/L. the Vine grows well. The high winter hardiness, resistance to frost to -26[degrees]C, has a good regenerative capacity, they can be grown in not covering culture.

Saperavi--old Georgian grape variety. A bunch of average size (length 13-17, of a width of 12-15 sm), leg clusters medium-length--up to 4,5 see Average grape cluster mass 93-99, Berry middle size (length 13-18, width 12-17 mm), oval, dark blue, with a thick wax coating, the pulp is juicy, the skin is thin, but durable. The average mass per 100 grapes 120-150. Saperavi to mildew and oidium weak, in rainy weather berries are affected grey mould. Significant damage wintering buds are seen in the temperature drops to -20[degrees]C, so the variety belongs to the group of relatively cold-resistant plant. Saperavi grow and bear good fruits on various soil types, except dry.

Levokumsky--technical grapes national selection. Ripens in the end of August. Production period 130 days. The growth of bushes above the average. Bunch of small or average weight of 90-120 g, cylindrical, average density. Berries are small, weighing 1.3 g, round, black. Flesh is juicy, simple taste, juice uncolored. The yield 130 kg/ha. Winter resistance is high.

Gurzufsky pink--generic grape variety early term of ripening. Bunch average, cylindrical, average weight of 250, Berry middle, dark red. The skin is thick, the pulp is juicy. The taste is harmonious, with pleasant strong Muscat flavor. The yield of 150 g/hectare lush Bushes, aging vine good. Sort of the grape Gurzufsky pink characterized field resistant to phylloxera [7].

Rkatsiteli--Georgian grape variety. Sheet of grapes of medium size. A bunch of medium size, cylinderconic. Grape cluster mass, 155-165 Berry average round. The average mass per 100 grapes 180-260, Berries Golden-yellow with bronze spots on the sun side. The skin is thin, durable, the pulp is juicy, with pleasant taste. From the beginning of Bud burst to removable maturity of grapes passes 155-160 days at the sum of active temperatures 2950-3000[degrees]C Buds bloom late. The ripening occurs in the first decade of October. Annual shoots grow vertically and Mature on 85-90 % of total growth.

Moldova--table grape varieties. Belongs to the late period of ripening. The leaf is large, roundish. A bunch of cylinder-conic, average density. The average weight of cluster 385 grams. Berries are large, oval, dark purple, with a thick wax coating. Skin is thick, dense, strong. Sort of like warm, well provided with food and moisture of the soil. [6].

The pulp [8], the skin and the grape seeds [9] were taken as objects for the analysis. For the analysis of the chemical composition and antioxidant activity determination, the following methods of analysis were used: measurement of total phenolics content [10], the total flavonoid content, the total content of tannins, the total content of anthocyanins [11], measuring of the level of free DPPH radical scavenging (2,2'--diphenyl picrylhydrazyl 1), the ability to scavenge ABTS radicals (2,2 '-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)), the total antioxidant power by the FRAP method (ferric reducing antioxidant power with the reagent 2,4,6-tripyridyl-s-triazine), antioxidant activity in a linoleic acid system.

MATERIALS AND METHODS

2.1. Chemicals and reagents:

Folin-Ciocalteu reagent in sodium carbonate, gallic acid, catechin, ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6- sulfonic acid) were purchased from Fluka (Germany). DPPH (2,2-diphenyl-1-picrylhydrazyl), Tween 40, hydrogen peroxide, sodium nitrite, aluminum chloride, thiobarbituric acid, trichloroacetic acid were purchased from Sigma-Aldrich Chem. mp. (USA).

2.2. Fruit collection:

The grapes collected on the territory of the Russian Federation at the enterprise wineries in the city of Pyatigorsk in 2013.

2.3. Determination of total phenols:

Total phenolic content of methanolic fruit extracts was assessed using a modified version of the Folin-Ciocalteu assay [12]. Gallic acid was used as a standard and the aqueous gallic acid solution (200 mg l 1) was di-luted with distilled water to give appropriate concentrations for a standard curve. For the analysis, 100 ll of methanolic fruit extract or gallic acid standard, 100 ll of methanol, 100 ll of Folin-Ciocal-teu reagent and 700 ll of Na2CO3 were added into 1.5 ml micro-centrifuge tube. The samples were vortexed immediately and the tubes were incubated in the dark for 20 min at room temperature. After incubation all samples were centrifuged at 13,000 rpm for 3 min. The absorbance of the supernatant was then measured at 735 nm in 1 ml plastic cuvettes using evolution 200 Series spectrophotometer. The results were expressed in mg gallic acid equivalent/100 g dry weight.

2.4. Determination of total flavonoids:

The flavonoid content of the methanolic extracts were measured using a assay [13]. A known volume (0.5 ml) of the extract or standard solution of quercetin was added to a 10 ml vol-umetric flask. Distilled water was added to make a volume of 5 ml. At zero time, 0.3 ml of 5% w/v NaNO2 was added to the flask. After 5 min, 0.6 ml of 10% w/v AlCl3 was added and after 6 min, 2 ml of 1 M NaOH was added to the mixture followed by the addition of 2.1 ml distilled water. Absorbance was read at 350 nm against the blank (water) and flavonoid content was expressed as mg querce-tin equivalents in 100 g of fresh material.

2.5 Determination of the anthocyanin profile:

The concept of determining the amount of anthocyanin present in a material by measuring the change in absorbance at 2 different pH values (3.4 and 2.0) [14]. Researchers have proposed using the pH values of 1.0 and 4.5 (2-5). Monomeric anthocyanins undergo a reversible structural transformation as a function of pH (colored oxonium form at pH 1.0 and colorless hemiketal form at pH 4.5. Thus, the difference in absorbance at the vis-max (520 nm) of the pigment is proportional to the concentration of pigment. Degraded anthocyanins in the polymeric form are resistant to color change with change in pH. Therefore, polymerized anthocyanin pigments are not measured by this method because they absorb both at pH 4.5 and 1.0.

2.6 Determination of condensed tannins:

In presence of concentrated H2 SO4 , condensed tanins were transformed by the reaction with vanillin to anthocyanidols [15]. 50 l of the methanolic seed extract appropriately dilute was mixed with 3 ml of 4% methanol vanillin solution and 1.5 ml of H2 SO4 . After 15 min, the absorbance was measured at 500 nm. Condensed tannin contents of seeds (three replicates per treatment) were expressed as mg catechin equivalents (CE) per gram of dry weight through the calibration curve with catechin. The calibration curve range was 50-600 mg ml-1.

2.7. DPPH radical scavenging activity:

The scavenging activity of samples was measured in accordance with the method [16]. The method was based on the reduction of methanolic DPPH in the presence of a hydrogen-donating antioxidant. DPPH solution was an intense violet colour and showed an absorption band at 515 nm. Adsorption and colour lowered when DPPH was reduced by an antioxidant compound. The remaining DPPH corresponded inversely to the radical-scavenging activity of the antioxidant. DPPH (2 mg) was dissolved in 54 ml of MeOH. Aliquots of investigated extract (50, 100, 200, 300, 500 and 1000 lg) were dissolved in 2 ml of MeOH. Then 1.0 ml of each solution was added to 2.0 ml of DPPH solution at room temperature. The absorbance at 515 nm was measured against a blank (2 ml MeOH in 2.0 ml of DPPH solution) using evolution 200 Series spectrophotometer. The results were expressed as percent-age of reduction of the initial DPPH adsorption by test samples:

% of reduction of the initial DPPH adsorption = ADPPH(t)_Asample (t)/ADPPH (t)_100,

ADPPH(t) is absorbance of DPPH at time t and Asample (t) is absorbance of sample at t the same time.

2.8. FRAP assay:

The FRAP assay was carried [17]. The FRAP solution was freshly prepared on the day of use, by mixing acetate buffer (pH 3.6), ferric chloride solution (20 mM) and TPTZ solution (10 mM TPTZ in 40 mM HCl) in a proportion of 10:1:1, respectively. Following this the FRAP solution was heated, while protected from light, until it had reached a temperature of 37 LC. Appropriate dilutions of methano-lic fruit extracts were prepared. One hundred microlitres of the di-luted sample extract (or for blank 100 ll methanol and for Trolox standard curves 100 ll Trolox of appropriate concentration) and 900 ll of FRAP solution were added into a micro-centrifuge tubes. The tubes were vortexed and left at 37 LC for exactly 40 min, and the absorbance was measured at 593 nm. The Trolox standard curves were used to calculate the antioxidant activity of the sam-ples in relation to Trolox and were expressed as mg Trolox equiv-alent/100 g dry weight sample (mg TE 100 g 1 DW).

2.9. ABTS free radical decolorization assay:

The total antioxidant capacity assay conducted using evolution 200 Series spectrophotometer. The procedure was based on a method [18] with some modification. ABTS_ + was generated by reacting ABTS (7.4 mM) with potassium persulphate (2.6 mM). The solution was diluted to obtain an absorbance of 1.4 units at 414 nm (molar extinction coefficient E=3.6_104 mol_1 l cm_1, Forni, Morla-Arellano, Packer, & Willison 1986) with 50 mM glycine-HCl buffer (pH 4.5) before use. Three millilitres of the solution were added to 20-80 ml of AA, trolox, hydroquinone, pyrogallol and fruit extracts separately. The changes in absorbance at 414 nm were recorded at 1, 3, 6, 10, 20, 30, 40, 60 and 90 min after mixing and until the absorbance reached a plateau. The antioxidant capacities, obtained by comparing the absorbance change at 414 nm in a test reaction mixture containing extract of fruit with that containing AA, were expressed as mg of AA equivalents per 100 g of homogenate (AEAC).

2.8. Determination of Antioxidant Activity in a Linoleic Acid System:

The total antioxidant activity of FEHP was carried out by use of a linoleic acid system. The linoleic acid emulsion was prepared by mixing 0.2804 g of linoleic acid, 0.2804 g of Tween 20 as emulsifier, and 50 mL of phosphate buffer (0.2 M, pH 7.0), and then the mixture was homogenized. A 0.5-mL ethanol solution of different concentration of FEHP (50-500 lg/mL) was mixed with linoleic acid emulsion (2.5 mL, 0.2 M, pH 7.0) and phosphate buffer (2 mL, 0.2 M, pH 7.0). The reaction mixture was incubated at 37[degrees]C in the dark to accelerate the peroxidation process. The levels of peroxidation were determined according to the thiocyanate method by sequentially adding ethanol (5 mL, 75%), ammonium thiocyanate (0.1 mL, 30%), sample solution (0.1 mL), and ferrous chloride (0.1 mL, 20 mM in 3.5% HCl). After the mixture was left for 3 min, the peroxide value was determined by reading the absorbance at 500 nm on a spectrophotometer.

3. Results:

3.1 Determination of phenolic compounds:

Based on Table 1, we can say that by the total content of phenols the undisputed leaders are the seeds of Rkatseteli variety and Risus. By the total content of phenols in the skin, the first place belongs to Moldova, the second place belongs to Gurzufsky pink. The lowest rates by the content of phenols in the skin belong to Rkatseteli variety. Among the pulp the first place belongs to Rkatseteli variety.

3.2 Flavonoids content:

Moldova seeds contain the greatest amount of flavonoids. Among the skin, the first place by the content of flavonoids belongs to Moldova variety. The pulp of all the considered grape varieties has the least amounts of flavonoids compared to the skin and seeds.

3.3 Determination of condensed tannins:

By the total content of tannins in the seeds, the leading variety is Moldova. The pulp of all the studied grape varieties differs slightly from one another and has low levels of the total content of tannins.

3.4 Determination of the anthocyanin profile:

By the total content of anthocyanins in the skin, the first place belongs to Levokumsky, with Moldova following. The final place belongs to Risus variety which has the lowest rates.

3.5 Total DPPH radical scavenging activity:
Fig. 1: Analyzing the experimental data in (figure 1), we can see
that the undisputed leaders in the ability to scavenge free DPPH
radicals are the seeds of Rkatseteli variety and Gurzufsky pink,
the second place belongs to Risus and Saperavi seeds. Among the
grape skin, by the ability to scavenge free DPPH radicals, the
first place belongs to Moldova skin.

[E.sub.c50] mg/[cm.sup.3]; (P-pulp, Sn-skin, S-seeds)

                 P       Sn     S

Rkatseteli       97.2    81     0,6
Moldova          153,7   8.3    0.9
Risus            121,1   71,9   0,8
Levokumsky       412     54     1
Saperavi         218,3   68,1   0,8
Gurzufsky pink   195,6   28,7   0.6

Note: Table made from bar graph.


3.6 Total antioxidant capacity of fruits by ABTS:
Fig. 2: By the ability to scavenge ABTS radicals (figure 2), the
first place belongs to Gurzufsky pink seeds, with Moldova seeds
differing slightly from them. Among the grape skin, by the ability
to scavenge ABTS radicals, the first place belongs to Gurzufsky
pink skin and the last place belongs to Rkatseteli variety. The
ability of the pulp to scavenge ABTS radicals is approximately
the same for all the varieties.

ABTS mmol trolox/g of material

                 P        Sn        S

Rkatseteli       2.26    4.79    46,49
Moldova          2.66    27.8    55.91
Risus            3.59    7,1     31.85
Levokumsky       3,71    9,46    15.83
Saperavi         3.73    7,52    39.58
Gurzufsky pink   3.38    36,68   57.64

Note: Table made from bar graph.


3.8. Determination of Antioxidant Activity in a Linoleic Acid System:

By the antioxidant activity in a linoleic acid system, the best results belong to the skin of Gurzufsky pink variety, the seeds of grapes Levokumsky and the pulp from the Saperavi grape variety with the highest rates.

4. Discussion:

The book "Grapes" by Gluschenko V.T. and Berezovsky Y.S. (Gluschenko, Berezovsky, 2008) studied structural components and bunches of grapes on their mechanical, chemical composition and antioxidant properties, and also made the dynamics of the ripening of berries and their organoleptic properties.

The value of the grapes is determined by the successful combination of taste of the fruit with their nutritional and dietary value. In the grape concentrate a large number of important organic and mineral substances for the body, as well as many digestible vitamins: A, B1, B2.Contained in the grape polyphenol substances help activate the body's metabolism that are associated with the development of the human body, especially the respiratory system. Flavonoids, catechins and anthocyanins contained in berries of grapes, can reduce the effects of radiation lesions, and to prevent their negative impact on the body.

These and many other useful properties of grapes glorified him as a high-value product, medical and simply necessary for beauty and health.

3.7. FRAP assay:
Fig. 3: In terms of FRAP (figure 3), Levokumsky seeds can be singled
out. Among the skin, the first place in terms of FRAP belongs to
Moldova variety, the last place belongs to Risus.

FRAP value, mmol [Fe.sup2+] 1 ru raw materials

                 P       Sn      S

Rkatseteli       2,16    2,52    14,76
Moldova          1,98    14,22   16,56
Risus            1,44    2,34    15,84
Levokumsky       1,53    5,4     17,28
Saperavi         1,26    6,48    16,02
Gurzufsky pink   1,62    9,72    16,92

Note: Table made from bar graph.


5. Conclusions:

Based on these results, we can say that all grape seeds contain the maximum values of the antioxidant activity and the chemical composition; and the grape pulp contains the minimum values.

Among the studied grape varieties, we can distinguish Gurzufsky pink grape variety as a variety with a maximum content of tannins, phenols, as well as with a high antioxidant activity.

Thus, it can be concluded that by the chemical composition the grape seeds and skin contain the highest number of phenols, tannins, anthocyanins, they also have the highest antioxidant effect and are excellent materials for the production of semi-finished products with antioxidant activity.

ACKNOWLEDGEMENTS

This study was financially supported by the Ministry of Education and Science of the Russian Federation within the basic part of the government task number 2014/199 FSBEI HPE Samara State Technical University on the project Creation of a scientific methodology for developing formulations and technologies of food products to control oxidative stress in humans code 974.

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[2] Potebnya, A.B., Skrobishevsky, 2010. Wine-growing Guide. Fiton, pp: 448.

[3] Stogova, N., 2006. Grapes against a Hundred Diseases. St. Petersburg.: Peter, pp: 96.

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[5] Solomonov, S.S., 2011. New Resistant Varieties of Grapes. Donbass, pp: 66.

[6] Dokuchaeva, E.S., E.S., Komarova and N.N. Pilipenko, 1986. Grape Varieties. Kiev: Urozhay, pp: 272.

[7] Troshin, L.P., N.A. Sviridenko, 1988. Resistant Varieties of Grapes. Simferopol: Tavriya, pp: 208.

[8] Pooja Doshi, Pangurang Absule and Kaushik Banerjee, 2006. Phenolic composition and antioxidant activity in grapevine parts and berries cv. Kismish black during maturation. Int. J. Food Sci. and Tehnol, 41(1): 1-9.

[9] Panasyuk, A.L., V.V. Zhirova, I.O. Mikhailov, N.M. Romanyuk and E.A. Nikulin, 2003. Extraction of Phenolic Compounds from Grape Seeds. Winemaking and Wine-growing, 1: 36-37.

[10] Baraboy, V.A., 1976. Biological Effects of Plant Phenolic Compounds. Kiev: Nayk. dumka, pp: 260.

[11] Chub, B., 2008 What are Anthocyanins Needed for. Floriculture, 6: 22-25.

[12] Rockenbach, I.I., E. Rodrigues, L.V. Gonzaga, V. Caliari, M.I. Genovese, G. Schmidt and R. Fett, (2011). Phenolic compunds content and antioxidant activity in pomace from selected red grapes (Vitis vinifera L. and Vitis Labrusca L.) widely produced in Brazil. Food Chem, 127(1): 174-179.

[13] Skerget, M., P. Kotnik, M. Hadolin, A.R. Hras, M. Simonic and Z. Knez, 2005. Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem, 89(1): 191-198.

[14] Harbourne, N., J. Christophe, D.J. Morgan and J.G. Lyng, 2008. Determination of the degradation kinetics of anthocyanins in a model juice system using isothermal and non-isothermal methods. Food Chem, 111(2): 204-208.

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Irina Andreevna Kustova, Nadezhda Viktorovna Makarova and Dinara Fanisovna Valiulina

The Samara State Technical University, Str. Molodogvardeyskaya, 244, Main Building, Samara, 443100, Russian Federation

Received 28 February 2015; accepted 26 March 2015; published 25 May 2015

Address For Correspondence:

Irina Andreevna Kustova, The Samara State Technical University, Str. Molodogvardeyskaya, 244, Main Building, Samara, 443100, Russian Federation.

E-mail: Batkova_ira7@mail.ru (I. Batkova)
Table 1: The results of the study of the chemical composition of the
grape pulp, skin and seeds.

Grade/item                 Total content of      Total content of
                            phenols, mg of      flavonoids, mg of
                         gallic acid/100 g of   catechine/100 g of
                               material              material

Rkatseteli       Pulp             51                    22
                 Skin            112                    56
                 Seeds           1274                  1280

Moldova          Pulp             30                    13
                 Skin            424                   329
                 Seeds           1022                  1332

Risus            Pulp             48                    21
                 Skin            131                    47
                 Seeds           1229                  1120

Levokumsky       Pulp             24                    14
                 Skin            197                   156
                 Seeds           1162                  669

Saperavi         Pulp             38                    19
                 Skin            187                    89
                 Seeds           1075                  1068

Gurzufsky pink   Pulp             29                    28
                 Skin            411                   194
                 Seeds           1123                  968

Grade/item                Total content of     Total content of
                           tannins, mg of     anthocyanins, mg of
                         catechine/100 g of       cyanidin/3/
                              material        glycoside/100 g of
                                                   material

Rkatseteli       Pulp           0,09               Not found
                 Skin           0,13               Not found
                 Seeds         19,08                  --

Moldova          Pulp           0,11                  9,7
                 Skin          36,80                 188,1
                 Seeds         46,72                  --

Risus            Pulp           0,11               Not found
                 Skin           0,14                 38,4
                 Seeds         13,96                  --

Levokumsky       Pulp           0,10               Not found
                 Skin           5,52                1365,6
                 Seeds          6,64                  --

Saperavi         Pulp           0,10               Not found
                 Skin           3,56                 85,7
                 Seeds         17,48                  --

Gurzufsky pink   Pulp           0,12               Not found
                 Skin          15,72                 140,7
                 Seeds         16,80                  --
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Author:Kustova, Irina Andreevna; Makarova, Nadezhda Viktorovna; Valiulina, Dinara Fanisovna
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Date:May 1, 2015
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