Polyphenolic Extracts of Red Grapes

Growth in radionuclide environmental background and chemicals use in food products are characterized by upset of antioxidant balance of human organism and pathologies of the immune system. Treatment and prevention of the mentioned pathologies and excretion of toxic substances from the body are possible only by using the drastic antioxidant, antitoxic and food born disease preventing plant preparations [1,2]. The best raw material base for producing such preparations and nutritional supplements is represented by environmentally safe, i.e. cultivated without using chemicals, colored grape varieties. Of particular interest is a grape stone, which contains 6570% of total amount of phenol compounds existing in grapes, and is mostly represented by bio-flavonoids. They prevent the development of thromobotic proceses, improve lipid metabolism that in turn reduces pathologies of cardiac ischemic diseases and extends life expectancy [3]. Proanthocyanidins, which represent the catechins’ polymeric chain were obtained for the first time by Professor Jacques Masquelier in 1936, and he named these compounds P vitamin.


Introduction
Growth in radionuclide environmental background and chemicals use in food products are characterized by upset of antioxidant balance of human organism and pathologies of the immune system. Treatment and prevention of the mentioned pathologies and excretion of toxic substances from the body are possible only by using the drastic antioxidant, antitoxic and food born disease preventing plant preparations [1,2]. The best raw material base for producing such preparations and nutritional supplements is represented by environmentally safe, i.e. cultivated without using chemicals, colored grape varieties. Of particular interest is a grape stone, which contains 65-70% of total amount of phenol compounds existing in grapes, and is mostly represented by bio-flavonoids. They prevent the development of thromobotic proceses, improve lipid metabolism that in turn reduces pathologies of cardiac ischemic diseases and extends life expectancy [3]. Proanthocyanidins, which represent the catechins' polymeric chain were obtained for the first time by Professor Jacques Masquelier in 1936, and he named these compounds P vitamin.
Therapeutic-preventive daily norm of flavonoids for adults is 80-85mg, but the upper acceptance limit in medicine is 120-130mg/per day. They strengthen eliminating signals for cancer cells in human organism, so that they do not harm the healthy cells. In 2006, American scientists found proteins in the grapeseed ethanol extract, which foster the destruction of cancer cells [4].
How to cite this article: TN   There are not studied in fact raw materials of colored chemical-free grape varieties (Izabela and Zeibeli) cultivated in the Imereti region's viticulture-winemaking micro-zone, each kilogram of which presumably contains about 10g of phenolic compounds, and most of them (3% of a dry mass) are concentrated in grape-stone [7,8] (Table 1).
The content of anthocyanin in general parts of the grape reaches up to 450-2200mg/dm 3 . The grape polyphenols represent drastic natural antioxidants and their biological activity in the presence of C vitamin it event grows further. They prevent free-radical destruction of bio-membrane cells structure, protect human organism in such pathological states, as atherosclerosis, stress, anemia, bronchitis, early ageing, intensive chemotherapy results, postoperative status, chronic fatigue syndrome and so on [9,10].
Therefore, of high topicality is the development of innovative technologies of extracting dissoluble total polyphenols from raw materials of colored chemical-free grape varieties cultivated in the Imereti region's viticulture-winemaking micro-zone, for producing biologiclly active drastic antioxidant, antiradiant antiradiation, antitoxic and food born diseases preventing preparations.
Within the World Health Organization program MONICA and according to subsequent studies carried out in France, USA and other countries, it has been established that the therapeutic effect of red grape wine on human organism and its recommended daily norm is 70-100ml, but not everybody can to intake this dose every day (children, patients, athlets, hardly working and religious people), consequently, the development of innovative technologies for producing from environmentally safe raw materials high antioxidant, antitoxic and foodborn diseases preventing polyphenol concentrate has a high pragmatic value [11,12].

Materials and Methods
The selected raw materials for the study were Zeibeli and Izabela color grape varieties cultivated in Baghdati viticulturewinemaking micro-zone.
The process of cultivating the mentioned grape varieties does not involve the use of pesticides, chemicals and inorganic enriching agents.
To provide maximum extraction of the phenolic complex from newly-squeezed grape skins and stone, we arranged a series of experiments.
Extraction of grape secondary resources was carried out in two stages separately, independently of each other.
Based on the carried out studies and analysis of batchtype extraction equipment, we developed the innovative batchtype industrial and experimental extractor, which was used for performing experimental works, the principal scheme of which is shown below in Figure 1

At the first stage
The newly-squeezed skins and stone, without any drying and crushing, are supplied to the extraction equipment, where per 1000g of the grape skins and stone composition were added with 1000 ml of 80-82% ethanol, which is oxidized to 1% by citric acid.

At the second stage
Per 1000 g of the extraction cake remaining in the extractor (i.e. skins and stone remaining after primary extraction) was added with 1000ml of 12-18% ethanol without oxidation, and extraction was carried out. At both stages, the extraction was carried out at the temperature of 45-50 °C within 4-4,5 hours under conditions of periodical pulsation ( Figure 2). The composition of extracts of both stages was centrifuged and filtered by means of German-made wine layered filters, and condensation-concentration of a part of the obtained extracts was carried out in a vacuum at the temperature of not higher than 50 °C until 57-63% of dry substances content.

Agricultural Research & Technology: Open Access Journal
For analysis, we used both uncondensed and condensed extracts for both grape raw materials separately.
Studies of physical-chemical characteristics of extracts were carried out in compliance with international standards (ISO-International Organization for Standardization). In particular, as follows: The content of water-soluble extracts: ISO 9768:1994, which envisages extraction in the conditions of reflux, filtration, drying of non-dissolved waste and weighing with further calculation of extracting substances; Determination of phenolic compounds in the extracts was carried out by colorimetric (Folin-Ciocalteu) method, which is based on the capacity of phenolic compounds to recover phosphatotungstic and phosphatomolybdic acids, And to measure the coloration intensity of a blue-color molybdenum to a rusting color by using colorimetric method.
1cm 3 of extract, 15cm 3 of water, 1 Folin Ciocalteu reagent and 20cm 3 of 20% sodium carbonate solution were placed in the flask with a capacity of 100cm 3 , and 30 minutes later we measured optical density by using the ditch with 10mm thick and the wavelength of 670nm. For comparison, we prepared the second solution, in which 1cm 3 of sample of the analyzed extract was replaced by water.
The concentration of phenolic compounds was determined by a calibration curve of standard solution of Gallic acid [13,14].
To determine antioxidant activity, i.e. radical binding activity, 1 ml of the analyzed extract is added with 3ml of DPPH alcohol solution (0,1mM DPPH -in 0,004g/100ml of ethyl alcohol), and 30 minutes later, there was carried out spectrophotometric determination of optical density of analyzed sample on 515nm. The reference solution is represented by DPPH solution, but the background -by 96% ethyl alcohol. During the research process, the optimal concentration of solution was determined for inactivation of DPPH radical for each standard compound [17][18][19].

Results and Discussion
An analysis of the research results has shown that many factors have a significant influence on the extraction process, including the ratio of extraction raw materials and extracting agent, extraction temperature, extracting agent polarity, degree of raw materials crushing, extraction method, pulsation frequency and so on.
Based on the analysis of available literature sources, it has been established that the extracts obtained from grape skins and stone without preliminary drying and crushing mostly possess health-promoting properties. Based on this, we selected for the extraction the newly-squeezed environmentally safe color grape skins and stone, and the extraction was carried out without drying and crushing.
The two-stage extraction at the temperature of 45-50 °C within 4-4,5 hours under pulsation conditions, is conditioned for increasing periodical pulsation and for maximal extraction of phenolic complex.
Enzyme preparations to optimize the extraction of juice from the grape mass, processing them and heated to 100 °C, do not reduce the high content of phenols with AO-activity, making grape pomade important potential source of food additives.
The majority of red grape varieties, cultural and hybrid varieties of anthocyans is only a thin skin, very rare, but there are some varieties (eg. As red grapes "Dirbula" and "Zeimberi") which is also painted with the juice. The content of anthocyanin in general parts of the grape reaches up to 2200mg/dm 3 .
The conducted research has shown a very high bio-flavanoid composition of eco-extracts, which in turn points to high antioxidant activity of extracts. The total phenols content is high enough in the grape skins and seed extracts of Zeibeli variety (≥423/100ml), and is relatively low in the similar extracts of Izabela (≥394,2/100ml).    Both types of extracts are characterized by high values of antioxidant activity (Figure 4).
The first and second samples illustrated in Figure 4 are the grape skins and seed extracts of Zeibeli variety, non-concentrated and concentrated, accordingly, but the third and fourth samples are the grape skins and seed extracts of Izabela variety, nonconcentrated and concentrated, accordingly.
Antioxidant activity of both non-concentrated and concentrated eco-extracts is very high, and therefore, of high relevance is the use of hydrophilic and lipophilic extracts of secondary resources of Zeibeli and Izabela varieties in production of food supplements fortified with biologically active compounds.
The average annual polyphenol reserves of the grape harvest in Georgia, which is mostly accumulated in a practically unused grape-seed, exceeds several hundreds of tons that in monetary terms makes up millions of US dollars. Because of fact that such natural wealth is not used, the health losses in the population are considerable and obvious, although it is difficult to assess them properly. In addition, we propose environmentally safe raw materials of colored chemical-free grape varieties cultivated in the region, which are practically unused, and the local population seriously sensible about that, since one of the main fields of their activities in viticulture

Conclusion
By using the method of high-pressure liquid chromatography, in the color grape skins and seed extracts cultivated without use of chemical pesticides, there have been established a high content of total phenols and anthocyanins and antioxidant activity.
These resources are the best, ecologically pure raw materials for production of the drastic antioxidant polyphenolic concentrates.

Author's Contribution
T.N.Gvinianidze, M.S. Karchava and P.H. Jabnidze made equal contribution to this paper.