New grape products with antioxidant properties

. The paper presents the results of mMonitoring of qualitative and quantitative composition of polyphenols in wines of trademarks of the Crimea and the Kuban made from Cabernet-Sauvignon, Merlot and Saperavi red grapes. It has been shown that total amount of polyphenols in wine exceeds the European average of 2.5 g/L. Antioxidant activity is also high, exceeding 2.0 g/L in units of the standard antioxidant trolox. Technology and regulatory documentation was developed for obtaining products with a standardized amount of polyphenols from grapes, grape pomace and grape vines. The presents the results of development and practical evaluation of products saturated with grape polyphenols (wine, grape drink, extract) having a high antioxidant activity confirmed by in vitro and in vivo.


Introduction
The present-day trends in the development of the domestic winegrowing industry are largely aimed at the development and implementation of technologies for obtaining new food products from grapes, enriched with biologically active substances.The results of epidemiological studies published in the early 1990s and conducted under a WHO program that revealed a relationship between the mortality rate due to coronary insufficiency and the level of saturated fat consumption in industrialized countries are widely known.With the same level of saturated fat consumption, the French had 36-56% lower mortality rate due to coronary insufficiency than the Americans.It has been shown that this result was directly related to regular moderate consumption of red grape wine by the French [1].The American nutrition specialists recommend regular consumption of red wine as a preventive measure to reduce the risk of cardiovascular diseases, 300 ml for men and 150 ml for women per day.We have evaluated the possibility of innovative production of domestic wine products with health-promoting properties for enotherapy.As it is known, a reduction of risk of cardiovascular system diseases and other disorders of human organism functioning is connected with the antioxidant activity of grape polyphenols present in wine.[2][3][4].The purpose of the study is to present the results of the development and practical evaluation under in vitro, in vivo conditions of the products saturated with grape polyphenols having high antioxidant activity.

Materials and methods
For conducting the study, the industrial samples of red dry wines made of grape varieties Cabernet-Sauvignon, Saperavi, Merlot were used as well as new types of products from red grape varieties: "Zdorovye" (health) red dry wine, "Zdorovye" (health) grape drink, developed by us.In the samples of wine products developed by us, the polyphenol content value is determined at a level of no lower than 2,5 g/L, and in the grape polyphenol extractno lower than 20 g/L [5].The mass concentration of phenolic substances in the extracts was determined by the Folin-Ciocalteu colourimetric method, according to Р 4. 1.1672-2003 [6].The qualitative and quantitative composition of polyphenols in alcohol extracts was determined by the HPLC method using an Agilent Technologies chromatography system (model 1100) with diode matrix detector [6].Calculation of quantitative content of individual components was performed using calibration graphs of the dependence of the peak area on the concentration of the substance, plotted on the solutions of individual substances.Anthocyanins content was determined expressed in terms of malvidin-3-O-glucoside chloride, caftharic acid content was determined expressed in terms of caffeic acid, polymeric and oligomeric procyanidins content -expressed in terms of (+)-D-catechin.All determinations were carried out in three replications.As standards, gallic acid, caffeic acid, (+)-D-catechin, malvidin-3-O-glucoside chloride, dihydroquercetin, isoquercitrin (Fluka Chemie AG, Switzerland) and trans-resveratrol, (-)-epicatechin, syringic acid (Sigma-Aldrich, Switzerland) were used.The results were processed using standard methods of mathematical statistics.To evaluate the antioxidant activity of wine and concentrates, the amperometric method for measuring mass concentration of antioxidants by the standard antioxidant trolox on the appliance "Tsvet-Yauza 01-АА" according to GOST Р 54037 was used [7].

Research of the results
Monitoring of qualitative and quantitative composition of polyphenols in wines of trademarks of the Crimea and the Kuban made from Cabernet-Sauvignon, Merlot and Saperavi red grapes as in experimental samples of wine products showed that in wines produced according to the technologies adopted in the domestic winemaking, polyphenols typical for red wines are found -flavonoids (anthocyanins, flavan-3-ols, flavones), hydroxy-cinnamic and oxybenzoic acids, oligomeric and polymeric proanthocyanidins (Table 1).Meanwhile, the total amount of polyphenols in wine exceeds the European average of 2.5 g/L.Antioxidant activity is also high, exceeding 2.0 g/L in units of the standard antioxidant trolox [8].
Thus, the traditional red wines of the Crimea, the Caucasus and the experimental samples of dry red wines "Zdorovye", by quantity, quality and antioxidant activity, are fully consistent with functional characteristics of European wines.
At the same time, domestic regulatory and technical documentation does not specify the quantitative determination of phenolic substances in wine products, which is connected apparently with underestimation of the role of polyphenols, whose functional properties are today recognized in the Russian Federation at the level of state standards.We have developed "Standards of Organization" (SO) and technological instructions for the experimental products "Zdorovye" (health) red dry wine, "Zdorovye" (health) grape drink, that determine the polyphenol content value at a level of no lower than 2.5 g/dm 3 , as well as for the grape polyphenol extract with the polyphenol content of no lower than 20 g/dm 3 .Achieving a standard value in phenolic substances in wine was ensured by traditional methods of fermentation on the pulp in fermenters and by control at the output of the finished wine material.When it was not possible to achieve the required value of the specified parameter of mass concentration of phenolic substances by traditional methods of fermentation on the pulp, mixing of wine materials with the estimated amount of an experimental sample of the extract of grape polyphenols was used.In this case, a grape drink with a guaranteed content of phenolic substances at the required level was obtained.The extract of grape polyphenols was obtained by alcohol extraction of grape pomace with subsequent dealcoholization to the strength of no more than 15 vol.% and a content of phenolic substances of no less than 20,0 g/dm 3 .
The studies of experimental samples of wines, drinks and extract obtained during the grape processing season of 2015 have shown that the polyphenol content in them was in wines 2.53-3.02g/dm 3 , in drinks -2.56-3.02g/dm 3 , in extracts -21.5 g/dm 3 .The antioxidant activity was 1.51-1.72g/dm 3 , and that of the extract -33.74 g/dm 3 , expressed as a standard antioxidant trolox, which indicated a high potential of biological activity of the experimental samples (Tables 2 and 3).

Discussion of the Results
In our paper, a possibility of innovative production of domestic wine products with health-promoting properties for enotherapy was experimentally demonstrated.We have developed "Standards of Organization" (SO) and technological instructions for the experimental products "Zdorovye" (health) red dry wine, "Zdorovye" (health) grape drink, that determine the polyphenol content value at a level of no lower than 2.5 g/L, as well as for the grape polyphenol extract with the polyphenol content of no lower than 20 g/L.The studies of experimental samples of wines, drinks and extract obtained during the grape processing season of 2015 have shown that the polyphenol content in them was in wines 2.53-3.02g/L, in drinks -2.56-3.02g/L, in extracts -21.5 g/L.The antioxidant activity was 1.75-2.26g/L, and that of the extract -13.6 g/L, expressed as a standard antioxidant trolox, which indicated a high potential of biological activity of the experimental samples.
Testing of in vivo biological activity of experimental samples of wine and extract were performed on the model of myocardial ischemic injury and the development of metabolic syndrome in experimental animals.The studies revealed that wine and extract of grape polyphenols possess a cytoprotective action, allowing to largely preserve the myocardial structure in rats under conditions of myocardial ischemia induced by administration of high doses of cobalt chloride.Polyphenols of experimental products showed a marked antihypertensive activity, efficiency in reducing blood glucose, increasing insulin secretion and sensitivity to it in presence of a suppression of the development of metabolic syndrome in rats [3][4].
The experimental samples of the wine, drink and extract that successfully passed the in vivo testing for the biological activity and containing grape polyphenols at the level of no less than 2,5 g/dm 3 in the wine und no less than 20.0 g/dm 3 in the extract, were used to conduct clinical studies of cardioprotective activity.
In the clinical sanatorium "Ai-Petri" in the city of Yalta, 259 patients were examined from May to October 2016.Out of them, 2 groups of patients were formed.One group of patients with coronary heart disease -96 persons, and one group with hypertensive vascular disease -163 persons.The study protocol was approved by the local ethics committee.All patients received an identical complex of basic sanatorium treatment, the patients of the study groups received, within a comprehensive treatment program, "Zdorovye" wine, "Zdorovye" drink, or extract of grape polyphenols for an average of 280 ml of wine and 36 ml of extract per day.It corresponded to 10 mg of grape polyphenols for 1 kg of body weight.The course of treatment was 14 days.The patients were examined at the beginning and at the end of the treatment.Functional status of cardiovascular system, biochemical blood parameters, status of lipid peroxidation and blood antioxidants, an objective status of the patients were analyzed as well as function tests of the hemodynamic heart function performed.
The health resort treatment resulted in a positive dynamic in the physical condition of the patients, at that, in the groups that received wine, drink or extract, more parameters improved compared to the groups that received basic treatment.These changes were most noticeable in the treatment of patients with coronary heart disease.There was a decrease in total cholesterol, atherogenic coefficient, and fibrinogen in the blood.
In such a way, the probability of formation of blood clots and cholesterol plaques in arteries was reduced.
Clinically, the need for intake of nitroglycerin in patients with coronary heart disease decreased by two times, restrictions on physical activity were lifted in a quarter of patients, in the majority of patients (more than 85%), reduced fatigue and increased tolerance to physical exertion compared to the groups with basic treatment were registered.On the basis of the results obtained in the course of the clinical study, the Instructional guidelines on the application of experimental products with optimal concentrations of phenolic compounds for enotherapy, as part of the health resort

Conclusion
Thus, a possibility to produce in the Crimea wine products saturated with grape polyphenols and allowing to carry out, within health resort treatment, a rehabilitation of patients with coronary heart disease and hypertension, has been demonstrated at the technological, experimental and clinical levels.
The results achieved allow to optimistically assess the efficiency of the application of a treatment-and-prophylactic course with wines, drinks, extracts saturated with grape polyphenols as part of health resort treatment of patients with coronary heart disease and hypertension.

Table 2 .
Results of tests of experimental product samples."Zdorovye" (health) red dry wine made of grapes; 2 -Grape drink made of grape raw materials

Table 3 .
Results of tests of the experimental sampleextract of grape polyphenols.