CORRECTIVE EFFECT OF RED WINE CONCENTRATE ENRICHED WITH NATURAL COMPLEX OF POLYPHENOLS ON ACTIVITY OF ANTIOXIDANT DEFENSE ENZYMES IN CARDIAC MUSCLE UNDER EXPERIMENTAL DIABETES MELLITUS of red concentrate enriched with natural complex of polyphenols on activity of antioxidant enzymes in cardiac muscle under

treated with a concentrate of wine polyphenolic complex. The activity of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase were detected to examine the corrective effect of the concentrate of red wine natural polyphenolic complex on the state of the enzymatic part of the antioxidant defense system. Results. The results have shown the normalization of activity of catalase, superoxide dismutase, and changes in the activity of enzymes of glutathione cycle after oral administration of polyphenolic complex concentrate for 14 days to rats with streptozo-tocin-induced diabetes mellitus. Conclusions. The results confirm a hypothesis about the antioxidant effect of the studied concentrate and the ability of natural polyphenolic complexes to serve as the basis for new drugs for treatment of diabetes-induced disorders.


INTRODUCTION
Diabetes mellitus (DM) is a chronic endocrine and metabolic disease caused by an impact of endogenous and exogenous factors with an absolute or relative absence of insulin that leads to disorders of all metabolic processes [4,25]. The number of people with this pathology increases dramatically every year, and according to the World Health Organization (WHO), there are more than 400 million people on the Earth with this disea se [5]. Approximately 10-15% of all people with this disease are people with type 1 DM.
Persistent hyperglycemia, which is typical of this pathology, not only activates the generation of free radicals, but also reduces the activity of cellular antioxidants, such as catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and others [14,21].
Therefore, it is important to develop new antidiabetic drugs. These agents are not only supposed to decrease the level of peripheral blood glucose, but also correct metabolic and morpho-functional disruption of cells. Additionally, they must help to maintain the oxidative-antioxidant balance and prevent the development of oxidative stress. Since the role of Reactive Oxygen Species (ROS) in the pathogenesis of DM and its complications is beyond doubt, the application of antioxidant therapy is one of the required parts of complex therapy of DM and its complications [13]. Today, scientists pay more and more attention to various herbal remedies, especially to natural polyphenolic complexes. Red grape wine contains a particularly large number of such compounds. Polyphenolic compounds of grape juice, including proanthocyanidins, flavan-3-ol derivatives, and other flavonoid derivatives, which have an effect in preventing cardiovascular diseases, have been subject to numerous studies [11].
Grape polyphenols interact with blood plasma proteins and blood cells to prevent premature oxidation of their molecules caused by oxidative stress. In addition, the powerful bactericidal and antiviral activities of these compounds were determined. The protective effect of grape polyphenols in some systems and organs under conditions of oxidative stress at diabetes mellitus was revealed [11].
It should be noted that almost 63% of all phenolic substances during the production of wine, especially large amounts of procyanidins of grape seeds and berry skins are converted into wine, so the multicomponent concentrate obtained from such wine can be considered as one of the most effective natural remedies [22].
The aim of this study was to determine biochemical mechanisms of action of red wine concentrate that contains a large amount of phenolic compounds, herein referred to as natural complex of polyphenols (NCP). The impact of NCP on the activity of antioxidant enzymes in heart tissue under experimental DM was studied.

MATERIALS AND METHODS
The experiments were performed on white outbred male rats weighing 100-150 g. Dry red wine for experiments was provided by Odesa National Academy of Food Technologies (Ukraine).
The concentrate was obtained by evaporating wine on a rotary evaporator Laborota 4001 (Germany) at a temperature of 40 °C and pressure of 0.8-0.9 kg/cm 2 . Evaporation was carried out from 1 L of wine to 300 mL of the concentrate. The concentrate was stabilized with ethanol up to 30% and with biogenic surfactants PS (surface-active products of biosynthesis of Pseudomonas sp. PS-17) up to 3% [28].
The total polyphenol content in the wine and the obtained concentrate was standardized using Folin-Ciocalteu reagent according to gallic acid equivalent [26]. The concentration of polyphenols in the obtained concentrate was 53.36 mg/mL. Experimental DM was induced by intraabdominal injection of Streptozotocin (Sigma, USA) dissolved in a 10 mM citrate buffer (рН 5.5) at a dose of 60 mg/kg of body weight. The induction of DM was controlled according to blood glucose level, which was measured 72 h after Streptozotocin injection. Animals with glucose concentration 12 mmol/L and higher were used for the research. Glucose concentration was determined by glucose oxidase method using a detection kit (Filisit diagnostics, Ukraine).
Experimental rats were divided into 4 groups: 1 st -control animals (C); 2 nd -control rats that were treated with red wine concentrate enriched with NCP with water for 14 days (C + NCP); 3 rd -rats with experimental DM (DM); 4 th -rats with experimental DM that were orally treated with red wine concentrate enriched with NCP with water for 14 days (DM + NCP).
The obtained concentrate was administrated per os with water at a dose of 45 mg of polyphenols per 1 kg of body weight, which corresponds to the theoretical average concentration of polyphenols contained in 300 mL of red wine (which is considered the daily norm for a person weighing 70 kg).
Rats from all experimental groups were decapitated under ether anesthesia on the 15 th day of the experiment. Extirpation of the heart was performed. The isolated tissues were immediately frozen at -70 °C.
Homogenization of cardiac muscle tissue was performed on an ice bath at 4°C using a manual Potter-Elvehjem homogenizer in the presence of hypotonic 50 mM Na-K-phosphate buffer (pH 7.4) at the rate of 10 mg of cardiac muscle tissue in 100 µL of a buffer. The studied parameters were determined in the supernatant obtained after centrifugation of the lysates for 5 min at 10,000 rpm.
Statistical analysis was carried out using Microsoft Excel. The calculation of the main statistical indicators was performed by direct quantitative data obtained from the research (arithmetic mean -M, the standard deviation of the arithmetic mean -m). To evaluate the reliability of the difference between the statistical characteristics of the two alternative data sets, the Student's ratio was calculated. The difference was considered significant at the indications of reliability p ≥ 0.95 (significance level P < 0.05).

RESULTS AND DISCUSSION
ROS are normally produced in cells of aerobic organisms [32]. Their generation intensifies in many pathological conditions, including diabetes [24]. Due to the high reactivity, ROS interact with various cellular components. Reacting with lipids, ROS initiate lipoperoxidation; with DNA -cause point mutations and breakage in the molecule; with proteins -break the peptide bonds and modify the side chains of amino acids. The negative effect of ROS in organisms is counteracted by the antioxidant defense system, the functioning of which prevents generation of free radicals, causes their neutralization and repairing of damages [17,30]. There is a certain ambiguity in the data about the state of the antioxidant system in diabetes. An increase of antioxidant enzymes activity and a decrease in the indices of others were detected [1,10,11], while in other investigations [8] changes of activity were not detected. It is possible that one of the reasons for such divergences may be fluctuations in blood sugar level, as well as tissuespecific activity of antioxidant enzymes. Cardiomyocytes are significantly affected in diabetes, this leads to the development of cardiopathies [1].
One of the key enzymes of the antioxidant defense system is SOD. This enzyme catalyzes the reaction of dismutation of superoxide radicals (О 2

•-
) with the formation of hydro gen peroxide and oxygen and, thus, participates in the regulation of free radical processes in living cells at an early stage [17]. According to obtained data ( Fig. 1), there was a tendency to reduction of SOD activity by 11% in cardiac muscle under experimental DM.
A decreased activity of SOD may be caused by glycation of the components of the active site of the enzyme [11]. A lowered SOD activity in DM may be also due to its inactivation by hydrogen peroxide (Н 2 О 2 ) [9]. Н 2 О 2 is formed as a result of glucose autooxidation, therefore, its level is high in diabetic patients. Furthermore, accumulation of superoxide-anion radical (О 2 •-) and overproduction of Nitrogen oxide (NO) contributes to the excessive formation of peroxynitrite (ONOO -) in diabetes, which is the mediator of oxidative cell damage. Subsequently, ONOOdeactivates SOD via nitration of the Tyr34 residue in the active site of the enzyme [15,7].
Under the conditions of concentrate enriched with NCP administration, the tenden cy to normalization of SOD activity was detected in animals with experimental DM, while no significant changes were identified in control animals (Fig. 1). Polyphenolic compounds are known as scavengers of ROS, thus, they have good protective properties [27]. They can "abduct" superoxide anions and other ROS, thereby reducing the generation of several dangerous molecules, including peroxynitrite [6].
CAT is another important antioxidant enzyme. This enzyme is involved in detoxication of Н 2 О 2. It catalyzes its decomposition into water and oxygen [17]. We detected a decrease in the activity of catalase by 16.8% (Fig. 2) in the cardiac tissue of animals with experimental DM compared to the control. A reduced activity of CAT can be associated with dysregulation at gene expression or a decrease in mRNA level. Besides, CAT is over-phosphorylated in diabetes, which can lead to a decrease in enzymatic activity [23]. It is known [28] that NO generation highly intensifies in the pathology. This may affect the antioxidant enzymes activity. For example, NO can bind to the iron-porphyrin complex of CAT to form nitroso-derivatives. The appearance of such complexes prevents binding of H 2 O 2 in the active site of CAT, as well as its decomposition. Nitrite ions are also able to bind directly to the iron of CAT heme, which can decrease the activity of the enzyme [28].
Under the conditions of administration of concentrate enriched with NCP to animals with DM, there was an increase in CAT activity by 27.7% compared to the data obtained in diabetes (Fig. 2). However, there were no significant changes detected in CAT activity of cardiomyocytes of control animals after concentrate administration. Polyphenolic compounds of red wine contain resveratrol. This polyphenol can decrease the overphosphorylation of CAT at diabetes, thus, enhancing CAT activity [23].
The glutathione system is also an important part of the antioxidant defense system. It can directly inactivate ROS, supplement and correct errors of "the first line of defense", formed by SOD and CAT [28]. GPx also neutralizes Н 2 О 2 and has much higher affinity for Н 2 О 2 than that of CAT [28]. GPx and GR activities are reduced in cardiomyocytes in DM by 9% and 25.5%, respectively (Fig. 3, 4). GPx activity depends on the content of reduced glutathione (GSH), the intracellular concentration of which is maintained by GR. In turn, the functioning of GR is determined by the level of reduced nicotinamide coenzymes. Under conditions of DM, energy depletion can be observed, which causes a shortage of energy substrates. Accordingly, this affects the effectiveness of the antioxidant system [11]. Under conditions of concentrate enriched with NCP administration, we detected a decrease in GPx activity by 23% and an increase in GR activity by 36% in cardiomyocytes of control animals, compared to control (Fig. 3, 4). However, a decrease in GPx activity by 44% and normalization of GR activity were detected in cardiac muscle of diabetic animals, compared to animals with the pathology after treatment with the concentrate (Fig. 3, 4).
Therefore, the influence of NCP on the activity of enzymes of the glutathione system was investigated. Under hyperglycemic conditions, glucose metabolism is intensified through the polyol and hexosamine pathways. NADPH is a cofactor of an enzyme of polyol pathway aldose reductase. NADPH is also a cofactor of GR. As a result, a decrease in GR activity can be caused due to a lack of the cofactor that is mainly used by enzyme of the polyol pathway in DM [31]. A decreased activity of GR has an impact on GPx activity, while the product of the reaction, GSH, is the substrate of GPx. In addition, an increased metabolism through the hexosamine pathway in diabetes leads to the  intensification of glycation. This can affect intracellular proteins, such as enzymes of different metabolic pathways [31]. Polyphenols reduce glucose concentration, which restores the level of NADPH available for GR. Hence, the activity of the studied enzyme increases. We can also hypothesize that GPx activity decreases after treatment with the studied concentrate because of the antioxidant action of polyphenols. These compounds due to scavenger activity reduce the level of peroxides that can serve as substrates for GPx. It can be assumed that the level of peroxides can be reduced because of an increased activity of other enzymes, such as catalase, under the same conditions. An antioxidant deficit in cardiac cells, especially a reduced activity of SOD, CAT, GPx, and GR, leads to myocardial infarction [2]. Intensification of fatty acid β-oxidation due to a high level of fatty acids in DM leads to an elevation of mitochondrial inner membrane potential and stimulates ROS production [20]. An increased generation of ROS also has a negative impact on cardiac cells [2]. Thus, the studied concentrate has not only the antioxidant, but also the cardioprotective properties, and can be the basis for the creation of food additives or drugs enriched with polyphenolic compounds for complex therapy of DM.

CONCLUSIONS
A decrease in the activity of enzymes of the antioxidant defense system in cardiac muscle under the conditions of DM was detected. That is the evidence of the development of oxidative-nitrative stress in cardiomyocytes in the studied pathology, which is one of the main causes of cardiovascular diseases.
Normalization of SOD and CAT activities after treatment with red wine concentrate enriched with NCP at diabetic condition indicates antioxidant, and, subsequently, cardioprotective effects of these compounds.
The glutathione system was violated at DM. Treatment with concentrate enriched with NCP caused an increase of GR activity and a decrease in GPx activity. Different effects of polyphenolic compounds on elements of the glutathione system in DM were found.
The molecular mechanisms of action of polyphenols from wine require further research. Besides, it is beyond doubt that such natural complexes of polyphenolic compounds show a corrective effect, and, therefore, can serve as a basis for new antidiabetic drugs.

Сonflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Human Rights: This article does not contain any studies with human subjects performed by the any of the authors.
Animal studies: All institutional, national and institutional guidelines for the care and use of laboratory animals were followed.