THE FRIGOPROTEСTIVE EFFECT OF “GLUCOSAMINE C-BCPP” DIETARY SUPPLEMENT AND THE ROLE OF ITS COMPONENTS

The effect of “Glucosamine C-BCPP” dietary supplement on the survival time of animals with acute hypothermia in experiments in mice has been determined. It has been found that with intragastric introduction of “Glucosamine C-BCPP” dietary supplement (its composition: glucosamine hydrochloride and ascorbic acid) to animals a significant increase in the the survival time compared to the the untreated group is observed on the model of acute hypothermia. The frigoproteсtive effect of “Glucosamine C-BCPP” is mainly due to glucosamine hydrochloride, the role of ascorbic acid is less.

Treatment and prevention of acute cold trauma is one of the most important problems of modern medicine and pharmacy both in peacetime and in wartime, and requires in-depth study of the mechanisms of the body adaptation to low temperatures [2, 5,6]. Cold trauma refers to serious injuries, and it is an important cause of disability [16,18,20].
In recent years there is a clear tendency towards increasing the number of injured victims of the cold in temperate regions. Every year more than 12 000 cases of such injury in Ukraine, more than 20000 cases in Russia are recorded. Most of the victims of the cold need hospital treatment; mortality exceeds 10% [6, 12,19].
In the pathogenesis of acute cold trauma hypoxia of the brain and other organs, disorders of the central nervous, cardiovascular, respiratory, excretory, endocrine systems, the gastrointestinal tract and the skin are involved. The course of these disorders causes the complexity of treatment and the consequences of hypothermia [1, 8,14,18,19].
Therefore, the search for new frigoproteсtive agents is an urgent task. In our previous studies it has been found that glucosamine hydrochloride (G h/chl), a drug with the anti-inflammatory activity and a number of other pharmacological properties, is an effective frigoproteсtive agent that exceeds another salt -glucosamine sulphate [3,4,9]. Glucosamine is an important component of biological membranes; it is contained in large quantities in the brain, kidney, liver and skin [7].
On the model of acute hypothermia G h/chl in the dose of 50 mg/kg improves survival rate of animals, motor activity, muscle tone, coordination and physical endurance, normalizes the renal function in the recovery period. All these advantages substantiate expediency of in-depth studies of G h/chl as a potential frigoproteсtive agent. Most glucosamine drugs registered at the Ukrainian market contain glucosamine sulphate ("Artron", "Dona", etc.). Only one dietary supplement includes G h/chl in its composition in combination with ascorbic acid ("Glucosamine C-BCPP" produced by "Borshchahivskiy CPP").
Vitamin C (ascorbic acid) has the metabolic action; it is involved in the regulation of redox processes, carbohydrate metabolism, blood coagulation, tissue regeneration, and reduces vascular permeability. This vitamin possesses antiplatelet and distinct antioxidant properties, regulates immunological reactions, promotes phagocytosis, and increases resistance to infection [17].
The aim was to study the effect of "Glucosamine C-BCPP" dietary supplement and its components -G h/chl and ascorbic acid on the survival time in mice under conditions of acute cold trauma.

Materials and Methods
The research was conducted in summer on white randombred female mice weighing 28-32 g; they were kept in standard vivarium conditions in accordance with the GLP rules. During our work the requirements of the EU Directive on the protection of animals used for experimental and other scientific purposes were performed [10].
To simulate cold injury mice were placed in individual plastic cases with the size of 8×8×15 cm, which do not restrict access to the air. The animals were placed in the cases to the "Nord Inter-300" freezer at -18°C, and the survival time was determined [15].
"Glucosamine C-BCPP", G h/chl, ascorbic acid or 0.9% solution of NaCl (control) were administered intragastrically (i/g) for 30 min prior to acute cold trauma, i.e. in the preventive mode. Laboratory animals were di- vided into 4 groups according to the drug that they received, the dose and the route of administration: group 1 -untreated animals (n = 8), NaCl solution (0.1 ml/10 g) was administered to these animals; group 2 -"Glucosamine C-BCPP" in the dose of 82.5 mg/kg (the dose is equivalent to 50 mg/kg of G h/chl) + cold injury (n = 8); group 3 -G h/chl /50 mg/kg + cold injury (n = 8); group 4ascorbic acid (Darnitsa) in the dose of 4 mg/kg + cold injury (n = 8). The dose of 4 mg/kg of ascorbic acid was equivalent to the dose that animals received with "Glucosamine C-BCPP" dietary supplement in the dose of 82.5 mg/kg. The survival time of mice was selected as a criterion of sensitivity to cold. For statistical analysis of the results Student t-test was used in normal distribution or White nonparametric criterion W -when it was absent. Differences were considered to be statistically significant at p<0.05.

Results and Discussion
In the test of acute hypothermia in mice the differences in the survival time listed in Table were observed.
Analysis of the data in Table indicates that "Glucosamine C-BCPP" in the dose of 82.5 mg/kg significantly increases the survival time of the animals with respect to the control pathology group by 33% and 16.1% compared to ascorbic acid. Under the action of G h/chl in the dose of 50 mg/kg the survival time of mice with acute cold trauma statistically significantly increases compared to the control (on average + 33.5%) and 16.7% compared to the value against the background of ascorbic acid.
Ascorbic acid in the dose of 4 mg/kg in animals with acute cold trauma significantly increases the survival time by 20.2% compared to the untreated group.
The changes identified, namely increase in the survival time of the animals against the background of acute cold trauma, show a marked frigoproteсtive effect of the investigated drug and its components. The highest effect (almost identical) is provided by G h/chl 50 mg/kg and "Glucosamine C-BCPP" in the dose of 82.5 mg/kg equivalent to G h/chl. The increased survival time under the action of G h/chl can be explained by a wide range of the pharmacological activity, such as anti-inflammatory, cerebroprotective properties [9] associated with the effect on neurotransmitter and metabolic processes in the brain, improvement of its blood supply, a possible positive impact on the systemic circulation, microcirculation, energy metabolism, etc. [4,7].
Almost all body systems participate in stress reaction, but to a greater extent stress is associated with the hypothalamus-pituitary-adrenal gland axis and the immune system [11]. Vitamin C plays a crucial role in the synthesis of steroids, which are important in the mechanism of resistance in cold injury. Thus, ascorbic acid is completely absent in the human adrenal glands after lethal hypothermia [13]. Vitamin C is also important for formation of norepinephrine -a mediator of the sympathetic nervous system, which activation is involved in the stress reaction caused by cold injury. These aspects of the mechanism of action of ascorbic acid explain its protective effect revealed in acute hypothermia. However, according to the results G h/chl plays a key role in the composition of "Glucosamine C-BCPP" dietary supplement. CONCLUSIONS 1. "Glucosamine C-BCPP" dietary supplement in the dose of 82.5 mg/kg possesses a marked frigoproteсtive effect increasing the survival time of mice with acute hypothermia by 33%.
2. The protective effect of "Glucosamine C-BCPP" dietary supplement is mainly due to the influence of glucosamine hydrochloride, which increases the survival time of animals by 33.5% in the equivalent dose of 50 mg/kg. Ascorbic acid, which per se has a moderate frigoproteсtive effect in the equivalent dose of 4 mg/kg, does not practically affect frigoproteсtive action of glucosamine hydrochloride when used in combination.
3. The results have experimentally substantiated applicability of "Glucosamine C-BCPP" dietary supplement as a protective agent on exposure to low temperatures.  Note: * -statistically significant differences when compared to the untreated group, p<0.05; ** -statistically significant differences when compared to the group receiving glucosamine hydrochloride, 50 mg/kg, p<0.05; *** -statistically significant differences when compared to the group receiving "Glucosamine C-BCPP", 82.5 mg/kg, p<0.05.