Membrane-protective preparation

FIELD: chemistry.

SUBSTANCE: application of 5% water solution of sodium and potassium salts of humic acids, obtained from leonardite brown coal, in dose 10.0 mg/kg as membrane-protective preparation.

EFFECT: application of composition described above makes it possible to extend arsenal of membrane-protective preparations from low-toxic, cheap, available natural raw material.

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The invention relates to medicine, pharmacology and pharmacy, in particular lipoic tools, and can be used for pathogenetic treatment and prevention of various diseases to protect cell membranes from the damaging effects.

Know the use of drugs with lipoic properties, in the treatment of various diseases. Used antihypoxant drug taurine (Nik), which has lipoic properties, has a positive effect on the phospholipid composition of cell membranes, improves metabolic processes in the heart, liver and other organs and tissues (Reference Vidal,

Know the use of Rexona as lipoic drug in the treatment of purulent-inflammatory diseases of maxillofacial area (EN 2006110681 A, IPC A61K 38/43, A61P 43/00, 2007). Drug Rext represents the enzyme superoxide dismutase, which is an endogenous acceptor is oxygen free radicals and prevents oxidative modification of proteins, and is also associated with activation of lipid peroxidation destruction of biological cells (Handbook Vidal,

Known drug oxitetraciclina succinate (Mexidol), which is an inhibitor of Svobodnoye the social processes, membraneelectrode, stabilizes the membrane structure of blood cells (red blood cells, and platelets) during hemolysis (Reference Vidal, Known hepatoprotective agent for the prevention and correction of toxic liver damage, according to the description as hepatoprotector use succinate 2-ethyl-6-methyl-3-hydroxypyridine known as Mexidol. Exchange application Mexidol has lipoic effect on liver cells, toxic affected by the prolonged use of tuberculostatics (U.S. Pat. EN 2189817, IPC A61K 31/4412, A61P 43/00, 2002).

Known for the preparation of alpha-tocopherol acetate (Tocopherol), exhibiting antioxidant effect, is involved in cell proliferation, tissue respiration, and other critical processes of tissue metabolism. It is known that the tocopherol prevents increased permeability and capillary fragility, prevents hemolysis of red blood cells (Handbook Vidal, Know the use of α-tocopherol in veterinary medicine to improve hemolytic stability of erythrocytes in thermal injury of animals (Bekyarova, I. and other Protection of α-tocopherol erythrocytes from hemolysis induced thermal injury. Bulletin of experimental biology and medicine. 1989, T. 107, No. 4, S. 413-415).

Know the use for which ysenia resistance to hemolysis of erythrocytes as a natural antioxidant carnosine (β-alanyl-L-histidine) or its N-acetyl derivative in a certain range of concentrations (patent RU 2162698, IPC A61K 35/18; A61K 38/06, 2001).

Known drug Galavit (aminodimethylaniline sodium), which is an immunomodulatory drug with anti-inflammatory effect, its mechanism of action is associated with the ability to influence the functional-metabolic activity of macrophages (Reference Vidal, Used to increase the resistance of erythrocytes to hemolysis induced by ethanol, by introducing into the erythrocyte suspension in ethanol drug Galavit, dissolved in physiological solution (patent RU 2315605, IPC A61K 35/18, A61P 25/32, 2008).

The ability to identify isobutylphenyl to protect the membrane of erythrocytes from oxidative stress, defined in terms of spontaneous and induced by hydrogen peroxide hemolysis of erythrocytes (Lipoic properties isobutylphenyl - a new class of antioxidants / Shevchenko O. G. [and others] // Biological membranes: a journal of membrane and cell biology, 2013. - So 30, No. 1. - S. 40.)

As a means with gepatoprotektoram action used ascorbate lithium with gepatoprotectornoe activity against the hemolysis of erythrocytes under the influence of alcohol and different lower toxicity compared to other drugs lithium (U.S. Pat. EN 2351326 C2, IPC A61K 31/375, A61P 43/00, A61P 39/00, 2009).

The task of nastoyascheevremya is expanding Arsenal of known lipoic funds for protection of biological membranes at various damaging effects.

The technical result consists in the use of the drug containing salts of humic acids, which are obtained from low-toxic, cheap, available natural raw materials.

The technical result is achieved by the fact that as lipoic tools use an aqueous solution of the sodium and potassium salts of humic acid derived from brown coal leonardite at a dose of 10.0 mg/kg

Conditional title of the proposed lipoic tools - HUMATE of leonardite. Substance powder is produced by LLC "HUMATE", , Irkutsk, name and designation of the products: "HUMATE" is a fertilizer based on humic acids for agricultural use, THE 2189-004-41764643-98. It lipoic properties are not known.

For production of the proposed lipoic tools - aqueous solution of the sodium and potassium salts of humic acids leonardite - use relatively cheap substance, which is obtained industrially available raw materials in large volumes in an environmentally safe manner (Perminova I. C. Humic substances - a challenge to the chemists of the XXI century / I. C. Perminova // Chemistry and life, 2008. No. 1. - 50 C.-55).

The proposed substance of HUMATE of leonardite belongs to the class of natural compounds, called humic substances. Humic substances and salts of humic acids (humates) have RA is nenapravlenie biological activity, in recent years, increasingly discusses the possibility of their application in medicine and pharmacy (Dry A. S. prospects of application of humic and humanophobia acids in medicine and pharmacy / A. C. Dry, P. C. Kuznetsov // Medicine in Kuzbass. - 2009. No. 1. - S. 10-14.). Features of the chemical structure of humic substances cause their surface-active properties (On the mechanism antidote action of humates in relation to the oil / A. C. Daurov [and other] // bull. WSNS WITH THE RAMS. - 2005. No. 6 - N-143-146; a Shrub D. I. Combined effect of petroleum products and "HUMATE" on Daphnia / A. I. Shrub, A. C. Daurov // Siberian ecological journal. - 2004. No. 1. - 35 C.-40), which leads to the ability to solubilisate and dispersing hydrophobic organic matter, increasing their solubility and increasing the hydrophilicity of the surfaces of cells (Possible mechanisms of biological action of humic substances / Shrub D. I. [and other] // Siberian journal of medicine. - 2008. No. 6. - S. 76-79). This feature of humic substances suggests the possibility of their influence on the properties of lipoproteins and other components of biological membranes.

Known application of humic substances and containing natural substrates, in different branches of medicine. For example, it is known the use of oxidate peat as a means for the treatment of patients with HRO is practical dermatoses (U.S. Pat. EN 2187315 C2, IPC A61K 35/10, A61P 37/04, 2002); use of products containing sodium HUMATE from wood-grass peat deposits "Dark" Tomsk region as a means of having antimicrobial and antifungal activity (U.S. Pat. EN 2340328 C1 IPC A61K 9/06; A61K 35/10; A61P 31/10, 2008); the use of humic acids and humates as biologically active additives in the cosmetic industry with antioxidant activity (U.S. Pat. EN 2094042 C1 IPC A61K 7/00, A61K 7/40, 1997); for care of hair and scalp on the basis of humic concentrate, which is a product of peat processing solution of sodium hydroxide or potassium when activated oxygen in the process of ozonation and subsequent neutralization with a solution of citric or acetic acid, containing the dry substance of humic acids not less than 2.8 wt.% (U.S. Pat. EN 2394556 C1 IPC A61K 8/97, A61Q 5/12, 2010); a method of treating gastric ulcer and duodenal ulcer by using medicines, obtained by processing of peat (U.S. Pat. EN 2357741 C1 IPC A61K 35/10; A61P 1/04, 2009).

In Fig.table 1 presents the results of determination of acute toxicity of HUMATE of leonardite after a single oral administration; Fig.2 presents curves of hemolysis in the test sample and under the influence of HUMATE of leonardite.

Example 1.

The study of acute toxicos and aqueous solution of sodium and potassium salts of humic acids, derived from brown coal (hereinafter referred to as the HUMATE of leonardite), the oral method of administration held on 30 white outbred mice-males weighing 22,0±5,3 g, divided into groups of 10 goals to 3 of the studied doses. HUMATE of leonardite was introduced in the form of a 5.0% aqueous solution once orally via gastric metal probe. Used doses which exceed the maximum therapeutic (10 mg/kg) 25 times (dose of 250.0 mg/kg, which corresponds to a volume of 0.1 ml per 20 g of body weight of a mouse), 50 times (dose of 500.0 mg/kg, a volume of 0.2 ml/20 g) and 100 times (dose 1000,0 mg/kg, volume of 0.4 ml/20 g).

Revealed that none of the studied doses (250.0 mg/kg, 500.0 mg/kg, 1000,0 mg/kg) found no deaths during the follow-up for the first night and over the next 14 days. There were no changes in behavioral responses (not identified increased excitability, nervousness, aggression, fearfulness), revealed no changes in neuromuscular excitability (tremors, convulsions, ataxia, changes in reflexes provisions changes reactions to touch, change the tone of the skeletal muscles). There has been no changes on the part of the autonomic innervation, because there were no changes in salivation, therasense coat; not identified changes in food and water consumption (from 1.4 to 3.2 ml per head per day) in experimental animals (Fig.). Enter oral dose greater than 1000 mg/kg does not seem technically feasible and impractical, because studied the maximum dose of 1000 mg/kg can be attributed HUMATE of leonardite to non-lethal compounds (moderately hazardous substances - median lethal dose LD50when the stomach is in the range 151-5000 mg/kg - III class of hazard) and makes possible its use as lipoic tools.

Thus, in accordance with GOST 12.1.007-76 HUMATE of leonardite is non-lethal and can be attributed to the III class of hazard (moderately hazardous substances - LD50when introduced into the stomach 151-5000 mg/kg).

Example 2.

As lipoic funds from natural raw materials, ensuring the protection of biological membranes with damaging effects, used an aqueous solution of the sodium and potassium salts of humic acid derived from brown coal (HUMATE of leonardite).

To study lipoic properties of HUMATE leonardite used the modified method of registration of chemical (acid) erythrogram on Terskov I. A., Gitelson I. I. (1957), allowing to assess structural and functional properties and the permeability of biological membranes (Gitelson I. I. Erythrogramma as a method of clinical studies of blood / I. I. Gitelson, I. A. Terskov. - Krasnoyars is: Publishing house of the Siberian branch of the USSR Academy of Sciences, 1959. - 247 S.; Terskov I. Method A. chemical (acid) erythrogram // I. A. Terskov, I. I. Gitelson // Biophysics. - 1957. - So 11, Vol.2. - S. 259-266; Workshop on Biophysics / Century, Artyukhov [and other] / edited Century, Artyukhova). - Voronezh: Publishing house of Voronezh state University, 2001. - S. 147-161). Cumulative to date actual data suggest that erythrocytes are involved in the pathological process not only with hematological diseases, but also undergo major changes in the structure and function in diseases of different Genesis. The choice of erythrocyte membranes as the object of study is dictated by the fact that it has common principles of molecular structure of plasma membranes. Therefore, the patterns of changes in the structure and function of the erythrocyte membrane with a certain degree of correction can be extrapolated to other membrane systems. In addition, the simplicity of the organization of the erythrocyte gives the opportunity to study the functional properties of the plasma membrane without interference imposed intracellular membrane structures and organelles (Molecular disrupt the membrane of red blood cells in the pathology of different Genesis are typical reaction: the contours of the problem / Novitskiy centuries [and other] // Bulletin of Siberian medicine, 2006. No. 2. - S. 62-69; Postnov Y. C. Primary hypertension as pathology of cell membranes / Y. C. Postnov, S. N. Orlov. - M.:Medicine, 1987. - 192 S.).

To study lipoic properties of HUMATE leonardite used the model of acid hemolysis of erythrocytes induced by hydrochloric acid. Estimated lipoic properties of an aqueous solution of HUMATE of leonardite at a dose of 10.0 mg/kg pre-incubated with a suspension of erythrocytes at 37°C for 40 min incubation Temperature of 37°C is selected as corresponding to the temperature of the human body, incubation duration 40 min is chosen as the average time required for admission introduction of medicinal substances in the blood plasma. The concentration of HUMATE leonardite, equivalent to a dose of 10.0 mg/kg, chosen experimentally based on the results of a series of preliminary experiments with doses of 1.0 to 5.0, and 10.0 and 20.0 mg/kg of incubation. Analysis of erythrogram was carried out by graphical and mathematical way. Expected constant maximum rate of hemolysis (Kmax, Rel. unit) is a parameter that characterizes the proportion of red blood cells simultaneously entered into the stage of hemolysis; determined the relative amount of hemolyzed cells (G, %) at different points in time monitoring, indicators of structural state of erythrocyte membranes when hemolysis was assessed by the estimated parameters in accordance with the methodology (Workshop on Biophysics / Century, Artyukhov [and other] / [edited Century, Artyukh the VA]. - Voronezh: Publishing house of Voronezh state University, 2001. - S. 147-161).

The HUMATE solution of leonardite at a dose of 10.0 mg/kg was added directly to the working suspension of red blood cells, after which the mixture of red and HUMATE of leonardite were subjected to pre-incubation in an incubator at 37°C for 40 minutes the Experiments were carried out in 3 series of experiments, the obtained results were subjected to statistical analysis with calculation of average values. In each series of experiments studies were performed in parallel samples, one of which was the control (erythrocytes and hemolytic) and the second experimental (red blood cells, HUMATE solution of leonardite, hemolytic).

It is established that the kinetic curve of the process of hemolysis in the test sample corresponds to the standard and is characterized by an S-shaped form. The offset of the S-shaped curve in the first few minutes below the zero line connected with ferulacea erythrocytes. Then the process of hemolysis slowly grows in its initial part and further ascending part of the kinetic curve of hemolysis reflects the destruction of the least sustainable of erythrocytes. Then curve sharply rises in the range from 2 to 5 minutes, describing the entry into the process of hemolysis main mass of red - registered phase own hemolysis. The period of plateau reflects the destruction of the most persistent erytrocyte is. The process ends by an average of 7 minutes with a total percentage of hemolysed cells (G, %) average 84,99±4,810%. Thus the estimated value of the constant maximum speed of hemolysis (Kmax) was 7,115±0,830 Rel. units (Fig.2).

HUMATE of leonardite at a dose of 10.0 mg/kg provided significant significant decrease in Kmax63.4%, indicating a decrease in the rate of hemolysis and the decrease in intensity of the process of destruction of cells. HUMATE of leonardite significantly reduced the percentage of collapsed cells during the observation period (Fig.2) 270 sec process revealed a reduction in the number of hemolyzed erythrocytes on 29,26%, 375 sec respectively 32,63% and by the end of the process of hemolysis on 735 s - a decrease in the total number of damaged cells in 20,45%, which was significantly lower compared with control. Analysis of the shape of the curve of hemolysis (Fig.2) clearly showed that despite the presence in the environment of HUMATE leonardite dynamics of the process of acid hemolysis of erythrocytes varies curve is shifted to the right and becomes more gentle, which should be interpreted as the rise time and the degree of stability of erythrocytes by srednestan and highly resistant populations to damaging action of acid hemolytica. This effect indicates an increase in resistance of memb the Academy of Sciences of cells to the action of damaging factors and linked to the ability of HUMATE of leonardite to induce conformational structural-functional changes of cell membranes, due to the interaction with lipoproteinemia components, resulting in reduced permeability to ions, providing increased osmotic resistance of the membrane.

Thus, for the first time revealed the ability of the HUMATE solution of leonardite at a dose of 10.0 mg/kg (with incubation at 37°C for 40 min) to normalize the permeability of cell membranes, as evidenced by the decrease in the rate of destruction of cells under the action of acid hemolytica and a decrease in the number of destroyed cells and indicates the presence of lipoic activity of HUMATE leonardite.

The use of a 5% aqueous solution of sodium and potassium salts of humic acids obtained from brown coal leonardite, at a dose of 10.0 mg/kg as lipoic funds.


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