RU2549449C2 - Method for preventing biological membrane damages - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: erythrocyte cell medium is added with an aqueous solution of sodium and potassium salts of humic acids prepared on brown coal of leonardite in a dose of 10.0 mg/kg. That is incubated at a temperature of 37°C for 40 minutes before treatment with acidic haemolytic.

EFFECT: invention enables normalising the cell membrane permeability and reducing the damaged cell count under acidic haemolytic.

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Description

The invention relates to the field of medicine and pharmacology, and in particular to methods of preventing damage to the biological membranes of red blood cells, and can be used to protect the biological membranes of red blood cells when exposed to chemical hemolytic agents.

It is known that damage to the biological membranes of cells is one of the universal pathogenetic mechanisms of various diseases (Vladimirov Yu.A. Role of violations of the properties of the lipid layer of membranes in the development of pathological processes // Pathologist. Physiology and Experimental Therapy. - 1989. - No. 4. - C . 7-19; Gennis R. Biomembranes. Molecular structure and functions: translation from English - 1st edition. - M.: Mir, 1997. - 624 p.). In this regard, the development of methods for restoring the functional properties of cell membranes may be pathogenetically substantiated in the strategy for the prevention and treatment of various diseases (Molecular disorders of the erythrocyte membrane in pathologies of different genesis are a typical reaction of the body: the contours of the problem / Novitsky V.V. [et al.] // Bulletin of Siberian medicine, 2006. - No. 2. - S. 62-69). It is known to use in the composition of pathogenetic therapy of diseases of medicinal substances having the ability to protect components of biological membranes of various cells from damage. For example, there is a method of treating purulent-inflammatory diseases of the maxillofacial region, including opening a purulent-inflammatory focus, medication, characterized in that intraoperatively or immediately after surgery and then daily, a membrane protector is administered intravenously to a patient once a day for 4-5 days - Rexod preparation in physiological saline, based on 0.2 μg / kg of patient’s body weight (application RU 2006110681; IPC A61K 38/43, A61P 43/00, 2007). Rexod is a superoxide dismutase enzyme that is an endogenous acceptor of free oxygen radicals and prevents oxidative modification of proteins, as well as destruction of cell biomembranes associated with the activation of lipid peroxidation (Vidal Handbook, http://www.vidal.ru/poisk_preparatov/rexod.htm )

Known hepatoprotective agent for the prevention and correction of toxic liver lesions, as described, hepatoprotector use 2-ethyl-6-methyl-3-hydroxypyridine succinate, known as mexidol. The course application of Mexidol has a membrane protective effect on liver cells that are toxicly affected by the prolonged use of tuberculostatics (Pat. RU 2189817; IPC A61K 31/4412, A61P 43/00, 2002).

A method of increasing resistance to hemolysis of red blood cells and restoring their damaged form is claimed (patent RU 2162698, IPC A61K 35/18; A61K 38/06, 2001). According to the description, a method of increasing resistance to erythrocyte hemolysis and restoring their damaged form consists in introducing hydrophilic natural antioxidants into the blood, and differs in that carnosine (β-alanyl-L-histidine) or its N-acetyl derivative in a certain range is used as antioxidants concentrations.

A method of increasing the resistance of erythrocytes to ethanol-induced hemolysis is claimed by introducing a halavit drug dissolved in physiological solution into a suspension of red blood cells in ethanol (patent RU 2315605; IPC A61K 35/18, A61P 25/32, 2008). Galavit (aminodihydrophthalazinedione sodium) is an immunomodulatory drug with anti-inflammatory action, the mechanism of action is associated with the ability to influence the functional and metabolic activity of macrophages (Vidal Handbook, http://www.vidal.ru/poisk_preparatov/galavit~22901.htm).

It is known that the combined use of low-intensity laser radiation and the complex compound of 5-hydroxy-6-methyluracil with succinic acid in experimental peritonitis increases the acid resistance of red blood cells to the hemolytic effect of hydrochloric acid, with this combination the most powerful membrane-protective effect is noted (Effect of antioxidant and laser therapy on the state of erythrocyte membranes in experimental peritonitis / Srubilin D.V. [et al.] // Medical Bulletin of Bashkortostan, 2009. - V. 4, No. 2. - S.102-106).

Close to the present invention (prototype) can be considered the use of lithium ascorbate as a agent with hematoprotective action, possessing hematoprotective activity against erythrocyte hemolysis under the influence of alcohol and having a lower toxicity compared to other lithium preparations (patent RU 2351326; IPC A61K 31/3755 , A61P 43/00, A61P 39/00, 2009). However, according to the description from this patent and according to well-known literature data, lithium ascorbate is a psychotropic drug that can be used mainly in narcology and psychiatry during chemotherapy.

One of the groups of compounds that are promising for the development of drug-based methods of protecting cellular biomembranes is natural compounds - humic substances and salts of humic acids (humates), which have bi-directional biological activity (Sukhikh A.S. Prospects for the use of humic and humic acids in medicine and pharmacy / A .S. Sukhikh, P.V. Kuznetsov // Medicine in the Kuzbass. - 2009. - No. 1. - S.10-14). Features of the chemical structure of humic substances determine their surface-active properties (On the mechanism of the antidote effect of humates in relation to petroleum products / A.V. Dagurov [et al.] // Bull. VSNS SB RAMS. - 2005. - No. 6 - P.143 -146; Stom DI The combined effect of petroleum products and "Humate" on daphnia / DI Stom, A.V. Dagurov // Siberian Journal of Ecology. - 2004. - No. 1. - S.35-40), which leads to the ability to solubilize and disperse hydrophobic organic substances, increasing their solubility and increasing the hydrophilicity of adhesive surfaces ok (Possible mechanisms of biological action of humic substances / Stom DI [et al.] // Siberian Journal of Medicine -. 2008. - №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.

The use of humic substances and natural substrates containing them is known in various branches of medicine.

In dermatology, it is known to use peat oxidate as an agent for treating patients with chronic dermatoses (patent RU 2187315; IPC A61K 35/10, A61P 37/04, 20020), use of an agent containing sodium humate from the wood-grass peat of the Tomnoe deposit of Tomsk area as an agent having antimicrobial and antifungal effects (patent RU 2340328; IPC A61K 9/06; A61K 35/10; A61P 31/10, 2008). It is known to use an extract from sapropel, obtained as the interaction of sapropel and potassium pyrophosphate in a mass ratio of 25: 1, exhibiting analgesic, anti-inflammatory and absorbable effects as a biologically active additive for cosmetic and therapeutic products (patent RU 2170094; IPC A61K 35/00, A61K 7/00, A61K 7/48, 2001). It is known to use humic acids and humates as a biologically active additive in the cosmetic industry with antioxidant activity (patent RU 2094042; IPC A61K 7/00, A61K 7/40, 1997). A means for hair and scalp care based on a humic concentrate is known, which is a product of treating peat with a solution of sodium hydroxide or potassium when activated with oxygen in the process of bubbling and subsequent neutralization with a solution of citric or acetic acid containing at least 2 humic acids on a completely dry substance , 8 wt.% (Patent RU 2394556; IPC A61K 8/97, A61Q 5/12, 2010).

The use of humates in rheumatology is known, for example, electrophoresis of a 0.09-0.15% sodium humate solution to increase the effectiveness of rehabilitation treatment of patients with osteoarthritis (patent RU 2196626; IPC A61N 7/00, 2003), a method of treating patients with osteoarthritis complicated by reactive synovitis , including a course of mud therapy with sapropel with a temperature of 35-37 ° C, magnetotherapy and exercise therapy (RU 96117924; IPC A61N 5/06, 1998).

It is known to use mud applications in neurological practice, for example, a method of reconstructive treatment of patients with moderate and low risk of recurrent stroke during the period of residual effects of acute cerebrovascular accident, including balneotherapy, manual massage of segmental zones, mud applications for paret limbs (using the sapropel of Lake Kirek on affected limbs with a temperature of 37-38 ° C for 20 minutes, every other day, the number of procedures 10) and physiotherapy exercises (patent RU 2009141660; IPC A 61H 1/00, 2011). There is a method of treating patients with persistent neurological manifestations of lumbar osteochondrosis, achieved by applying mud applications to the lumbosacral region and affected limb at 40-42 ° C for 30 minutes, after which hypobarotherapy is additionally carried out (SU 1711883; IPC A61G 10/02, 1992).

The use of various substrates containing humic substances in gastroenterology is known. For example, the antiulcer effect of mud applications and electrophoresis of peat mud is known (Effect of mud applications on the state of acetate ulcers in white rats / NN Abushinova [et al.] // Issues of balneology, physiotherapy and physiotherapy. - 2001. - No. 3. - P.27-28; The experience of treating patients with peptic ulcer of the duodenum with SMT electrophoresis of peat mud in a day hospital / Korepanov AM [et al.] // Issues of balneology, physiotherapy and physiotherapy. - 2003. - No. 5. - S.22-25); use in case of gastric ulcer of the electrophoresis of the medicinal product humisol, which is a biogenic stimulant and represents a 0.01% solution of the fractions of humic acids of the Haapsalu marine therapeutic mud of the Baltic Sea in an isotonic sodium chloride solution (discontinued in 1997) (Lurie G.V. The effectiveness of various methods of electrophoresis of humisole from dimexide in gastric ulcer / G.V. Lurie // Modern aspects of resort gastroenterology: collection of scientific papers - Pyatigorsk, 1988. - P. 46-49). It is known that by using inside the extract from sludge therapeutic mud (the drug Peloidin, contains a complex of natural biologically active substances) for the treatment of peptic ulcer of the stomach and duodenum, the drug belongs to the group of biogenic stimulants (Mashkovsky M.D. Medicines: in 2 t. - T.1. - 14th ed., Revised., Revised and supplemented. - M .: New Wave Publishing House LLC: Publisher SB Divov, 2002. - P.174). A known method of treating gastric ulcer and duodenal ulcer, characterized in that they use a drug obtained by treating peat containing a complex of biologically active substances, including humic acids. According to the description, the oral administration of the above peat preparation once a day daily intragastrically in a single dose of 50-100 mg / kg provides a complex effect on various pathogenetic links in the development of gastric ulcer, showing both local and general healing effect (patent RU 2357741; IPC A61K 35/10; A61P 1/04, 2009).

A known method of treating cancer in the experiment, including chemotherapy of tumors with cyclophosphamide and the introduction of peat products containing humic acids, fulvic acids, etc., moreover, the peat product is introduced into the feed or drinking water based on a daily dose of 100-200 mg / kg (RU patent 2396967; IPC A61K 35/10; A61P 35/00, 2010). It is known to use suppositories for the treatment of chronic prostatitis, consisting of 2.0 ml of a 1% aqueous humic acid solution (pH 7.4) of low mineralized peloids and a sufficient amount of cocoa butter to obtain a suppository weighing 2.0-2.2 g (RU patent 2241471; IPC A61K 35/10, A61K 35/02, A61K 9/02, A61P 13/08, 2004).

The use of humates from peat and sapropels in veterinary medicine is also known, for example, a method for the prevention of liver diseases in cows, which includes introducing into the diet a humic agent from peat, used in the form of a solution with 1.0% humic acid content once a day at a dose of 0.125- 0.25 ml / kg body weight in two courses lasting 25-28 days (patent RU 2372926; IPC A61K 35/10, 2009).

However, the known methods of protecting the biological membranes of mammals and humans with the help of salts of humic acids, in particular salts of humic acids obtained from leonardite, were not previously reported. The objective of the present invention is to expand the arsenal of known methods for protecting biological membranes of red blood cells under damaging effects by chemical hemolytic agents.

The technical result consists in the use of a drug based on low-toxic, cheap, affordable natural raw materials for the prevention of damage to biological membranes of red blood cells.

The technical result is achieved by the fact that a method of preventing damage by chemical hemolytic agents of the biological membranes of red blood cells consists in using an aqueous solution of sodium and potassium salts of humic acids obtained from brown coal leonardite, introduced at a dose of 10.0 mg / kg in a medium with red blood cells and subsequent incubation at a temperature of 37 ° C for 40 minutes before the damaging effects of acid hemolytic. As an acid hemolytic, for example, 0.1 Η hydrochloric acid solution is used. The method provides a decrease in the rate of cell destruction under the action of acid hemolytic and a decrease in the number of destroyed cells.

The conventional name for an aqueous solution of sodium and potassium salts of humic acids is Leonardite humate. The powder substance is produced by Gumat LLC, Irkutsk, product name and designation: Gumat is a fertilizer based on humic acids, for agricultural use,

TU 2189-004-41764643-98, pharmacological properties have not been studied previously, membrane-protective properties are not known.

The advantages of the proposed method for the prevention of damage to biological membranes using an aqueous solution of sodium and potassium salts of humic acids obtained from brown coal of Leonardite are the relative cheapness of the substance, widely available raw materials in large volumes for environmentally safe production of the substance, the possibility of industrial production (Perminova I.V. Humic substances - a challenge to chemists of the 21st century / IV Perminova // Chemistry and Life, 2008. - No. 1. - P. 50-55, 10), low toxicity (Low toxicity and anti-fire the suppressive activity of humates isolated from peat and sapropel of the Tomsk region / R.R. Ismatova, A.U. Ziganshin, S.E. Dmitruk, I.V. Fedko // Kazan Medical Journal. - 2006. - Volume 87, No. 6 . - S. 454-455) and the absence of serious side effects, high efficiency.

In FIG. 1 shows the results of the influence of a solution of humate leonardite (10 mg / kg, incubation for 40 min at 37 ° C) on the indicators of acid hemolysis of red blood cells.

Example 1

To prevent damage to the biological membranes of erythrocytes when exposed to chemical hemolytic agents, a agent from natural raw materials was used, which is an aqueous solution of sodium and potassium salts of humic acids obtained from brown coal of Leonardite (hereinafter referred to as Leonardite humate).

To study the ability of Leonardite humate to change the structural and functional properties of biological membranes, we used a modified method for recording chemical (acid) erythrograms according to I. Terskov and I. I. Gitelzon. (1957) (I. Gitelzon, erythrograms as a method of clinical blood testing. / II Gitelzon, I. Terskov. - Krasnoyarsk: Publishing House of the Siberian Branch of the USSR Academy of Sciences, 1959. - 247 p .; Terskov I .A. Method of chemical (acid) erythrograms // I.A. Terskov, I.I. Gitelzon // Biophysics. - 1957. - V. 11, Issue 2. - P. 259-266; Workshop on Biophysics / V.G. Artyukhov [et al.] / Edited by V.G. Artyukhov. - Voronezh: Publishing House of Voronezh State University, 2001. - P. 147-161). It is proved that red blood cells are involved in the pathological process not only with hematological diseases, but also undergo serious changes in the structure and function in diseases of different genesis. The choice of the erythrocyte membrane as an object for testing is dictated by the fact that it has the general principles of molecular organization 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 red blood cell makes it possible to study the functional properties of the plasma membrane without interference

intracellular membrane formations and organelles (Molecular disorders of the erythrocyte membrane in pathologies of different genesis are a typical reaction of the body: the contours of the problem / Novitsky VV [et al.] // Bulletin of Siberian medicine, 2006. - No. 2. - P. 62-69 ; Postnov, Yu.V. Primary hypertension as a pathology of cell membranes / Yu.V. Postnov, S.N. Orlov. - M .: Medicine, 1987. - 192 p.).

The ability of Leonardite humate to provide protection of biomembranes under damaging effects was assessed by changing the parameters of the chemical resistance of erythrocyte cells to an acid hemolysis inducer, experiments were carried out on a 3-component model system, including: 1 - “modifier” - a solution of humic acid salts derived from Leonardite (humate leonarditis); 2 - “membrane” - suspension of rat erythrocyte membranes; 3 - "destructor" - a component that causes acid hemolysis of red blood cells, i.e. 0.1 H hydrochloric acid solution.

The effect of Leonardite humate on the change in the resistance of cell membranes was evaluated using the method of automatic registration of acid erythrograms based on the photometric registration of the hemolysis process in time; the method was developed at the Department of Biophysics and Biotechnology, Voronezh State University. The principle of the methodology used is based on the method of recording acid erythrograms proposed by I.I. Gitelzon and N.A. Terskov (Gitelzon I.I. Erythrograms as a method of clinical blood testing. / II Gitelzon, I.A. Terskov. - Krasnoyarsk: Publishing House of the Siberian Branch of the USSR Academy of Sciences, 1959. - 247 p .; Terskov I.A. The method of chemical (acid) erythrograms // I.A. Terskov, I.I. Gitelzon // Biophysics. - 1957. - T.11, Issue 2. - P.259-266; Workshop on Biophysics / V.G. Artyukhov [et al.]. / Under the editorship of VG Artyukhov. - Voronezh: Publishing House of Voronezh State University, 2001. - P.147-161).

The principle of the method for recording the acid resistance of red blood cells is photometric registration of the dynamics of cell breakdown when 100 μl of 0.1 N hydrochloric acid solution is added to the erythrocyte suspension (acid hemolysis method). A measure of resistance for each red blood cell is the time during which its destruction occurs. The data obtained make it possible to construct an erythrogram - a kinetic curve that reflects over time the sequential entry of red blood cells of different resistance into the hemolysis phase.

Analytically, this process can be represented by an equation of the form:

G (%) = f (t),

where G (%) is the number of destroyed red blood cells in percent, t is the time, expressed in seconds.

Red blood cells for research were obtained from the blood of white outbred rats of males with a total of 30 animals weighing 210.0 ± 9.5 g. Blood was taken by resection of the distal part of the tail, in each series of experiments, manipulation was performed at the same time between 8.30 and 9.30 hours. Blood was collected by gravity into a tube with a stabilizer (heparin). In order to obtain a suspension of red blood cells, heparin stabilized blood was centrifuged in a laboratory medical centrifuge (model OPN-8, manufactured by TNK Dastan OJSC, Kyrgyz Republic) for 15 minutes at a speed of 3000 rpm with intermediate washing from a stabilizer and plasma 0 9% sodium chloride solution. For this, a 0.9% NaCl solution was added to the erythrocyte sediment, carefully mixed and centrifuged again under the same conditions, the procedure was repeated 3 times.

The erythrocyte hemolysis was performed in cuvettes with external dimensions of 20 × 40 × 10 mm and a working volume of 5.0 ml. The light scattering was measured with a light filter (No. 5) with a maximum transmission in the region of 490 nm (at a wavelength of λ = 490 nm), because at a given wavelength, it is not the hemoglobin release to the incubation medium that is tested, but the light scattering of the samples, the intensity of which is directly proportional to the volume, that is, the surface of the erythrocyte and the content of erythrocyte cells in the medium. In this way:

G = f (V _i × N),

where G (%) is the number of destroyed red blood cells in percent, V _i is the volume of one red blood cell, N is the number of red blood cells in the medium. Therefore, the hemoglobin yield is a secondary phenomenon and, at the used wavelength (490 nm), practically does not affect the recorded signal.

Photometric registration of the dynamics of the process of induced hemolysis of erythrocytes was carried out using a spectrophotometer (model PE-5400 VI, manufactured by Ecros Group of Companies, Russia).

Acid hemolysis was induced by adding 100 μl of a 0.1 N hydrochloric acid solution to a 5 ml suspension of erythrocytes in a working cuvette (in an isosmotic 0.9% NaCl solution). The choice of this substance as an acid hemolytic is due to the stability of the solution during storage and the presence of both ions (H ⁺ and Cl ^- ) in the blood plasma. Indicators were recorded every 15 seconds for 735 seconds.

The dynamics of the process of erythrocyte hemolysis was recorded immediately after the addition of hemolytic (0.1 M HCl) to the erythrocyte suspension (0.9% NaCl).

Digital data were displayed on a graph; as a result, an S-shaped integral kinetic curve (erythrogram) was recorded, the shape of which reflects the total change in the light scattering (τ) in the test solution over time, i.e., τ = f (t). In the process of hemolysis, the decay rate of red blood cells reaches a maximum value (V _max ) approximately in the middle of the curve.

The analysis of erythrograms was carried out graphically and mathematically, indicators of the structural state of erythrocyte membranes (Gennis R. Biomembranes. Molecular structure and functions: translation from English. - 1st edition. - M .: Mir, 1997. - 624 p.) During hemolysis was evaluated according to the calculated parameters in accordance with the methodology (Workshop on Biophysics / VG Artyukhov [et al.]. / Edited by VG Artyukhov. - Voronezh: Publishing House of the Voronezh State University, 2001. - P.147-161) .

The constant hemolysis rate constant (K _max rel. Units) was calculated - a parameter characterizing the proportion of red blood cells that entered the hemolysis stage at the same time was determined by the formula (1):

$$K_{max}=tg\alpha ,(\mathrm{one})$$

where K _max (rel. units) is a constant of the maximum hemolysis rate; tan α is the tangent of angle α; α is the angle of inclination of the linear part of the curve to the abscissa axis, was determined graphically.

The relative amount of hemolyzed cells (G,%) was calculated at various points in time of observation according to the formula (2):

$$G_n=K\times \left(T_{\mathrm{about}P.\mathrm{from}Redn}-T_0{}_{\mathrm{from}Redn}\right),(2)$$

where G _n - the number of red blood cells involved in the hemolysis process for n second of the process, expressed as a percentage, was determined every 5 seconds for 735 seconds; K is the hemolysis rate constant per t second of the process, calculated by the formula (3); T _{od Avg} - the average value of light scattering in the experimental sample for n second process; T _{0 avg.} - the average value in the experimental sample is initial, i.e. for 0 sec.

$$K=\frac{\mathrm{one}00}{\mathrm{one}00-T_{0\mathrm{from}Redn\mathrm{.,}}}(3)$$

The relative number of spherocytes (G _sf ,%) was determined - an indicator reflecting the number of red blood cells (mainly a low-resistance population) involved in the initial stage of the hemolysis process - the stage of spherocytosis. The maximum number of hemolized red blood cells in percent (G _max ,%) was determined.

The time of the latent period of hemolysis (t _lat , sec) was determined — the period of time after the addition of the hemolytic agent and before the start of registration of the hemolysis phase.

To assess the ability of Leonardite humate to protect biomembranes under the damaging effects of acid hemolytic, an aqueous solution of Leonardite humate was used at a dose of 10.0 mg / kg, which was added directly to the test cell with erythrocytes, then a mixture of a suspension of red blood cells and Leonardite humate was subjected to preliminary incubation in a thermostat at a temperature 37 ° C for 40 min, and then the actual acid hemolysis was induced by adding 100 μl of 0.1 H hydrochloric acid solution to the medium with red blood cells and Leonardite humate you. The incubation temperature of 37 ° C was chosen as corresponding to the human body temperature, the incubation time of 40 min was chosen as the average time required for the drug to enter the blood plasma. The dose of Leonardite humate 10.0 mg / kg was selected experimentally based on the results of a series of preliminary experiments with doses of 1.0, 5.0, 10.0 and 20.0 mg / kg.

The experiments for a dose of 10.0 mg / kg leonardite humate were carried out in 3 series of experiments, the results were subjected to statistical processing with the calculation of average values. In each of the series of experiments, studies were carried out in parallel samples, one of which was a control (red blood cells + hemolytic), and the second experimental (red blood cells + Leonardite humate solution + hemolytic).

It was found that the hemolysis process in the control sample corresponds to the parameters of the standard norm and the hemolysis curve has an S-shape. The process ends on average at 7 minutes with a total percentage of hemolyzed cells of 84.99 ± 4.810%. In this case, on average, the latent period of hemolysis (t _lat ) was 5.00 ± 1,000 sec, the relative number of spherocytes (G _sf ) 0.32 ± 0.800% and the maximum hemolysis rate constant (K _max ) was 7.115 ± 0.830 rel. units (Figure 1).

It was established that when Leonardite humate was introduced into the medium with red blood cells at a dose of 10.0 mg / kg with preliminary incubation for 40 minutes at a temperature of 37.0 ° C, there was no change in t _lat , a slight increase in the number of spherocytes G _sf by 0 , 37%. A significant significant decrease in K _max by 63.4% was revealed, which indicates a decrease in the rate of hemolysis and a decrease in the intensity of the process of cell destruction. During the observation period, the ability of Leonardite humate to significantly reduce the percentage of destroyed cells was revealed, for example, a decrease in the number of hemolyzed red blood cells by 29.26%, by 375 sec, by 32.63%, respectively, by the time of the end of the hemolysis process by 735 sec, was revealed by 270 sec. - a decrease in the total number of destroyed cells by 20.45%, which was significantly less in comparison with the control (Figure 1).

Therefore, against the background of the presence of leonarditis in the humate medium, an increase in the time and degree of resistance of red blood cells to the damaging effect of acid hemolytic was revealed. The revealed changes are associated with the ability of Leonardite humate to cause conformational structural and functional changes in cell membranes due to interaction with lipoprotein components, which leads to a decrease in permeability for hydrogen ions and provides an increase in the osmotic resistance of the cell membrane.

Thus, for the first time, the ability of a solution of humate leonardite at a dose of 10.0 mg / kg was revealed upon preliminary incubation with a suspension of red blood cells at a temperature of 37 ° C for 40 min to normalize the permeability of cell membranes, which is manifested by a decrease in the rate of destruction of cells under the influence of acid hemolytic and a decrease in the number collapsed cells. The established changes indicate the ability of Leonardite humate at a dose of 10.0 mg / kg to protect the biological membranes of red blood cells, which can be used to prevent the violation of the integrity of the biological membranes of red blood cells under the influence of chemical hemolytic agents.

Claims (1)

A method for the prevention of damage by chemical hemolytic agents of biological membranes of red blood cells, which consists in using an aqueous solution of sodium and potassium salts of humic acids obtained from brown coal of Leonardite, administered at a dose of 10.0 mg / kg in a medium with red blood cells and subsequent incubation at a temperature of 37 ° C within 40 minutes before the damaging effects of acid hemolytic.

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