RU2756363C1 - Anti-inflammatory humic agent - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to the field of pharmaceuticals, namely to a humic agent exhibiting anti-inflammatory activity and an application thereof for treatment and/or prevention of inflammatory diseases. The humic agent exhibiting anti-inflammatory activity is produced from leonardite, lignin, coal, peat, sapropel, by means of ultrasonic dispersion of the pre-ground raw materials in a mixture with water at a certain temperature and a certain pressure, followed by cooling the solution to room temperature and diluting with water until reaching a content of humic substances of 1 to 20% wt., wherein the humic substances include humic and fulvic acids and salts thereof, as well as hydroquinone in an amount not exceeding 3% wt. of the weight of the humic substances.

EFFECT: group of inventions ensures production of a humic agent exhibiting anti-inflammatory activity, containing a wide range of humic substances and containing no chemical impurities, usable in the field of medicine and pharmaceuticals.

2 cl, 2 tbl, 2 ex

Description

The invention relates to the field of medicine and pharmaceutics, namely to humic preparations, and can be used in the complex therapy of inflammatory diseases.

Humic acids, fulvic acids have a powerful effect on any living organism due to their rich composition. They contain a full set of amino acids, macro- and microelements, minerals, as well as naturally occurring polysaccharides, vitamins, peptides, fatty acids, polyphenols, ketones, catechins, etc. There are about 70 useful components in total. This rich composition explains the positive biological effects of humic acid (see https://fb.ru/article/288472/guminovyie-kislotyi-chto-eto-takoe-i-kak-oni-vliyayut-na-organizm).

It is known that humic acids are able to stimulate some functions of human neutrophils. Antiviral activity has been revealed for preparations based on humic and humic-like substances, for example, for the herpes simplex virus - HSV. Humic compounds can be used as microbiocides, prophylactic agents against the spread of HIV / AIDS. The cytotoxic and antiviral properties of humic acids and fulvic acids isolated from coal and peat were studied. Studies have shown that all studied compounds were low-toxic and had a sufficiently high inhibitory effect against HIV infection (AI POPOV, VN ZELENKOV, TV TEPLYAKOVA Biological activity and biochemistry of humic substances. Part 2. Medico- biological aspect (literature review) Bulletin of the Russian Academy of Sciences, 2016/5, pp. 9-15). It has been shown that Oxygumate increases the activity of Th-1 cells and reduces the production of cytokines by Th-2 cells [Mariette et al., 2002]. The observed stimulation of the proliferation of human lymphocytes was associated with an increase in the production of IL-2 and the expression of IL-2 receptors, together with a decrease in the amount of IL-10 under the action of Oxygumate [Joone et al., 2003]. In vivo, it has been shown that oral administration of humic substances improves the parameters of innate immunity in experimental animals: there is an increase in the antibacterial activity of blood serum, phagocytic activity, lysozyme activity and bacterial agglutination [Sanmiguel et al., 2016]. This makes humic substances potential products for the development of multi-target immunoactive microbicides.

Research in the development of new biologically active compounds has shown that humic substances of various origins have immunomodulatory and anti-inflammatory effects, which makes it possible to use them for the prevention and treatment of chronic dermatoses, atopic dermatitis, allergic rhinitis and other diseases accompanied by inflammatory and allergic reactions. The use of humic substances as antifungal and antiviral agents is promising. The anti-inflammatory activity of humic acids has been studied in models of acute and chronic inflammation. A possible mechanism of anti-inflammatory action is explained by the ability of humic acids to reduce the generation of oxygen radicals and reduce oxygen consumption by activated phagocytes (IA Savchenko et al. BIOLOGICAL ACTIVITY OF HUMIC SUBSTANCES: PROSPECTS AND PROBLEMS OF THEIR APPLICATION IN MEDICINE No. ) April, 2019, pp. 54-60, DOI: http://dx.doi.org/10.21145/2225-0026-2019-1-54-60).

The anti-inflammatory effect of humic compounds is associated with their ability to reversibly inhibit the excessive synthesis of interleukin-1β by hyperactivated macrophages, level the increased release of neutrophilic granulocytes from the bone marrow depot into the blood, reduce oxygen consumption by activated phagocytes with a subsequent decrease in the generation of oxygen radicals, which ultimately leads to a decrease reactions (Poluyanova I.E. Biological activity of humic substances obtained from peat, and the possibility of their use in medical practice. International reviews: clinical practice and health, No. 4, 2017, pp. 114-122).

It is known from the prior art to use water-soluble humic acids isolated from high-moor peat magellanicum, with a number average molecular weight of 4871.2 Da, a weight average molecular weight of 18755.3, a polydispersity of 3.9, and a median of 9558.3 Da, as a means of stimulating the development of Th1 reactions -dependent type of immune response by regulating the balance of activation of the pro-inflammatory properties of macrophages secreting the cytokines IL-1β, IL-12 and TNF-α, and the anti-inflammatory properties of macrophages that produce IL-10 cytokines (patent RU 2716504, 03/12/2020). The disadvantage of the known solution is that it contains only a narrow fraction of humic acids and does not contain other active components of humic substances.

The objective of the present invention is to obtain a humic agent containing a wide range of humic substances and having anti-inflammatory activity, without the use of chemical reagents.

The technical result of the claimed invention is to obtain an anti-inflammatory humic agent containing a wide range of humic substances and not having chemical impurities, which can be used in the field of medicine and pharmaceuticals.

The specified technical result is achieved due to the fact that the claimed humic agent having anti-inflammatory activity contains water and humic substances obtained from humic raw materials, a selected group, including leonardite, lignin, coal, peat and / or sapropel, by ultrasonic dispersion at a temperature of 30 -80 ° C and a pressure of 0.05-0.8 MPa, while the mass of humic substances is from 1 to 20 wt. %, and humic substances include humic and fulvic acids and their salts, as well as hydroquinone in an amount not exceeding 3 wt. % of the mass of humic substances.

Also proposed is the use of the obtained humic agent for the treatment and / or prevention of inflammatory diseases.

To obtain the claimed agent, pre-crushed raw materials (leonardite, lignin, coal, peat, sapropel) were used mixed with water, which was placed in an ultrasonic unit. Placed in the ultrasonic unit, a mixture of humic raw materials with water was heated to 30-80 ° C, and when the required temperature was reached, ultrasonic treatment was performed at a pressure of 0.05-0.8 MPa. After sonication, the solution was cooled to room temperature. The resulting product was diluted with water to the content of humic substances ranging from 1 to 20 wt. %.

The study of the composition of the obtained product was carried out by the GC-MS method on an analyzer "Chromatek", consisting of a gas chromatograph "Chromatek-Crystal 5000" and a liquid dispenser DAZH-2M. For the identification of derivatives, an automatic database for the search and identification of gas chromatography-mass spectrometry data NIST17MS Library was used.

Research conditions:

The results of the study are shown in table. 1

According to the above data, in addition to humic and fulvic acids, the composition of the claimed agent also includes phenolic derivatives, in particular hydroquinone, flavanoids (chrysin) and other active substances. Therefore, the claimed agent is characterized by a wide range of active substances.

Toxicity study.

Determination of indicators of acute toxicity included experiments on mice. Animals were randomly assigned to groups by randomization. The criteria for the acceptability of randomization were the absence of external signs of diseases and the homogeneity of groups by body weight (± 10%). The drug was administered intragastrically in increasing doses according to Litchfield-Wilcoxon. The highest dose was limited to the maximum possible volume of drug administration. To study each dose of the drug, groups of 10 animals of different sex were used. The observation period was 14 days. With the introduction of the drug in doses of 4000-8000 mg / kg (for humic acid), the animals showed a change in the response to picking up, changes in respiration, motor activity, and muscle tone; in some animals, a change in the consistency of feces was observed. When the drug was administered at a dose of up to 4000 mg / kg (for humic acid), all animals survived, when the drug was administered at a concentration of 6000 mg / kg (for humic acid), 5 animals out of 10 died, and when the drug was administered at a dose of 8000 mg / kg ( for humic acid), 8 animals out of 10 died. The surviving animals tolerated intoxication satisfactorily and after the end of the drug action for 14 days of observation, no signs of delayed influence were noted. There were no signs of prolonged clinical intoxication. The dynamics of the body weight of the experimental animals did not differ from the control. At the end of the observation period - on day 14, the surviving animals were slaughtered in order to determine possible pathological changes after a single dose of the drug. Examination of the experimental and control groups showed that all the animals in them were normally well-fed, had the correct constitution, smooth and shiny hair, shiny mucous membranes of normal color, clean and tidy natural openings. Macroscopic examination of the internal organs did not reveal any peculiarities. The analysis of the values of the mass coefficients did not reveal any significant differences between the groups of animals that received different doses of the drug. Thus, the results obtained allow us to assume that the obtained drug can be attributed to the V class, i.e. practically non-toxic medicinal substances.

Study of the anti-inflammatory activity of the claimed agent.

The studies were carried out on male white rats with an average body weight of 320.0 ± 5.0 g. To simulate exudative inflammation, subplantar injection into one of the hind limbs of 3.0% formalin aqueous solution in a volume of OD ml was carried out. The claimed humic agent (hereinafter referred to as HS) investigated for anti-inflammatory activity was administered 1 hour before the administration of formalin in the form of a 0.1% aqueous solution at a dose of 1.0 mg / kg orally once.

The criteria for assessing the anti-inflammatory properties of the claimed HS were:

1 - the presence of antiexudative activity;

2 - the presence of antipyretic activity - assessed by the ability to prevent an increase in body temperature; and

3 - the ability to prevent a shift of the leukocyte formula to the left.

To assess the anti-inflammatory activity, 1 and 3 hours after formalin administration, the volume of the limb was measured by the oncometric method, the volume of the limb was expressed in cm ³ . To determine the presence of antipyretic activity, rectal body temperature was measured using an electronic thermometer. To confirm the presence of anti-inflammatory activity, blood smears were prepared to calculate the leukocyte formula.

Efficiency was assessed by comparing the absolute and relative data in the experimental and control groups between the initial and subsequent indicators. Mathematical processing of the results was carried out in accordance with the generally accepted methods of biomedical statistics with the determination of the reliability of changes using the Student's t-test.

As a result of the research, the following was determined. Initially, the volume of a healthy limb for the control and experimental groups was 1.92 ± 0.03 cm3. The introduction of a formalin solution in the control group was accompanied by a significant increase in the volume of the limb, which indicates the development of a local exudative inflammatory process - after 1 hour by 19.6% and after 3 hours by 12.0% in relation to the initial values of the volume for the control group. Against the background of the introduction of the declared HS, a decrease in edema was noted after 1 hour by 12.2% and after 3 hours by 24.7%.

The average initial body temperature corresponded to the normal values for healthy animals and averaged 36.6 ± 0.22 ° C. The hyperthermic inflammatory reaction in the control group of animals was detected 24 hours after the injection of the formalin solution and was characterized by a significant increase in rectal temperature by 3.6% (37.5 ± 0.62 ° C) in relation to the initial values. The use of GS according to the invention at a dose of 1.0 mg / kg contributed to a decrease in body temperature after 3 hours by 5.1% and after 24 hours by 8.4% in relation to the control.

The composition of the leukocyte formula in healthy animals was represented by the following ratio: young neutrophils 0.10 ± 0.08%, stab neutrophils 2.40 ± 0.45%, segmented neutrophils 21.7 ± 2.07%, eosinophils 0.15 ± 0 , 10%, basophils 1.30 ± 0.17%, monocytes 3.60 ± 0.14%, and lymphocytes 67.0 ± 3.9%. These indicators correspond to the values of the average species-age norm for healthy adult rats.

In the control group, there was a significant increase in the number of neutrophils in relation to healthy animals - segmented by 6.90% (significant, P <0.05), young by 1.53% and stab by 1.64% (P <0.05) with a decrease in the percentage of lymphocytes by 9.4% (P <0.01), which indicates the presence of neutrophilic leukocytosis with a shift of the leukocyte formula to the left and indicates a systemic reaction of the lymphoid system of the blood to the inflammatory process.

Against the background of the use of the HS of the invention, a relative decrease in the number of young and stab neutrophils by 0.8% and 0.92%, segmented neutrophils by 8.2% (P <0.01) was observed. This result indirectly confirms the ability of the HS to reduce the severity of the inflammatory process.

As a result of the studies, it was found that the claimed HS, when administered at a dose of 1.0 mg / kg, has anti-inflammatory activity in local exudative inflammation. The antipyretic activity of GS is confirmed by the ability to reduce subfebrile temperature, which is a systemic manifestation of the inflammatory process. The antiexudative activity of GS is manifested by a decrease in the severity of the limb edema induced by formalin solution. The anti-inflammatory properties of GS are objectively confirmed by the ability to prevent the shift of the leukocyte formula to the left, i.e. to reduce the migration of immature forms of leukocytes to the focus of inflammation.

Thus, the claimed humic agent has a high biological activity, which is manifested in the reduction of excessive inflammatory reactions. Therefore, the claimed agent can be used as an anti-inflammatory agent for the prevention and treatment of inflammatory diseases, which confirms the achievement of the claimed technical result.

Claims (2)

1. A humic agent with anti-inflammatory activity, containing water and humic substances obtained from humic raw materials, selected from the group including leonardite, lignin, coal, peat and / or sapropel, by the method of ultrasonic dispersion of pre-crushed raw materials in a mixture with water at a temperature of 30 -80 ° C and a pressure of 0.05-0.8 MPa, after which the solution is cooled to room temperature and diluted with water until the content of humic substances is from 1 to 20 wt. %, while humic substances include humic and fulvic acids and their salts, as well as hydroquinone in an amount not exceeding 3 wt. % of the mass of humic substances. 2. The use of a humic agent according to claim 1 for the treatment and / or prevention of inflammatory diseases.