RU2716504C1 - Humic substance having immunomodulatory activity - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, namely to pharmacology. Disclosed is the use of water-soluble humic acids recovered from highmoor peat Magellanicum with average molecular weight 4,871.2 Da, weight-average molecular weight 18,755.3, polydispersity 3.9 and median 9,558.3 Da, as an agent for stimulating development of Th1-dependent immune response by controlling the balance of activation of pro-inflammatory properties of macrophages secreting IL-1β, IL-12 and TNF-α cytokines, and anti-inflammatory properties of macrophages, which produce IL-10 cytokines.EFFECT: invention provides an immunomodulatory agent of natural origin, having low toxicity and ability of selective stimulation of production by macrophages of interleukin-1 beta, interleukin-12, tumor necrosis factor-alpha and lymphocytes of interferon-gamma.1 cl, 2 dwg, 8 tbl, 2 ex

Description

The invention relates to medicine, specifically to pharmacology, the results of which can be used to correct disorders in the immune system in pathological conditions associated with a deficiency of the Th1-dependent immune response (chronic, sluggish and recurring infectious and oncological diseases).

Peloid therapy (mud therapy with natural organomineral colloidal formations (silt, peat, hill), which have high plasticity, heat capacity and heat transfer, containing active biological substances (salts, gases, vitamins, enzymes, hormones, etc.) and living microorganisms) is one of the most ancient methods of treating chronic diseases, as mentioned in the treatises of scientists of ancient China, Egypt, Greece, India, dated as early as 4-5 century BC In the 2nd century, the writings of Claudius Galen contained recommendations on the use of sludge for the treatment of inflammation, swelling and pain, in the 15th century Paracelsus pointed to 5 main effects of mud on the body systems. Peloid therapy today is widely used to treat diseases of the skin, musculoskeletal system, endocrine and genitourinary system, adhesions, rheumatoid arthritis [1, 2, 3].

A study of the composition of natural high-molecular biologically active compounds (peat, sapropel, mummy, coal) formed as a result of the decay and humification of biological objects showed that their organic part consists of humic substances, lignin, polysaccharides, lipids, pectins, hemicellulose and cellulose. Humic substances - a group of natural biopolymers that are mainly found in plant objects (caustobiolites) - peat, brown coal, bottom sediments (sapropels, peloids), consist of 80-90% of humic and fulvic acids. The biological properties of humic acids (HA) have been studied quite extensively, it has been shown that humic and fulvic acids are used to increase the resistance of the human body to the action of various adverse factors, including as adjuvant therapy in HIV-infected patients [4]. Humic substances suppress a delayed-type hypersensitivity reaction, a transplant versus a host reaction, reduce contact hypersensitivity and swelling of rat paws, the level of C-reactive protein in patients with osteoarthritis, hay fever, and also have cardioprotective and pro-angiogenic properties [5]. It has been proven that peat HA show a pronounced pro-inflammatory activity, stimulating the proliferation of macrophages, neutrophils and lymphocytes (T-killers) [6, 7] and the secretion of IFN-α and -γ, TNF-α in the culture of human lymphocytes and IFN-β and TNF-α α peritoneal macrophages of mice [8, 9]. In Poland, an immunomodulator with interferonogenic effect and inducing tumor necrosis factor is produced on the basis of HA [10]. In this case, HA do not show toxic effects in a wide range of doses in experimental animals after oral or cutaneous use, and potassium humate is safe for humans in a daily dose of up to 1 g / kg [5, 11].

It is known that an effective immune response depends on the coordinated functioning of various cells of innate and / or adaptive immunity, in which macrophages, as antigen-presenting cells, occupy a special place [12]. The macrophage population is characterized by significant functional and phenotypic heterogeneity and plasticity, which are regulated by the surrounding microenvironment. Macrophages are most often represented by two different types: 1) pro-inflammatory - classically activated macrophages (M1), which are polarized by lipopolysaccharide (LPS) or in combination with Th1-cytokines, such as interferon gamma (IFN-γ), granulocyte-macrophage colony stimulating factor and respectively produce pro-inflammatory cytokines; and 2) anti-inflammatory and immunoregulatory - alternatively activated macrophages (M2) polarized with Th2 cytokines (IL-4 and IL-13) produce anti-inflammatory cytokines. Macrophages M1 and M2 have opposite functions and transcriptional profiles, have unique abilities, destroying pathogenic microorganisms - M1 or repairing an injury associated with inflammation - M2. The polarization of the balance of macrophages M1 / M2 determines the direction of the immune response in case of inflammation or trauma. In case of massive infection or inflammation, macrophages polarize into the M1 phenotype, secrete interleukin-β (IL-1β), IL-6, IL-12, IL-23 and tumor necrosis factor alpha (TNF-α), activating antimicrobial protection. However, delaying the M1 phase, which can lead to depletion and pathological changes in tissues and organs, causes an increase in the activity of M2, which secrete large amounts of IL-10 and transforming growth factor beta (TGF-β) to suppress inflammation and promote tissue repair, remodeling, vasculogenesis and preservation homeostasis [12]. Thus, substances capable of suppressing excessive activation of both M1 and M2, regulating the balance of M1 (IL-12 and TNF-α) and M2 (IL-10) mediated cytokines are of significant interest as pharmacological regulators for the restoration of body homeostasis in various pathologies associated with insufficiency of the Th1-dependent immune response (chronic, sluggish and recurrent infectious, as well as cancer). [13, 14].

In previous studies, we have shown that HAs isolated from sodium pyrophosphate from peat Magellanicum peat, taken from a depth of 100-120 cm from the ridge-urea complex of the oligotrophic bog of the Bakcharsky bog massif of the southeastern spurs of the Vasyugan bog in the Iks and Bakchar interfluves, enhance oxide production nitrogen peritoneal macrophages of mice do not contain endotoxin impurities, inhibit arginase activity, which suggests the presence of their potential immunomodulating activity [15].

The objective of the invention is to expand the arsenal of immunomodulating agents of natural origin, with low toxicity and the ability to selectively stimulate macrophage production of interleukin-1 beta, interleukin-12, tumor necrosis factor-alpha, and interferon-gamma lymphocytes.

The problem is solved by the use of water-soluble humic acids, isolated by sodium pyrophosphate solution from peat magellanicum selected from a depth of 100-120 cm from the ridge-urea complex of the oligotrophic bog of the Bakcharskoye bog massif of the south-eastern spurs of the Vasyugan bog in the Iks and Bakchar interfluves, the ability to stimulate the secretion of IL-12, TNF-α and IFN-γ, for the correction of disorders of the immune system in pathological conditions requiring activation of a Th1-dependent ty and immune response, i.e. chronic, sluggish and recurrent infectious and oncological diseases.

A fundamentally new use in the invention proposed by the authors for the effective immunomodulation of water-soluble humic acids isolated from peat Magellanicum taken from a depth of 100-120 cm from the ridge-urea complex of the oligotrophic bog of the Bakcharsky bog massif of the south-eastern spurs of the Vasyugan bog in the interfluve of the Iks and Tomsk rivers , was discovered as a result of experimental studies and for the specialist does not explicitly follow from the prior art, and the description of this property is not revealed Assigned by the authors in the patent and medical literature. Thus, the proposed technical solution meets the criteria of the invention, namely, “novelty”, “inventive step” and “industrial applicability”.

The invention will be clear from the following description and the attached figures 1 and 2. In FIG. Figure 1 shows the electronic spectrum of humic acids isolated from peat Magellanicum peat taken from a depth of 100-120 cm from the ridge-urea complex of the oligotrophic bog. In FIG. Figure 2 shows the infrared (IR) spectrum of humic acids from peat magellanicum peat.

A representative average peat sample, which maximally reflects the heterogeneity of the chemical composition of the entire batch of the analyzed material, was taken with the TBG-1 drill in the genetic center of the ridge-ridge structure of the oligotrophic bog complex of the Bakcharsky bog massif of the southeastern spurs of the Vasyugan bog in the interfluve of Iks and Bakchar Bakcharsky region of the Tomsk region the middle of the horizon, uniform in botanical composition, from a depth of 100-120 cm, in the summer period (June-August) according to GOST 17644-83 “Peat. Methods of sampling from a deposit and processing them for laboratory tests. " In the well from the indicated depth, each sample (weighing at least 600 g, the number of wells at least 50) was taken only once, the average weight of the peat sample was more than 30 kg. According to the general technological characteristics, botanical and gravimetric analysis, the selected peat is characterized as highland magellanicum peat, the degree of decomposition of plant residues 10-15%, the content of mineral impurities (total ash) is not more than 2.7%, the content of biologically active humic acids is not less than 4.2 wt.%.

The obtained peat sample was dried at room temperature in air in a well-ventilated room, periodically stirring, to an air-dry state (humidity 15-20%), ground in a rotary knife mill and sieved through a sieve with a hole diameter of 1-3 mm, processed 0 , 1 mol / l solution of sodium pyrophosphate (Na ₄ P ₂ O ₇ ) in a mass ratio of 1: 100 for 8 hours with constant stirring in a P-100 reactor at a temperature of 25-27 ° C, the liquid phase was separated (humic acid extract) from sediment (peat residue) by filtration using vacuum filtration systems (suction filter), then, to precipitate humic acids from the extract, the liquid phase was treated with a 10% hydrogen chloride solution to pH 1-2, the released humic acids (HA) were separated by centrifugation, washed with cold water purified on a Buchner funnel to pH 7 and dried at room temperature.

GCs obtained from the peat magellanicum peat obtained by the above method were characterized by physicochemical parameters of the molecular structure: elemental (C, H, N, O) composition, electronic and molecular spectral properties, and molecular weight distribution.

1. The elemental composition of HA was determined by burning on a C, H, N analyzer EuroEA 300 (manufactured in Italy), the oxygen content was determined by difference. Mass fractions of elements (% mass) were calculated from calibration dependences constructed using the Sulfanilamide Reference standard material. The atomic fractions of elements (% atom), atomic ratios, and the ratio of aromatic and aliphatic carbon (Caromatic / Caliphatic) were calculated according to generally accepted methods [16].

The elemental composition of HA from Magellanicum peat, presented in Table 1, shows the distribution of the main constitutional elements of the molecular structure of the substance and is a diagnostic indicator for characterizing the active pharmaceutical substance. The atomic ratio of carbon and hydrogen elements (H / C = 1.11) indicates the aromatic nature of the HA molecule, because the hydrogen content is only 11% higher than carbon, therefore, the aliphatic part of the molecule is slightly expressed and / or is substituted by various oxygen and nitrogen-containing functional groups. The ratio of the oxygen content in HA to carbon of the order of 44% indicates a large number of oxygen-containing functional groups of their molecular structure, mainly phenolic, carbonyl and carboxyl groups. The nitrogen content in the structure of humic acids relative to the amount of carbon is negligible (about 6%).

2. The electronic absorption spectra of 0.001% aqueous HA solutions were recorded on a Unico 2800 spectrophotometer (manufactured in the USA) in the wavelength range 190-800 nm in a 1 cm thick quartz cuvette. Absorption maxima and extinction coefficients (E 0.001% C) were determined in the obtained spectra. which characterize the optical density of humic acid solutions at a concentration of 0.01 mg / ml for a 1 cm layer at wavelengths of 465 nm (A ₄₆₅ ) and 650 nm (A ₆₅₀ ) and the color coefficient Q _{4/6 was} calculated by E. Welt [ 16] as the ratio of optical densities at wavelengths of 465 and 650 nm (A ₄₆₅ / A ₆₅₀ ).

According to the electronic GC spectrum of the upper magellanicum peat (Fig. 1) in the visible region (400-800 nm), the spectrum does not have distinct absorption maxima, and looks like a gentle curve characterizing continuous absorption with a gradual decrease in optical density with increasing wavelength. In the ultraviolet region of the spectrum (200-400 nm), light absorption sharply increasing in the short-wavelength direction is observed, while in the region of 255-275 nm a shoulder is characteristic of phenolic, carbonyl, carboxyl groups and polyene chains. A higher absorption intensity in this region (255-275 nm) most likely corresponds to π → π * and n → π * transitions characteristic of aromatic aldehydes, ketones, carboxylic acids, their functional and heterofunctional derivatives, kata-condensed and pericondensed aromatic systems such as “acenes”, “phenes”, pyrenes, perylene, etc. In addition, the absorption of HA in the visible and ultraviolet regions has some specificity: in addition to the slope of the light absorption curve relative to the abscissa axis, ins optical density in certain wavelengths and calculated on the basis of their color and extinction coefficients that depend on the number and location of the electrons in the absorbing molecules and ions.

The most objective indicator characterizing humic acids as a substance as a whole is the extinction coefficient at A ₄₆₅ — a generalized indicator — a certain conditional absorption coefficient calculated in relation to a conventional unit of molecular weight [16]. Another important indicator characterizing the optical properties of humic acids is the color coefficient Q _4/6 according to E. Welt (A ₄₆₅ / A ₆₅₀ ). The value of Q _4/6 shows the steepness of the decrease in optical density with increasing wavelength, due to the peculiarities of the chemical structure of HA, and is a quantitative value of the aromaticity of HA macromolecules, showing the relative content of aromatic fragments of their molecular structure to aliphatic substituents.

The obtained HAs isolated from the upper magellanicum of peat taken from a depth of 100-120 cm from the ridge-urea complex of the oligotrophic bog of the Bakcharsky swamp massif are characterized by low extinction and color coefficients (Table 2), which indicates their complex molecular structure and high content more voluminous cyclic part of the molecule, where polycondensed aromatic structures predominate, as well as the high stability of their molecules.

3. The molecular parameters of the HA structure were studied by infrared (IR) spectroscopy on an FSM 1201 IR-Fourier spectrometer (Infraspec LLC) in KBr tablets (1: 100 ratio, respectively), in the frequency range from 500 to 4000 cm ^-1 . In the obtained spectra, absorption maxima were determined. A relative quantitative assessment of the parameters of the structure of humic acid molecules according to IR spectroscopy was given on the basis of the ratios of the optical density of the absorption bands (OPP) of oxygen-containing functional groups (υОН ₃₄₀₀ cm ^-1 , υC = O ₁₇₂₀ cm ^-1 , υC-O, С-О- From ₁₂₂₅ cm ^-1 , υС-О ₁₀₃₅ cm ^-1 ) to the OPPP corresponding to aromatic (υC = C ₁₆₁₀ cm ^-1 ) and aliphatic (υAliphatic ₂₉₂₀ cm ^-1 ) fragments.

The infrared spectrum of HAs of the upper magellanicum peat (Fig. 2) revealed a wide intense absorption band with a maximum at 3500-3300 cm ^-1 , due to variable stretching vibrations of hydroxyl groups (ion) of aliphatic and aromatic nature, mainly associated with hydrogen bonds, the presence of which is manifested by a maximum absorption in the range of 1270-1220 and 1170-1040 cm ^-1 . On the long-wavelength wing of the main band at 3250-3200 cm ^-1 , absorption of medium intensity was found, having the form of a step (kink) and corresponding to vibrations of NH (υ _NH ) in the structure of amide, amines, connected by hydrogen bonds. The medium-intensity bands are clearly visible at 2928-2921 cm ^-1 and 2855-2842 cm ^-1 due to variable stretching vibrations of -CH ₃ and -CH ₂ groups of side chains in humic acid molecules, including those associated with aromatic fragments, judging by the presence of absorption bands in the region of 1385 cm ^-1 . There is a weak absorption at 2700-2400 cm ^-1 inherent in carboxylic acid dimers. A distinct maximum of strong intensity was detected in the range of 1725 cm ^-1 -1710 cm ^-1 , which is characteristic of stretching vibrations of carbonyl groups (υ _{С = О} ), which can be represented by ketones, aldehydes, carboxylic acids and their functional derivatives. A strong band was found in the IR spectrum of the HA in the region of 1650–1600 cm ^–1 , caused by plane stretching vibrations of conjugated carbon – carbon (aromatic, υ _{С = С} ) and carbon – oxygen bonds (carbonyls bound by hydrogen bonds, carboxylate ions, υ _{C = O} ) in the aromatic skeleton and quinones. An absorption band of about 1513 cm ^-1 (υ _С-С ) indicates the presence of non-condensed monoaromatic structures. In this area, there are also fluctuations in the bonds of polypeptides in the composition of humic acids bound to nitrogen and oxygen atoms (NH, NC = O): primary (1580-1632 cm ^-1 ) and secondary (1512-1560 cm ^-1 ) amino groups. Fluctuations in the region of 1264-1225 cm ^{-1 are} determined mainly by valence (υ _С-О ) and deformation vibrations (δ _OC ) of bonds of undissociated carboxylic acids and their functional derivatives (mainly esters of both aryl and alkyl types). The valence vibrations of the hydroxyl groups (υ _OH ) of alcohols and carbohydrates are responsible for the absorption of radiation in the short-wave part of the spectrum in the region of 1175–1000 cm ^–1 . In the range of about 1075-1013 cm ^-1 υ _CO ) primary alcohols absorb radiation, at 1125-1100 cm ^-1 (υ _С-О ) secondary alcohols and at 1175-1150 cm ^-1 (υ _С-О ) tertiary alcohols . Absorption in this area (1175-1000 cm ^-1 ) can also be due to stretching vibrations (υ _С-О-С ) of glycosidic bonds of carbohydrates, lactones, С-О-С-bonds of cyclic and aliphatic ethers, with the presence of С- O bonds of polysaccharides is also confirmed by fluctuations in the region of 830-1100 cm ^-1 . In the range of wave numbers from 800 to 600 cm ^-1 , weak absorption bands are observed, possibly due to out-of-plane deformation vibrations (δ _С-Н ) in aromatic rings having two or more unsubstituted hydrogen atoms, including the presence of condensed multinuclear arenes (755- 760 cm ^-1 ).

When studying the IR spectra of HA, spectral coefficients are usually used as diagnostic parameters, an increase in the intensity of the absorption bands of which indicates a large content of certain functional groups or fragments. In the description of polymorphic and macromolecular compounds of non-stoichiometric composition, for the quantitative assessment of the intensity of absorption bands and the content of functional groups, in addition to the baseline method, the method of optical density ratios of absorption bands (OPP) is also used. the organization of oxygen-containing functional groups, the presence of protein and carbohydrate components.

A relative quantitative assessment of the content of functional groups in HA molecules according to IR spectroscopy based on an OPPI indicates that some of the main oxygen-containing functional groups in the humic acid molecule of the upper magellanicum peat are: hydroxyl (υ _OH 3400-3300 cm ^-1 , υ _{С -O} 1150-1000 cm ^-1 ) groups, carboxyl groups and their functional derivatives (υ _{С = О} 1725-1700 cm ^-1 , υ _С-О 1260-1225 cm ^-1 ), as well as ether groups (υ _{С- C-O} 1050-1035 cm ^-1). Considering the ratios of the OPPP of aromatic fragments of the structure to aliphatic (А _{С = С 1610} / A _Alif2920 ), the prevalence of aromatic structures over alkyl substituents and over oxygen-containing functional groups is revealed. An inverse relationship is also observed in the relationship between the OPPP of oxygen-containing functional groups and aliphatic fragments of the structure, where the groups of carboxylic acids and esters predominate (Table 3).

4. The molecular weight was determined by size exclusion HPLC using an Ultimate 3000 instrument (Thermo Scientific, USA) using an Ultrahydrogel 250, 300 × 7.8 mm column, the mobile phase was 0.1 M NaNO ₃ , 0.01% NaN ₃ in water, flow rate 1 ml / min. Spectrophotometric detection at a wavelength of 200 nm. The molecular weight was calculated according to the calibration graph log M _w = f (t _R ) constructed according to the PSS standards (polystyrene sulfonate) 891 - 976000 Da (Polymer Standarts Service Service GmbH, Germany). The number average molecular weight of humic acids was 4871.2 Da; mass average molecular weight was 18755.3; polydispersity - 3.9; median - 9558.3 Yes.

The immunomodulating properties of humic acids were studied on linear C57BL / 6 mice [17]. Animals (males / females) aged 8-10 weeks were obtained from the Department of Experimental Biological Models of the NIIIFiRM them. E.D. Goldberg Tomsk NIMC RAS. All procedures (content, administration of the test substances, killing) were carried out in accordance with GOST 33215-2014 “Rules for the equipment of premises and organization of procedures for working with laboratory animals”.

To isolate macrophages (MF) and splenocytes, animals were killed with a cervical spine. Splenocytes were obtained from mouse spleen homogenate twice washed with physiological saline (RF), the MFs were fractionated by culturing peritoneal exudate cells (1.5-2.0 × 10 ⁶ / ml) in plastic Petri dishes at 37 ° С (in an atmosphere of 5% СО ₂ and absolute humidity) in a complete culture medium (RPMI 1640 (Sigma), 10% ETS (Huclcone), 20 mM HEPES (Sigma), 0.05 mM 2-mercaptoethanol (Sigma), 50 μg / ml gentamicin (Sigma), 2 mM L-glutamine (Sigma)) and after 2 hours collecting the fraction adhered to the plastic. Mononuclear cells (Mn) were isolated from the peripheral blood of healthy donors by layering heparinized blood (10 U / ml) on Histopaque-1077 cell separation fluid (Sigma-Aldrich) with a density of 1.077 mg / ml, centrifuging for 15 minutes at 400 g , collecting cells that formed a ring on a density gradient. All types of cells were resuspended in the culture medium, viability was evaluated in the test with 0.1% trypan blue (cell suspensions with a viability of at least 95% were used). MF (2.5-3.0 × 10 ⁶ cells / ml), splenocytes (2.5-3.0 × 10 ⁶ cells / ml) or Mn (2.5-3.0 × 10 ⁶ cells / ml) incubated in 96-well plates under the above conditions in the presence of HA (10 μg / ml); 1 μg / ml LPS (serotype O111: B4, Sigma); 4 μg / ml of concanavalin A ("Sigma") or 5 μg / ml of the pituitary mitogen ("Sigma"). After 24 hours from the start of cultivation, the supernatant was collected from the wells and the cytokine concentration was measured in it by enzyme-linked immunosorbent assay on a ChemWell®Combo automated analyzer using test systems according to the attached protocols: mouse cytokines IL-1β, IL-4, IL-10, IL- 12, TNF-α and IFN-γ ("eBioscience", "InVitroGen"), human cytokines IL-12 and IFN-γ ("R @ D Systems"), TNF-α ("Vector-Best").

Indicators of the cellular and humoral immunity links in mice were evaluated at the end of intraperitoneal administration of HA for 10 days at a dose of 1 mg / kg / day, the LPS analog - pyrogenal was used as a control drug (Medgamal Branch of NF Gamalei Federal Research Center for the Economics and Medicine) - 5 μg / kg day in 0.1 ml of RF, the animals of the control group were injected with a similar volume of RF. To induce a Th1-dependent type of immune response, 5 days after the start of administration, the animals were immunized with an intraperitoneal injection of ram erythrocytes (5 × 10 ⁷ ) in 0.1 ml of RF. To assess the effect of HA on the cellular immunity in the delayed-type hypersensitivity reaction (HRT) on the 5th day after immunization, the animals received a second (permissive) injection of sheep erythrocytes into the paw pad of the hind paw - “experimental paw” (10 ⁸ sheep erythrocytes at 0.05 ml of isotonic sodium chloride solution). 0.05 ml of RF was injected into the contralateral paw (“control paw”). After 24 hours, the animals were sacrificed, both paws were cut off along the protrusion of the bone below the junction of the tibia and tibia and above the heel joint, the local inflammatory response was evaluated by the difference in weight of the experimental and control paws. The effect of HA on the humoral immunity was evaluated by the number of antibody-forming cells (AOK) in the spleen and the titer of antibodies in the blood serum in the hemagglutination reaction (RGA), expressed as log2. For this, animals treated with PS were killed on the 5th day after ram erythrocyte immunization (at the peak of IgM-AOK and IgG-AOK), the spleen was isolated and blood serum was collected [17].

The experimental data were processed using the Statistica 8.0 statistical software package, checking the normality of the distribution using the Shapiro-Wilk criterion, calculating the arithmetic mean (X), arithmetic mean error (m), and arithmetic mean deviation (σ) for each sample. Comparison of sample averages was carried out according to the Dunnet criterion to compare several experimental samples with one control.

Example 1

HA isolated from peat Magellanicum peat taken from a depth of 100-120 cm of the ridge-urea complex of the oligotrophic bog of the Bakcharsky bog massif of the southeastern spurs of the Vasyugan bog in the Iks and Bakchar rivers of the Bakcharsky region of the Tomsk Region significantly increased the secretion of the main Th1-type polarization cytokine response: 16.7 times IL-12 and 168 times TNF-α relative to spontaneous control (table. 4). Incubation of cells with HA also led to an increase in the production of IL-1β by macrophages and IFN-γ lymphocytes by 10.9 and 32.8 times, respectively. The secretion of cytokines by macrophages under the influence of HA was significantly higher than the LPS-stimulated control 154 (IL-12), 118 (TNF-α) and 6.5 (IL-1β) times. The stimulating effect of mitogen and HA on the production of IFN-γ by splenocytes of mice was relatively equivalent and 26.6-32.7 times higher than the control values under the influence of both Con A and HA.

Addition of humic acids to mitogen-activated cells (in vitro inflammation model) revealed a significant rise in secretory activity by macrophages of IL-1β by 2.1 times and by IFN-γ lymphocytes by 6.7 times relative to the corresponding LPS and ConA-stimulated control (table. 5).

Against the background of the revealed increase in the concentration of Th1-type cytokines of humic acids from the upper magellanicum peat, taken from a depth of 100-120 cm of the ridge-urea complex of the oligotrophic bog of the Bakcharsky swamp massif, 1.8 times mitogen-stimulated production of IL-10, the key monokine Th2 type and did not affect the secretion of IL-4 (table. 6).

In another model — human peripheral blood mononuclear cells — it was shown that the studied HA also significantly increased the secretion of the cytokines, which are important for the polarization of the Th1-type immune response (Table 7). The production of IL-12 increased 8.3 times, TNF-α 93.6 times, IFN-γ 170 times relative to the intact control. Moreover, the concentration of IL-12 was significantly 2.7 times higher than the values of LPS-activated monokine production, and the amount of TNF-α and IFN-γ was significantly 1.4 and 167 times lower than the secretion of cytokines during incubation with the corresponding mitogen.

Example 2

The course introduction of hepatitis A isolated from the upper magellanicum peat, selected from a depth of 100-120 cm of the ridge-urea complex of the oligotrophic bog of the Bakcharsky swamp massif, to C57 / BL6 mice against the background of the development of the Th1-dependent immune response induced by the introduction of ram red blood cells, led to the suppression of marker reactions of cellular Th1 immune response of HRT reaction (table. 8). The magnitude of edema with pyrogenal and HA decreased by 1.6 and 1.9 relative to the control.

The effect of humic acids on the humoral link of the Th1-dependent immune response was evaluated by the amount of AOA and RGA. It was revealed that the course administration of HA isolated from the upper magellanicum peat, taken from a depth of 100-120 cm of the ridge-urea complex of the oligotrophic bog of the Bakcharsky swamp massif, led to a significant increase in the number of AOKs by 3.8 times and 2.5 times in mice, treated with pyrogenal. Moreover, the titer of hemagglutinins increased 1.6 times only in mice treated with humic acids.

Thus, it was experimentally established that humic acids isolated from peat Magellanicum peat taken from a depth of 100-120 cm of the ridge-urea complex of the oligotrophic bog of the Bakcharsky swamp massif with a number average molecular weight of 4871.2 Da, a weight average molecular weight of 18755.3, and a polydispersity of 3 , 9 and a median of 9558.3 Yes, with direct exposure, they reduce the production of IL-10 and do not affect the secretion of IL-4 against the background of significant stimulation of the production of key pro-inflammatory cytokines of IL by antigen-presenting cells -12, TNF-α, IL-1β and IFN-γ in both mice and IL-12, TNF-α and IFN-γ in humans. The course administration to mice of humic acids isolated from peat Magellanicum peat, taken from a depth of 100-120 cm of the ridge-urea complex of the oligotrophic bog of the Bakcharsky swamp massif, enhances the humoral immunity indices by increasing the number of antibody-forming cells in the spleen and the antibody titer in the blood serum in the hemagt reaction . The data obtained indicate that humic acids isolated from peat Magellanicum peat, taken from a depth of 100-120 cm of the ridge-urea complex of the oligotrophic bog of the Bakcharsky bog massif, have the ability to regulate the balance of pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory ( IL-10) cytokines are of significant interest as the basis for the creation of pharmacological regulators for the restoration of homeostasis in various pathologies associated with deficiency of Th1-zavis imogo immune response (chronic, sluggish and recurrent infections and cancers).

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Claims (1)

The use of water-soluble humic acids isolated from peat Magellanicum peat, with a number average molecular weight of 4871.2 Da, a mass 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-dependent type of immune responses response by adjusting 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.

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