Humic acids isolated by sodium pyrophosphate extraction from various types of peat activate macrophages in the classical proinflammatory pathway and stimulate nitric oxide production by these cells. This effect is mediated by activation of intracellular signaling pathways involving MAPK p38, PI3K, MEK 1/2 kinase, cAMP, and NF-κB via TLR-2 and TLR-4 receptors.

Physicochemical and pharmacological properties of the humic substances from coal of the Kansk-Achinsk coal basin, the Pereyaslovskoe deposit, have been studied. The presence of polycyclic aromatic structures and aliphatic fragments with various functional groups as substituents (carboxyl, carbonyl and quinoid, phenolic, alcoholic, ether and ester, amino and amide) was established by means of UV, IR, 13C NMR spectroscopy, fluor-imetry, and elemental analysis. It has been determined by HPLC that coal humic substances are highly hydro-philic polydisperse biopolymers with medium molecular weights. According to the indicator of microbiological purity (category 3B), the content of microorganisms does not exceed the indicators required by the State Pharmacopeia of the Russian Federation (SPh XIV edition). The content of radionuclides, toxic metals (lead, cadmium, mercury, arsenic) corresponds to the norms (according to SPh XIV edition). The presence of 11 essential elements has been established. The studied humic substances belong to the V class of hazard (low-hazard sub-stances, according to GOST 32644-2014), do not have allergising properties and cytotoxic effect within a broad concentration range. Antioxidant, immunotropic, cytoprotective properties were established in the studies of specific pharmacological activity with various experimental models. The ability to inhibit free radicals such as hydroxyl radicals (HO.) and superoxide-anion radical (O2-. should be stressed, as these radicals are able to by-pass the endogenous antioxidant defense systems of the body. It is concluded that the studied humic substances of the Kansk-Achinsk coal basin, the Pereyaslovskoe deposit, are safe and effective biologically active sub-stances of natural origin for use as a promising biologically active substance in the food and pharmaceutical industries.

The current article describes the biological activity of new biomaterials combining the “green” properties of humic substances (HSs) and silver nanoparticles. The aim is to investigate the antioxidant activity (AOA) of HS matrices (macroligands) and AgNPs stabilized with humic macroligands (HS-AgNPs). The unique chemical feature of HSs makes them very promising ligands (matrices) for AgNP stabilization. HSs have previously been shown to exert many pharmacological effects mediated by their AOA. AgNPs stabilized with HS showed a pronounced ability to bind to reactive oxygen species (ROS) in the test with ABTS. Also, higher AOA was observed for HS-AgNPs as compared to the HS matrices. In vitro cytotoxicity studies have shown that the stabilization of AgNPs with the HS matrices reduces the cytotoxicity of AgNPs. As a result of in vitro experiments with the use of 2,7-dichlorodihydrofluorescein diacetate (DCFDA), it was found that all HS materials tested and the HS-AgNPs did not exhibit prooxidant effects. Moreover, more pronounced AOA was shown for HS-AgNP samples as compared to the original HS matrices. Two putative mechanisms of the pronounced AOA of the tested compositions are proposed: firstly, the pronounced ability of HSs to inactivate ROS and, secondly, the large surface area and surface-to-volume ratio of HS-AgNPs, which facilitate electron transfer and mitigate kinetic barriers to the reduction reaction. As a result, the antioxidant properties of the tested HS-AgNPs might be of particular interest for biomedical applications aimed at inhibiting the growth of bacteria and viruses and the healing of purulent wounds.

The effect of humic acids and substances with similar action - derivatives of succinic acid (ethylmethylhydroxypyridine succinate) and combined agent consisting of succinic acid, nicotinamide, riboflavin, and riboxin on the performance and stress resistance of experimental rats was studied. Performance was assessed in the test of exhaustive forced swimming with a load, stress resistance was evaluated by the serum level of corticosterone and open field behavior, and the state of anaerobic metabolism was estimated by the serum level of lactate after swimming test. Humic acids from peat showed anti-stress activity comparable to that of the officinal preparation and preventive effect on fatigue during physical exercise. They can be recommended as a component for the development of drugs that increase human performance and stress resistance.

A comparative pharmacognostic study of two lowland wood-grass peats of different origin from the Tomsk oblast deposits and of the physicochemical parameters of the structure of humic acids (HAs) isolated therefrom was carried out in order to substantiate the interchangeability of peat raw materials classified into the same peat type and to select a promising source of biologically active HAs for development of new drug candidates with non-specific biological activity. It was found that two lowland wood-grass peats of different origin from the Tomsk oblast deposits (Klyukvennoe and Tagan peatlands) have similar botanical compositions and pharmacognostic parameters. Humic acids were isolated from the peats by alkaline extraction and, according to a combination of physicochemical methods (UV and IR spectroscopy, elemental CHNS analysis, 13 C-NMR spectroscopy, titrimetry), have common structural features due to the presence of the aromatic structure substituted with aliphatic radicals and of the polyconjugated system, identical aromaticity degrees, the predominance of hydrophilic over hydrophobic moieties, polydispersity, and pronounced reducing properties. This indicates the interchangeability of the peat raw materials, provided that the technology for HAs extracting from the peat is observed. The revealed distinctions between the HA samples, mainly in terms of the nitrogen content and the content of phenolic and quinoid moieties, the degree of condensation of aromatic structures, and the contents of lignin methoxy group and of carbohydrate moieties, made it possible to conclude that the HAs extracted from the Tagan deposit peat are more promising raw materials for the development of safe plant-based medicines effective in the prevention and treatment of a wide range of pathologies.

The course administration of humic acids isolated by the sodium pyrophosphate method from pine-sphagnum-cotton sedge peat reduced the general anaphylaxis reaction in mice and guinea pigs immunized with ovalbumin and decreased serum content of IgG1 and IgE in mice. The serum from mice treated with humic acids and sensitized with ovalbumin did not increase the rate of degranulation of mast cells isolated from intact Wistar rats in the presence of ovalbumin in comparison with the serum of control animals.

Peat humic acids are well known for their wide range of biological effects which can be attributed to the complex chemical structure of naturally occurring humic substances. One of the promising tools is an ontology-based quantitative analysis of the relationship between physical and chemical parameters describing a chemical structure of peat humic acids and their biological activity. This article demonstrates the feasibility of such an approach to estimate the antioxidant and cell protective properties of the peat humic acids. The structural parameters of the peat humic acids were studied by electronic, fluorescence, infrared, 13C-NMR spectroscopy, titrimetric analysis, elemental C,H,N, and O- analysis, and gel chromatography. Antioxidant and antiradical activities were assessed by physicochemical methods of analysis: electronic paramagnetic resonance, cathodic voltammetry, ABTS•+ scavenging, assay of DPPH radical-scavenging activity, assay of superoxide radical-scavenging activity, iron chelating activity, and scavenging of hydroxyl radicals. Cytoprotective activity was evaluated by the neutral red-based cytotoxicity test in 3T3-L1 cell culture in a wide range of concentrations. Assessment of intracellular ROS production was carried out using a 2,7-dichlorodihydrofluoresceindiacetate (DCFDA) fluorescent probe. Intracellular ROS production was induced using two common prooxidants (tert-butyl hydroperoxide, Fe2+ ions). We suggested an ontology-based model for the antioxidant and cytoprotective activity of humic acids based on experimental data and numerical models. This model establishes the way to further research on the biological effects of humic acids and provides a useful tool for numerical simulation of these effects. Remarkable antioxidant and cell protective activity of humic acids makes them a promising natural source of new pharmaceutical substances that feature a wide range of biological effects.

Aim. To assess the effect of iron-rich humic substances on hematological parameters in acute post-hemorrhagic and iron deficiency anemia.Materials and methods. Materials for the study were samples of iron-rich active pharmaceutical ingredients based on humic substances (Fe(III) hydroxide complexes with humic substances and polymaltose): HA-Fe3+, HA-PMFe3+, FA-Fe3+, and FA-PM-Fe3+. The anti-anemic activity of the substances was studied on 53 female Wistar rats of the conventional rat line in the model of acute posthemorrhagic and iron deficiency anemia. Anti-anemic activity was assessed by the hemoglobin level, erythrocyte count, hematocrit, and serum iron level.Results. The studied substances HA-Fe3+ and FA-Fe3+ are the most effective in correcting the consequences of both experimental acute posthemorrhagic anemia and iron deficiency anemia. Their effect is comparable to that of the positive control drug Ferrum Lek.Conclusion. Fe(III) hydroxide complexes stabilized by humic and fulvic acids exhibit anti-anemic activity.

Background . Antigen-presenting cells (APCs), especially macrophages, play an important role in the body defense against various pathogens. Their dysfunction and polarization are associated with most inflammatory and autoimmune diseases. The inflammatory process is regulated by activation and / or inhibition of genes differentially expressed by macrophages. Successful correction of inflammation leads firstly to elimination of inflammatory stimuli and then to remodeling and restoration of tissues and organs. It was experimentally confirmed that silvercontaining bionanocomposites based on natural humic substances (HS) obtained from coal of different origin, as well as initial matrices of these HS, are capable of activating pro- and anti-inflammatory properties of macrophages. Aim . To study cytotoxic, pyrogenic, and immunomodulatory properties (arginine balance) of initial HS samples and samples of silver nanoparticles ultradispersed in these HS matrices (HS-AgNPs) in the cell culture of peritoneal macrophages, as well as their effect on pro- and anti-inflammatory properties of APCs. Materials and methods . Cultural and biochemical methods were used in the study. Results . The study showed that the samples CHE-K, CHE-AgNPs, CHS-K, and CHP-K increased M1 macrophage polarization due to stimulation of the NO-synthase activity and inhibition of arginase. The samples CHI-K, CHIAgNPs, CHP-AgNPs, and CHS-AgNPs modulated an alternative M2 or M2-like state of macrophage activation. At the same time, HS are not cytotoxic at effective concentrations, and three out of four studied samples did not contain pyrogenic impurities. Conclusion . The use of HS and their silver-containing bionanocomposites, which have the ability to greatly affect the polarization of antigen-presenting cells, is a promising research area in correction of the inflammatory response for solving an important social and medical problem of treating chronic wounds.

The study substantiated the possibility of using peat humic acids for improving endurance during extreme physical exertion. The mature outbred Wistar rats weighing 200-250 g ( n =40) were subjected to forced swim test until complete exhaustion. The humic acids (1%) were administered intragastrically (0.5 ml/100 g body weight) 30 min prior to the test. Chronic administration of peat humic acids for 5 days increased physical capacity and endurance of rats in exhaustive forced swim test without the changes in serum lactate and corticosterone.

Purpose: This article focuses on new method to estimate biological activity of peat humic acids (HAs) using artificial neural network (ANN) to process spectroscopic measurements in infrared and visible ranges. Conventional approaches generally rely on biological models and direct detection of chemical substances related to bioactivity. These methods proved to be accurate and reliable, but at the expense of speed and simplicity. Materials and methods: Recently, a conception of quantitative structure-activity relationship (QSAR) has been introduced and successfully implemented to predict effects of HAs on toxicity of polycyclic aromatic hydrocarbons. Our research stems from this conception, but employs multilayer perceptron (MLP) model to improve overall performance. The developed MLP model allowed us to estimate biological activity of the complete vertical peat cores collected from oligotrophic peat bog, located in southern taiga zone of West Siberia (northeastern spurs of the Great Vasyugan Mire, 56 degrees 58' N 82o36' E). In total, 42 samples taken from the cores were collected. The protocol included spectroscopy (in infrared and visible ranges) and biological model with peritoneal activated macrophages as a reference method to directly measure biological activity of HAs. Results: and discussion. Numerical experiments confirmed consistency of the measured and estimated bioactivity, coefficient of determination R2 = 0.97. These experiments also showed that the MLP model significantly outperforms conventional linear multiple regression models, mainly due to essential nonlinearity of structure-activity relationships. Conclusions: Our research demonstrates that biological activity of HAs extracted from peat samples can be estimated using an artificial neural network model trained on infrared and visible spectra.

The cardioprotective and inotropic effects of standardized active natural substance based on high-molecular-weight compounds of humic origin were studied on the model of global ischemia (40 min) and reperfusion of isolated perfused rat heart. Preventive administration of the test substance (0.1 mg/ml) before ischemia/reperfusion modeling reduced reperfusion contracture and necrotic death of cardiomyocytes and promoted recovery of myocardial contractility. Blockade of NO synthase with L-NAME (100 μM) abolished the above effects of the test substance. It was hypothesized that NO synthase plays an important role in the development of the cardioprotective and inotropic effects of the test natural substance.

Humic acids (HAs) positively affect many body systems: excretory, digestive, nervous. In a few studies, the effect of HAs on the activity of the cardiovascular system is proved [Zykova, 2017; Lasukova, 2018]. With pathology of the heart and blood vessels, which annually take the lives of about 17 million people, there is a decrease in exercise tolerance due to the development of hypoxia. Due to the high mortality from cardiovascular disease, it is important to investigate the actoprotective activity of HAs. Materials and methods. HAs were isolated by alkaline extraction from sample of lowland wood-grass peats collected from «Tagan» deposit of Tomsk region [Zykova, 2013]. Physicochemical study of HAs carried out [Zykova, 2013]. Bioactivity of HAs was tested on 40 utbred male rats “Wistar” 250-300 g in autumn. The animals were divided on 4 groups of 10 animals each (Table 1). Solution of NaCl and HAs was administered intragastrically to experimental animals before 30 min swimming at the rate of 0.5 ml per 100 g of mass. As forced exercise used a forced swimming test [Pat. 2617206]. Upon completion of the five-day testing, all animals were withdrawn from the experiment by simultaneous decapitation under CO2-anesthesia. Decapitated animals collected blood to obtain serum. To assess the level of glycolytic processes during exercise, the rat serum lactate content was determined [Brancaccio, 2010; Brooks, 1999]. The concentration of lactate in the serum of the experimental and intact groups was determined by the colorimetric method [Dolgova, 2012; Kamyshnikov, 2000].

Although humic acids (HAs) from peat exhibit various therapeutic properties, there is little information available concerning their physicochemical and antioxidant properties. To address this issue, nine different types of peat, including oligotrophic, mesotrophic, and minerotrophic peat samples, were used for isolation of HA fractions by basic (HAb) and pyrophosphate (HAp) extractions. Physical parameters of the HAs were analyzed by UV-Vis, fluorescent, infrared (IR), and electron paramagnetic resonance (EPR) spectroscopy. Average Mr of the fractions ranged from 17.2 to 39.7 kDa, while their humification index (HIX) varied from 0.49 to 1.21. HAp fractions had a higher content of aromatic structures compared to HAb fractions. Moreover, HAp fractions had a significantly higher content of phenolic OH groups (3.6 ± 0.5 mmol/g) versus HAb (3.1 ± 0.5 mmol/g). All HA fractions exhibited antioxidant activity in radical scavenging and electrochemical assays, and their EPR signal had a single line with g = 2.0035, which is consistent with semiquinone type radicals. Furthermore, the HIX was found to be important in determining the number of semiquinone-type free radicals in the HA structures. Overall, these data provide a molecular basis to explain at least part of the beneficial therapeutic properties of peat-derived HAs.

The active ingredient extracted from the peat humic substances was characterized by physicochemical parameters evaluated by UV- and IR-spectroscopy, titration, and elemental (C, H, N) analysis. The cardiovascular effects of this ingredient were examined on isolated Langendorff-perfused rat heart. It was found that the active substance in a concentration range of 0.01-0.1 mg/ml produced a vasodilating effect; in addition, it decreased the end-diastolic and left-ventricular developed pressures.

Materials and methods. 18 native humic acids (HAs) were received from nine representative types of peat of the Tomsk region. Two extraction methods were used: sodium hydroxide and sodium pyrophosphate. Molecular structure parameters were investigated by IR-spectroscopy. The assessement of qualitative and quantitative features of the IR-spectra of 18 different humic acids was made. When HAs with mouse macrophages were cultured their ability to influence the NO-stimulation was determined. Thus, the biological activity of HAs and its dependence on the parameters of the molecular structure were studied.Results. The results of infrared spectroscopy showed that the HAs of upland types of peat contain more carbonyl, carboxyl, and ester groups, and HAs of lowland types of peat contain more aromatic carbon, phenolic and alcoholic hydroxyl, ether and carbohydrate fragments. The results of biological activity showed that HAs from upland types of peat induce the formation of nitrogen oxide, wherein the cell activation decreases with HAs obtained by alkali. All types of HAs from lowland types of peat contain an admixture of endotoxin. Some HAs obtained by sodium pyrophosphate have higher immunotropic activity; the HAs can cause antigen-specific stimulation of cells. The activity of HAs does not depend on endotoxin admixture. The results of molecular spectroscopy showed that the most biologically active HAs have higher aromaticity and higher concentration of oxygen-containing functional groups. This result can be used as a marker factor in the standardization of HAs.