Scientific researches confirm the variety of positive effects of humic substances and their great potential for pharmacological application. Several pathogenically interrelated main effects of humic substances can be distinguished in order to comprehend and explain the wide range of their pharmacological properties.
Thus, humic substances have complexing, anti-inflammatory, immunomodulatory, antiviral, anti-hypoxant and antioxidant effects.
Taking into account the variety of chemical, physiological and biochemical effects of Humic acids, it can be summarised that they have:
One of the most common properties of humic substances is their ability to bind various substrates, usually described as detoxifying activity.
Carbohydrates, amino acids and steroids can form complex compounds with humic substances through hydrogen and covalent bonds. Due to chelation properties humic substances are able to bind ions of various metals into strong complexes.
Other compounds are reactive with humates through ionic interactions, electron loss or addition, Van der Waals force, ligand exchange as well as hydrophobic binding. Due to macrocolloid properties, after being injected humic substances can form a barrier layer on the surface of the gastrointestinal mucosa, retain water and prevent its loss through the intestine, providing mucosa protection from various aggressive factors.
Thus, the detoxifying, enterosorbent, gastroprotective, antidiarrheal effects are achieved. Due to the fact that the mechanism of detoxifying action of humic compounds evolves not only at the physical level (adsorption), but also at the level of physical and chemical interactions (complexation, ion exchange), their higher efficiency and greater antitoxic coverage as compared to simple physical adsorbents should be assumed.
This is proved by a wide list of intoxications for which the efficiency of humic substances has been confirmed. For example, experimental studies have established the ability of sodium humate obtained from peat to reduce the death rate of animals caused by the introduction of various toxic agents: lethal doses of strychnine, in case of toxic anemia caused by phenylhydrazine, hepatitis caused by tetrachlormethane.
The detoxifying effect of humic substances in relation to heavy metal ions is connected with the presence of a wide set of functional groups (carboxyl, hydroxyl, carbonyl, etc.) in humates, responsible for binding metal ions with formation of insoluble complexes. Binding of humic substances to toxic substances leads to reduction of their concentration in the dissolved form, and, consequently, to reduction of toxicity of these substances in relation to various organisms. Many research results show that various living organisms have a positive reaction to the presence of small doses of humic substances in the environment, and at high concentrations humic substances show a bactericidal effect in relation to living organisms. In addition, humic substances can perform a protective function, defending living organisms against the effects of various toxicants.
The hepatoprotective properties of humate have been proved in a model of toxic hepatitis caused by tetrachlormethane, which is manifested by a decrease in liver transaminase activity, preservation of ceruloplasmin activity and prevention of hypoproteinemia.
Due to the fact that humate injection leads to a decrease in the duration of hexenalum sleep, one of the mechanisms of the antitoxic effect of humates is an increase in the activity of liver microsomal enzymes. It is also established that synergism and significantly increased hepatoprotective activity are observed on the model of hepatitis caused by tetrachlormethane, at combined application of the Eplir preparation obtained from sulfide silt muds, together with enterosorbents Polyphepan and experimental peat preparation ECT-1, that is confirmed by normalization of biochemical and morphological parameters of liver functional state.
It has been shown that Humic acids of peat prevent hepatotoxic effect of tetrachlormethane at intragastric injection (25-100 mg/kg), reducing intensity of lipoperoxidation processes, cytolysis and cholestasis syndrome severity, being not less effective than the comparison standard – karsil.
In experimental resection of two-thirds of the rat liver, long-term application of peat humic substances at a daily dose of 20 mg/kg increases ornithine decarboxylase activity, raises spermidine, RNA and DNA levels in hepatocytes and results in increased regenerating liver weight.
Thus, hepatoprotective activity of humic substances is realized due to their detoxifying and antioxidant properties, ability to act as inducers of microsomal enzymes, influence metabolic processes and increase biosynthesis of polyamines, such as spermidine, involved in formation of ribosome structure and protein biosynthesis processes in hepatocytes.
Humic substances have a regulatory effect on various parts of the immune system, increasing the activity of cellular and humoral components of natural resistance. Thus, it has been proved that the elements of humate immunostimulating activity include the increase of phagocytic activity of leukocytes and blood serum lysozyme.
A peat-derived preparation containing humic, uronic and amino acids has been developed, which is called Tolpa Peat Preparation (TRP). At the end of the 1990s, a series of researches were conducted on the immunostimulating, regenerative and anti-inflammatory properties of this preparation, which resulted in the authorisation for the use of TPP as a medicinal product in Poland. TRP has been shown to be an interferon inducer and increase the content of IgM and IgG immunoglobulins and the phagocytic and bactericidal activity of human leukocytes.
It was found that potassium humate derived from bituminous coal, conventionally named oxyhumate, increases the proliferative activity of human lymphocytes stimulated by phytohemagglutinin, both in Vitro at 20 µg/ml and ex vivo when prescribed for a fortnight at a dose of 4 g/day for HIV-infected immunodeficient patients. A probable mechanism for oxihumate immunostimulatory effect is its ability to increase the activity of the T-helper subpopulation of ТН1 lymphocytes, responsible for interleukin-2 synthesis, and to raise the expression of interleukin receptors.
According to experimental studies, humic substances demonstrate antibacterial and fungicidal properties.
The most probable mechanism for these types of humic substances activity is the disruption of protein and carboanhydrate metabolism by a catalytic mechanism, as well as the ability to form inter-ionic bonds with high-molecular substrates of microorganisms. The combination of immunomodulatory, detoxifying, antibacterial and antiviral effects determines the ability of humic substances to increase the body's resistance in a non-specific manner. This complex of effects is a premise for further research into the possibility of using humic substances in the treatment of infectious diseases and intoxications of various etiologies.
One of the best known pharmacological activities of humic substances is the anti-inflammatory effect. Bituminous coal oxyhumate has been found to inhibit neutrophil adhesion and degranulation in a dose-dependent manner and to suppress the expression of complement type 3 receptors (CR3-receptor) on activated neutrophils.
Since CR3 receptor expression is associated with increased synthesis of proinflammatory cytokines, reactive oxygen intermediate, nitrogenous metabolites and proteolytic enzymes, this effect may be one of the mechanisms of anti-inflammatory and immunomodulatory activity of humic substances. It has been proved that potassium humate derived from bituminous coal at a concentration of 40 mg/ml in vitro significantly reduces the release of proinflammatory cytokines - tumour necrosis factor alpha (TNF-α), interleukins (IL-1- β, 6 and 10) in human mononuclear lymphocytes stimulated by phytohemagglutinin A, and also inhibits classical and alternative ways of complement system activation.
In a model of rat paw edema, humic substances show anti-inflammatory properties acting as inhibitors of 5-lipoxygenase. At a concentration of 100 µg/ml peat Humic acids lead to a tenfold decrease in TNF-a release in human cell culture I937 stimulated by lipopolysaccharide, which should be regarded as a significant anti-inflammatory activity. The anti-inflammatory and immunocorrective effects of Humic acids of sulphide silt muds were proved on the model of experimental adjuvant polyarthritis. Decrease in the activity of autoimmune reactions was manifested in reduction of ESR, leukocytosis, content of myeloperoxidase, IL-1-β, TNF-α, fibronectin, normalization of properties of monocytic-macrophage system and leveling the imbalance of immunoregulatory subpopulations of T-lymphocytes.
Leonardite humate, prescribed orally at a daily dose of 61 mg/kg for 6 days, significantly reduces the severity of inflammatory auricular edema by suppressing contact hypersensitivity caused by dinitrofluorobenzene pre-sensitisation to a degree comparable to prednisolone.
Sodium humate, derived from Tomsk peat, reduces the severity of anaphylactic shock and the intensity of the delayed-type hypersensitivity reaction to red blood cells. 1 and 5 % humate ointment accelerates the healing of skin burns caused by ultraviolet radiation. Its efficiency is similar to that of the reference reparant, Solcoseril. The application of humate ointment induces inhibition of passive cutaneous anaphylaxis in whole blood serum, to a degree comparable to Beloderm ointment.
According to the authors of these researches, the antiallergic properties of sodium humate are related to the previously detected mechanisms of its anti-inflammatory action - antihistamine, antiserotonin, anti-bradykinin and antiprostaglandin effects.
One of the mechanisms of the regenerative and wound-healing action of humic substances is their ability to form hydrogen and covalent bonds with collagen-type biopolymers. It has been proved that in vitro incubation with Humic acids significantly increases the tensile strength of the heel tendon of animals, increases the mechanical and chemical resistance of collagen fibres and accelerates the process of their maturation.
Humic substances are also known to accelerate the healing of ulcerative defects in the mucosa of the gastrointestinal tract.
The best known practical application of the humate anti-ulcerogenic effect in Russian medicine is the application of various electrophoresis techniques with humate solutions derived from peat (sodium humate) and sapropel (humisol) in gastric and duodenal ulcers.
The mechanism of the humate anti-ulcerogenic effect is interesting due to the fact that substances with an anti-inflammatory effect often, on the contrary, exhibit the ability to ulcerogenic action. It can be assumed that the anti-ulcerogenic effect of humic substances is realized due to their macrocolloid properties, as mentioned above, and the ability to reduce the permeability of histohematic barriers.
One of the mechanisms of humic substances effect at the cellular level is the ability to regulate the processes of oxidative phosphorylation. Peat Humic acids in doses of 25 - 100 mg/kg have a significant antihypoxic effect in models of histotoxic and hypobaric hypoxia. Under the conditions of normobaric hypercapnic hypoxia, Humic acids prevent the uncoupling of oxidative phosphorylation by normalizing the activity of succinate and NAD-dependent energy production processes in brain and liver mitochondria, and their effectiveness is comparable with the antioxidant and antihypoxant dihydroquercetin.
The antioxidant effect appears to be one of the important properties of humic substances, since it is a mediating mechanism of many of their biological effects. The antioxidant effect of humic substances is realized due to the restorative properties [32] of the direct free radical scavenging, increased activity of antioxidant defense enzymes, participation in the recovery of endogenous antioxidants, and the ability to iron-chelating effect. For example, peat-derived alkaline humate has a significant antiradical and antioxidant effect comparable to that of the standard ascorbic acid and dihydroquercetin.
The peat preparation TPP and its fractions reduce the severity of lipoperoxidation in mitochondria and decrease the MDA level to a degree comparable to vitamin E.
Peat Humic acids show antioxidant and antiradical properties acting as catalysts of superoxide disproportionation.
A research on the antioxidant activity of the humic complex has shown that it is able to neutralise various forms of active oxygen and other free radicals formed during metabolism by direct interaction, and represents a promising natural source for the production of drugs with antioxidant activity.
The accumulative function involves the accumulation of chemical elements and energy required by living organisms. In practice, this means that humic substances are responsible for the sustenance of the soil biota and the hydrobiota, but since they persist for a long time (hundreds or thousands of years according to radiocarbon dating) due to their stability, they thereby guarantee a continuous supply of energy and building material for plants and microorganisms.
Humic substances contain from 40 to 60% of C, 3-5% of N, 30-40% of O, as well as hydrogen, sulphur, phosphorus, many metal cations, including the so-called microelements. It is not by chance that dark grey and black-coloured soils have always been regarded as fertile and called, though not always correctly, chernozem. The colouring of such soils is given by humic substances.
Humic substances give nutrients to living organisms gradually as they are consumed, thus maintaining the necessary supply of these elements for future generations. This makes them very different from many mineral compounds, which can supply plants with nutrients, but usually are readily soluble substances that are quickly consumed or washed out of the soil. At the same time, some of the mineral elements are part of the crystalline lattice of the aluminosilicates, which are not accessible to the living organisms and are only consumed by plants after the breakdown of the minerals.
The participation of Humic acids in the regulation of the immune response during intravenous injection has been proved. Humic acids can stimulate the factors of innate antiviral immunity, being active inducers of endogenous interferon-α, inhibitors of complement system activation and enterosorbents of LPS. Humic acids effectively inhibit the replication of enveloped viruses (HIV, VSV, TBEV) and have no effect on the replication of nonenveloped viruses (enterovirus A71, poliovirus and Coxsackie virus).
Thus, the detected antiviral activity of Humic acids, consisting in the inhibition of envelope virus fusion proteins, enables to reduce the viral load on the body and increase the body's own production of antiviral vaccine and antibodies. Humic acids are antiviral components for microbicides with a multipurpose mechanism for inhibiting viral replication: inhibiting the fusion of HIV with the cell and inhibiting HIV reverse transcription. Humic acids show a synergistic effect with other antiretroviral substances: azidothymidine, lectin protein griffithsin and chitosan sulfates. The prospects of using microbicides based on Humic acids of natural origin for topical pharmacological treatment of HIV infection have been experimentally proved. Microbicides based on Humic acids of natural origin are classified as Hazard Class 4 chemicals - substances of low toxicity (or substances of extremely low toxicity). It is also proved that microbicides made on the basis of humic substances, which were extracted from natural brown coals and leonardites, are superior in their antiviral activity to microbicides, which were obtained from humic substances extracted from peat.
Researches have shown that Humic acids do not have a negative effect on the components of innate and acquired immunity, do not produce a local irritant and allergic effect and do not induce allergic reactions such as anaphylactic shock.
The greatest anti-inflammatory activity of Humic acids was found at a dose of 2.0 mg/kg. When studying the nature of changes in immunological and haematological parameters in suppressing acute immune inflammation by Humic acids, it was found that the anti-inflammatory effect was primarily caused by the action on the activity of neutrophil granulocytes and monocytes.
Leonardite humate has been shown to exhibit antiexudative, antipyretic and analgesic properties and to be comparable in efficacy to diclofenac sodium, with activity at a lower dose of 1.0 mg/kg versus 8.0 mg/kg for diclofenac.
The results of studies suggest that leonardite-extracted humate may be considered as an alternative to classic non-steroidal anti-inflammatory drugs.
Free-radical oxidation is a non-specific unifying link in the pathogenesis of various diseases, which include diabetes mellitus, hepatitis, cardiovascular pathologies, many oncological and autoimmune diseases, including rheumatoid polyarthritis, rheumatism, scleroderma.
Humic substances can exhibit antitumor and antiproliferative properties. The research results on the antioxidant activity of Humic acids in the model of oxidative stress demonstrate that Humic acids have antitoxic activity in relation to the metabolic products. They mobilise the body's endogenous systems by increasing the level of key antioxidant enzymes in both liver and blood plasma. In research by S.K. Aydin et al. (2017) the effect of humic substances on various cancer cells was evaluated. Cells (Hep3B, HT29 and PC3) were treated with different concentrations of humic substances for 48 and 72 hours and proliferation was assessed by MTT assay. The investigation found that it inhibited the proliferation of all applied cell lines. It was also discovered that Hep3B cells exhibited the highest sensitivity for 48-hour applications at IC50 = 1.58-2.43 µg/μl. It is worth noting that apoptotic genes regulation at the mRNA level was also significantly increased during its application compared to the untreated control group.
A similar effect was observed in the work of R.G.P.T. Jayasooriya et al. (2016) The research also showed that humic substances enhanced the death of various cancer cells, such as Hep3B, LNCaP and HL60, and induced apoptosis of MCA-102 fibrosarcoma cells.
This information is confirmed by the work of K. Pant et al. (2015), which evaluated the antitumour and antiproliferative properties of humic substances. In the experiment, Huh7 cells were treated with different concentrations of Humic acids (10-1000 µg/ml) for 24 hours. The results were evaluated by MTT assay and Tunel assay. The MTT assay demonstrated a dose-dependent inhibition of cell proliferation. At a concentration of 1000 µg/ml, humic substances inhibited proliferation by 73.23%, while at a concentration of 500 and 200 µg/ml – by 56.4% and 45.36%, respectively. NO production also showed a dose-dependent effect. At a concentration of 1000 µg/ml an increase in production of 45.59% was observed, and at concentrations of 500 and 100 µg/ml an increase of 26.17% and 24.04%, respectively. The Tunel assay also revealed an enhancement of apoptosis and an increase in DNA damage with higher concentrations of humic substances.