Potential of mushroom bioactive compounds for use in pharmacology

. Mushrooms contain many different biologically active compounds. The immunomodulatory action of mushroom β -glucans has been shown. Native liquids of mushroom cultivation can be sources of different valuable enzymes with significant potential for use in medicine and pharmacology.


Introduction
Traditionally, mushrooms are considered a valuable food product reach in proteins and essential amino acids.They are also rich in dietary fiber, B vitamins and are a source of ascorbic acid, niacin and biotin.However, mushrooms have not only nutritional value, but also healing properties [1].Mushrooms have been used in traditional oriental medicine in China and Japan.
The healing properties of mushrooms were also known in Europe.So the use of mushrooms for the treatment of various chronic diseases is reported in his writings by the "father of medicine" the ancient Greek physician Hippocrates [2].
Mushrooms have long been known among the Slavic peoples, including in the territory of Ancient Rus'.Mushrooms acquire special significance in the 9th century.after the baptism of Rus', when mushrooms began to replace fast food during fasts.
Along with nutritional value, mushrooms have a number of other beneficial properties.They synthesize a wide range of biologically active substances and, due to the developed enzymatic apparatus, are able to consume a wide range of substrates.
Currently, much attention is paid to the medicinal properties of mushrooms and their metabolites.
Studies have shown that many edible mushrooms are able to strengthen the immune system by influencing cellular activity and promoting the synthesis of chemical compounds that stimulate the immune system, as well as helping to treat diseases and restore immunity destroyed by radiation and chemotherapy [3,4].This effect is mainly associated with fungal polysaccharides -β-glucans [5].In addition to glucans, mushrooms also contain a number of other compounds with therapeutic effects [6,7].
According to modern concepts, β-D-glucans, together with LPS, MDF, teichoic acids and other archetypal components of microorganisms, are united by the concept of "pathogenassociated patterns" (PAMPs).This group of substances has one common property: to activate the innate immune system through interaction with genetically determined receptors specific for each archetype [8].The action of β-glucans includes strengthening the immune system by stimulating lymphocytes, NK cells, and macrophages, improving cytokine production, inhibiting cancer cell proliferation, stimulating apoptosis, and blocking angiogenesis [9].
Of considerable interest is the study of fungal proteinases in order to develop a technology for obtaining enzyme preparations applicable in the medical and food industries.Many fungal species synthesize extracellular proteinases.
Plasminogen activators that promote the conversion of plasminogen to plasmin, the main component of the plasma fibrinolytic system, belong to serine proteinases.Clinical studies of plasminogen activators have shown that they are the most active thrombolytic agents.Plasminogen activators have significant advantages over other thrombolytic drugs, in particular with regard to the effect on the clinical course of the acute period of myocardial infarction and long-term prognosis, as well as after acute ischemic stroke, but the high cost of drugs limits their use.
Collagenases are metalloproteinases.These enzymes have collagenolytic, elastolytic, trypsin and chymotrypsin activities.Collagenase is a unique enzyme that is able to selectively hydrolyze an insoluble protein -collagen, which is part of the connective tissue of a living organism.Cleaving collagen fibers that are extremely resistant to the action of other proteases with the formation of high-molecular fragments, collagenase performs the most important functions in the human and animal body, participates in the transformations of connective tissue during growth and morphogenesis, as well as in the elimination of some pathological processes in arthritis, tumor metastases, etc.
Collagenase selectively acts on collagen, the main component of connective tissue, causing its destruction.Viable muscles, granulation tissue and epithelium are not affected by collagenase.In purulent wounds, collagenase contributes to the rapid evacuation of nonviable tissues and exudate, the early appearance of granulation tissue, and epithelialization.Its use prevents the development of rough (like keloid) scars, while maintaining the mobility of the skin and soft tissues, and maintaining the function of the joints.
Collagenase is recommended for cleansing wounds of various etiologies, any localization and at any stage of the wound process, both immediately after surgical debridement and hemostasis, and in the treatment of long-term non-healing wounds with wet necrosis (especially of connective tissue origin) or excess fibrin that interferes with healing.
Milk-clotting enzymes are widely used in cheese making as substitutes for scarce and expensive renin.suaveolens L. : Fr), pure cultures, which are stored in the Collection of the Department of Microbiological Synthesis Technology, St. Petersburg State Institute of Technology.
The fungal biomass was separated from the native solution by filtration through a paper filter under vacuum and dried in an oven at a temperature of 500C.The amount of dry biomass was determined by the gravimetric method.
The extraction of β-glucans was carried out based on the method described in the works of T. Mizuno [10].The protein content in the extracts was determined by the Lowry method [11].Determination of the content β-glucans in preparations was determined using the βglucan Assay Kit(Yeast & Mushroom) (Megazyme, USA) [12].
When analyzing the immunomodulating effect of glucans, the production of reactive oxygen species was determined by the method of luminol-dependent chemiluminescence [13].
Thymus and spleen cell suspensions were used in the blast transformation reaction (RBTL) of lymphocytes.Spleens from CBA mice were collected under aseptic conditions, homogenized in RPMI-1640 medium, and filtered through two layers of sterile gauze.The obtained homogenate was centrifuged and the erythrocytes were lysed using 0.83% ammonium chloride solution.Splenocytes were washed twice with RPMI-1640 medium supplemented with 2mM L-glutamine and 80 μg/ml hetamycin.The number of cells was counted in the Goryaev chamber.The concentration of thymus cells was adjusted to 10*10 in one ml of RPMI-1640 medium supplemented with 2mM L-glutamine, 80 μg/ml gentamicin and 4% fetal serum inactivated by heating at 56°C for 40 minutes.The concentration of spleen cells was adjusted to 3*10 in 1 ml of RPMI-1640 medium supplemented with 2 mM L-glutamine, 80 μg/ml gentamicin, and 20% fetal serum inactivated by heating at 56°C for 40 minutes.To stimulate the proliferation of thymus and spleen cells, Conconavalin A was used at a final concentration of 1 μg/ml and the resulting mushroom extracts.96-well culture plates (Costar) were used to set up RBTL.The cell culture was incubated at 37°C in an atmosphere containing 5% CO for 72 hours.16 hours before the end of the culture period, N-thymidine was added to all wells of the plate at a final concentration of 5 μm per ml.At the end of cultivation, cell cultures were transferred to filters using a harvester.The filters were dried and the amount of H-thymidine included was measured using a liquid scintillation counter.The results were expressed in pulses per minute [14].
The cytokine-inducing activity of fungal mycelial extracts was studied using a suspension of mononuclear cells resuspended in RPMI 1640 medium, including 10% fetal serum, placed in 96-well flat-bottomed microplates (manufactured by Kostar, St. Petersburg), 0.1 ml per well .Simultaneously, study drugs were added to the same wells.Prodigiosan, a lipopolysaccharide drug known to be a strong inducer of pro-inflammatory cytokines, was used as a positive control.The microplates were placed in a CO2 incubator for 24 hours, after which the amount of cytokines was determined by the immunoenzymatic method.For these purposes, preparations produced by Cytokin (St.Petersburg) were used.
The study of antitumor properties of aqueous extracts of higher fungi on the model of grafted Ehrlich ascitic carcinoma was carried out on same-sex mice of the C57BL line, obtained from the nursery of the Russian Academy of Medical Sciences, pos."Rappolovo" (St.Petersburg).The animals were kept in a typical vivarium on a normal diet, and their body weight was measured weekly.
The studied extracts were dissolved in water and the food was impregnated with the prepared solution, which was dried at 50°C.The daily dose of drugs was 10 mg/kg of animal body weight.The control group of animals received normal food, without additives.
Before the start of the experiment, mice were kept for 7 days (10 individuals in standard polypropylene cages) in order to adapt to vivarium conditions.
On the 7th day from the start of the drug administration, the animals of the control and experimental groups received 1 x 106 cells of Ehrlich's ascitic carcinoma subcutaneously into the left paw (0.2 ml of a suspension of tumor cells at a dilution of 1:10 in physiological sodium chloride solution).
A week after inoculation and then weekly, the size of the tumors was measured.During the experiment, the death of animals was also recorded, the survival and average life expectancy of mice with Ehrlich's ascitic carcinoma were determined.
The study of the antitumor properties of aqueous extracts of higher fungi on the model of transplanted melanoma B-16 was carried out on female mice of the C57BL line with an average weight of 23 g (nursery of the Russian Academy of Medical Sciences, Rappolovo village, St. Petersburg).Animals were kept in standard polypropylene cages, 8 animals each.
The test drug was dissolved in water and the food was impregnated with the prepared solution, which was dried in the open air (25°C).Animals were divided into several groupscontrol and experimental.Animals of the control group (n=8) received the usual granulated feed, the experimental group (n=8) received feed with extract.The daily dose of the drug in terms of dry extract averaged 10 mg/kg of body weight.
On the 15th day from the beginning of the administration of the extract, the animals of the control and experimental groups received 1 x 106 B16 melanoma cells subcutaneously in the right side (0.2 ml of tumor cell suspension diluted 1:10 in physiological sodium chloride solution).From the 7th day after inoculation, the parameters of tumor growth were assessed: the volume of tumors and the life span of animals.
Membrane methods (dialysis, ultrafiltration) were used to isolate and purify enzymes.The proteolytic activity of enzymes will be determined by changing the concentration of proteins using the Lowry method.
The activity of the plasminogen activator was determined in vitro using a photometric method using the Chromotech-plasminogen kit (Technology Standard LLC), in vivo using a model of experimental photothrombosis of the femoral artery in rats.

Results and discussion
The immune stimulating activity of the extracts was evaluated by the results of their influence on the phagocytic activity of human whole blood cells.The functional activity of phagocytic cells was judged by their formation of reactive oxygen species (ROS) in the luminoldependent chemiluminescence of whole blood.The chemiluminescence light sum was chosen as the measured parameter.
From the results presented in Figure 1, it can be seen that most of the extracts at a concentration of 1 mg/ml increased the chemiluminescence light sum, which indicates an increase in the functional activity of phagocytes, which indicates the manifestation of immunostimulatory activity by the extracts.The highest activity was observed in extracts from: Bjerkandera adusta, Cerrena unicolor, Coprinus lagopides, Hypsizigus ulmarius, Panus conchatus, Pleurotus ostreatus, Pleurotus cornucopiae, Trametes ochracea.With the introduction of extracts from the mycelium of these fungi, the level of blood chemiluminescence increased in comparison with the control by 3-10 times.These fungi were selected for further study.
For a deeper study of immunomodulatory activity, it was decided to investigate the effect of mushroom extracts on the production of cytokines.Cytokines are involved in the regulation of hematopoiesis, immune response, inflammatory processes, angiogenesis, apoptosis, chemotaxis, embryogenesis, and also communicate between the immune, nervous, endocrine, hematopoietic and other systems and serve to involve them in the organization and regulation of a single protective reaction.About 200 individual polypeptide substances are currently referred to the cytokine system [15].
In this work, we evaluated the effect of mushroom extracts on the induction of proinflammatory cytokines -interleukins 1-β and 8, which are powerful autocrine stimulators of the protective functions of mononuclear cells, as well as the production of interleukin 2, which is involved in stimulating the immune response due to the activation of T-cell populations, stimulation of natural killer growth [16].The results of studying the effect of the studied extracts on the production of IL 1-β (interleukin 1-β) and IL-8 (interleukin 8) are presented in Table 1.The level of spontaneous production of cytokines IL 1-β: 670 pg/ml, IL-8: 2169 pg/ml Level of production of LPS-induced IL 1-β: 1653 pg/ml, IL-8: 6668 pg/ml When compared with the level of spontaneous production of cytokines: IL 1-β and IL-8, it can be seen that the studied mushroom extracts stimulated the production of proinflammatory cytokines: IL-1-β and IL-8.The level of cytokine production: IL 1-β, caused by extracts from Bjerkandera adusta, Cerrena unicolor (at concentrations of 5.0 mg / ml), Pleurotus ostreatus, Pleurotus cornucopiae (at concentrations of 0.5-5.0mg / ml), exceeded the level production of IL 1-β induced by lipopolysaccharide.When using an extract from Bjerkandera adusta at a concentration of 5.0 mg/ml, the production of IL-8 was practically on the same level as the production of IL-8 caused by LPS.
The extracts studied by us practically did not affect the activity of IL-2.
The mitogenic properties of the extracts were studied using the leukocyte blast transformation reaction, which makes it possible to reveal the effect of extracts on the proliferation of immunocompetent cells.
It was shown that all extracts significantly (by one to two orders of magnitude, depending on concentration) stimulated the proliferation of B-lymphocytes (Figure 2).The extract from Pleurotus cornucopiae at a concentration of 100 μg/ml stimulated proliferation by 400% compared to the control.The rest of the studied extracts at the same concentration increased the proliferation of B cells by 200-250%.Significant stimulation was also observed when exposed to extracts at lower doses.
When studying the effect of extracts on T-lymphocytes, it was found that in doses of 25 to 50 µg/ml they stimulated proliferation by 110-240% compared to the control.The maximum effect was observed with the influence of the Pleurotus cornucopiae extract at a concentration of 50 µg/ml.With a further increase in the concentration of the extract, the stimulation effect decreased.Some polysaccharides and polysaccharide-protein complexes are able to activate antitumor activity.The best known of them are: lentinan from Lentinus edodes, PSK from Trametes versicolor, schizophyllan from Schizophyllum commune.Basically, the antitumor activity of β-glucans from fungi is manifested as a result of the immunomodulatory action associated with the stimulation of macrophages, the synthesis of cytokines, DC, NK-and Th1.
We studied the antitumor properties of the extracts on tumor models: B-16 melanoma and Ehrlich's ascitic carcinoma.These tumor models have a short incubation period, rapid growth and, unlike the widely used sarcoma-180, are less treatable.During the study, the volume of tumors, body weight and life expectancy of animals were recorded.
When studying the effect of extracts on the development of Ehrlich's ascitic carcinoma, the best results were obtained for the extract from Cerrena unicolor (Figure 3) when 10 mg of extract per kg of animal body weight was added to the daily diet.The average lifespan in the experimental group receiving the extract from Cerrena unicolor was 45±4 days, and the maximum was 51 days, which is significantly higher compared to the control group -the average lifespan was 29±3 days and the maximum was 39 days.In other groups, life expectancy did not significantly differ from the control group.When studying the effect of mushroom extracts on the development of B-16 melanoma, the best results were obtained by adding 10 mg of Pleurotus ostreatus extract (Figure 4) per kg of body weight to the daily diet of animals.The average life expectancy in the control group was 23±2 days, the maximum -27 days, which is significantly lower compared to the experimental group, where the average life expectancy was 30±4, and the maximum -44 days.In other groups, life expectancy did not significantly differ from the control group.
The idea of how the β-D-glucans of edible mushrooms interact with human and animal cells came from the study of antifungal immunity.It is known that all living organisms at different stages of development have a defense system against pathogenic fungi.The mechanisms of antifungal protection have evolved from simple humoral reactions (for example, hemolymph coagulation in crustaceans) to complex intercellular interactions in the immune system [17].Factors that can strongly influence the antitumor and immunomodulatory activity of glucans are their structure, molecular weight, degree of branching, and conformation [18,19].Recent studies indicate that innate immune populations may have a form of memory called trained immunity (TRIM), in which innate immune cells undergo metabolic, mitochondrial, and epigenetic reprogramming after exposure to an initial stimulus, resulting in a memory phenotype with enhanced immune responses when subjected to secondary, heterologous stimulus.It has been shown that TRIM induction using inducers such as the BCG vaccine or β-glucans can confer protection through altered immune responses against a range of viral infections.Geller and Young [20] hypothesized a potential role for β-glucans in reducing morbidity and mortality due to COVID-19 and put forward several concepts explaining how TRIM might actually effect the observed epidemiological events associated with COVID-19.They suggested that oral use of β-glucan for protection could be an effective way to enhance the immune response and alleviate symptoms of COVID-19, although clinical trials are necessary to confirm the effectiveness of this treatment and further study the various effects of β-glucan.
B-glucans have shown remarkable efficiency against viruses that infect the upper and lower respiratory tract, as well as against viruses that cause viral pneumonia [21.22].
While, of course, the symptoms and outcomes of COVID-19 are much more severe than the "common cold," there is evidence that β-glucan administration can reduce severity and improve outcomes, especially in the most vulnerable populations.
Although β-glucans themselves cause direct stimulation of immune responses, they are also known to act as a training agent that results in enhanced immune responses when these trained cells are exposed to a secondary, heterologous stimulus.From an evolutionary standpoint, living multicellular organisms have long struggled with fungal and bacterial pathogens, so over time it became logical that organisms lacking adaptive responses developed a way to defend against these recurring infections.This antifungal and bacterial TRIM was likely conserved in higher vertebrates, with the result that TRIM was observed after administration of β-glucan or other elements resembling fungal and bacterial antigens.
Animal studies using β-glucans support the TRIM paradigm that exposure to β-glucans followed by secondary infection with Staphylococcus aureus results in protection against the pathogen.In addition, various examples of BCG and β-glucan vaccines that provide protection against secondary infections such as Candida albicans in a macrophage-specific manner ultimately lead to the idea that exposure of innate immune cells, in particular myeloid cells, to specific training stimuli leads to nonspecific immune protection [25][26][27][28].
The study of native liquids of culture fluids of many fungi showed that native liquids of cultivation of Coprinus lagopides, Funalia trogii, Flammulina velutipes and Cerrena unicolor had good fibrino-and thrombolytic activity and in the future these cultures can become producers of thrombolytic enzymes.
Native liquids of cultivation of Coprinus lagopides and Funalia trogii are also a source of collagenolytic enzymes that can be used in the treatment of scars, burns and trophic ulcers, as well as for the use of meat processing waste.

Conclusion
Mushrooms have a great and as yet little explored biomedical potential.β-glucans of mushrooms have a pronounced immunomodulatory effect.By influencing TRIM, they can be effective in protecting the population during various epidemics.Native solutions of submerged cultivation of fungi can be sources of valuable enzymes such as collagenolytic, fibrino-and thrombolytic or milk-clotting enzymes.

Fig. 1 .
Fig. 1.The effect of extracts from higher fungi on the functional activity of phagocytes

Fig. 2 .
Fig. 2. Influence of extracts of deep mycelium of fungi on spontaneous proliferation of spleen cells

Table 1 .
Effect of extracts from higher fungi on the production of cytokines