RU2741801C2 - Pharmaceutical composition from galleria mellonella larvae and a method for production thereof - Google Patents

Abstract

FIELD: pharmaceutical industry.SUBSTANCE: invention refers to a pharmaceutical composition possessing antioxidant activity. Pharmaceutical composition, having antioxidant activity, obtained from Galleria mellonella larvae, including thymogen, thymosin-beta, lysozyme, superoxide dismutase, aspartic acid, glutamic acid, glycine, alanine, citric acid, isocitric acid, alpha-ketoglutaric acid in a certain amount.EFFECT: composition described above is characterized by high content of biologically active components and antioxidant activity.7 cl, 50 dwg, 23 tbl, 13 ex

Description

The technical field to which the invention relates

The invention relates to biochemistry, medicine and pharmacology, in particular to a pharmaceutical composition obtained from wax moth larvae, including biologically active peptides, organic acids, amino acids and antioxidants, which has a cardioprotective, anti-tuberculosis, anti-stress effect, as well as a method for its production.

State of the art

The large wax moth Gallerla mellonella L. (Lepldoptera. Pyralldae), hereinafter the wax moth (WM), is a ubiquitous parasite of the bee colony. The wax moth is named for its extremely rare ability for animals to digest and absorb beeswax. Caterpillars (larvae) of VM are bred and used for various purposes: as a laboratory host for the cultivation of insects - entomophages, used for biological plant protection in agriculture; as a test object for assessing the activity and quality of bacterial preparations; and also as an object of biochemical and physiological research in entomology. Empirical studies on the biological activity of VM caterpillars and the use of an extract from them can be considered analogs of our invention.

1. Application of alcoholic extract from VM caterpillars in folk medicine.

The alcoholic extract of VM caterpillars has been used as a folk remedy for tuberculosis in European countries neighboring Russia, especially in Germany, Poland, and also in Russia, in particular, in Ukraine since the 15th-17th centuries. The conditions for obtaining drugs by handicraft methods are not strictly specified, they vary arbitrarily, do not contain criteria for the quality of drugs. As shown by our sample analyzes of commercially available drugs, the content of individual components in them is much lower than in the claimed FC. There may also be samples in which the biologically active substances identified by us are completely absent (Fig. 9, 16).

Thus, the methods used by "artisanal" manufacturers are not suitable for the production of products with guaranteed activity.

2. Scientific research of the biological activity of preparations from VM caterpillars.

2.1. Study of the role of lipases.

Initial and modern studies of the biological activity of preparations from VM caterpillars are mainly aimed at understanding the mechanism of their anti-tuberculosis action and do not affect the main direction of our invention - the experimental reproduction and understanding of the mechanism of a broader therapeutic effect on the human body in other pathological conditions that are outwardly different from tuberculosis.

The direction of research into the immune activity of VM caterpillars was discovered by the works of the outstanding Russian scientist I.I. Mechnikov and his follower S.I. Metalnikov [1]. The main idea of these works was the assumption that the essence of the therapeutic effect of VM preparations in tuberculosis consists in damaging tuberculosis microbes by digesting their stable membranes containing wax-like substances of the lipid group.

The antimicrobial effect of VM caterpillar extracts against Mycobacterium tuberculosis was confirmed by a number of other authors [2-5]. It has been shown that VM larvae have extremely high immunity against a number of microorganisms pathogenic for humans: pathogens of tuberculosis, diphtheria, tetanus, plague, and some others, which also contain wax-like lipids in their membranes.

Attempts have been made to study the biochemical mechanism of this action by isolating an enzyme that breaks down lipids. Previously, digestive esterases were considered as such. Later, the process of lipid digestion came to be understood as the process of lipolysis - oxidative breakdown of lipids. In the hemolymph of larvae, a highly active lipase was found and purified, which initiated, in the modern understanding, the process of lipid peroxidation [6]. Indeed, it was capable of destroying mycobacteria in the body of guinea pigs, which was revealed by tissue cultures following infection [7]. Isolation of lipase for therapeutic use was carried out. The disadvantage of the method for producing lipase is the complexity of the multistage purification process, the spectrum of the biological activity of the drug and the need for use in the form of injections, which revealed its high toxicity. The drug is highly pyrogenic, which in some cases can lead to the death of animals. In addition, the drug does not have a wide spectrum of biological activity on the human body, which, in our opinion, is essential for the implementation of the therapeutic effect in tuberculosis. Another method is described for the isolation of the preparation of lipases, which are digestive enzymes of an insect, which have antimicrobial action against Mycobacterium tuberculosis, from VM caterpillars. This method is also laborious, it includes obtaining a tissue powder and its extraction with an emulsifying mixture containing 50% glycerol and 1.5% carboxel, then the lipases are precipitated with acetone and subjected to lyophilization [8]. The disadvantage of this method is also the limited spectrum of biological activity of the resulting drug.

2.2 Exploring the role of peptides

Another, more recent and modern direction of research of a possible antimicrobial effect was the detection of antimicrobial peptides in the hemolymph of VM caterpillars after immunization with various bacteria. Cecropin-like peptides were also found in Galleria [9-11]. In recent years, more than ten new peptides with antibacterial and antifungal activity have been isolated and characterized from wax moth larvae [11-13]. The method described in [11] included the following stages: immunization of larvae with bacteria, obtaining hemolymph, methanol-acetic extraction, lyophilization, delipidization, lyophilization, separation on a Supelcosil LC-18-DB column, gel electrophoresis, determination of the antibacterial activity of fractions, chromatographic Purification on Superose 12 HR 10/30 and Supelcosil LC-18-DB columns.

The disadvantage of this method is the high cost and laboriousness of obtaining peptide preparations, low yield of the active product and the limited spectrum of biological activity.

Thus, all the considered methods do not lead to the production of drugs with a broader biological activity than just antimicrobial action. The preparations obtained in the process of studying are not considered as a form of medicinal products, since the procedure for obtaining them is laborious. Therefore, the modern direction in the field of medical use of peptides is their synthesis, and not isolation from living objects. In addition, as noted above, the purpose of the studies considered was to substantiate the mechanism of only the antimicrobial action of VM caterpillars. At the same time, their broader biological effect on the human body was not affected.

2.3 Study of the therapeutic effect of alcoholic extract from VM caterpillars

Attention to the broader therapeutic effect of handicraft preparations made from VM attracted S.A. Mukhin. He was shown the therapeutic effect of the drug VM after myocardial infarction [14].

The subject of the study of the authors of the present invention is precisely the new aspects of the biological action of VM preparations, which significantly expand their effect on the human body and higher animals: first of all, an increase in the defenses of a sick person, which can be called general strengthening, adapagenic or restorative.

As an analogue of the drug, a recently published patent should be considered for obtaining a complex of substances in propylene glycol with keratolytic action for external use from larvae of the Big Wax Moth, as well as other insect species [32].

The drug is very different from Voskovit - a pharmaceutical composition according to this invention. It is obtained by growing larvae on artificial feed. To obtain, a solvent that is sharply different in its extracting properties was used. It does not show the presence of substances found by the inventors of this invention. It is intended to be used for other purposes in cosmetology. It is only analogous to the method of obtaining.

3. Characteristics of the closest analogue

In collaboration with S.A. Mukhin in our laboratory undertook the first experimental study of the fortifying effect of an alcoholic extract from VM caterpillars on the body of warm-blooded animals in normal conditions and on experimental models of pathology. The initial stages of this work are reflected in a patent jointly with S.A. Mukhin [16] is the closest analogue.

In contrast to the works - analogs considered above, where the components contained in the tissue of caterpillars were investigated, in this analogue, a preparation obtained from VM, which is used in treatment, was studied. It was not known to what extent the substances found in caterpillar tissues enter the extract.

First of all, the analogue investigates a seemingly obvious circumstance, the role of feed quality for the biological activity of preparations from caterpillars. This question has not been raised earlier either. The food ration of the larvae developing in the bee hive is unique, it is bee bread, honey, as well as wax, from the spent honeycomb - dark waxy dryness containing propolis - components rich in biologically active substances. However, in commercial breeding, the larvae grow well on a simplified feed - mostly pure wax with added sugar. Due to the convenience of this method, it is used for the production of larvae in large quantities. Beekeepers usually use spent honeycomb, a dark wax dry, to grow larvae in small quantities. Before our analogue, there was no data on the effect of food on the biological activity of the extract obtained from the larvae. In this work, experimental studies of the dependence of biological activity on the quality of feed were carried out. We compared the activity of the preparation from caterpillars grown on standard artificial food, grown on dark waxy sushi and the extract of the waxy sushi itself. The product obtained by the proposed method contained several biologically active components. Its chemical composition differed from the composition of the extract obtained in a similar way from VM caterpillars grown on artificial feed, as well as from the composition of the dark waxy sushi extract.

Studies carried out on biological models showed that the extract of VM caterpillars obtained by the proposed method did not have a sufficiently high physiological activity: adaptogenic, cardioprotective and growth-stimulating effects, and also did not sufficiently activate the oxidative metabolism of cardiac and vascular tissues.

Was described the standard regulations for growing larvae and obtaining an alcoholic extract. To obtain larvae were selected, the last before pupation age, weighing 150 mg or more, grown outside the hive on a dark wax land at a temperature of 20-25 degrees. The extraction was carried out at room temperature. Using this technique, a stable product controlled by activity was obtained, which distinguished it from artisanal alcohol extracts. The resulting product is a transparent reddish-yellow liquid containing about 1% dry matter. The extract was stored in the dark at 4 ° C, while the physiological activity was maintained for a year. Only free amino acids were found in the composition of the product in a significant amount as a percentage of dry weight: 50-60%. The rest of the organic components are contained in minor amounts, mainly 1-2% and are not identified to individual substances.

However, the following disadvantages of this analog should be noted.

1. The mechanism of a broad restorative restorative action, which was not explained by the discovered composition, remained unclear.

2. The revealed physiological activity was found on models that are insufficient to demonstrate the therapeutic effect:

- studies of adaptive action were carried out on healthy animals during swimming, which shows rather resistance to physical activity.

- cardioprotective effect was revealed on the outdated model of strophanthin toxicity for the cold-blooded heart - frog.

3. There is no express standardization of the product obtained.

In the analogue, the product was standardized by the characterization of its chemical composition, including minor components without understanding their role in physiological action. Standardization included the use of several laborious and expensive research methods: fractionation on columns, use of thin layer and gas chromatography, electrophoresis, amino acid and elemental analysis, NMR, UV and luminescence spectroscopy.

4. The analogue method is inconvenient for industrial production due to the need to constantly maintain the larvae in culture and daily processing of small portions without the possibility of storing raw materials and creating stocks.

Disclosure of invention

The purpose of the invention is to obtain a standardized physiologically active product (pharmaceutical composition (FC)) from the larvae of the Big Wax Moth in industrial quantities, which has a therapeutic effect in pathological conditions caused by overstrain of the working functions of the body due to hyperactivation of sympathetic regulation, leading to a weakening of recovery processes that support biological stability organism. Such conditions are characteristic of constant intense activity without an appropriate volume of recovery and underlie a number of socially significant diseases of our time - acute stress, cardiovascular diseases, acute inflammatory diseases, including tuberculosis, deterioration in the quality of life of the elderly, the number of which is increasing in Russia and the world. ...

The objective of the claimed invention is to obtain a standardized pharmaceutical composition (PC) suitable for industrial reproduction by the method of wax moth larvae and preparations based on it with a unique set of physiologically active compounds that determine the therapeutic effect detected in models of a number of pathological conditions.

The technical results provided by the claimed invention are:

a) obtaining from the caterpillars of the wax moth standardized FC and preparations based on it of a higher quality compared to the prototype (and handicraft samples) with an increased content of components, in particular biologically active peptides and antioxidants;

b) obtaining FA with a physiological effect, including the normalization of energy processes in mitochondria: prevention of hyperactivation of succinate dehydrogenase and maintenance of the activity of alpha-keto-glutarate dehydrogenase;

c) obtaining FC with a therapeutic effect in pathological conditions such as myocardial infarction, tuberculosis, psycho-emotional stress, purulent wounds;

d) obtaining FA with high safety in a wide range of doses.

The method for obtaining the declared FC, providing the specified technical results, includes the stages:

1) growing larvae outside the hive in specially equipped, thermostatically controlled rooms;

2) growing the larvae on a certain artificial feed, providing an increased content of biologically active ingredients in the declared FC;

3) additional feeding of the larvae with a sugar solution;

4) pretreatment of larvae before extraction;

5) extraction of FA components with an organic solvent with an additional procedure for re-extraction with a part of the extraction solution;

6) adding the antioxidant dihydroquercetin to the extraction solution; and

7) obtaining a dry alcohol-free FA preparation by drying using sorbitol.

Pretreatment of larvae before extraction consists in preliminary freezing or lyophilization. These procedures make it possible to use the claimed method for industrial production of FA, since does not require immediate extraction of the collected larvae, but allows to accumulate, store and transport raw materials (frozen or lyophilized larvae). In addition, the pretreatment procedure accelerates the yield of extractables. To obtain FA, extraction is carried out mainly with an ethyl alcohol solution. Part of the original extractant solution is added to the larvae remaining after the first extraction and a secondary extraction is carried out. This makes it possible to significantly increase the antioxidant activity of FA.

The mechanism of the broad biological action of FA is not limited to the presence of antimicrobial activity in it, but is explained by the synergistic effect of several groups of substances that reduce the state of sympathetic hyperactivity of the human body, which underlies a number of seemingly different diseases that are currently socially significant. These include - constant overstrain (stress), cardiovascular diseases, hypertension, myocardial ischemia, broncho-pulmonary pathologies, including tuberculosis.

In the presented experiment on the healing of an infected wound, the regenerative regenerative effect of the BM preparation on the heart and lungs in case of heart attack and tuberculosis, as well as the growth-stimulating effect described in another work of our laboratory, was reproduced [17].

Strengthening the recovery processes is important in itself and reduces the hyperactivation of the cells by adrenergic stimulation.

Differences from the closest analogue

In contrast to the closest analogue in the present invention, the following results are obtained.

1. An increase in the yield of extracted substances has been achieved. A clear indication of an increase in yield is an increase in dry weight per unit volume of extract. In the Prototype, it was about 1%, and with the claimed method more: 1.5% and up to 2,%. These data are shown in example 2 when describing the amino acid content.

2. The development of a convenient express method for monitoring the physiological activity of drugs in terms of its antioxidant activity (AOA). AOA activity is a chemical indicator that reflects the biological activity of drugs, and is also convenient for measurement. With the help of this method, it is convenient to select improvements in the method of obtaining active drugs. A detailed description of this method is given in example 3.

3. Achieved reproducible production of preparations with high activity by methods suitable for industrial use, due to the improvement of the composition of the feed, the improvement of the processing procedures for the caterpillars of the BM and the creation of dry preparations, in addition to the known alcohol extract. These results are presented in examples 11, 12.

4. The composition of the claimed FC includes new important substances with regulatory physiological activity, which makes it possible to consider the resulting product as a pharmaceutical composition that can be used as a pharmaceutical.

PK includes three groups of highly active physiological regulators

1) Peptides, including thymogen, thymic peptide of higher animals, which we first discovered in insects, thymosin, low molecular weight proteins: superoxide dismutase (SOD) - the most active antioxidant in the body, as well as lysozyme - a protein of animal tissues and apolipophorin - a BM protein, which have a lyzing effect on membranes, including bacteria and mitochondria. Several other insect peptides with a lytic effect on membranes are also present.

The content of biologically active peptides in the declared PK is:

- thymogen - mainly 4-5 μg / ml, but the range can vary from 0.5 to 6 μg / ml;

- thymosin-beta 4 - mainly 2 μg / ml, but the range can vary from 0.5 to 3 μg / ml;

- lysozyme - mostly 1 μg / ml, but the range can vary from 0.1 to 2 μg / ml;

- superoxide dismutase - mainly 8 μg / ml, but the range can vary from 1 to 15 μg / ml.

2) The second group of FA substances are amino acids corresponding to those in the closest analogue, but their yield is higher. The data are presented in example 2.

In the present invention, a group of 4 amino acids participating in the transamination process and two substances closely related by interconversions, aparagine, gutamic {glutamine, proline}, glycine, alanine, (ASP, GLU {GLN, PRO}, GLI, ALA ), which are contained in greater quantities. They differ from other amino acids in that they have a strong regulatory effect on the oxidative processes of energy supply in mitochondria, thereby ensuring the resistance of various tissues to hypoxia and a sedative, sedative, anti-stress effect, especially pronounced on the brain.

Despite the significant differences between analogous studies carried out on other samples of feed and caterpillars, and on preparations according to this invention, the same ratio of acids - participants in transamination and ABM was found with an increase in yield (example 2). They grow in the sequence of ASP, GLU, GLI, ALA, and ABM is adjacent to them in terms of content.

amino acids:

aspartic - mainly 500 μg / ml, but the range can vary from 100 to 800 μg / ml;

glutamic - mainly 700 μg / ml, but the range can vary from 200 to 1000 μg / ml;

glycine - predominantly 300 μg / ml, but the range may vary from 50 to 500 μg / ml;

alanine - mainly 1000 μg / ml, but the range can vary from 300 to 1500 μg / ml;

3) The third group is represented by carboxylic acids: citric (LIM), isolimonic (IZL), α-ketoglutaric (CHL), linked by interconversions. They are known as oxidation substrates in mitochondria. Earlier, we also showed a pronounced regulatory role of ILD and CHL, which reduce the hyperactivation of the oxidation of succinic acid (CNT) in mitochondria, increasing the resistance of tissues to stress and hypoxia, which is discussed below. For a description of these data, see example 2. The carboxylic acids included in the FA are contained in the following amounts:

lemon - mainly 1000 μg / ml, isolimone - mainly 250 μg / ml, α-ketoglutaric - mainly 60 μg / ml, but the range can vary from 10 to 100 μg / ml.

5. The claimed FC has high antioxidant / antiradical activity.

Its antioxidant activity, measured by the method of inhibition of adrenaline autooxidation, is not less than 20 conv. units / ml, and when measured by the method of inhibition of luminescence accompanying the thermal decomposition of α, α'-azodiisobutyroamidine dihydrochloride, is not less than 100 conv. units / ml. The data are presented in example 3.

6. In the claimed invention, it is proposed to use the value of antioxidant activity for testing the quality and standardization of the resulting FA preparations (both liquid extracts and dry preparations), tk. it was shown that its value is consistent with the content of other active components of FA, in particular peptides (examples 1, 3, 11, 12).

7. On experimental models of pathological conditions of higher animals, for the first time, the therapeutic effect of FC in acute psychoemotional stress close to life, experimental tuberculosis and myocardial infarction, as well as accelerated healing of infected wounds, has been shown. For a description, see examples 4-6, 8.

8. Shown the absence of toxicity PK (example 7)

9. Pharmacological composition can increase the threshold of sensitivity to the tuberculosis pathogen. An increase in the anti-tuberculosis activity of blood when taking PK has been experimentally shown, which allows it to be used for prophylaxis (example 5).

With the modern threatening spread of tuberculosis infection, due to both the acceleration of the emergence of strains of mycobacteria resistant to antimicrobial drugs, and the weakening of the human body's resistance to pathogens, the search for and creation of means that increase the body's defenses is acute.

According to existing concepts, the activity of neutrophils largely determines the innate resistance to tuberculosis infection. This resistance is provided by the production of antibacterial peptides with anti-tuberculosis activity [18]. It has been shown that the risk of tuberculosis infection is inversely related to the number of neutrophils in the peripheral blood. Human neutrophil peptides 1-3 kill M. tuberculosis, and neutrophil peptides such as cathelicidin LL-37 and lipocalin 2 restrict the growth of microorganisms.

The amount of antimicrobial peptides in the blood depends not only on the number of circulating neutrophils, but also on their activity. Vitamin D is one of these regulators of peptide production [19]. It is known that people with dark skin color have a low content of vitamin D in blood serum and a low content of antibacterial peptides [20]. They are characterized by increased susceptibility to tuberculosis infection and its more severe course [21]. The claimed PK promotes the activation of neutrophils (example 9, Fig. 40), and also significantly increases the bacteriostatic activity of the blood against M. tuberculosis (example 5, table 10). Thus, PK can be used not only for treatment, but also as a means of preventing people in contact with patients from contracting tuberculosis. The use of FC as a means of disease prevention is no less important for the fight against tuberculosis than its therapeutic use. The possibility of prophylactic use is also determined by the absence of PK toxicity (example 7).

7. Elucidation of the mechanisms of biological action based on the influence of the identified new components.

Based on the results obtained, a new mechanism of the broad biological action of VM preparations is proposed, which is not reduced to its known antimicrobial activity, but is explained by the synergistic effect of several groups of substances that reduce the state of sympathetic hyperactivity of the human body, which underlies a number of externally different diseases that are socially significant at present. time. These include - constant overstrain (stress), cardiovascular diseases, hypertension, myocardial ischemia, broncho-pulmonary pathologies, including tuberculosis, as well as the weakening of the elderly.

In addition, a new explanation of the role of substances with antimicrobial activity in influencing the physiological state of the human body has been proposed. It is due to the ability of mainly peptides with antimicrobial activity to increase the permeability of biological membranes. This kills microbes, but it can have an important protective effect on cells and mitochondria of a macroorganism, discarding excess potential, which stops the formation of ROS, that is, it exhibits antioxidant activity (AOA) and normalizes important physiological functions, in particular, insulin resistance.

Used model of sympathetic hyperactivity.

In this work, the state of sympathetic hyperactivity was experimentally simulated by a modern model of stress, close to the conditions of life, consisting in creating only emotional stress in rats by placing them in a box that restricts movement without pain. However, this procedure leads to intense excitement - psycho-emotional stress. We have previously shown that this is accompanied by hyperactivation by the stress trigger hormone adrenaline (ADR), the most powerful oxidation enzyme in the mitochondria, SDH. This high activity can lead to strong inhibition of the enzyme. It has been shown that both the hormone and the oxidation substrate - JNT - interact in the realization of the effect of ADR on mitochondria. Therefore, these interactions were designated by us as the substrate-hormonal system [22-24], (Fig. 1). This pair of ADR and YNT represent the sympathetic half of the regulation of tissue activity, enhancing energy supply and supporting the external work of cells. The second half is represented by a pair of acetylcholine (ACH) - CHL, which maintains the opposite state - an increase in the recovery processes of disturbances caused by work, which will be discussed below.

In this study, the antistress effect was determined quantitatively according to the effect of FC on the hyperactivity of SDH. This was a sensitive indicator. As shown in example 4, PK has antistress activity for this indicator, consistent with the responses of the immune system.

The state of the initial phase of (acute) stress is always associated with an increase in the level of reactive oxygen species (ROS), primarily superoxide. Therefore, antioxidants are protective agents against stress. Identification and measurement of the antioxidant activity of FA and its components is both an indicator of its quality and an indicator of anti-stress action.

The acute phase of arousal is also associated with hypoxia; therefore, antihypoxic activity can also be considered as protective against stressful sympathetic hyperactivation.

The noted adverse metabolic changes determine the characteristic development of damaging stress, represented by the left curve in Fig. 2. The response is characterized by a large amplitude with a decline to the adaptation response, which ends in exhaustion. In terms of SDH activity, this is manifested by strong inhibition.

The kinetics of the development of a response to a non-physiological load differs sharply from the action of a physiological load, which increases the working capabilities of the tissue and is a training one. Under loads in the physiological range, the response is less in amplitude, but it is replaced by a stable phase of adaptation and, most importantly, it stimulates recovery processes, which provide an increase in the efficiency of the tissue, coupled even with cell division - the right curve in Fig. 2.

Reducing the severity of the first phase of stress is very important to prevent pathological tissue disorders caused by overexertion. The use of substances that protect against adverse metabolic shifts helps to weaken the harmful effects of a strong load. As noted above, hormones, ADR and ACH, and some substrates, metabolites of mitochondria, such as NNT, CHL, ILL acids, and proline, interact with CHL, also participate in this regulation.

In terms of physiological manifestations, the antistress effect is manifested in a sedative, sedative effect on the nervous system, which is known for GLI in the composition of FC and, according to our data, is likely for ILD and CHF.

Thus, the FA contains several components with a synergistic antistress effect, however, differing in mechanisms.

Mechanisms of action of FC components on sympathetic hyperactivity

FC as a whole has an antistress effect according to the parameters considered (a decrease in SDH hyperactivity in combination with the normalization of immune responses of lymphocytes and neutrophils). It also exhibits pronounced antioxidant activity, which is usually associated with antihypoxic action. The discovery of new components in the composition of the FA makes it possible to understand what substances determine the action of the composition and what are the mechanisms of these effects.

Antioxidant Activity (AOA) PK components have two types of AOA activity. The first type is more widely known, which can be called the elimination of ROS after their formation in the form of superoxide. The most powerful biological antioxidant of this type is the enzyme superoxide dismutase (SOD), the presence of which in FA is detected by electrophoresis (example 1). This type of antioxidants includes some amino acids, for example, glutamic acid, many peptides (examples 1, 2).

An equally important role can be played by antioxidants of the second type, which prevent the formation of superoxide, which is especially important in the case of hyperactivation of energy processes. In this state, the membrane potential of mitochondria rises to a maximum that is not normally achieved. Hyperpolarization of the membranes leads to a sharp increase in the formation of superoxide. Such conditions are fraught with critical pathogenic shifts - the development of myocardial infarction or epileptic seizure. A very quick way to prevent a pathological build-up of superoxide is even a small dump of the mitochondrial membrane potential. This is clearly manifested under the action of uncouplers - substances that form pores in the lipid regions of membranes [25]. In this case, the formation of superoxide drops very strongly. There are many substances with a similar action in FC. All of them damage microorganisms, as they increase the permeability of their membranes. The mitochondrial membranes of animals and microbes have similarities, and substances of this type have a similar effect on the mitochondrial membranes. The action of uncouplers on mitochondrial membranes is normally regarded as damaging. But in states of hyperactivity of energy processes, excess potential leads to a number of disorders. In addition to those mentioned above, they include such a scourge of our time as insulin resistance, which leads to type 2 diabetes, as well as other metabolic diseases. Metabolic diseases are a new class of diseases caused by an imbalance in the use of nutrients, especially fats and glucose, in conditions of lack of physical activity. It turns out that with this and an excess of nutrition, which is characteristic of modern life, the above-discussed hyperpolarization of mitochondrial membranes develops, which induces a surge in the formation of superoxide, which directly induces a loss of insulin sensitivity and the inability of cells to use glucose - that is, type 2 diabetes [26]. It has been shown that uncouplers, harmful to normal tissue, rapidly stop superoxide formation and restore insulin sensitivity.

There are many uncouplers in FC. Among them, first of all - lysozyme, lipoforin, a whole group of peptides that increase membrane permeability (example 1). We identified such activity of FA by its effect on the mitochondrial membrane potential, measured by the accumulation of Ca ions (example 4).

Antihypoxic action in the composition of FC. This action is carried out by amino acids - participants in the transamination of ASP, GLU, GLI, ALA, since they switch the oxidation to a path less dependent on NADH.

Reconjugating effect of IDL-CHL-GTP An important regulator of SDH hyperactivity under stress is the ability of IDL to reduce it, bringing it closer to the normal level [23]. This is based on the formation of CHL, leading to the synthesis of GTP, a substance that prevents the uncoupling of mitochondrial respiration under stress and reduces respiratory hyperactivation.

Sedative action. In terms of physiological manifestations, the antistressor effect is manifested in a sedative, sedative effect, which is known for GLI in the composition of FA and probably according to our data for ILD and CHF.

Multiple duplication of regulatory action in a reliable system

As indicated above, several components with a synergistic antistress effect, however, differing in specific mechanisms, were found in the composition of FA.

This shows that the resulting FA is not a random mixture, but a biologically active combination of substances with different mechanisms of action, but synergistic in their effect on pathogenic sympathetic hyperactivity, namely, discarding this dangerous state of energy excess.

Apparently, the ratios of the amounts of substances are not accidental either. So it turned out that the ratio of the amounts of isolated amino acids - participants in the transamination retains the same sequence in the data on the prototype, the samples of which were obtained from larvae grown on a different food, and in our modern preparations (example 2).

In living systems, as in good technical devices, the regulation mechanisms are carried out by a system of parallel fuses. This provides the required control reliability. Therefore, the multiplicity of mechanisms is not redundant, but necessary. And the work of the composition cannot be replaced by the action of one pair of controls. There is a second reason for the multiplicity of regulation of cell activity - the dissimilarity and even the opposite of the sign of regulation in different states. This position is illustrated in FIG. 3. In this diagram, the different state of cells is presented by the activity of SDH. The two left columns represent SDH activation within normal limits. In a normal state, according to microscopic studies, the state of cells in an organ or mitochondria in a cell is basically the same. This is shown by a dotted bar whose height is equal to the total number of microscopic objects. The average value of activity that is measured is characteristic of all of them. However, with hyperactivity, which is the basis of many pathologies, the state of objects falls into two groups, represented by dotted columns. The initial changes are expressed in the hyperactivation of a part of the cells or mitochondria, which, as the exposure continues, is gradually replaced by the inhibition of a certain proportion of them. Thus, despite one average total tissue hyperactivity, it contains two different populations of cells or mitochondria in a cell - hyperactive and strongly inhibited. To normalize them, opposite shifts in their state are needed: in order to return overactive cells to normal, it is necessary to reduce their activity, and to increase it for inhibited cells, which is shown by arrows. Undoubtedly, more flexible regulation will be carried out by a richer composition of substances.

It should be noted that the method used for determining the activity of SDH in blood lymphocytes of humans and animals makes it possible to measure the activity of mitochondria in the body [23]. This is not available with accepted biochemical methods, in which this important state is lost due to the rough isolation procedure, and only the difficult to regulate inhibition of SDH is observed, shown by the last pair of bars in FIG. 3. However, it does not reflect the activity in the body but is the result of damage to mitochondria in the study [27]. The damage masks the real meaning of the state of hyperactive energy surplus, which is a threat to many diseases, and closes the possibility of studying its regulation.

Influence of PK on recovery processes

Strengthening cholinergic regulation. The property of FC is to influence the parameters of tissue hyperactivity. According to physiological concepts of the regulation of the vital activity of cells, work and recovery, including reproduction, cell division cannot occur simultaneously, just as physical work cannot be performed in sleep [28].

To turn on the second side of regulation - to enhance recovery processes, it is necessary to turn off sympathetic activation and open the pathway for the regulation of ACH, shown in Fig. 1. It should be noted that the presence of CHL and its precursors - IZL, and LIM shown by us in FC (example 2), which participants and stimulants of ACC regulation, is consistent with the idea that PK suppresses the adrenergic sympathetic branch of regulation, stimulating work and activates cholinergic, parasympathetic, stimulating cell regeneration and reproduction. Such opposite effects of ADR and ACC have been shown by the regulation of insulin synthesis in the pancreas [29].

Thus, LIM-ILL-CHL in FC promotes the activation of cholinergic regulation and, through it, recovery processes.

The cholinergic effect of the prototype VM is strongly pronounced. The studies performed on PK show that the cholinergic effect is due to the influence of a complex of substances with myotropic action. First of all, these are amino acids and carboxylic acids - stimulators of energy processes in mitochondria. The cholinergic effect can also be manifested by the activation of the immune functions of neutrophils and an increase in their number in the blood. This effect was observed as protection against stress (example 4). As noted above, the activation of the immune defense of neutrophils also determines the anti-tuberculosis effect. However, when analyzing the action of drugs from the VM, the anti-tuberculosis effect is usually not associated with an increase in cholinergic regulation. It turns out that these are two sides of the same mechanism.

Peptide regulation is the central link in the biological activity of FA

A powerful factor supporting the restorative effect of PA is apparently the thymus peptide, thymogen, found in its composition, and, possibly, other peptides with similar effects (see below), the main function of which is to activate tissue repair and regeneration in normal and damaged. An important discovery among thymus peptides is thymosin-beta4, which has the ability to activate stem cells that can develop into cardiomyocytes. This ability has been shown for other tissues as well [30]. In connection with our work, it should be noted that this peptide also has high antioxidant activity.

The identification of peptides as the main link in the biological activity of FA also clarifies some of the features of the action of VM preparations that had no scientific substantiation. For example, its effectiveness in doses that are low in the scale of allopathic pharmacology. A remarkable phenomenon was revealed in the mechanism of action of peptides, showing an increase in their activity at very strong dilutions up to ultra-low [31]. This is due to the existence of a single peptide-water continuum in tissues. Due to this, the initial action appears in “infinitely” (infinitesimal) low concentrations.

The considered growth-stimulating effect of VM, due to the content of peptides, has not previously been used to understand the biological activity of VM caterpillars and their antimicrobial action.

It is important to note that the combination of peptides with proline is important for the completeness of the implementation of growth-stimulating activity. It has been noted that its content is also high in FC. Proline plays a key role in ensuring the structural and morphological stability of tissues. It is indispensable for creating a connective tissue framework for the resistance of growing cells - collagen. Collagen creation involves the interaction of proline and CHL, which, as noted above, is also present in PK. Based on the study of the mechanism of the biological action of FA, the authors came to the conclusion that its basis is the enhancement of the recovery processes in the tissues of the body, and not the actual antibacterial effect on the causative agents of tuberculosis.

The studies carried out have led to a new understanding of the mechanism of biological action of VM preparations as a stimulator of regenerative processes in tissue, including cell division. This conclusion draws attention to a group of publications not related to VM and anti-tuberculosis action. However, it is very similar in composition and mechanism of action.

This is a group of drugs, the main property of which is precisely the stimulation of regenerative processes in tissues, including the enhancement of cell division to replace damaged ones. This group includes tissue extracts from higher animals, mainly calves. Among them, thymalin is a total thymus extract, its separate peptide, an isolated and synthetic thymogen - betahistine, thymosin-beta 4.

Extracts from other endocrine glands, especially the pineal gland and other tissues, also have high regenerative biological activity. It is recommended to use extracts from organs that are affected by the pathological process for treatment, since the extract also contains regulators specific to individual tissues. For them, it is the combination that is important - the composition of many synergistically active substances [31].

There are widely known drugs of this type with a broad effect on different tissues - Actovegin, Cerebrolysin. The main active ingredients are peptides, amino acids and low molecular weight proteins. According to these parameters, the group of substances - stimulators of cell regeneration is close to the mechanism of action of FA that we have identified. In the description of Actovegin, there is also a high content of amino acids - participants in transamination, which we also identified for PK. Despite the undoubted closeness of the main action of the substances of this group to FA, they are not considered analogs, since they differ in sources and methods of preparation. In addition, they are generally not characterized by antimicrobial action, which is considered to be the main property of preparations made from VM.

It should be noted that, as participants in the substrate-hormonal system, ordinary metabolites, as substrates for biochemical transformations, participate in much lower concentrations than is known for substrates - on the order of millimoles, and act in signal concentrations, closer to the action of hormones - micromolar and lower. This is described by the example of the interaction of CNT with the regulation of ADR, which was designated as a signaling action [33-37]. Later, receptors for the JNT and CHL substrates were discovered, which made it possible to understand the mechanism of the physiological action of low doses of substrates, as mediated by receptors [38–43].

Currently, drugs are widely used, the action of which is directed at various receptors, mainly adrenergic receptors, or histamine, which is characteristic of antihypertensive, diuretic and neurological agents. These drugs are also used in much lower concentrations than drugs with a different mechanism of action. Therefore, the efficacy of low doses of PK does not seem to be as unusual as before.

The studies carried out allow us to believe that the bacteriolytic effect of VM on microbes and the stimulation of the regenerative processes of the macroorganism are two sides of the same mechanism.

Throughout the study of the biological effect of drugs from VM larvae, the main mechanism was mainly considered - antimicrobial activity against tuberculosis pathogens. It was explained by the splitting of wax-like and other lipids of microbial membranes. In the initial period of research, this action was associated with lipases. Since the end of the last century and, especially since the beginning of the new, antimicrobial and bacteriolytic action is associated with a group of peptides and proteins apolipophorin III, lysozyme. Thus, the proposed explanations focused on the destruction of microorganisms and did not include the effect on the host's macroorganism - a sick person.

Currently, first in the thymus, and then in other tissues, peptides, thymus thymogen and thymosin - beta 4, which are stimulators of cell division of the heart, as well as other organs, have been found. Based on their own research on the protection of thymus tissue from damage under stress, the authors suggested and found the presence of these peptides, which are inherent in the endocrine gland of higher animals, in the FA from wax moth caterpillars. This explains the restorative effect of VM preparations on heart and lung tissue in pathology. In addition, using the model to accelerate the healing of an infected wound, the authors showed the presence of a growth-stimulating effect of PK. These data forced to pay more serious attention to the therapeutic effect of FC on the body of the sick person.

To elucidate the mechanisms of such an action, a study was carried out of the effect of PK on the main system of nervous regulation of organ tissues - the vegetative one. It controls the switching between the two main states of living tissues - work and rest, in which the regenerative processes of work spending take place. Accordingly, the system consists of two parts - sympathetic, stimulating work, and parasympathetic, stimulating recovery processes. At the hormone level, this is done by the action of ADR or ACH, respectively. Previously, the authors showed that their action includes the effect on processes in mitochondria with the participation of previously known only as substrates of acids - JNT and CHL as signaling molecules - synergists of ADR and CHL. The action is directed to enzymes that oxidize these substrates - SDH and CDH. They are known only as ordinary equal participants in the energy supply processes. However, free gas movement plays a leading role in the energy supply of vigorous activity, and the free gas movement - in ensuring recovery processes.

The maximum activity of SDH is carried out at a high level of energization of mitochondria and a high membrane potential. These conditions inhibit and suppress the activity of CDH. However, it is activated when the mitochondria become “tired”, which is associated with a drop in energization and potential.

Such an approach to the analysis of the mechanism of action of PK made it possible to assume that the well-known - "killing" effect of SDH on a unicellular microbe can have a therapeutic effect on the state of a multicellular organism.

It was shown that FA has the same strong membranolytic effect on the state of mitochondria in higher animals as it does on microbes. It develops in seconds when added to the mitochondria (example 10). Typically, this action is also considered "deadly" for the mitochondria.

However, in modern life conditions, the harmfulness of excess energy accumulation by mitochondria is revealed. It is caused by excess food and lack of physical activity in humans. This condition is seen as the scourge of civilization and the basis of diseases that increase in number and rejuvenate with age. These include: loss of insulin sensitivity (diabetes), hypertension, cancer. The basis of the metabolism of these pathologies is the disorder of the mobile switching of the oxidation of fats and carbohydrates - metabolic syndrome. It represents - stagnation of energy metabolism. For cells in a hyperergic state, the release of potential is a curative effect. It helps to overcome the mechanisms that hold this state, and move to a low-energy state that triggers the recovery processes.

This is similar to the problem of "losing weight", when it is difficult to break free from the captivity of fat accumulations in the body without additional stimulus.

This understanding of the PK action allowed the authors to offer their own explanation for its restorative action, which is the reverse side of the long-discussed antibacterial action of PK.

A powerful group of membranolytic peptide-protein substances present in PK, increasing the permeability of membranes, kills the microbe organism, consisting of one cell. However, a decrease in membrane permeability in some regulatory cells of a macrocellular organism can play an effective regulatory role in pathologies caused by constant stress.

Thus, FC has a therapeutic effect not due to the "spurring" of functions, which is impossible when weakened, but by strengthening recovery, which requires weakening - turning off the impulses that stimulate work and energy supply.

At the hormonal level, this means the well-known reciprocal suppression of the adrenaline impulse by the action of acetylcholine. In essence, this reflects a duality in the response to stress. It is perceived more as an external reaction to a stimulus: muscle contraction, visual arousal, etc. It is known, but more hidden from observation, that the response to stress carries at the same time an adaptive, adaptive response, in fact, anti-stress. It is not so obvious to observe. At the mitochondrial level, its quantitative measurement is assessed by the ratio of the activity of SDH and CDH (example 4).

Let us summarize the above as follows: the membranolytic effect of FC allows one to reduce the intensity of work - the stress component of the response to stimulation, and thereby enable recovery, that is, the anti-stress part of the tissue reaction, while the working output “weakens” in the sense of the work performed. However, it becomes more complete for life in the sense of maintaining the biological stability of the tissue.

Thus, FC (VOSKOVIT) really supports the basis of life - the process of self-reproduction of living structures, their restoration.

Brief Description of Drawings

FIG. 1 schematically illustrates the concept of a physiological regulation system that combines the action of two oppositely affecting the activity of tissues of the sympathetic (adrenergic) and parasympathetic (cholinergic) branches of autonomic regulation with the processes of substrate oxidation in mitochondria. The system includes the action of the hormones adrenaline (ADR) and acetylcholine (ACH) on dehydrogenases, which oxidize only two substrates - succinic (YNT) and α-ketoglutaric (CHL) acids - succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH), respectively. This effect on mitochondria is synergistic with the new signaling effect of only these two substrates on mitochondria through receptors specific for JNT and CHL.

FIG. 2 schematically shows the relationship between work and recovery under pathogenic and physiological stress (stress, activity, activity)

Damage Load - Left

Developing (training) load - right

The answer develops, according to Selye's terminology, from the initial, acute stage - an anxiety reaction, through the stage of adaptation to the final phase, which is sharply different under different loads.

The vertical axis shows the amplitude and nature of the response. Under a damaging load, the range of activity is larger, but a significant part of the reaction causes damage in the tissue, which are marked by the gray area between the curves. The stage of adaptation in this case is replaced by a stage of depletion, requiring long-term recovery, or partially irreversible. The lower curves show the level of response that does not damage tissue. With reactions in the physiological range of compensatory capabilities, the amplitude of the response, working activity is less, but with such activity, the damage zone is very small or absent, but instead of damage in the tissue, recovery processes are much more enhanced, the light zone, which overlap the initial level. This physiological exercise training is at the heart of progressive development.

FIG. 3 shows the average activity of SDH in the mitochondria of lymphocytes and the proportion of cells in the population with different activities (solid and dotted contour of the bars, respectively) under different states of the body - normal, sympathetic hyperactivity - stress, nonphysiological inhibition.

FIG. 4 shows the fine separation of low molecular weight peptides and proteins using our developed electrophoresis system.

The natural preparation thymalin (far left lane) and synthetic taps (far right lane) were used as molecular weight taps. Their mass is indicated in KDa. Nos. 1-5 show 5 different FA samples with high antioxidant activity according to the chemiluminescent method (upper inscription above the lanes) and by the adrenaline method (lower inscription above the lanes).

FIG. 5 shows the separation of the protein-peptide fraction of FA by electrophoresis in a tris-tricine buffer system (pH 8.45). 16.5% acrylamide gel. Lanes: 1 - molecular weight markers (Fermentas, Lithuania), 2-3 - FA obtained from live larvae, 4-6 - extracts stored in the refrigerator for 3 months, 4 - from live larvae, 5 and 6 - from lyophilized ones. 7 - a commercial preparation of thymalin, 8-10 - samples similar to 4-6, but stored at room temperature.

FIG. 6 shows the identification of the thymogen band in PK by comparison with natural drugs - thymalin, thymogen (T) and its synthetic analogue bestim (B), as well as the assessment of the effect of various extraction methods on the protein-peptide composition of PK using standard markers (M). No. 1 - the extract for FA is obtained from live larvae; No. 2-A - from lyophilized larvae: No. 2 - from freshly obtained, No. 3 and 4 - from those stored in the freezer for 1 and 3 years, respectively.

FIG. 7 shows the identification of the thymosin band in PK in comparison with the synthetic analogue of thymosin beta-4 (44 amino acids, MW 4.8). 1-3 - different PK preparations, M - marker.

FIG. 8 shows the effect of storage under various conditions (12 months) on the protein-peptide composition of FA and its antioxidant activity. Above are the values of AOA according to the chemiluminescent method. Each FC variant was stored in a refrigerator or at room temperature as indicated above the lanes.

FIG. 9 shows the assessment of the effect of potential protectors on the preservation of the properties of FA during long-term storage. The preparations were kept warm or refrigerated for 21 months. Carbon dioxide and dihydroquercetin (DHQ) in concentrations of 0.1 and 1 g / L were used as possible protectors. The last lane is coated with a commercial preparation of wax moth larvae.

FIG. 10 shows protein-peptide components detected by polyacrylamide gel electrophoresis in FA preparations obtained by different methods.

1, 2 - FA preparations obtained from pretreated caterpillars

3, 4 - preparations from live caterpillars

5, 6 - preparations from frozen caterpillars.

1, 3, 5 - preparations from caterpillars grown on wax sushi

2, 4, 6 - preparations from caterpillars grown on a mixture of merva-dry.

Each of the six preparations shown in the figure is a mixture of equal amounts of 4 preparations separately obtained by this method.

The numbers above the drug numbers indicate:

bottom row - AOA, conventional units / ml;

top row - amino acid content, mg / l.

Tim - thymalin, pharmaceutical preparation, peptide fraction of calf thymus, M - markers.

FIG. 11 shows the graphs of the dose-dependent inhibitory effect of five separately obtained samples of the pharmaceutical composition on the autooxidation of adrenaline.

FIG. 12 shows the dose-dependent inhibitory effect of PA on the rate of reduction of nitroblue tetrazolium by superoxide anion radicals formed during the oxidation of xanthine by xanthine oxidase.

FIG. 13 shows the inhibitory effect of FA on the intensity of luminol-dependent chemiluminescence of activated human blood neutrophils.

From top to bottom: without adding FC; 0.22%; 0.9%; 2.3% FC (V / V). The average values are given for 8 experiments in which the blood of four donors was used. In each experiment, chemiluminescence measurements were carried out in 2-3 repetitions.

FIG. 14 shows AOA in two different FA preparations when stored cold (top) or room temperature (bottom). Determination by autooxidation of adrenaline (left) or chemiluminescent method in the ADBA-luminol system (right). The abscissa shows the storage time in days. Y-axis - AOA in conventional units / ml FA. The unit of AOA in the figures on the left is taken as such an amount of the drug in μl, which inhibits the autooxidation of adrenaline by 50%. The AOA unit in the figures on the right is taken as such an amount of the drug in μL, which leads to the same change in the latent period of chemiluminescence in the model system, as trolox at a concentration of 1 μM.

FIG. 15 shows the effect of storage conditions on FA AOA as measured by inhibition of adrenaline autooxidation.

FIG. 16 shows the antioxidant activity of FA preparations obtained by different methods (3-5), FA preparation during prolonged storage in warmth (6), as well as extracts from wax moth larvae obtained by handicraft method (1, 2, 7) * AOA unit is taken as the amount of μl of the drug required for 50% inhibition of adrenaline autooxidation. The unit of prooxidant activity is taken as the number of μl of the drug required for 50% activation of adrenaline autooxidation.

FIG. 17 shows the results of gel electrophoresis separation of proteins and peptides contained in different PK samples.

Lane 1 (T): thymogen preparation (glutamyltryptophan dipeptide); Lanes 2-5 - PK preparations:

2 (-4%) - FA from live larvae after 8 months of storage;

3 (11%) - FA, 5 years of storage, from live larvae;

4 (70%) - FC from raw materials stored for 4 years;

5 (86%) - FC from fresh raw materials; 6 track (M) - markers.

Above the lanes, the degree of inhibition of adrenaline autooxidation is indicated when 50 μl of the test extract is added to the standard incubation mixture.

FIG. 18 shows the anti-stress effect of PK on the activity of SDH and DRH in animals that are moderately excited and calmer.

A: Changes in the activity of enzymes (measured by the total area of formazan granules in 30 cells, μm ² ) in the lymphocytes of control and those treated with FA (1 ml according to the scheme: 5 days of feeding - 2 days off - 1 day of feeding) rats with adrenaline (50 μg) / 100 g weight). In the presented experiment, a group of animals had a slightly increased activity of SDH at rest.

B: Identical experiment on staging animals with low SDH activity at rest.

FIG. 19 shows the anti-stress effect of PK on the activity of SDH and DRH in rats using different stress models - adrenaline administration and 24-hour psychoemotional stress.

Changes in enzyme activity (measured by the average area of formazan granules in a cell, μm ² in lymphocytes of control and those receiving FA (1 ml according to the scheme: 5 days feeding - 2 days off - 1 day feeding).

FIG. 20 shows the antistress effect of PA in terms of the accumulation of calcium ions in mitochondria upon administration of ADR (above) or upon 30-min psychoemotional stress (below).

FIG. 21 shows the anti-stress effect of PK to prevent spleen weight gain upon administration of epinephrine.

FIG. 22 shows the intensity of blood chemiluminescence induced by 200 μg / ml zymosan in intact animals or in animals receiving FA after administration of adrenaline (top) or after 30 minutes of RPE.

FIG. 23 shows the intensity of zymosan-induced (200 μg / ml) chemiluminescence per 10,000 blood neutrophils in intact or FC-treated rats after adrenaline administration (top) and after 30 minutes of RPE (bottom). The calculated values are given (the ratio of the chemiluminescence of 20 μl of blood to the number of neutrophils in this volume) * p <0.05; compared to PES.

FIG. 24 shows the number of neutrophils in the blood of intact or FC-treated rats after administration of adrenaline (top) and 30 min RPE (bottom). Each graph represents the average of three independent experiments. * p <0.005 compared to epinephrine or RPE.

FIG. 25 shows the leukocyte indices of intact and FC-treated rats after administration of adrenaline (top) and 30 min RPE (bottom). Each graph shows the average of three independent experiments.

FIG. 26 shows the intensity of spontaneous chemiluminescence of rat blood with intraperitoneal injection of adrenaline (top) and with a 30-minute RPE. Data are presented as% of control.

FIG. 27 shows micrographs of stained sections of the lung tissue of mice infected with MBT. Group No. 4 (no treatment). Magnification × 100.

Scattered large and medium lymph - macrophage granulomas are visible in: A - interalveolar septa; B - peribronchial region; C - perivascular area. In the lung tissue along the interalveolar septa, in the peribronchial and perivascular tissue, massive, diffuse focal, mainly lymphocytic, cell clusters and lymphocytic granulomas are found. Expressed plethora of blood vessels. There are foci of the acinar type of emphysema.

FIG. 28 shows micrographs of stained sections of the liver tissue of mice infected with MBT. Group No. 4 (no treatment).

Multiple lymph - macrophage infiltrates are visible: A - large; B - medium. In the course of the veins, triads and bile ducts, massive cell clusters are located with a predominance of lymphocytes in them. In the parenchyma of the liver there is an abundance of lymphocytic granulomas with "loose" distribution. Discomplexation of the hepatic tracts was expressed with the death of a number of groups of hepotacites.

FIG. 29 shows micrographs of stained sections of the lung tissue of mice infected with MBT. Group No. 3 (Treatment with PK 150 μl / mouse / day). Magnification × 100. Sparse, submicroscopic lymph - macrophage accumulations are visible in: A - interalveolar septa; B - peribronchial region; C - perivascular area; D - normal lung tissue.

In the lung tissue, there are single granulomas, infiltrative manifestations, including interstitial, are regarded as scarce. A decrease in the activity of a tuberculosis-specific inflammatory process and the presence of large areas of normal lung tissue inherent in intact mice were expressed.

FIG. 30 shows micrographs of stained sections of the liver tissue of mice infected with MBT. Group No. 3 (Treatment with PK 150 μl / mouse / day).

Visible: A - submicroscopic epithelioid - macrophage granulomas; B - lymphocytic infiltration.

The liver contains areas with a high content of submicroscopic epithelioid-macrophage granulomas; along the septa, only here and there minimal manifestations of lymphocytic infiltration are found.

FIG. 31 shows the ECG of a rat from group I (normal)

FIG. 32 shows an ECG of a group II rat (EIM without treatment) 1 hour after surgery.

Decrease in the ST segment below the isoline (depression) characterizes postinfarction ischemic changes and indicates insufficient blood supply and myocardial hypoxia.

FIG. 33 shows an ECG of a rat from group II (EIM without treatment) 3 days after surgery.

FIG. 34 shows the ECG of a rat from group V (PK therapy at a dose of 500 μl / rat) 3 days after surgery.

The magnitude of ST segment depression is lower than in group II (no treatment, Fig. 32).

FIG. 35 shows an ECG of a rat from group II (EIM without treatment) 7 days after surgery. The magnitude of ST segment depression in group II increased compared to the 3-day period.

FIG. 36 shows the ECG of a rat from group V (PK therapy at a dose of 500 μl / rat) 7 days after surgery.

The magnitude of ST segment depression in group V (therapy with PK at a dose of 500 μl / rat) was significantly less pronounced than in the group without treatment (group II, Fig. 35).

FIG. 37 shows micrographs of stained sections of the myocardium of the left ventricle of the rat heart: intact (a) and group II after 24 h (b), 3 (c) and 7 (d) days after EIM. Van Gieson staining, magnification 40 × 7.

FIG. 38 shows micrographs of stained sections of the myocardium of the left ventricle of the heart of rats from groups III (a), IV (b), V (c) after 3 days and in group V (d) 7 days after EIM. Van Gieson staining, magnification 40 × 7.

FIG. 39 shows the dynamics of skin wound healing in a dog.

Above: PK treatment.

Bottom: treatment without FC (control).

FIG. 40 shows the priming effect of a FA preparation obtained by cold extraction with a buffered saline solution from pretreated larvae: the effect on the chemiluminescence intensity of whole blood neutrophils activated by zymosan.

FIG. 41 shows the effect of FA on the accumulation of calcium ions in mitochondria (A) and the membrane potential of mitochondria (B).

A - changes in the membrane potential of mitochondria in rat liver homogenates (50 μl) in response to successive additions of CaCl ₂ at a final concentration of 50 μM each in the presence of 20 μl of PA (curve 1) and in the control (curve 2).

B - the effect of the peptide fraction of FA (curve 1) and thymalin (curve 2) on the membrane potential of isolated liver mitochondria. The peptide fraction was obtained from 25 μl of the extract; thymalin is a pharmaceutical preparation in the form of a lyophilized powder, 10 mg in an ampoule, supplements of 100 μg each.

Homogenates or isolated mitochondria were incubated in 125 mM KCl, 10 mM Hepes, 1.5 mM phosphate, pH 7.2 in the presence of 4 mM succinic acid. Changes in the membrane potential were determined by a TFP ⁺ - selective electrode.

FIG. 42 shows a general view of FA preparations obtained using live (1) or pretreated larvae (2). In the panel on the left, the extraction time has been cut in half.

FIG. 43 shows photographs of gels after separation of peptides in FA preparations in PAGE and staining with silver nitrate. Lanes 1 and 2 - PK - from live and pre-treated larvae, respectively (the same preparations as shown in Fig. 42 and in Table 22). 3 - FA preparation obtained from pretreated larvae stored for a year at -20 ° C; 4 - Preparation of FA from pretreated larvae stored for 4 years at -20 ° C.

On the left panel, extraction time has been halved.

The numbers above indicate the AOA, determined by the inhibition of adrenaline autooxidation (conventional units / ml of extract).

FIG. 44 shows the results of a study of the AOA of FA preparations obtained as a result of 4-day cold extraction of components from pre-treated larvae or from a lyophilized homogenate of larvae in comparison with a 20-day-old extract from native larvae.

FIG. 45 shows the antioxidant activity of liquid FA (LPA) (a) and sorbitol (b). Antioxidant activity was assessed by the degree of inhibition of adrenaline autooxidation.

FIG. 46 shows the inhibitory effect of LPA on adrenaline autooxidation in the presence of different amounts of sorbitol. To construct the presented graphs, the indicated volumes of sorbitol solution were introduced into the spectrophotometer cuvette, and the rate of adrenaline autooxidation was determined at each sorbitol concentration in the presence of different amounts of added LPA. The reaction rate of adrenaline oxidation without additives was taken as 100% (inhibition is 0%).

FIG. 47 shows AOA LPA in the presence of sorbitol. Determination of AOA was carried out by measuring the degree of inhibition of adrenaline oxidation by the pharmaceutical composition in the presence of different concentrations of sorbitol. The rate of autooxidation of adrenaline in the presence of an appropriate amount of sorbitol was taken as 100%. The percentage of inhibition by the added extract was determined relative to this rate. The upper curve is 200 μl of sorbitol (169 mg of dry matter), the dotted curve is 50 μl (42 mg), the middle curve is 100 μl (84 mg).

FIG. 48 shows the AOA of the dry FA preparation.

A certain volume of a dissolved sample containing from 42 to 169 mg of dry FA preparation was introduced into the measuring cuvette. A carbonate buffer was added, followed by an adrenaline solution, and the rate of adrenaline oxidation was determined by the change in optical density. The rate of adrenaline oxidation in the presence of an appropriate additive of the control sample was taken as 100%. Each point is the average value of the measurement of 3 samples.

FIG. 49 shows the antioxidant activity of a dry FA preparation during 24-day storage under various conditions.

FIG. 50 shows the antioxidant activity of a liquid FA preparation during 24-day storage under various conditions.

Implementation of the invention

Example 1

Detection of peptides and low-molecular-weight proteins in PK by polyacrylamide gel electrophoresis

The purpose of this stage of the work was to study the peptide and (partially) protein composition of FA to identify the main active substances and standardize the quality of the drug. The qualitative and quantitative composition of peptides and proteins was determined by electrophoresis in polyacrylamide gel of various percentages and buffer composition.

For optimal separation of peptide bands, several electrophoresis systems were tested, including native electrophoresis and standard Laemmli electrophoresis in gels of various percentages (15-20% SDS-PAGE in a Tris-glycine buffer system (pH 6.8-8.8)). The best result was obtained in 15-20% SDS-PAGE in a tris-tricine buffer system (pH 8.25-8.9) [44]. This analysis system allows to obtain well reproducible results and, therefore, can be used to standardize the quality of the preparation.

To carry out electrophoretic studies, we used the following buffers and reagents:

Buffers:

cathode: 0.1 M Tris-HCl (high purity, Amresco, Spain, pH 8.25); 0.1M tricine (Ultra pure grade, Amresco, Spain); 0.1% SDS (ultra high purity, Dia-M, Russia)

anode: 02 M Tris-HCl (ultra high purity, Amresco, Spain, pH 8.9);

solubilizing: 0.063 M Tris-HCl (pH 6.8); 5% 2-mercaptoethanol (ultra high purity, Dia-M, Russia); 2% SDS; 0.004% bromophenol blue; 10% glycerin (USP grade, Panreac, Spain).

Reagents:

Acrylamide (Ultra pure grade, Amresco, Spain);

N, N'-methylenebisacrylamide (Ultra pure grade, Amresco, Spain);

ammonium persulfate (high purity grade, Fisher Scientific, USA);

TEMED (OSCH, Helikon, Russia);

Two different approaches were used to stain the gels - standard staining with Coomassie R-250 solution and more sensitive staining with silver nitrate solution. Such a comparative staining at the first stages was necessary for confidence in the protein origin of the bands, since silver nitrate is also capable of staining DNA bands.

In the case of using Coomassie, the gel after electrophoresis was stained for 10 hours in 0.25% Coomassie Brilliant Blue R-250 solution (Sigma, USA) (with the addition of 45% ethanol and 10% acetic acid (high purity grade, Himmed, Russia)) ... The gel was washed in a solution containing 40% ethanol and 7% acetic acid (high purity grade, Khimmed, Russia). The gels were stained with silver nitrate according to the standard procedure [45].

RESULTS

FIG. 4 shows an example of the quality of separation of the fraction of low molecular weight peptides and higher molecular weight proteins using a high-percentage polyacrylamide gel and a tris-tricine buffer system. It can be seen that in such a system, both the low molecular weight fraction of peptides weighing up to 10 kDa and higher molecular weight proteins are clearly separated; a qualitative resolution of peptides and proteins with molecular weights from several hundred Daltons to 50 kDa is provided, which makes it possible to identify the putative active components of FA.

First of all, it was elucidated whether peptides and proteins identified in caterpillar tissues are included in the extract and retained in it, according to literature data, and whether a peptide close to thymogen of higher animals is present in PK. The latter assumption is based on the fact that caterpillars are usually used before pupation to obtain active drugs from VM. This stage consists in the transformation (metamorphosis) of an insect into a new form of life - a caterpillar into a butterfly. This is analogous to the birth of higher animals. At the stage of neonatal and early intensive development, the thymus plays a very important role. It is known that the thymus is the carrier of the young properties of the organism. A decrease in its functions occurs with the maturation of the organism, and in an adult state it greatly decreases. Thymus preparations are used to increase resistance during aging or weakening of the body.

Thymogen is a highly active immunostimulating peptide that has a regulating effect on the reactions of cellular, humoral immunity and nonspecific resistance of the body, and stimulates regeneration processes. Thymogen enhances the processes of differentiation of lymphoid cells, inducing gene expression in lymphocytes, normalizes the number of T-helper cells, T-suppressors and their ratio in patients with various immunodeficiency states. Its detection in PK explains the high biological activity of the drug.

To test this assumption, in addition to standard synthetic molecular weight markers during electrophoresis, three markers were used containing thymogen itself, the dipeptide glutamyl-tryptophan (mw - 0.31 kDa). First of all, this is a preparation from the thymus - thymalin. Timalin - a pharmaceutical preparation, is a total extract of peptides and proteins from the calf thymus. Timalin is produced as a source of therapeutic thymogen, the content of which is very high and prevails over other components. We compared the position of the thymogen band in thymalin with the band of pure thymogen or its synthetic analogue, bestim, which were the same.

Taps with thymogen or bestim were applied in 20 μl at a concentration of 10 μg / ml. The rest of the lanes were loaded with 20 μL of PK. The natural preparation of thymalin (Fig. 4, leftmost lane) and synthetic taps (Fig. 4, far right lane) were used as molecular weight taps. Their mass is indicated in kBa. FIG. 4 numbered 1-5 show 5 different FA samples with high antioxidant activity according to the chemiluminescent method (upper inscription above the lanes) and by the adrenaline method (inscription below the upper one).

Consider an electrophoregram from the leading front of movement from the smallest peptides. This front brings a major discovery that confirms our assumption of the presence of thymogen in the VM preparation. Thymogen in thymalin is represented by a huge thick band at the very leading front of the solution movement along the path. Its position and large amount in thymalin is known from the literature and is confirmed in our definitions by comparison with the band of added thymogen or its analog bestim given in other figures (5 and 6). FIG. 4, it can be seen that exactly at this level in all investigated active preparations of FC there is a clear strip in the form of a cut nail. The amount of this peptide in PK is large, since the band is of the same order of density as the thymogen band. The performed determination shows that the concentration of thymogen in PK is about 5 μg / ml. This strip is reproduced in all preparations with high antioxidant activity (AOA) for adrenaline (Figs. 5 and 6), but its intensity decreases or it disappears in preparations with low AOA (Figs. 7, 8, 9). Thus, the presence of thymogen is beyond doubt, since it is confirmed by three natural taps, a high content, the presence of FAs with high antioxidant activity in the samples, and disappearance with the loss of this activity.

It turned out that the content of peptides with a mass of between 4 and 8 kDa in FA, which are known for VM caterpillars, is lower than that of thymogen: from 0.5 to 5 μg / ml. However, their presence is observed in all preparations. It is minimal in the last sample with minimal antioxidant activity. Peptides of this group have very high lytic activity and therefore can make a significant contribution to the biological activity of PA. According to the literature, this group may include the following peptides:

1. Cecropin-D-like peptide, Mw = 4.256 kDa, 39 amino acids.

2. Lebocin-like antimicrobial peptide 1, Mw = 4.819 kDa, 42 amino acids.

3. Defensin-like peptide, Mw = 4.949 kDa, 44 amino acids.

4. Anionic antimicrobial peptide 2, Mw 6.980 kDa, 60 amino acids.

5. Defensin (Haliomycin), Mw = 7.823 kDa, 72 amino acids.

All these components have high lytic and antibacterial activity against a number of gram-positive (Micrococcus luteus, hysteria monocytogenes, Sternbergia luted) and gram-negative (E. coli) bacteria. In addition, they are able to exhibit fungicidal action against Aspergillus niger, Candida albicans, Candida fructus, Candida wickerhamii, Pichia pastoris, Pichia tannophilus, Trichoderma harzianum and Zygosaccharomyces marxianus. [46, 47]

Further, at the level of the tap strip of 18 kDA and slightly below, one major strip (above) is noted, somewhat weaker (below). They are almost absent in the last sample with lower antioxidant activity.

The relative concentrations of these substances in the preparation ranged from 0.5 to 5 μg / ml of active peptides, from 1 to 10 μg per ml of SOD, and from 0.1 to 1 μg / ml of lysozyme.

Considering this group in the same order from bottom to top, according to the literature [46-49], it can be assumed that the following substances found in the tracks of VM are identified in FA;

1.14.027 kDa. Lysozyme, 121 amino acids. First of all, it has bacteriolytic activity. In addition, it is associated with the macrophage system and enhances the activity of immune cells. 0.1-1 μg / ml.

2.16.3-17 kDa. Monomer Cu-SOD insect superoxide dismutase, which has a pronounced antioxidant activity. 1-10 μg / ml.

3. ~ 17.5 kDa - Apolipoforin III. A specific protein of the wax moth, which binds to lipopolysaccharides of the bacterial cell wall and enhances the lytic action of lysozyme [49]. Up to 100 μg / ml.

The presence of apolipophorin III is especially likely in the form of the most intense band at the level of the 18 kDa marker corresponding to the mass of this peptide. Due to its high lytic activity and high content, apolipophorin can play a leading role in the regulation of the state of the human body, which is discussed further in example 10.

Studies have shown that the preparation from BM obtained by the method according to the invention contains a wide variety and in sufficient quantities of biologically active peptides and proteins, mainly with antioxidant and lytic activity.

The dependence of the FA composition on the preparation and storage method was monitored separately. These results are shown in FIG. 5 when stained in two ways.

FIG. 6 shows the results of electrophoresis of samples after 20 days of extraction obtained from live larvae (1), from lyophilized fresh larvae (2), lyophilized larvae stored for 1 year at -20 ° C (3) and from lyophilized larvae after three years of storage at - 20 ° C (4). FIG. 6 that the optimal quality was obtained in the third variant of FA preparation, which was confirmed by the maximum AOA of this FA (400 cu / ml for adrenaline versus 286 in variant 2 and 47 in variant 4). Also, electrophoresis of samples was carried out after 10-day extraction, where the intensity of the bands was much lower, like the AOA of PK. Therefore, in the future, the preparation was extracted using the third method.

FIG. 7 shows the identification of the thymosin beta-4 band in PK when compared with a synthetic analogue (44 amino acids, molecular weight 4.8).

FIG. 6 shows the identification of the thymogen band in FA when compared with natural taps - thymalin, thymogen and its analogue - bestim.

FIG. 6, like others, also shows that the most mobile band of the fraction of low molecular weight peptides corresponds to the band of commercial thymogen, thymogen in thymalin, and bestim. As in other definitions, the content of thymogen is about 5 μg / ml.

In this definition, as in FIG. 4, several proteins with molecular weights between 14-18 kDa are present, which corresponds to the lysozyme, insect SOD and apolipophorin III described in HM.

These and other data show that the AOA of the drug correlates well with the concentration of peptides in it, and prolonged storage at room temperature reduces both indicators. An additional experiment was performed with multiple parallel samples that were stored at room temperature or in a refrigerator for 12 months. In parallel with the electrophoretic studies of the protein-peptide composition, the antioxidant activity was measured by two different methods - by adrenaline and by chemiluminescence. The result of this study is shown in Fig. 8. A natural preparation "Timalin", standard markers, was used as a tap.

FIG. 8 shows that the AOA activity of PA correlates with the content of the low molecular weight fraction of peptides - the brighter it is, the higher the AOA activity - and also with the content of Cu-SOD superoxide dismutase in the preparation: on all gels - a band with a molecular weight of 17 kDa - the second band of markers from the bottom. At the same time, it should be noted that when stored in heat, AOA disappears by the adrenaline method, and when stored in cold, it slowly decreases (Fig. 14, 15, 17).

These data show that long-term storage of FC should be carried out in the refrigerator. In addition to this method of preserving active substances, we investigated the possibility of their protection with the help of approved protectors.

FIG. 9 shows the result of a similar experiment - the preparations were stored in a warm or refrigerator for 21 months. Carbon dioxide and a special protector - dihydroquercetin in concentrations of 0.1 and 1 g / l were used as possible protectors.

FIG. 8 and 9 clearly show that prolonged storage in heat leads to a decrease in the protein-peptide composition of FA.

The addition of carbon dioxide even further degrades the FA components. The addition of dihydroquercetin at a lower concentration is not very effective. But at a concentration of 1 g / l, it significantly increases the safety of proteins and peptides.

The importance of controlling the storage conditions and preparation of the preparation to ensure its high quality follows from the analysis of the last sample shown in FIG. 9. This electrophoresis was applied to an artisanal preparation sold on the market without proper documentation, designated as “foreign”. Even trace amounts of possible active protein-peptide components are not observed in this preparation.

Example 2

The content of amino acids, peptides, AOA activity and carboxylic acids in FA, depending on the method of production

General conditions for growing caterpillars

To obtain a pharmaceutical composition, the wax moth caterpillars were used, cultivated in the laboratory and production conditions of the Wax Moth Nursery, ITEB RAS, in compliance with the year-round temperature, light and humidity conditions of the premises. Caterpillars were grown in open containers. The food used was dark waxy dry land and merv in a 1: 1 ratio. In some cases, caterpillars were grown only on dark waxy land without additional feeding. The containers with the larvae were periodically sprayed with a 20% solution of sugar syrup. For the preparation of PA, caterpillars were collected that were at the last stage of development, before pupation. Further, the extraction of FA components was carried out either directly from live caterpillars or from caterpillars subjected to preliminary freezing or lyophilization. In the first case, the selected caterpillars, after removing the cobweb and the remnants of the wax land, were placed into glass vessels and a solution of rectified ethyl alcohol was added (at the rate of 450 ml of extractant solution per 100 g of caterpillars). Extraction was carried out at 20-25 ° C in the dark, with periodic stirring for 21 days. The extract was filtered. Then a secondary extraction was carried out by adding the extractant at the rate of 20% of the initial volume. Extraction was carried out for 2-3 days, followed by filtration. The same method was used for extraction from pre-treated caterpillars: frozen at -20 ° C or lyophilized caterpillars. Lyophilization was performed on an LS-500 unit (Russia). Six variants of PK were prepared as indicated in Tables 1 and 2, differing in the methods of growing caterpillars and preparing extracts. For each FC variant indicated in Tables 1 and 2, as well as in FIG. 10, 4 separate preparations were obtained, which were then mixed in equal proportions and the dry matter content was determined in the obtained extracts (according to GOST R 51437-99).

Determination of amino acids

The amino acid content was determined at IL Test-Pushchino LLC using the HPLC method on the YL-9100-Pinnacle PCX amino acid analyzer (according to the Comission Regulation (EC) protocol No. 152/2009). The amino acid content in the above FA preparations is shown in Table 1.

As follows from Table 1, PK contains a wide range of different amino acids. It draws attention to the fact that the highest concentrations are presented by amino acids - participants in the transamination process: ASP, GLU-Glutamine, GLI, ALA. The activation of transamination leads to protection against hypoxia, since it bypasses the stages in mitochondria that require the participation of oxidized NAD. ABM is also associated with the regulation of tissue hypoxia (in interaction with CHF), the content of which is also increased. GLI has a sedative effect in the body, which is important for the anti-stress action of PK.

As follows from table 2, with any method of obtaining FA, the total amino acid content is higher when growing caterpillars on a combined feed. The total amino acid content is slightly higher (1.2 times) in FA preparations obtained from live caterpillars, while AOA in them is significantly lower (up to 2 times) than in FA preparations from pretreated caterpillars. This may indicate the degradation of peptides in preparations from live caterpillars, since the AOA value was shown to correspond to the level of peptides (Fig. 10, as well as example 1). In comparison with the prototype, the content of amino acids with the proposed modifications of the preparation increases, since the dry residue increases - about 1% in the prototype and on average 1.7% in the declared FC.

To determine the dry residue and alcohol content, extracts obtained on different types of food were combined. * For 1 conv. Unit a volume of extract was adopted that, when added to the model system, leads to the same changes in the CL latency period as the addition of trolox at a concentration of 1 μM.

The dry matter content in the resulting FA preparations averages 1.7%, which exceeds the dry matter content (about 1%) in the wax moth extract described in the analogue [16].

Determination of peptides and AOA

In addition to amino acids in the obtained extracts, the content of peptides was determined by electrophoresis in PAGE as indicated in example 1, as well as antioxidant activity (AOA). AOA was determined by inhibition of luminescence accompanying thermal decomposition of α, α'-azodiisobutyroamidine dihydrochloride (ADBA) as described in example 3.

The unit of antioxidant activity of the studied drug was taken to be its volume (in μL) which, when added to the model system, leads to the same changes in the CL latency period as the addition of trolox at a concentration of 1 μM. AOA was expressed in arbitrary units in 1 ml of FA. The results are shown in FIG. 10. As shown in FIG. 10, the content of peptides is higher when pretreated caterpillars are used to obtain FA as compared to live ones. The maximum content of peptides is observed when the stage of freeze drying is included in the procedure for obtaining FA. In FA preparations obtained by this method, the maximum AOA is also observed. This once again shows the relationship between the content of peptides and AOA activity, which was noted in a number of comparisons.

Determination of carboxylic acids

To detect organic acids in the extract, it was analyzed by high performance liquid chromatography on Aminex HPX-87H columns for the separation of organic acids (300 × 7.8 mm; Bio-Rad, United States). 4 mM H2SO4 was eluted at a rate of 0.5 ml / min at a temperature of 35 ° C. Acids were detected spectrophotometrically at 210 nm. The column was calibrated with standard solutions of the expected acids: isolicic, citric, alpha-ketoglutaric, (Boehringer; Mannheim, Germany). Enzymatic methods were used to quantify the content of organic acids in the extract. To determine α-ketoglutaric acid, a reaction catalyzed by glutamate dehydrogenase was used [50]. Under the action of the enzyme in the presence of ammonium ions, α-ketoglutaric acid is converted into glutamate. NAD ^{+ is} reduced to NADH in an amount equivalent to the amount of α-ketoglutaric acid present in the medium. To determine isocitric acid, NADPH was measured, which is formed as a result of the conversion of isocitric acid into α-ketoglutaric acid under the action of the enzyme NADP + - dependent isocitrate dehydrogenase [51].

The oxidation of isocitric acid is accompanied by the formation of reduced NADPH ₂ , the amount of which was measured on a Spekol spectrophotometer at a wavelength of 340 nm. The measurement accuracy of isocitric acid by this method is 90-95%. Table 3 presents data on the content of carboxylic acids in FA. The table shows the results of determination in five samples obtained in parallel FA (prepared using pretreated caterpillars).

As follows from Table 3, the content of alpha-ketoglutaric acid (which due to the presence of the keto group is known as an unstable substance) decreases during storage of FA. Cold storage slows down the decline in alpha-ketoglutaric acid. Isolic acid content remains constant even when stored for a long time at room temperature.

Example 3

Antioxidant activity (AOA) of the pharmaceutical composition

It is generally accepted that for a complete characterization of antioxidant properties in preparations that are a mixture of various compounds, it is necessary to use different methods for determining AOA, since this makes it possible to reveal in the mixture components with antioxidant activity of different mechanism of action [52]. Therefore, in this work, AOA studies were carried out using several methods described below, and for express quality control of PK, two of the most informative and convenient ones were chosen.

The antioxidant activity of the pharmaceutical composition (PK) was tested in various model systems.

The following methods were used to characterize the antioxidant activity of FA:

1. Determination of antioxidant activity (AOA) by inhibition of adrenaline autooxidation;

2. Determination of AOA by inhibiting the reduction of nitro blue tetrazolium in the xanthine oxidase reaction;

3. Determination of AOA by inhibition of luminescence accompanying thermal decomposition of α, α'-azodiisobutyroamidine dihydrochloride (ADBA) using the antioxidant trolox as a standard;

4. Determination of AOA by inhibition of luminol-dependent chemiluminescence of activated neutrophils.

1. Determination of AOA by autooxidation of adrenaline

To determine AOA by autooxidation of adrenaline, the method of T.V. Sirota [53], which is a modification of the method of Misra NR, Fridovich I. [54]. The antioxidant activity of AOA was measured by the degree of inhibition of adrenaline autooxidation under standard conditions when different volumes (5-200 μL) of the drug were added to the reaction mixture. The required sample volume was added to 2 ml of 0.1 M carbonate buffer pH 10.6; 100 μL of 0.1% adrenaline solution and the rate of adrenaline oxidation was measured at 347 nm at room temperature (20 ° C). The rate of adrenaline oxidation without added FA sample was taken as 100%. The inhibition of the rate of adrenaline oxidation was plotted against the amount of sample added and the amount of sample required for 50% inhibition of the rate of adrenaline autooxidation was determined (FIG. 11). This amount (in μL) was taken as a conventional unit of AOA.

In the examples shown in FIG. 11 graphs - curves of inhibition of adrenaline autooxidation by different doses of FA samples. From these curves, we find the amount of FA required to inhibit the rate of the autooxidation of adrenaline by 50%, which is taken as 1 unit of AOA. FIG. 11 shows five separately obtained in the same way, but from different batches of raw materials of FC samples. Table 4 shows the calculated AOA values for these samples in arbitrary units.

As shown in FIG. 11 and Table 4, the AOA in the obtained samples is very close. During the autooxidation of adrenaline in an alkaline environment, a large number of superoxide anion radicals are formed, therefore this reaction is used to determine the activity of superoxide dismutase.

2. Determination of AOA in xanthine oxidase reaction.

To confirm the presence of superoxide dismutase activity in FA, a test with a xanthine oxidase reaction was used, which is used to generate superoxide radicals, which can be detected by the formation of a colored product of their interaction with nitro blue tetrazolium. The determination was carried out according to the method described [55]. In this reaction, PK also exerted a dose-dependent suppression of the formation of a colored product, i.e. showed the ability to inactivate superoxide anion radicals (Fig. 12).

Thus, the ability to inactivate superoxide anion radicals was found in FA by two methods. The results obtained indicate the presence of superoxide dismutase in FA, which is consistent with the data of electrophoresis (example 1)

3. Determination of AOA by inhibition of ADBA luminescence.

The ability of FA to inactivate organic radicals was tested by inhibition of luminescence accompanying thermal decomposition of α, α'-azodiisobutyroamidine dihydrochloride (ADBA). Upon heating, ADBA molecules undergo thermal degradation, decomposing into two radicals, the interaction of which with oxygen forms peroxyl radicals [56]. The interaction of these radicals with luminol leads to its oxidation, accompanied by chemiluminescence (CL). When a radical interceptor is introduced into the system, competition for a radical between luminol and the interceptor is created in the system, which is accompanied by CL quenching and the appearance of the so-called latency period. The duration of the latency period is determined by the time during which the antioxidant is completely consumed. In other words, as long as there are interceptors of peroxyl radicals ADBA in the model system, luminescence will not develop until then, and the duration of the latency period determines the antioxidant activity of the inhibitor.

To test or quantify the antioxidant activity of the substances under study, model oxidation systems are calibrated using a standard antioxidant [57]. This approach was applied in this study. Trolox, a water-soluble analogue of vitamin E, which is a good interceptor of peroxyl radicals, was chosen as the standard antioxidant [58].

Taking into account the fact that the dependences of the change in the latent period of CL on the volume of the studied drugs and the concentration of trolox were linear, the activity of the drugs was expressed through the equivalent concentration of trolox. So if the equation of the dependence of the change in the latent period of CL on the volume of the studied drug has the following form:

- константа изменения латентного периода ХЛ для исследуемого вещества; V - объем исследуемого раствора, который добавляется к модельной системе;where t / to is the change in the latent period of CL;

- constant of change in the latent period of CL for the test substance; V is the volume of the test solution, which is added to the model system;

and a similar dependence for trolox:

- константа изменения латентного периода ХЛ для тролокса; C_T - концентрация тролокса в модельной системе; то можно записать:where t / to is the change in the latent period of CL;

- constant of changes in the latent period of CL for trolox; C _T is the concentration of trolox in the model system; then you can write:

Consequently, as a measure of the antioxidant activity of the studied drug, one can take such its added volume into the model system, which leads to the same changes in the CL latency period as the addition of trolox at a concentration of 1 μM:

The following reagents were used in the work: luminol, trolox (Serva, Germany), α, α'-azodiisobutyroamidine dihydrochloride (Fluka, Germany).

CL in the model system of luminol oxidation induced by peroxyl radicals formed during the thermal decomposition of the azo compound ADBA (luminol – ADBA system) was recorded as follows [59]. A certain volume of the test preparations or trolox solution (final concentration 1 μM) was added to the reaction medium containing a buffer solution with luminol. Then the ADBA solution was added to a final concentration of 10 mM, and the CL kinetics was recorded for 10-60 minutes. Measurements were carried out using a SmartLum-5773 chemiluminometer and PowerGraph software. The CL latent period (t) was used as a measured parameter, which is the time from the moment of ADBA administration to the onset of luminescence development. The latent period of chemiluminescence was found for each drug (t _X ) and trolox (t _T ).

Using this approach, the antioxidant activity of several separately obtained FA samples was evaluated. We used the same FA samples, which are presented in Table 4. For liquid FA samples containing a food solvent, control samples with corresponding volumes of solvent were set. The results are shown in Table 5.

* the volume added to the model system, which leads to the same changes in the CL latency period as the addition of trolox at a concentration of 1 μM.

It can be seen from Table 5 that the volumes of samples that must be added to the model system in order to obtain an antioxidant activity equivalent to 1 μM of trolox, averaged 1.98 ± 0.13. The right column shows the calculated AOA of the samples, expressed in arbitrary units per 1 ml of the sample. For 1 conventional unit of the antioxidant activity of the studied drug, the volume added to the model system is taken, which leads to the same changes in the CL latency period as the addition of trolox at a concentration of 1 μM.

From table 5 it follows that the average AOA of the investigated FA samples is 506 ± 33 conv. units / ml.

Thus, as follows from table 4 and table 5, the method of obtaining the declared PK allows you to obtain drugs with high and reproducible AOA values.

Estimation of AOA using this method is close to determining AOA using the well-known ORAC method, since also uses the azo compound AAPH (2,2'-azobis (2-amidino-propane) dihydrochloride) as a source of radicals and trolox as an antioxidant standard, although the AOA values obtained cannot be directly converted to the units used in the ORAC method.

4. Determination of antioxidant activity ex vivo

Antioxidant activity was also determined in the ex vivo system by inhibition of luminol-dependent chemiluminescence accompanying the respiratory burst of neutrophils upon their activation by zymosan. Heparin-stabilized human capillary blood was added to a chemiluminometer cell containing Hanks solution with luminol. A certain volume of the Pharmaceutical Composition or the corresponding solvent was also introduced into the cell. Opsonized zymosan was added and chemiluminescence recorded. At the same time, spontaneous chemiluminescence was recorded in another cell of the chemiluminometer. The integral intensity of chemiluminescence was expressed in arbitrary units and was determined by measuring the area under the chemiluminescence curve (Fig. 13).

As shown in FIG. 13, when added to the reaction mixture 0.22%; 0.9%; 2.3% FC (V / V), the luminescence intensity decreased by 25%, 37%, 52%, respectively. It has been established that PK dose-dependently reduces the chemiluminescence of luminol during respiratory burst of neutrophils.

Advantages of the AOA method with adrenaline

As indicated above, the AOA pharmaceutical composition has been confirmed by various methods. The method for determining the autooxidation of adrenaline turned out to be the most convenient for rapid assessment of the quality of the obtained FA samples. it is informative, the simplest and does not require expensive reagents. In addition, this method is better than the chemiluminescent method in the ADBA-luminol system, reveals differences in AOA in different FA preparations, as well as changes in AOA that occur during storage of preparations (Fig. 14).

As shown in FIG. 14, when stored in the cold, the AOA of preparations does not change, while when stored at room temperature, the ability to inhibit the autooxidation reaction of adrenaline decreases, but the ability to inhibit chemiluminescence accompanying the thermal decomposition of ADBA does not decrease during storage at room temperature.

The use of both methods for the determination of AOA in PK makes it possible to differentiate two components of AOA: thermolabile with superoxide dismutase activity and thermostable with antiradical activity.

It was found by the method with adrenaline that when stored in the light, an even more rapid decrease in AOA occurs. (Fig. 15).

Determination of AOA with adrenaline made it possible to reveal differences in the preparations obtained by different methods (Fig. 16).

Determination of AOA with adrenaline reveals the differences between the preparations obtained by different methods, from different raw materials, and also allows us to identify changes in the composition of FAs that occur during improper storage (not only FA (Fig. 8, 16, 50), but also raw materials (Fig. 17)). At the same time, for the express analysis of the quality of the obtained samples, one can do without plotting the dependence of the degree of inhibition of adrenaline autooxidation on the amount of the applied drug, and carry out the measurement at one applied dose, for example, 50 μl. In this case, the degree of inhibition in high-quality drugs should be at least 60% (usually 70-85%).

The differences detected correlate with the differences detected by the electrophoresis method (Fig. 17, as well as example 1).

Example 4

Antistress effect of PK on the model of adrenaline administration and psychoemotional stress in rats

This example presents the results of a study of the anti-stress action of FC. Investigated the effect of stress on the energy functions of mitochondria, as well as the immune functions of the blood. The studies were carried out on a model of physiological stress on animals before reaching maturity with intraperitoneal adrenaline (ADR) injection and also on a modern model of painless psychoemotional stress (PES) of rats when placed for a short time (30 min) in a plastic ventilated box that restricts movement. The effect of stress was studied by the responses of mitochondria (the activities of succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (CDH), as well as the accumulation of calcium ions by mitochondria, and the responses of the blood system (by the change in the cellular composition of the blood induced by zymosan and spontaneous chemiluminescence, which are described below). presented in the links of the section "Disclosure of the invention" and in publication [23].

Model protocol.

The experiments were carried out on Wistar rats, breeding the ITEB vivarium, 6 weeks old, weighing up to 200 g. A group of animals (6-8) for one series was selected from litters of the same date of birth and jigging. During deposition, animals of the same series were kept in one cage and got used to each other. In all other respects, the maximum standardization of conditions was carried out to maintain the state of rest of the animals. The study of the effect of stress and the effect of the studied drug on it was carried out simultaneously on the entire group of animals, which were prepared for the experiment within one calendar period and euthanized in one day, in a compressed period of 10-12 hours. This minimizes the variation in animal conditions on different days and times of the day. According to previous personal and literary observations, the state of a group of homogeneous animals is similar on one day, but changes on different days, and the changes in individual animals of the group are similar. Experimental animals received 0, 25, or 1 ml of FA applied to bread according to several schemes: 5 days of feeding - 2 days off - 1 day of feeding, or for 10 days without a break (see figure captions). The preparation was fed after drying in air to remove alcohol.

Stress was also simulated by administering to animals a moderately high dose of ADR 50 μg / 100 g of body weight intraperitoneally 30 minutes before slaughter. Alternatively, the animals were subjected to a 30-minute RPE by placing them in a chamber restricting movement, but without pain. This pattern corresponds to physiological emotional stress.

The animals were euthanized in a sealed chamber pre-filled with carbon dioxide, which ensured the preservation of a calm state in the animals - euthanasia without excitation. After 15-25 s. the animals stopped breathing, but the heart continued to work, then the rat was quickly removed from the chamber, decapitated, and, after draining the first drops of blood, blood was taken for research.

Research methods

Weighing organs. Immediately after decapitation, the thymus, adrenal glands and spleen were weighed, not allowing the organs to dry out.

Research on the energy functions of mitochondria

1. Cytobiochemical determination of dehydrogenase activity.

The activity of two dehydrogenases, succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (CDH), was determined, reflecting the regulation of the sympathetic (adrenaline) and parasympathetic (acetylcholine) nervous systems. The measurements were carried out in blood lymphocytes on a smear according to a previously developed cytobiochemical method, which is highly sensitive to changes in the body due to the combination of the advantages of cytochemical and biochemical methods. The measurement of the activity of dehydrogenases for the reduction of nitroblue tetrazolium (NBT) was carried out in a freshly prepared blood smear after incubation for 1 hour at 37 ° C in a cytobiochemical medium containing 125 mM KCl, 10 mM HEPES, 1.22 mM NBT, pH 7.2 ± 0.05 as well as substrates. For all procedures, bidistilled water was used. After incubation, the smears were rinsed with bidistilled water, and the nuclei were counterstained with 0.5% neutral red for 8 min. The smears were examined under oil immersion on a Mikmed-2 microscope (Lomo, Russia), at a magnification of 6.3 × 100 / 1.30, equipped with a MINTRON CCD MTV-1 802 SV video camera (Candela, Russia). 30 lymphocytes were selected from blood smears, their video photographs taken through a red filter were placed on one tablet in a computer, and the whole picture was subjected to quantitative morphometric analysis using the Image Tool software, version 2 (The University of Texas Health Science Center in San Antonio, USA) ...

The advantage of the method, carried out by computer video microscopy of many stained objects in 30 lymphocytes, is the high statistical reliability of each individual value. The value of the enzyme activity is determined automatically by scanning the areas of many colored reaction products - formazan granules contained in 30 cells and calculating the average of them. The number of colored objects n is 250-350 for samples with average activity up to 600-700 for samples with high activity and about 100 for samples with low activity. The results are presented for each individual as the average values of the area of colored objects (M) in each sample. For all the obtained mean values, the standard deviation (σ), the error of the mean (± m) are calculated, and the reliability of the found mean values (р) is assessed using the Student's t-test using Microsoft Office Excel 2003. Due to the large sample size, in all presented cases, the mean values are statistically significant 99.9% (p <0.001). Thus, the data obtained for each individual animal is statistically representative. Thanks to this, it is possible to compare the individual data of individual individuals with each other.

2. Absorption of calcium ions

These studies were performed on a liver homogenate by measuring the absorption of calcium ions by mitochondria - the capacity of mitochondria - which was determined by the change in membrane potential in response to successive additions of calcium ions at concentrations of 50 μM. The mitochondrial membrane potential was measured with a TFP + -selective electrode with computer registration. Mitochondria were incubated in a medium of 120 mM KCl, 10 mM HEPES, and 1.5 mM phosphate, pH 7.25 at a TFP + concentration of 1 μM.

3. Study of the cellular composition of blood and immune function of neutrophils. In addition to the treatment of rat blood smears by the cytobiochemical method, the blood of the same rats was taken to measure the cellular composition of the blood by the conventional method and the immune activity of neutrophils (NF) by recording the chemiluminescence induced by the addition of zymosan [60]. The blood obtained during decapitation was mixed in a 1: 1 ratio with Hank's medium, which does not contain calcium, pH 7.45, and contains 40 units / ml of heparin. The blood was stored at 4 ° C. Measurement of chemiluminescence was started 1 hour after blood collection. Into the cells of a chemiluminometer (IBK RAS) containing 200 μL of Hanks medium with calcium (CaCl ₂ 1 mM) and 50 μM luminol, pH 7.45, 20 μL of blood was added, after 10 minutes opsonized zymosan was added (the final concentration in the cell was 217 μg / ml) and recorded the activated chemiluminescence at 37 ° C. At the same time, spontaneous chemiluminescence was recorded in a separate cell. The integral intensity of chemiluminescence, expressed in arbitrary units, was determined by calculating the area under the chemiluminescence curve.

Statistical analysis was performed according to Student's t test; mean values ± S.E.M.

In total, 6 experiments were carried out in which 24 animals were studied, of which 10 received FA.

The following results were obtained.

The effect of FA on the activity of mitochondrial dehydrogenases

This section presents data from the study of the effect of PK on the activity of SDH and CDH in the mitochondria of blood lymphocytes. It is known that these enzymes are participants in the substrate-hormonal regulation system that binds mitochondria and the autonomic nervous system through the signal interaction of the substrates YNT and CHL and mediators - adrenaline (ADR) and acetylcholine (ACH) [For more details, see the section - "Disclosure of the invention"] ... SDH activity reflects energy expenditure for cell functions, as well as the activity of sympathetic regulation, the activity of CDH - providing recovery processes and the activity of parasympathetic regulation.

Control animals at rest are characterized by low SDH activity. Under the action of adrenaline or stress, its strong increase is observed - by 2-3 times or more. However, even without controlled stimulating effects in control animals, a moderate increase in SDH is found. Such animals can be seen as more agitated.

In the example shown in FIG. 18 shows the effect of PK on two identified groups with moderately high and low SDH activity in the control.

Panel A. of FIG. 18 shows the data for the group, which, judging by the control indicators, is in a state of less complete rest with slightly increased enzyme activity. In this group, the classic manifestation of acute stress is observed by the administration of the stress trigger hormone ADR. It is expressed in a strong activation of functions, in this case, the activity of SDH, which ensures work. The dose of ADR used is high in terms of its content in the body. In addition, unlike excitation in the body, when ADR is released accompanied by ACH, an artificial predominance of ADR, not balanced ACH, is created in the experiment. A sharp activation of SDH is visible. However, it exceeds the adaptive capabilities of the tissue, which is indicated by the catastrophic decrease in DRG.

This indicates a violation of normal recovery and an increased risk of pathology.

The protective effect of pre-consumption of FA is extremely pronounced. It protects against stress by restoring DRG activity. As we have shown earlier, this mechanism also lies in the normalization of SDH activity, which is manifested in a decrease in its hyperactivation. It should be emphasized that moderate SDH activity after FA feeding is observed against the background of ADR addition. Thus, PK helps to resist strong stressful effects, that is, it exhibits an anti-stress effect and is an indication of the cholinomimetic (similar to ACH) action of PK.

Panel B of FIG. 18, the effect of PK was tested against the opposite background - groups with initially low SDH activity. This group is an example of an organism in a calmer, more balanced state. He does not respond to the introduction of ADR with a violent reaction, as in the first group. On the other hand, CDG, although it decreases, nevertheless remains active, despite a sharp violation of the regulation of ACH by the introduction of ADR. The strengthening effect of FA on balanced animals is manifested in the provision of a full-fledged response to ADR, which even exceeds the response in less calm animals. This is very pronounced in both the SDH activity and the DRH activity. This action is also anti-stress, since it restores an active response to stress, and without disturbing the recovery processes supported by CDH activity.

Figure 19 shows the protective effect of pre-consumption of FA on mitochondrial function in a milder, life-like stress model of nervous tension caused by placing a rat in a capsule. With this effect, the response of the organism is regulated by the combined effect of the release of ADR and ACH. The answer is presented in comparison with the impact against the background of the introduced ADR. It can be seen that the anti-stress action of PK is manifested in both cases.

In the above experiment, a long-term storage in a box was used - for 24 hours. During this time, according to the data of other experiments, the activation of SDH passed its maximum and passed into the phase of inhibition. DRG was completely inactive, as in the case of ADR. However, the consumption of FA led to an increase in the activity of this enzyme important for recovery. Its activation, as noted above, leads to a decrease in SDH hyperactivation.

Thus, the study of the effect of PK against the background of various stressors shows a decrease in adverse reactions in the body, that is, an anti-stress effect.

Effect of FA on the absorption of calcium ions

The influence on the energy functions of mitochondria was also measured by the consumption of calcium ions by them, which requires a large expenditure of energy. This measurement is shown in FIG. 20.

In this study (in contrast to the measurement of enzyme activity without a workload (Fig. 18 and Fig. 19), the enzymes are studied under a high load on the transport of calcium ions. Therefore, when ADR is administered in combination with a high calcium load, no activation of energy supply is observed, as in Fig. 18 and 19, and its decrease. In relation to control, the work of mitochondria decreases with the introduction of ADR, which is evident from the decrease in the number of portions of accumulating calcium. The consumption of FA has a strongly pronounced protective effect, providing with the introduction of ADR: This is significantly higher than in the control - 5.

The lower panel shows the action of FC, investigated against the background of an even softer RPE than discussed above - placed in a box for 30 minutes. A milder stress and calcium loading retains an activating effect on mitochondria, which is manifested by an increase in the accumulation of ions in comparison with the control. PK removes stress-induced activation. This indicates the preservation of calmness during arousal, that is, anti-stress action. This action is aimed at hyperactivation caused by stress, since PK does not affect work in control, at rest. The shown action of PK can be considered as the preservation of calmness during excitation - sedative or anti-stress.

Thus, according to the responses of mitochondria when feeding FA in groups with activation of ADR or RPE, a pronounced anti-stress effect of the drug was revealed: its administration reduces hyperactivation of SDH (absolute and relative to CDH), which corresponds to sympathetic hyperactivation in the body. The inhibitory effect of FC on the activity of SDH is based on the preservation of the activity of CDH during exercise, which supports the recovery processes. In terms of meaning for the body, this corresponds to the regulation of ACH. This action should help reduce the risk of developing pathologies of sympathetic hyperactivity (in particular, chronic stress, hypertension).

Anti-stress action of PK on the effect on spleen weight

A clear, quick and convenient indicator for assessing acute stress is a rapid change in the weight of the spleen, which depends on a noticeable increase in blood circulation, but also on the activation of stress reactions, in particular, the formation of uncoupling protein [23]. In this work, the weight of the spleen was measured in parallel with the accepted indicators of stress - changes in the weight of the thymus and adrenal glands. However, changes in the weight of these glands appear under stronger and less natural influences than the models used. Spleen weight changes were most pronounced. The same trend was observed in all groups (Fig. 21). In an animal under stress, caused by the administration of epinephrine 50 μg / 100 g of body weight, a significant increase in the weight of the spleen was observed, and the intake of PA in all cases prevented this increase. The detected increase in the blood supply to the spleen may have something in common with hemorrhage of the gastrointestinal tract, which develops under stress and leads to the appearance of gastric and duodenal ulcers.

Effect of FA intake on cellular composition and parameters of blood chemiluminescence during psychoemotional stress (PES) and adrenaline administration

It is known that stress changes the cellular composition of the blood and the state of the hematopoietic organs [61-63]. Therefore, the effect of PK on the cellular composition and functional activity of leukocytes in whole blood was investigated under stress caused by the administration of ADR and at 30 min RPE. The functional activity of neutrophils was assessed by the value of chemiluminescence arising in response to the stimulator of the immune response of phagocytes - zymosan.

Effect of stress and PK on induced blood chemiluminescence

Zymosan-induced chemiluminescence reflects the magnitude of the immune response of blood cells, mainly neutrophils (respiratory burst). Administration of ADR and 30 min RPE led to a weakening of the immune response, as evidenced by a decrease in induced chemiluminescence. PK, as well as RPE, reduced the chemiluminescence response in control animals. However, at the same time, in animals receiving FA, not only did the decrease in chemiluminescence in response to stress (lower panel of Fig. 22) did not occur, but on the contrary, it was restored almost to the level of control. This action is similar to example B in Fig. 18, when the response of SDH to stress in animals with initially low SDH activity was enhanced by FA intake. The decrease in chemiluminescence caused by the administration of ADR (lower part of Fig. 22) was not attenuated, but increased in animals treated with PK. This can be explained by the sharper, nonphysiological effect of ADR, noted above, compared to the model of psychoemotional stress, when stress in the body is supported by the joint influence of ADR and ACH. These data show that NF activity is higher with the participation of ACC regulation.

Since the value of the chemiluminescent response of blood is determined by both the number of neutrophils and their functional activity, the effect of stress on the number of neutrophils was investigated and their functional activity was assessed by the intensity of chemiluminescence per 10 thousand cells.

Effect of stress and PK on induced neutrophil chemiluminescence

As shown in FIG. 23, administration of ADR reduced the functional activity of NF, assessed by the value of the chemiluminescent response to a high dose of zymosan per 10 thousand NF, while 30 min RPE activated it, i.e., ADR suppresses NF activity, and ACC activates it ... The FA intake did not restore activity against the background of ADR administration, but prevented the increase caused by RPE. This can also be explained by the more gross stressor effect of the administered ADR in comparison with the natural response regulated by the balanced influence of the physiological concentrations of ADR and ACH.

The decrease in blood chemiluminescence observed with RPE (Fig. 22) is determined by a decrease in the amount of NF in the blood (see below). The functional activity of NF reduced by the introduction of ADR is not compensated even by an increase in their amount in the blood (Fig. 24).

Effect of stress and PK on the number and ratio of leukocytes

The intake of FC leads to a decrease in the amount of NF in control animals. This indicates an increase in the state of rest.

Intraperitoneal administration of ADR after 30 minutes leads to an increase in the amount of NF in the blood of rats. The intake of PK relieves the stress response to ADR: the amount of NF does not increase, but even decreases to the control level with the introduction of PK. The introduction of adrenaline does not lead to an increase in the number of neutrophils.

A short RPE leads to a decrease in the amount of NF, to a level lower than the intake of PK. The intake of FC prevents the decrease in the amount of NF in the blood caused by RPE, which remains at the control level. Thus, the course of FC administration prevents changes in the amount of NF in the blood caused by the administration of ADR and RPE.

Multidirectional changes in NF in the blood under various stress models are described in the literature. An increase in the amount of NF under stress is considered a classic response to stress, including immobilization stress [61]. In this case, the works used, as a rule, long-term immobilization stress (3 hours or more) with fixation by the limbs. In our experiments with a soft RPE model, a decrease in the amount of NF in the blood is observed when measured immediately after the end of the 30-minute stress. Such a decrease was also observed by other authors who used RPE in a cramped chamber without fixation by limbs [64]. According to modern concepts, the direction of the response to stress (cell increase or decrease) depends on many factors (duration, strength, and nature of stress exposure) [64-66], since it is the result of the redistribution of immune cells between tissues during the stress response. In addition, the direction of the response is biphasic. This is due to the inclusion of different regulatory systems: first, catecholamines (hormonal and neurotransmitter) cause the release of leukocytes from the depot (marginal pool, spleen, lungs, etc.) into the blood and lymph. Then, if stress continues, the hypothalamic-pituitary-adrenal system is activated, released glucocorticoids, which lead to the release of leukocytes from the blood to possible sites of damage: lungs, skin, gastrointestinal tract [5 63]. Thus, the sign of changes in the amount of NF in response to stress is determined by the intensity of exposure. It is important that the intake of FC relieves the stress response of NF by quantity under the two forms of stress we studied.

Changes in the number of leukocytes in general and LF are poorly expressed (data not shown), since in rats they strongly prevail over NP. As shown above, changes in response to stress are mainly determined by the activity and amount of NF. This can be seen when determining the indicators for the NF themselves or by the ratio of the percentage of the amount of LF to the amount of NF - the lymphocyte index (LI).

As shown in FIG. 25, 30 minutes after the administration of ADR and after the end of RPE, the ratio of different types of immune cells changed, in particular, the ratio of the amount of Lf to the amount of NF-LI. As shown in FIG. 25, LI decreases with the introduction of ADR and increases with RPE. Apparently, as shown above, the change in LI is associated with a change in the amount of NP. In animals receiving PK, in response to stress, there is no corresponding change in the lymphocyte index. It is believed that the change in LI is a nonspecific response to various influences. Thus, PK intake prevents LI changes in both stress models.

Spontaneous chemiluminescence

As shown in FIG. 26 ADR and RPE increased spontaneous blood chemiluminescence.

Administration of epinephrine and 30 min RPE caused an increase in spontaneous blood chemiluminescence. This increase may be due to an increase in free radicals from oxidative stress. In animals treated with FA, spontaneous chemiluminescence did not increase in response to stress. At the same time, PK intake did not change spontaneous chemiluminescence in the control. Spontaneous chemiluminescence in whole blood is determined by many factors, including free radical processes that occur both inside and outside cells and are not associated with immune functions. These processes, such as lipid peroxidation or hemoglobin oxidation, can be enhanced by stress. Prevention of spontaneous chemiluminescence of blood by the intake of FC is apparently associated with its antioxidant effect.

Thus, with a 30-minute RPE and stress caused by the administration of adrenaline, preliminary intake of PK for 5-6 or 10 days prevented stress-induced changes, normalizing the activity of SDH and CDH enzymes and the accumulation of calcium ions in mitochondria, as well as the cellular composition. and the immune function of the blood. Studies have shown that PK, when taken as a course, has an anti-stress effect, preventing or weakening the effect of stress on mitochondria and blood cells.

Example 5

The effect of the pharmacological composition on tuberculosis infection during infection in mice and outside the body

1. Anti-tuberculosis action of the pharmacological composition in the experiment in vivo.

The study was carried out on 60 nonlinear sexually mature mice weighing 36-40 g. The model of generalized tuberculosis of mice was reproduced by intravenous (into the venous sinus of the eye) injecting a suspension of mycobacterium tuberculosis of the laboratory strain H37Rv at a dose of 0.05 mg in 0.2 ml of saline. The indicated dose of MBT for infection was used to obtain a model of generalized non-acute tuberculosis, which excludes the death of animals within 1 month. This model makes it possible to determine the optimal dose of a drug for which its minimum bacteriostatic and bactericidal characteristics are not known.

2 weeks after infection, the animals were divided into 4 groups of 15 mice each (table. 6).

* - for every 20 grams of mass. Mice of the 4th group (control) did not receive treatment.

During the experiment, the general condition of the treated mice, their activity and changes in weight were observed and compared with those in the control group.

2 days after the end of treatment, animals of all groups were euthanized by dislocation of the cervical vertebrae. At autopsy, macroscopic changes in the lungs (the number and size of tuberculous foci) were determined, the lungs, liver, kidneys, spleen were weighed to calculate the weight indices, and the material (lungs, liver, spleen) was taken for microbiological and histological studies.

Under conditions of strict sterility, pieces of the lungs, spleen and liver were weighed, then homogenized and the suspension was inoculated on a dense Levenshtein-Jensen nutrient medium. The growth of mycobacteria was monitored for 3 months, the number of grown colonies in test tubes was counted and then the number of colony forming units of MBT (CFU) per gram of organ tissue was calculated.

For histological studies, pieces of the lungs, spleen, and liver were fixed in 10% formalin. After fixation and placement, the material was embedded in paraffin, and stepwise histological sections were prepared from the paraffin blocks. Used staining with hematoxylin and eosin.

The results were statistically processed; calculated (M ± m). The significance of differences between the observation groups was assessed by the Student's test.

results

At the end of the experiment, death was not observed in any of the groups. By the end of the experiment, the increase in body weight was observed in mice of the 2nd and 3rd experimental groups. However, it was significantly higher in comparison with the control only in group 3 of animals (table 7).

Note: * - differences with the control (4) group are statistically significant (p <0.05).

The results of macroscopic pathomorphological studies are presented in table 8.

Note: * - differences with the control (4) group are statistically significant (p <0.05).

As follows from table 8, significant differences in indicators of the degree of lung damage and the weight index of the spleen were determined between the control group and the experimental groups. Among the experimental groups, significant differences were found in terms of the degree of lung damage between groups 1 and 3 and groups 2 and 3 of mice (p <0.05). The smallest degree of lung damage was in mice of the 3rd experimental group.

Results of histological examination.

In 4, control, observation group: in the lungs there were scattered multiple small lymph-macrophage granulomas and foci, located both in the interalveolar septa, and in the peribronchial and perivascular tissue. The presence of areas of the interstitial type of pneumonia in the subpleural areas was noted (Fig. 27). The liver showed multiple, small granulomas. Lympho-macrophage infiltrates were found in the central sections of the hepatic lobules and along the septa (Fig. 28). In the spleen, the presence of follicles of different sizes without clear boundaries, the formation of granulomas, and intense histiocytic proliferation in the sinuses were noted.

In the 1st group of observations: limited areas of interstitial pneumonia were found in the lungs, lymphocytic infiltrates were found subpleurally, peribronchially and perivascularly. Lymph - macrophage granulomas in moderate amounts were found more often subpleurally and in the pleura. The liver tissue contained scattered multiple, small lymph macrophage granulomas. There were almost no cell clusters along the septa. The manifestations of hepatosis were minor. Reactive follicular hyperplasia took place in the spleen. Macrophage infiltration was more intense in the marginal sinuses. Single granulomas were found.

In the 2nd group of observations: in the lung tissue, the interstitial component of inflammation was not preserved in all animals. Lympho-macrophage infiltrates along the vessels and bronchi were moderate. Granulomatous manifestations were scanty. In the liver, granulomas in moderate amounts were localized mainly subcapsularly. In the course of the triads, scanty lymphocytic infiltrates were encountered. The manifestation of protein dystrophy of hepatocytes was weak. In the spleen tissue, the follicles were of different sizes, the macrophage reaction in the sinuses was active.

In the 3rd group of observations: in the lung tissue, there were single granulomas, infiltrative manifestations, including interstitial, were regarded as scarce (Fig. 29). In the liver, there were areas with a high content of submicroscopic epithelioid-macrophage granulomas; along the septa only in some places minimal manifestations of lymphocytic infiltration were found (Fig. 30). In the spleen, there was a moderate hyperplasia of the follicles. Macrophage proliferation in the sinuses was preserved.

Thus, the intensity of manifestations of specific inflammation fell from group 1 to group 3, which was especially significant in terms of the change in the “indicator of granulomatosis” and lymph-macrophage infiltration in the lung tissue. In the 3rd group of observations, the decrease in the activity of a specific process in the liver was realized by the submicroscopic size of granulomas, the minimal severity of lymphocytic infiltration within the septa, the severity of reactive hyperplasia in the spleen decreased. There were no fundamental differences between groups 2 and 3.

Results of microbiological studies of tissues

Table 9 shows the results of microbiological studies of mouse tissues.

Notes: * - differences with the control (4) group are statistically significant (p <0.05).

As follows from table 9, the seeding rate of MBT from the organs of mice in the experimental groups is significantly less than in the control. The lowest rates of the seeding of colonies among the mice of the experimental groups were in the mice of the 3rd group. There were no significant differences in microbiological efficacy in mice of groups 3 and 2.

Thus, in the model of generalized non-acute tuberculosis, pronounced manifestations of the therapeutic effect of FC were observed.

According to the data of clinical, microbiological and pathomorphological studies, it was established that the administration of the pharmacological composition to mice infected with mycobacterium tuberculosis for 1 month at a dose of 150 μl / mouse was more effective than at doses of 75 and 15 μl / mouse. No significant differences in microbiological and histological criteria were found in mice of groups 3 and 2.

Comparative characteristics of the degree of morphological manifestations of the activity of tuberculous inflammation in groups 3 and 4 of experimental animals indicate the presence of a pronounced reduction of tissue specific changes in group 3, which, in terms of severity, approach the level of the vaccine process. Specific changes were most clearly traced in the lungs and in the liver, where sparse, submicroscopic lymph-macrophage accumulations remained, which did not have a pronounced and permanent character.

In the 4th control group of observations, multiple medium and large lymph-macrophage granulomas were found in the lungs. The presence of areas of the interstitial type of pneumonia was noted. Multiple granulomas were detected in the liver, and lymph-macrophage infiltrates were found in the central parts of the hepatic lobules and along the septa.

The presented data are experimental evidence of in vivo anti-tuberculosis action of the developed Pharmacological composition.

2. Increasing the threshold of sensitivity to the tuberculosis pathogen

The resulting pharmacological composition (FC) can increase the threshold of sensitivity to the tuberculosis pathogen, which was demonstrated in an experiment to increase the bateriostatic blood activity (BAC).

To determine the BAC, we used blood samples from mice after administration of FA at a dose of 150 μl per mouse weighing 20 grams. The drug was administered once intragastrically using a special probe. Whole blood was used, which was taken from the carotid arteries of animals immediately after the dislocation of the cervical vertebrae in 30 minutes or 1 hour after the administration of FC. Bacteriostatic activity of blood (BAC) was determined in accordance with the methodological recommendations "Modification of the determination of bacteriostatic activity of blood of patients with tuberculosis on a solid nutrient medium by the method of serial dilutions", 1983. From blood samples, serial dilutions from 1: to 1:64 were prepared and inoculated on a dense nutrient medium. Wednesday Lowenstein-Jensen together with the Office. The laboratory strain H37Rv was used as a test culture. The value of the bacteriostatic activity of the blood was determined by the dilution of blood, where there was no MBT growth. A delay in MBT growth is the complete absence of colonies or the presence of single (up to 20) small colonies on a solid nutrient medium. Each row of blood dilutions was controlled by MBT growth. For comparison, a similar series of blood dilutions from intact mice was supplied.

results

Table 10 shows the data of accounting for the growth of the culture of mycobacteria, which was carried out 3 weeks after sowing.

As follows from Table 10, the LHC of intact mice was low already at a blood dilution of 1: 2. The absence of MBT growth in test tubes with blood of mice that received FC was observed at blood dilutions of 1: 8 (30 min after administration of FC) and 1:16 (after 1 hour). Thus, the LHC in mice that received FA at a dose of 150 μl per mouse increased 4-8 times as compared to the natural LHC in intact animals.

3. Strengthening the therapeutic effect when used together with anti-tuberculosis drugs.

The study of the effectiveness of the combined use of PK and anti-tuberculosis drugs was carried out on a model of tuberculosis in mice infected with drug-resistant mycobacteria (according to clinical, microbiological and pathomorphological criteria)

Material and research methods:

The study was carried out on 70 nonlinear sexually mature mice weighing 30-34 g. The model of generalized tuberculosis of mice was reproduced by intravenous (into the venous sinus of the eye) injecting a suspension of mycobacterium tuberculosis (MBT) at a dose of 0.1 mg in 0.2 ml of saline. The MBT strain was isolated from a sick patient and was resistant to 10.0 μg / ml of isoniazid and 40.0 μg / ml of rifampicin.

Animals in this experiment were divided into 7 groups of 10 mice each (table 11), depending on the treatment regimen.

* Note: H * - isoniazid; R ** - rifampicin; DC *** (drug composition) - pharmacological composition (FC)

Thus, mice of groups 2, 4, and 6 received anti-tuberculosis drugs in combination with PK. Groups 1, 3 and 5 were comparison groups (anti-TB drugs only). Mice of the 7th group of drugs did not receive (control group). Treatment of mice was started 2 weeks after infection. The drugs were administered intragastrically 5 times a week for 1 month. During the experiment, the general condition of the treated mice, their activity and change in weight, death was observed and compared with similar indicators in the control group.

2 days after the end of treatment, animals of all groups were euthanized by dislocation of the cervical vertebrae. At autopsy, macroscopic changes in the lungs (the number and size of tuberculous foci) were determined, the lungs, liver, kidneys, and spleen were weighed to calculate the weight indices. The weight indices of the internal organs were calculated by dividing the organ weight by the mouse weight and expressed in relative units. The material was collected (lungs, spleen) for microbiological and histological (lungs, liver, spleen) studies.

Under conditions of strict sterility, pieces of the lungs and spleen were weighed, then homogenized and the suspension was inoculated on a dense Levenshtein-Jensen nutrient medium. The growth of mycobacteria was monitored for 3 months, the number of grown colonies in test tubes was counted, and then the number of colony-forming units of mycobacteria per gram of organ tissue (CFU / g) was calculated.

For histological studies, pieces of the lungs, spleen, and liver were fixed in 10% formalin. After fixation and placement, the material was embedded in paraffin, and stepwise histological sections were prepared from the paraffin blocks. Used staining with hematoxylin and eosin.

The results were statistically processed; calculated (M + m). The significance of differences between the observation groups was assessed by the Student's test.

Research results.

The death of single animals in the control and experimental groups was observed in the last week of chemotherapy (table 12). By the end of the experiment, the most significant increase in body weight in the remaining animals was observed in mice 2, 3 and 4 of the experimental groups.

Note: * - differences with the control (7) group are statistically significant (p <0.05).

The remaining animals were euthanized. At autopsy, a visual assessment of internal organs was performed and material was taken for histological and microbiological studies.

The results of macroscopic pathomorphological studies are presented in table 13.

Note: * - differences with the control (7) group are statistically significant (p <0.05).

As follows from Table 13, significant differences in the indicators of the degree of lung damage and the weight index of the lungs were determined between the control group (7) and some experimental groups: 2, 3 and 4. However, there were no significant differences in the indicator of the degree of lung damage between these experimental groups.

Histological examinations

The following results were obtained during histological examination of the lungs, spleen and liver of mice.

In the control group (7):

In the lungs, focal-infiltrative changes are expressed, but the type of interstitial pneumonia and a pronounced intensity of lymph-macrophage accumulations in the peribronchial tissue, foci of the type of large-focal dissemination and plaque-type cell accumulations in the pleura.

In the liver tissue: pronounced hydropic dystrophy of hepatocytes with discomplexation of the hepatic tracts. The presence of the miliary type of lymph-macrophage granulomas, including in septa, is noted.

In the spleen: follicles of different sizes with varying degrees of delineation. The tissue contains a significant amount of megakaryocytes.

In the 1st group (H):

The presence of irregularly located infiltrative-pneumonic foci was noted in the lung tissue. There were limited peribronchial lymph-macrophage infiltrates, as well as single large lymph-macrophage foci.

The presence of small disseminated lymph macrophage granulomas was noted in the liver tissue against the background of meager manifestations of hydropic degeneration of hepatocytes and slight desquamation of the hepatic tracts. In the course of the septa, limited lymphoid accumulations were present here and there.

In the spleen, manifestations of hyperplasia of lymphoid tissue in the follicular type are expressed.

In the 2nd group (H + DC):

In the lung tissue, the presence of areas of the infiltrative-pneumonic type was revealed against the background of plethora and areas of emphysema. In the peribronchial tissue there were scanty lymph-macrophage accumulations. There are no granulomatous changes.

In the liver, slight manifestations of hydropic degeneration of hepatocytes are expressed. Granulomas are small, not numerous.

In the spleen, the presence of large and medium hyperplastic follicles with blurred outlines is noted. The subcapsular zone contained a significant amount of megakaryocytes.

In group 3 (H + R):

In the lungs, the manifestations of specific inflammation were weaker: there were limited areas such as interstitial pneumonia. Peribronchially, there were single lymph-macrophage nodular accumulations, including subpleurally in the form of "stripes".

In the liver, dystrophic changes in hepatocytes were weak and lymph-macrophage granulomas were isolated.

In the spleen: follicles of different sizes with an expanded, indistinct marginal zone.

In the 4th group (H + R + DC):

The lungs showed limited interstitial-pneumonic changes. Peribronchial and perivascular lymph-macrophage cell accumulations were minimal in volume. Single plaque-like granulomatous foci were identified.

In the liver, the manifestations of hydropic degeneration of hepatocytes and desquamation of the hepatic tracts are insignificant. Lympho-macrophage granulomas are single, point-like in size. Cellular infiltration along the septa was absent.

Spleen: follicles are large, with a slight expansion of the marginal zone; subcapsular-moderate number of megakaryocytes.

In the 5th group (R):

The presence of areas of infiltrative changes was noted in the lung tissue. Peribronchial and perivascular lymph-macrophage accumulations are expressed, sometimes merging with parenchymal foci. Focal-like cell clusters were also found in the subpleural zone.

In the liver, hydropic degeneration of hepatocytes with foci of necrobiosis and desquamation of the hepatic tracts were sharply expressed. Small lymph-macrophage granulomas are scattered in the liver tissue in a moderate amount. There were limited lymphoid accumulations along the vessels in the septa.

In the spleen: follicles of different sizes with a predominance of large ones with blurred boundaries. The number of megakaryocytes in the subcapsular zone is moderate.

In the 6th group (R + DC):

In the lungs, areas of infiltrative-pneumonic changes and lymph-macrophage peribronchial infiltrates were small (expressed in separate areas). Lympho-macrophage foci are large, irregular in shape, mainly with subpleural localization. The tissue is full of blood.

In the liver, sharp dystrophic changes in hepatocytes with areas of necrobiosis and necrosis were detected, desquamation of the hepatic tracts was expressed. Scanty lymphoid accumulations along the septa. Lymph-macrophage granulomas are small in moderation.

In the spleen, there is a plethora of red pulp. There were large follicles with blurred boundaries.

Thus, involute changes in the components of specific inflammation were monitored both in volume and in nature. Thus, histological preparations from organs of mice in the control (7) group showed widespread focal infiltrative changes similar to interstitial pneumonia, and massive lymph-macrophage infiltrates in the peribronchial tissue, and focal and subpleural plaque cell accumulations were also found.

A decrease in the severity of the process was noted in the preparations of mice from the experimental groups that received various treatments: infiltrative-pneumonic changes subsided, the volume of peribronchial cell accumulations decreased, and disseminated foci disappeared. In the liver, granulomas became punctate, the intensity of hydropic degeneration of hepatocytes decreased. However, the involution of specific inflammation in the experimental groups differed in "effectiveness". According to the effectiveness of reducing the manifestations of the tuberculous process, the groups were arranged as follows: 4, 2, 3 and 1. In mice in groups 5 and 6, the manifestations of specific inflammation were preserved in a larger volume, and dystrophic changes in hepatocytes were accompanied by the development of necrobiosis and necrosis.

Thus, according to histological studies, when combined with the anti-tuberculosis drug isoniazid, as well as together with isoniazid and rifampicin, FC enhanced the therapeutic effect in the treatment of drug-resistant tuberculosis. When FC was used with rifampicin without isoniazid, such an increase was not observed.

Conclusion.

According to clinical, microbiological and pathomorphological studies, it was established:

1. When mice were infected with MBT culture resistant to isoniazid and rifampicin, a specific process developed, characterized by widespread focal-infiltrative changes in the lung and peribronchial tissues and massive seeding of MBT colonies from the lungs and spleen;

2. When mice were administered FC in combination with isoniazid for 1 month, a significant decrease in the degree of lung damage, MBT sowing from the lungs and spleen was observed in comparison with the control group. Compared with the use of isoniazid alone, a significant decrease in lymph-macrophage granulomas in the lung tissue was observed.

3. When mice were administered FC in combination with isoniazid and rifampicin for 1 month, there was a significant decrease in the degree of lung damage, the weight index of the lungs, MBT sowing from the lungs and spleen compared to the control group. In the lung tissue, a more pronounced decrease in infiltrative-pneumonic changes was noted in comparison with the control group and the rest of the experimental group.

Example 6

Study of the pharmacological activity of the pharmaceutical composition in experimental myocardial infarction

This study presents materials on the study of the specific pharmacological activity of the pharmaceutical composition (FC) in experimental myocardial infarction (EMI) in comparison with the standard antihypoxanth preparation Riboxin.

1. Experimental part

Animals. We used sexually mature male rats of the SD line weighing 180-200 g, obtained from the nursery of the FIBC RAS.

Quarantine. The duration of the quarantine (acclimatization period) was 14 days. During the quarantine, each animal was examined daily. Before the start of the study, animals meeting the criteria for inclusion in the experiment were assigned to groups using a randomization method.

Keeping animals. The cages with animals were placed in separate rooms with a light regime of 12 h - light, 12 h - darkness, air temperature in the range of 19-25 ° С and relative humidity - 50-70%. Air temperature and humidity were recorded daily. The ventilation mode was set, providing about 15 volumes of the room per hour, the concentration of CO ₂ not more than 0.15% by volume, ammonia - not more than 0.001 mg / l.

Daily inspection. During the study, each animal was examined daily. The examination included an assessment of the behavior and general condition of the animals. The examination was carried out approximately 1 hour after the administration of the drugs.

Experimental myocardial infarction. Modeling of myocardial infarction was performed by ligation of the left coronary artery. The skin and subcutaneous adipose tissue were dissected under ether raush anesthesia, the tissues were separated along the fascia, the pectoral and intercostal muscles were separated along the course of the fibers. After opening the chest, the heart was taken out into the surgical wound, the left coronary artery was sutured and tied with a ligature. The onset of myocardial ischemia was monitored by electrocardiography. After the operation, the wound was sutured in layers. Experimental myocardial infarction (EIM) is characterized by the formation of a necrosis focus, insufficiency of coronary blood flow, inhibition of myocardial contractile activity, impaired atrioventricular and intraventricular conduction, tissue respiration and metabolism, activation of lipid peroxidation and a decrease in antioxidant protection.

ECG registration. ECG registration was performed on non-anesthetized animals using a RM-6000 polygraph (Japan). The rats were placed in special transparent plastic cases. Thin steel needles inserted subcutaneously were used as electrodes. An ECG was recorded in lead I. An electrocardiographic sign of EIM in rats is the recording of the ST segment elevation 1 hour after surgery.

The dynamics of the body weight of the rats was determined on a VLR-500 balance. The hearts of the animals were weighed on a 1602 MP electronic balance of the German company "Sartorius".

Study drugs.

Riboxin (9-β-D-ribofuranosylhypoxanthin, LLC "Nizhpharm", Russia, No. 001382 / 01-2002, 06.05.2002), widely used in the complex therapy of coronary heart disease, myocardial infarction, myocardial dystrophy (decreased functional capacity of the heart muscle) , rhythm disturbances associated with the use of cardiac glycosides, were administered intragastrically at a dose of 10 mg / kg once a day for 7 days after EIM. The FA preparation at doses of 200 and 500 μl / rat was administered according to the same scheme as Riboxin. The rats of the control groups were injected with a solvent - distilled water. Comparative efficacy of drugs was assessed by several indicators: mortality and clinical presentation; functional - ECG (1 h, 3 days and 7 days), biochemical (examination of blood and heart tissue 24 hours after EIM), histological (24 hours, 3 days and 7 days).

Experimental groups. Five experimental groups of 15 animals each were formed: group I - sham-operated animals (negative control), group II - animals with EIM without treatment (positive control), group III - animals with EIM, therapy with Riboxin (10 mg / kg) , group IV - animals with EIM, therapy with PK (200 μl / rat), group V - animals with EIM, therapy with PK (500 μl / rat).

Blood for biochemical studies was obtained by puncture of the tail vein. The animals killed by decapitation were subjected to postmortem dissection and histological examination of the heart tissue. The heart was fixed in 10-15% formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin-eosin and Van Gieson's method.

The content of reduced glutathione in the liver was determined iodometrically [67], the glycogen content in the liver was determined by the Samogyi method [68], the level of glucose in the blood and organ homogenates was measured by the orthotoluidine method [69]. The activity of ALT, ACT, ALP, CF, LDH blood serum was determined using Bio-Lat-Test kits (Lahema, Czech Republic).

The intensity of tissue respiration in organ homogenates was determined by the Warburg manometric method [70]. The lactic acid content in the blood was determined by the Barker method [71], the succinate dehydrogenase activity - by the Vogels method [72].

Statistical processing of the results was carried out using the Student's t-test.

2. Infarction characteristics of experimental groups

Experimental modeling of myocardial infarction was accompanied by approximately 20-26% mortality; thus, 11-12 animals from each group were observed in the study (Table 14). The results of the study of the ECG dynamics did not reveal practically any differences between the studied groups before the start of the RIM modeling (Table 15, Fig. 31).

* I - negative control; II - positive control - EIM without therapy; III - EIM + Riboxin; IV - EIM + FC (200 μl / rat); V - EIM + FC (500 μl / rat).

ECG analysis revealed the distribution of animals in the experimental groups depending on the registration of the ST segment elevation, which characterizes the presence of myocardial infarction, with the allocation in each experimental group of subgroups with registered myocardial infarction (MI) and without electrocardiographic signs of myocardial infarction (BMI) 1 hour after surgery. According to the data obtained, after modeling EIM, there were no significant differences in the incidence of MI in the experimental groups: the frequency of MI was on average 83.2%, and that of FIM was 16.8%.

n is the number of animals in the group; p <0.05 compared with group I. Arrhythmia in the experimental groups was not registered.

In 1 hour after modeling EIM, the magnitude of the ST segment elevation did not significantly differ between the groups (Table 16, Fig. 32). In group I (negative control), no ST segment elevation was observed. As evidenced by the data obtained, after experimental myocardial infarction, there were no significant differences in the incidence of MI in the experimental groups. The frequency of IS averaged 83.2%, BIM - 16.8%.

* p <0.05 compared with group II.

Decrease in the ST segment below the isoline (depression) characterizes postinfarction ischemic changes and indicates insufficient blood supply and myocardial hypoxia. The results shown in Table 16 show that the value of ST segment depression 3 days after EIM in groups III (therapy with Riboxin) and IV (therapy with FC at a dose of 200 μl / rat) was 1.5, and in group V (therapy with FC in dose of 500 μl / rat) is 2 times less than in group II (EIM without therapy). Figures 33 and 34 show ECGs of rats of groups II and V.

7 days after EIM, the ST segment depression in group II significantly increased by 1.2. times in comparison with a period of 3 days (table. 16, fig. 35; 36). On the background of therapy with Riboxin and PK at a dose of 200 μl / rat (groups III and IV), the magnitude of ST segment depression did not change, and in group V (therapy with PK at a dose of 500 μl / rat) it significantly decreased 2 times and was 4 times less expressed than in the control (group II). The results obtained indicate the ability of FC to increase myocardial resistance to ischemia. It should be noted that the anti-ischemic effect of FC was dose-dependent (Table 16).

3. ECG characteristics of experimental groups

Table 17 shows data on the effect of Riboxin and FC on the dynamics of heart rate (HR) and ECG parameters in rats after EIM. It can be seen that in group II (EIM without treatment) there was a pronounced negative chronotropic effect (a significant decrease in heart rate by 30-40% after 1 hour and 7 days compared with group I). In addition, in rats of this group, the following significant ECG changes were observed after 7 days: a 2-fold decrease in the amplitude of the R wave compared to the observation period of 1 h and group I, which characterizes the inhibition of myocardial contractile activity in the postinfarction period; as well as lengthening of PQ and QT by 2.4 and 2 times compared to the observation period before EIM, which indicates a persistent progressive violation of atrioventricular (PQ) and intraventricular (QT) conduction.

During therapy with Riboxin (Table 17, group III), a significant decrease in heart rate, a decrease in the amplitude of the R wave and an increase in QT after 7 days compared with the norm (group I) and with a follow-up period of 1 hour persisted. At the same time, there was no significant PQ lengthening, which indicates the normalization of atrioventricular conduction.

p<0.05 по сравнению со сроком наблюдения 1 ч

p <0.05 compared with 1 hour follow-up

•p<0.05 по сравнению с нормой - до ЭИМ

• p <0.05 compared to normal - before EIM

FC (groups IV and V) also did not affect heart rate - bradycardia was at the same level as in groups II and III (Table 17). At the same time, after 7 days there was no significant decrease in the amplitude of the R wave and a significant (2 times) increase in the amplitude of the T wave was observed, which indicates an increase in myocardial contractile activity against the background of FC therapy at doses of 200-500 μL / rat. As in the case of Riboxin therapy, in groups IV and V, significant disturbances in intraventricular conduction were observed (QT prolongation by 1.3 and 1.5 times after 1 hour and 7 days compared to the observation period before EIM) and normalization of atrioventricular conduction.

4. The influence of the investigated drugs on the morphometric and biochemical parameters of experimental animals.

The results of morphometric and biochemical studies against the background of experimental myocardial infarction are presented in table. eighteen.

* - the differences are significant compared to group II (p <0.05).

It turned out that EIM is characterized by a significant increase in the relative mass of the heart. In addition, reliable signs of cytolysis in the myocardium were observed: an increase in the activity of ALT, ACT, CP and LDH in the blood plasma. Disturbances in the processes of tissue respiration and metabolism in the myocardium were recorded: the reserves of ATP and glycogen decreased; the content of under-oxidized metabolic products - lactic acid increased; activation of free radical reactions (an increase in the concentration of MDA - the end product of lipid peroxidation) and a decrease in the level of antioxidant protection (catalase, SDH, VG) were observed. FC (groups IV and V) significantly reduced the activity of ACT, CP and LDH in the blood; normalized the relative heart mass, as well as the content of lactic acid and MDA, and increased the content of glycogen and ATP in the myocardium (Table 18). In all cases, PK was more active than Riboxin (group III).

5. Histological examinations

The results of histological studies are shown in FIG. 37 and 38. FIG. 37-a shows a typical histological picture of the myocardium of the left ventricle of the heart of an intact rat. The transverse striation of myofibrils and the nucleus of cardiomyocytes are clearly visible. 24 hours after the operation, in the myocardium of the left ventricle of rats of group II (control), general circulatory disorders in the form of plethora, minor hemorrhages, blood stasis in the capillaries, and edema were observed. Mainly under the epicardium, muscle fibers are visible, intensely stained with eosin or picric acid (Fig. 37-b). After three days (Fig. 37-c), the circulatory disorder was no longer general, but local in nature. FIG. 37-c shows that cardiomyocytes are homogeneously stained with eosin, i.e. devoid of cross striation and nuclei. Most of the necrotic fibers are in a state of lysis and are very pale in color. In the zone of necrosis, hemolyzed erythrocytes and polymorphonuclear leukocytes are visible. The myocardial stroma outside the necrosis zone was edematous. On the 7th day (Fig. 37-d), resorption of necrotic fibers and replacement of areas of necrosis with young connective tissue rich in cellular elements of predominantly fibroblastic series was observed. At a later date, a fibrous scar formed at the site of these proliferation foci.

In rats of experimental groups (III-V), 24 h after EIM, the general circulatory disorder of the left ventricular myocardium was less pronounced compared with group I. In the ischemic area, eosinophilia of muscle fibers and slight edema were observed. After three days (Fig. 38-a, b, c), the formation of myocardial infarction was observed, mainly under the epicardium of the anterior wall of the left ventricle. The size of the necrosis focus was significantly smaller than in control rats. FIG. 38-in (group V) the transverse striation of myofibrils is discernible. Leukocyte infiltration around the necrosis focus is poorly expressed. There is no interstitial edema outside the ischemic zone.

On the 7th day, a proliferative reaction of the stroma with a large number of fibroblastic cells was observed at the site of the necrotic fibers. The size of the proliferation site was smaller compared to the control. FIG. 38-g in the infarction zone, young connective tissue with a large number of fibroblasts is visible (group V).

Summarizing the results obtained, the following conclusions can be drawn: 1) PK has a pronounced anti-ischemic effect: improves coronary blood flow in the postinfarction period (at a dose of 200 μl / rat - 1.5 times at a dose of 500 μl / rat - 4 times); 2) PK increases the contractile activity of the myocardium in the post-infarction period by 2 times, contributes to the normalization of violations of atrioventricular conduction of the myocardium, and this effect is achieved already at a dose of 200 μl / rat; 3) PK has a pronounced cardioprotective effect, reduces the intensity of myocardial cytolysis, the intensity of lipid peroxidation, increases the level of antioxidant protection, and is more effective than the reference drug Riboxin.

Example 7

Investigation of the toxic effect of FC

Experimental studies of general toxicity (acute, subacute, and chronic) and allergenicity with intragastric administration of FA were carried out to identify the possible toxic, irritant, and allergenic effects of FA.

The volume of preclinical trials was determined by: "Rules for the preclinical safety assessment of pharmacological agents (GLP)" (RD 64-126-91. M., 1992) and "Methodological guidelines for the preclinical study of new drugs developed from natural raw materials" (in the book. on experimental (preclinical) study of new pharmacological substances ”, M., 2000, pp. 346-348). In addition, the "Methodological instructions on the procedure for preclinical and clinical study of preparations of natural origin and homeopathic medicines" were used (M., 1994, 64 pp.). In accordance with the requirements of these regulatory documents concerning the study of the safety of a preparation (substance) from a known natural raw material, the following types of toxicity were studied:

- acute toxicity;

- subacute and chronic toxicity;

- local irritant effect;

- allergenic effect.

In addition, we used the regulatory documents of the Ministry of Health and Social Development of the Russian Federation:

- "Guidelines for the study of the general toxic effect of pharmacological substances" (in the book "Guidelines for experimental (preclinical) study of new pharmacological substances", M., JSC "Medicine" Publishing House, 2005, pp. 41-53);

- "Guidelines for the assessment of allergenic properties of pharmacological substances" (ibid., Pp. 54-69). The main results of these studies are summarized in Table 19.

* V class - a class of practically non-toxic medicinal substances [73, 74]

Experiments have shown that even this insignificant acute toxicity of FA is determined only by the presence of the extractant in the final product. It was shown that the claimed pharmaceutical composition is practically non-toxic and, under conditions of long-term (90 days) continuous use, does not cause disturbances in the biochemical parameters of the blood of rats and dogs, does not have a negative effect on the main physiological systems and internal organs. PK when administered intragastrically does not cause irritating effect and does not possess allergenic properties.

Example 8

Wound healing effect of PK in the model of acute infected wounds in dogs

The experiment was carried out on 10 dogs, which were divided into 2 groups (5 animals per group) according to the analogy principle, experimental and control. The animals of both groups received a burn wound. A purulent wound was obtained by infection of a burn wound with manure (staphylococcal and streptococcal infection).

In the experimental group, the treatment of infected wounds was performed with FC at a dose of 0.1 ml per 1 kg of live weight; in parallel, a wet tamponade of the wound was carried out with the application of a bandage after the corresponding toilet. For wet tamponade, FA was diluted with water 1: 1.

In the control group, treatment was carried out according to the generally accepted method using balsamic liniment according to Vishnevsky. During the experiments, clinical, microbiological, hematological and histological studies were carried out, as well as cellophaneograms and photographing of wounds. In the experimental group, the healing process of infected wounds was accelerated, and the discharge of purulent exudate stopped on the 5th day. Along with this, hematological parameters were normalized. Complete healing of infected wounds occurred on the 13th day, while in the control group, healing of infected wounds did not occur even on the 15th day. The results are shown in FIG. 39.

The results of cellophaneographic studies of wound healing are presented in table 20.

Notes: a / P≤0.001; b / P≤0.01; s / P≤0.05; d / P≤1.0

Example 9

Immunomodulatory effect of the pharmaceutical composition

The priming activity of the preparations was assessed by the effect of the FA preparation obtained by extraction with a buffer saline solution of pretreated larvae or lyophilized homogenate of wax moth larvae on the respiratory burst of neutrophils.

As shown in FIG. 40 preincubation of blood with non-alcoholic extracts causes an increase in chemiluminescence accompanying the response of whole blood neutrophils to zymosan. An increase in the concentration of the non-alcoholic extract of lyophilized larvae in the incubation medium leads to a dose-dependent activation of the respiratory burst of neutrophils in response to zymosan (priming effect), reaching its maximum CL (by 62% relative to the control) at a concentration of 0.006 mg of crude substance. With a further increase in concentration, CL decreases. Non-alcoholic extract of lyophilized homogenate also leads to an increase in the CL response of neutrophils to zymosan, however, to a lesser extent - by 20% at a concentration of 0.005 mg.

Example 10

Membranolytic action of PK on mitochondria

The detection of the presence of a complex of peptides and proteins with high bacteriolytic activity in PK indicated that the drug can also have a membranolytic effect on the mitochondria of higher animals and humans.

The commonality of the physicochemical organization of various biological membranes is known. The membranes of microbes and mitochondria are especially close, since it is believed that mitochondria in animals originated as a result of symbiosis with microorganisms.

An increase in the permeability of bacterial membranes by substances that activate the transfer across the mitochondrial membrane, as shown in [75].

The supposed membranolytic action of PA was verified by us by measuring the value of the membrane potential by the absorption of the TPP ⁺ cation by an ion-selective electrode. The measurements were carried out either with the addition of calcium ions, which increase the load on the mitochondria (panel A), or without loading, with the addition of FA compared with the standard peptide preparation thymalin (panel B).

The data obtained are shown in FIG. 41 and tab. 21

FIG. 41 show that for 5-100 seconds of contact with FA, the permeability of mitochondrial membranes is greatly increased, since they accumulate approximately half the amount of calcium ions.

An increase in the permeability of the mitochondrial membrane is associated with a decrease in the membrane potential. This is shown in panel B in comparison with a thymalin formulation containing more peptides in added amounts. Also, rather quickly, during the interaction of FA with mitochondrial membranes of 100-300 sec, a decrease in the membrane potential by about half is observed.

Table 21 shows the average results of the influence of the studied samples containing peptides according to the initial effects of influence with a brief interaction with mitochondria, 100 sec. It can be seen that the action is developing rapidly.

The studies carried out show that the well-known bacteriolytic action of a complex of highly active peptide-protein components of FA can exhibit a completely unaccounted for previously effect on the membrane potential of the mitochondria of a human macroorganism. This action consists in reducing the membrane potential of mitochondria, which may have an important regulatory role in some common pathologies (for more details, see the Disclosure section).

Example 11

Increasing the rate and degree of extraction of active components of the composition

The method of obtaining FA significantly reduces the time required for the extraction of active components from raw materials, and also increases the concentration of peptides, amino acids and antioxidants in FA. FIG. 42 shows photographs of FC variants obtained by different methods. From FIG. 42 it follows that the use of pre-treated larvae reduces the extraction time of the components of the composition. At a half incubation period, a significant release of the pretreated larvae into the solution is already observed, while when FA is obtained from live larvae, the release of components practically does not occur. Table 22 shows the yield of some components of FA in the extract with the shortened and claimed extraction method using live or pre-treated larvae.

FIG. 43 shows the results of the separation of proteins and peptides in FA preparations obtained from live and pretreated larvae, as well as from pretreated larvae with different shelf life. As shown in Table 22 and FIG. 43, pretreatment of larvae allows faster extraction of FA components from raw materials, and also increases the content of FA components in the final product. The amino acid content in FA depends to a greater extent on the method of growing and feeding the larvae (Example 2). The proposed method of obtaining allows you to increase the content of antioxidant components and stimulating the activity of phagocytes components in FA and can be carried out using different extractants.

FIG. 44 shows the results of a study of the AOA of FA preparations obtained as a result of 4-day cold extraction of components from pre-treated larvae or from a lyophilized homogenate of larvae.

The FA preparations were obtained using freeze-drying of whole larvae or previously homogenized in a glass Potter-Elwegeim homogenizer (36 hours, in a chamber -80 °, system in flasks). To assess the AOA and the priming activity of the preparations, the lyophilized larvae were extracted in two ways: with ethanol solution (30 mg / 1 ml) or calcium-free Hanks solution (30 mg / ml) for 4 days at 4 ° C in the dark. After extraction, the AOA of each sample was measured. As can be seen from Figure 2, for 50% inhibition, 0.45 mg (calculated on the wet weight of larvae) of the extract obtained from the lyophilisate of whole or homogenized larvae (curves coincide) and 4.5 mg (calculated on the wet weight of larvae) alcoholic 20 daily extract from live larvae. Thus, the AOA of alcohol extracts obtained from lyophilized larvae significantly exceeds the AOA of FA. In this case, as follows from FIG. 44, preliminary lyophilization significantly reduces the extraction time (4 days instead of 21 days) required for the release of antioxidant components of FA into the extract.

Example 12

Preparation of a dry preparation based on a pharmaceutical composition

To obtain a dry preparation, sorbitol can be used as a thickener. To obtain a dry alcohol-free form of FA, different amounts of sorbitol, previously ground into a powder, were mixed with 0.45 ml of FA in a mortar.

A dose of 0.45 ml corresponds to 15 drops (1 drop of about 0.03 ml) - the average recommended single dose per person, when calculating 2 drops of FC per 10 kg of body weight (the maximum recommended dose in severe conditions is 1 drop per 1 kg of body weight ).

To assess the activity of the preparations obtained, their antioxidant activity (AOA) was determined by inhibition of adrenaline autooxidation. The AOA of the initial liquid PC (LPA), the AOA of the thickener solution were measured, and the effect of the addition of the thickener solution on the AOA of the LPA was also investigated. The determination of AOA was carried out as described in Example 3. The results are shown in FIG. 45, 46. As seen in FIG. 45-b, 46, sorbitol inhibits the reaction of adrenaline oxidation, but to a much lesser extent than PK (Fig. 45-a). FIG. 46 shows the inhibitory effect of LPA on the oxidation of adrenaline in the presence of various amounts of sorbitol in the reaction medium. To determine the inhibitory effect of LPA on the rate of adrenaline oxidation in the presence of sorbitol, the spectrophotometer indicated in FIG. 46 on the right, the volumes of sorbitol solution (2 g / ml of water), and at each sorbitol concentration, the rate of autooxidation of adrenaline in the presence of different amounts of added LPA was determined. The reaction rate of adrenaline oxidation without additives was taken as 100% (inhibition is 0%). To determine the AOA of LPA in the presence of sorbitol, the degree of inhibition of adrenaline autooxidation by the LPA preparation was measured in the presence of different concentrations of sorbitol in the incubation mixture as indicated above, but the rate of adrenaline autooxidation in the presence of an appropriate amount of sorbitol was taken as 100%. The percentage inhibition by the added FPA preparation was determined relative to this rate (FIG. 48).

As shown in FIG. 46, 47, sorbitol increases the maximum degree of inhibition of the adrenaline autooxidation reaction by the pharmaceutical composition. In the presence of sorbitol, it is possible to achieve complete inhibition of adrenaline oxidation.

To determine AOA in dry FA preparations, test samples were obtained from LPA with known AOA. To prepare the samples, sorbitol was ground in a mortar to a powdery state, and LPA was added to this powder. Mixed until smooth, and when granules formed, placed them in Petri dishes.

The samples were dried for a day, then transferred to closed vials. In the control sample, instead of LPA, a solvent at the appropriate concentration was used, which was mixed with sorbitol in the same proportion as LPA and dried to the state of granules.

Determination of antioxidant activity.

Immediately before determining the activity, the test and control samples were dissolved in bidistilled water. The AOA of the dry PK-based formulation is shown in FIG. 48.

As shown in FIG. 47, 48, upon mixing liquid FA with a thickener and drying the AOA of the resulting dry FA preparation, FA remains.

To determine the storage conditions under which their AOA is preserved, samples of a dry FA preparation with the same AOA were stored for 24 days in closed glass vials under the following conditions:

1) in the refrigerator

2) at room temperature, in the dark,

3) at room temperature in the light.

Then the samples were dissolved in water and the AOA was determined in relation to the control sample (prepared using a solvent instead of FA) as described above. The results are shown in FIG. 49. FIG. 50 shows the effect of the same storage conditions on the AOA of the liquid FA that was used to prepare dry forms.

As seen in FIG. 49, 50 AOA decreases on storage in heat and light. It should be noted that the LPA preparation before the start of the experiment was already stored in the refrigerator for 3 months from the date of manufacture without reducing its activity, but when it was placed at room temperature, it began to rapidly lose its activity. The activity of dry preparations during storage differs little from liquid preparations. Thus, for dry preparations obtained using sorbitol, a prerequisite for the preservation of AOA in them is their storage in the cold, in the dark. The use of sorbitol makes it possible to obtain a dry preparation that does not contain alcohol with preserved AOA. The ratio of FA and sorbitol, optimal for obtaining solid granules, corresponds to the extract amount equal to the dose of the drug for one dose and the amount of sorbitol to the dose optimal for the preparation of the tablet.

Example 13

Extraction in the presence of dihydroquercetin

To increase the antioxidant activity, the content of peptides in the pharmaceutical composition and increase the stability during storage, the food antioxidant dihydroquercetin was added to the extraction solution and treated with carbon dioxide.

Conditions for obtaining extracts

Fresh frozen larvae were used to obtain extracts. The larvae were frozen immediately after collection, in small batches, and stored in a refrigerator freezer until extraction began. Before the start of extraction, all batches of frozen larvae were pooled and thoroughly mixed. For the preparation of each extract, the larvae were poured with ethanol solution with or without additives. The pH of the extracts containing dihydroquercetin was adjusted to 4.6. To obtain extract No. 4, the extraction solution was purged with carbon dioxide for 10 minutes, larvae were placed in it, before closing the container, the space under the lid was filled with carbon dioxide.

Extraction was carried out at room temperature in the dark for 20 days. The resulting extracts were stored in 50 ml dark vials at + 4 ° C or + 20 ° C for 21 months.

For 1 conventional unit of AOA it is taken:

by the adrenaline method: the amount (μl) of the extract required for 50% inhibition of the autooxidation of adrenaline under standard conditions [54];

according to the chemiluminescent method: such a volume of the drug added to the model system that leads to the same changes in the CL latency period as the addition of trolox at a concentration of 1 μM [59].

As follows from table 23, the addition of DHQA to the extraction solution increases the AOA of the resulting FA by 1.5-2 times.

FIG. 9 shows the results of electrophoresis in PAAG of FA obtained using DHAQ. It is clearly seen that prolonged storage in heat leads to a decrease in the protein-peptide composition of FA. The addition of carbon dioxide even further degrades the FA components. The addition of dihydroquercetin at a lower concentration is not very effective. But at a concentration of 1 g / l, it significantly increases the safety of proteins and peptides. Thus, the use of DHQA in the preparation of FA prevents the destruction of peptides during storage, especially during storage in the cold.

List of sources used

de I'infection tuberculeuse// Archives des sciences biologiques publiees par l'Institut Imperial de medicine experimentale a St.-Petersburg - 1906 - Vol. 12, N 1 - P. 300-317.1. Metalnikoff SJ Contribution a rimmunite de la mite des ruches d'abeilles (Galeria melonela)

de I'infection tuberculeuse // Archives des sciences biologiques publiees par l'Institut Imperial de medicine experimentale a St. Petersburg - 1906 - Vol. 12, No. 1 - P. 300-317.

2. Oliver H.R. Antibiotic action of an extract of Galleria mellonella // 1947 - Nature - V. 159. - N 4046. - P. 685-687.

z larw Galleria mellonella na wzrost

Medicina, Doswiadczalna I Microbiologia// 1950. - Vol. 2 - N 2 - P. 246-249.3. Kuzniecow A. & Wojciechowski E. Wpliw

z larw Galleria mellonella na wzrost

Medicina, Doswiadczalna I Microbiologia // 1950. - Vol. 2 - N 2 - P. 246-249.

Lublin - Polonia, sectio C. - 1959. - V. 14 - N 20. - P. 435-438.4. Paszevski A. Influence of an enzyme extract from larvae of Galleria mellonella together with penicillin or sulphatiazole on the Growth of Micobacterium tuberculosis 607 // Annales Universitatis Mariae

Lublin - Polonia, sectio C. - 1959. - V. 14 - N 20. - P. 435-438.

5. Muftic M.K. Progress in pharmacological study of cerase, a new preparation against tuberculosis // 1958. - Brit J. of Tuberc - V. 52. - N. 4. - P. 308-312.

6. Muftic M.K. Isolation properties of the cerase // 1957. - Enzimologia Acta Biocatalytica. - V. 18. - N 1. - P. 7-21.

7. Mankiewicz E. The lipidilytic enzymes of larvae of galleria mellonella // 1949 - Canadial Journal of Research. - V. 27. - sec E. - P. 195-201.

8. Powning, R.F., Davidson, W.J. Studies on insect bacteriolytic enzymes - 1. Lysozyme in haemolymph of Galleria mellonella and Bombex mory // Comp. Biochem. Phisiol. - 1972. - Vol. 45 B. - P. 669-686.

9. Jarosz, J. Simultaneous induction of protective immunity and selective Synthesis of hemolymph lysozyme protein in larvae of Galleria mellonella // Biol. Zbl. - 1979. - Vol. 98. - P. 459-471.

10.10 Hoffmann, D., Hultmark, D., Boman, H.G. Insect immunity: Galleria mellonella and other lepidoptera have cecropiaP9-like Factors active against Gram negative bacteria // Insect Biochem. - 1981. - Vol. 11. - P. 537-548.

11. Cytrynska M., Mak P., Zdybika-Barabas A., Suder P., Jakubowicz T. purification and characterization of eight peptides from galleria mellonella immune hemolymph // Peptides. - 2007. - Vol. 28. - P. 533-546.

12. Bergin D., Murphy L., Keenan J., Clynes M., Kavanagh K. Pre-exposure to yeast protects larvae of Galleria mellonella from a subsequent lethal infection by Candida albicans and is mediated by the increased expression of antimicrobial peptides / / Microbes and Infection - 2006. - Vol. 8. - P. 2105-2112.

13. Mack P.T., Chmiel G.J., Gacek J.W. Antibacterial peptides of moth Galleria mellonella // 2001. - Acta Biochim. Polon. - Vol. 4. - P. 1191-1195.

14. Mukhin S.A. (1961) On some issues of homeopathic treatment of heart diseases // E. Velichko / In search of truth. - M. - 2005 .-- S. 225-264.

15. Studitsky A.N. Muscle recovery in higher mammals. Moscow, USSR Academy of Sciences, 1961

16. Patent RU 2038086. A method of obtaining a biologically active product from the larvae of a large wax moth / Spiridonov N.A., Rachkov A.K., Mukhin S.A., Kondrashova M.N.// Bulletin No. 18 dated 27.06.1995.

17. Spiridonov N.A., Arkhipov V.V., Narimanov A.A., Kondrashova M.N. Effect of Galleria mellonella larvae preparation on cell cultures // 1992. - Comp. Biochem. Physiol. - Vol. 102 C. - P. 205.

18. Martineau A. R, Newton S. M., Wilkinson K. A., Kampmann B., Hall B. M., Niga Nawroly N., Packe G. E., Davidson R. N., Griffiths C. J., Wilkinson R. J. Neutrophil-mediated innate immune resistance to mycobacteria // J. Clin. Invest. 2007, 117: 1988-1994.

19. Adams J.S., Martin Hewison M. Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity // Nat Clin Pract Endocrinol Metab. 2008 February; 4 (2): 80-90.

20. Matsuoka L.Y., Wortsman J., Haddad J.G., Kolm P., Hollis B.W. Racial pigmentation and the cutaneous synthesis of vitamin D //, Arch. Dermatol. 1991, 127, 536-538.

21. Stead W.W., Senner J.W., Reddick W.T., Lofgren J.P. Racial differences in susceptibility to infection by Mycobacterium tuberculosis // N Engl J Med. 1990 Feb 15; 322 (7): 422-427.

22. Kondrashova M.N. Interaction of transamination and oxidation processes. Biochemistry 56, 388-406, 1991.

23. Zakharchenko MV, Zakharchenko AV, Khunderyakova NV, Tutukina MN, Simonova MA, Vasilieva AA, Romanova OI, Fedotcheva NI, Litvinova EG, Maevsky EI, Zinchenko VP, Berezhnov AV, Morgunov IG, Gulyaev AA, Kondrashova MN. Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria. Int J Biochem Cell Biol. 2013, 45, 190-200.

24. Kondrashova MN, Zakharchenko MV, Zakharchenko AV, Khunderyakova NV. Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria inside Glass-Adhered Lymphocytes under Physiological Conditions. The Two Dehydrogenases as Counterparts of Adrenaline and Acetylcholine Regulation. In: Dehydrogenases. Janeza Trdine, Croatia, 2012.354 p. ISBN 978-953-307-019-3. Rosa Angela Canuto Ed. p. 235-265. http://www.intechopen.com/articles/show/title/study-of-succinate-dehvdrogenase-and-ketoglutarate-dehydrogenase-in-mitochondria-inside-glass-adhere

25. Korshunov SS, Skulachev VP, Starkov AA. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 1997 Oct 13; 416 (1): 15-18.

26. K.L. Hoehna, A.B. Salmonb, C. Hohnen-Behrensa, N. Turnera, A.J. Hoya, G.J. Maghzalc, R. Stockerc, H. Van Remmenb, E.W. Kraegena, G.J. Cooneya, A.R. Richardsonb, D.E. Jamesa. Insulin resistance is a cellular antioxidant defense mechanism. PNAS, October 20, 2009 vol. 106 no. 42, 17787-17792.

27. Kondrashova MH, Zakharchenko MB, Khunderyakova HB, Fedotcheva NI, Litvinova EG, Romanova OI, Gulyaev AA. The state of succinate dehydrogenase in the body is "out of balance" or hyperactive. Biophysics. 2013, V. 58, no. 1, 108-116.

28. Reznik N.L. Sleeping Brain Activities. Chemistry and Life 2014, 3, 36-40.

29. Begg D.P., Woods S.C. Interactions between the central nervous system and pancreatic islet secretions: a historical perspective. Adv Physiol Educ 2013, 37, 53-60.

30. Tian-Jiao Xu, Qi Wang, Xiao-Wen Ma, Zhen Zhang, Wei Zhang, Xiao-Chang Xue, Cun Zhang, Qiang Hao, Wei-Na Li, Ying-Qi Zhang, Meng Li. A novel dimeric thymosin beta 4 with enhanced activities accelerates the rate of wound healing. Drug Des Devel Ther. 2013; 7: 1075-1088.

31. Khavinson B.X., Anisimov B.H. Peptide bioregulators and aging. St. Petersburg, Nauka, 2003, 223 pp.

32. MD 4198 B1 2013.02.18. Complex of entomological origin in propylene glycol with keratolytic action, process for its preparation, pharmaceutical and cosmetic products based on it () / Ciuhrii Veaceslav // BOPI nr. 2/2013.

33. V.M. Dilman, V.N. Anisimov, M.N. Kondrashov. The effect of succinic and glutamic acids on the sensitivity of the hypothalamic-gonadotropic system to the inhibitory effect of estrogens in old rats. Pharmacology and Toxicology 3, 540, 1976.

34. M.N. Kondrashova, N.M. Doliba Polarographic observation of substrate-level phosphorylation and its stimulation by acetylcholine. FEBS Lett. 243, 153-155b 1989.

35. M.N. Kondrashova, G.D. Kuznetzova. Succinic acid as a physiological signal molecule. In; Signal molecules and behavior. Eds: W. Winslow, O.S. Vinogradova. Manchester University Press, Manchester & New York. 1991, 295-301.

36. M.N. Kondrashova. Mechanisms of physiological activity and cure effect of a small doses of succinic (amber) acid. Eur. Journ. Med. Res. 5, 10, 58, 2000.

37. M.N. Kondrashov. Hormone-like action of succinic acid. Question Biol. Honey. Pharmac. Chemistry 1, 2002, 7-12.

38. Not W, Mlao F, Lin D, Schwandner RT, Wang Z, Gao J, et al. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature 2004; 429: 188-193.

39. Gonzalez NS, Communi D, Hannedouche S, Boeynaems JM. The fate of P2Y-related orphan receptors: GPR80 / 99 and GPR91 are receptors of dicarboxylic acids. Purinergic Signal. 2004 Dec; 1 (1): 17-20.

40. Sadagopan N, Li W, Roberds SL, Major T, Preston GM, Yu Y, Tones MA. Circulating succinate is elevated in rodent models of hypertension and metabolic disease. Am J Hypertens. 2007 Nov; 20 (11): 1209-15.

41. Correa PR, Kruglov EA, Thompson M, Leite MF, Dranoff JA, Nathanson MH. Succinate is a paracrine signal for liver damage. J Hepatol. 2007 Aug; 47 (2): 262-9.

42. Regard JB, Sato IT, Coughlin SR. Anatomical profiling of G protein-coupled receptor expression. Cell. 2008 Oct 31; 135 (3): 561-71.

43. Toma I, Kang JJ, Sipos A, Vargas S, Bansal E, Hanner F, Meer E, Peti-Peterdi J. Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney. J Clin Invest. 2008 Jul; 118 (7): 2526-34.

H., Von Jagow G. Tricine-sodium dodecil sulfate - polyacrylamide gel electrophoresis for the separation of proteins in the range from1 to 100 kDa// Anal. Biochem. - 1987. - Vol. 166. - P. 368-379.44.

H., Von Jagow G. Tricine-sodium dodecil sulfate - polyacrylamide gel electrophoresis for the separation of proteins in the range from1 to 100 kDa // Anal. Biochem. - 1987. - Vol. 166. - P. 368-379.

45. Pokusaeva B.O., Antipov S.S., Shvyreva US, Tutukina M.N., Ozolin O.N. (2012) Superproduction, isolation and purification of functionally active bacterioferritin DPS E. coli // Sorption and chromatographic processes, vol. 12, no. 6, p. 1011-1017.

46. Cytrynska M., Mac P., Zdybicka-Barabas A., Suder P., Jakubowicz T. Purification and characterization of eight peptides from Galleria mellonella immune hemolymph. // Peptides. - 2007. - Vol. 28. P. 533-546 [PubMed: 17194500].

47. Poppy P., Chmiel D., Gacek G. J. Antibacterial peptides of the moth Galleria mellonella. // Acta Biochim. Pol. - 2001. - Vol. 48. P. 1191-1195 [PubMed: 11995991].

48. UniProt database (http://www.uniprot.org/).

49. Halwani A.E. and Dunphy G.B. Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity // Developmental & Comparative Immunology. - 1999. - Vol. 23. - Issues 7-8. - P. 563-570.

50. Korff von RW (1969) In: Methods in Enzymology Lowenstain L.M. (Ed.), N.-Y., Academ. Press, 13, pp. 519-523.

51. Stern JR (1957) In: Methods in Enzymology Colowick S.P., Kaplan N.O. (Eds.), N.-Y., Academ. Press, 3 ,; Biochem. Catalog Boehringer, 1995, pp. 425-443.

52. Khasanov V.B., Ryzhova G.L., Maltseva E.B. Research methods of antioxidants // Chemistry of vegetable raw materials, 2004, No. 3, pp. 63-75.

53. Sirota TV, RU 2144674, "Method for determining the antioxidant activity of superoxide dismutase and chemical compounds", priority from 24.02.1999).

54. Misra H.R., Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 1972, v. 247, 3170-3175.

55. Beauchamp C., Fridovich I. Superoxide dismutase: improved assay and assay applicable to acrylamide gels. Anal. Biochem., 1971, v. 44, 276-287.

56. Lissi A., Salim-Hanna M., Pascual C., Castillo M.D./Evaluation of total antioxidant potential (TRAP) and total antioxidant reactivity (TAR) from luminol-enhanced chemiluminescence measuriment // Free Rad. Biol. Med. (1995) V. 18, No. 2, P. 153-158.

57. Klebanov GI, Teselkin YO, Babenkova IV, Lyubitskiy OB, Vladimirov YA / Antioxidant activity of blood serum // Bulletin of Ross. acad. honey. Sciences (1999), No. 2, pp. 15-22.

58. Wayner D.D.M., Burton G.W., Ingold K.U., Locke S. Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. The important contribution made by plasma proteins. // FEBS Letters (1985) V. 187, No. 1, P. 33-37.

59. Klebanov G.I., Lyubitsky O.B., Vasilieva OB, Klimov Yu.V., Penzulaeva O.B., Teplyashin A.S., Tolstykh M.P., Promorenko V.K., Vladimirov Yu. AND. Antioxidant properties of 3-hydroxypyridine derivatives: mexidol, emoxypine and proxypine // Problems of medical chemistry (2001), V. 47, pp. 288-300.

60. Safronova V.B., Matveeva N.O., Avkhacheva N.V., Sidelnikova M.M., Vanko L.V., Sukhikh G.G. Change in the regulation of oxidase activity of peripheral blood granulocytes in women with recurrent miscarriage // Byull. experimental biol. and honey. - 2003. - T. 136, No. 9. - 295-98.

61. P.D. Gorizontov, O. I. Belousova, M. I. Fedotova. Stress and the blood system - M., 1983.

62. Schedlowski M., Jacobs R., Stratman G., Richter S., Hadike A., Tewes U., Wagner T.O.F., Schmidt R.E. Changes of natural killer cells during acute psychological stress // J Clin Immunol - 1993 -Vol. 13. - P. 119-126.

63. Dhabhar F.S. Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathology // Neuroimmunomodulation. - 2009. - Vol. 16 - P. 300-317.

64. Bovers S.L., Bilbo S.D., Dhabhar F.S., Nelson R.J. Stressor-Specific alterations in corticosterone and immune responses in mice // Brain Behav Immun. - 2008 - Vol. 22 - P. 105-113.

65. Schwab C.L., Fan R., Zheng Q., Myers L.P., Hebert P., Pruett S.B. Modeling and predicting stress-induced immunosuppression in Mice using Blood Parameters // Toxicological Sciences. - 2005. - Vol. 83. - P. 101-113.

66. Rinner I., Schauenstein K., Mangge H., Porta S., Kvetnansky R. Opposite effects of mild and severe stress on in vitro activation of rat peripheral blood lymphocytes // Brain Behav Immun - 1992 - Vol. 6. - P. 130-140.

67. Ellouk-Achard S., Levresse V., Martin C, Pham-Huy C, Dutertre-Catella H., Thevenin M., Warnet J.M., Claude J.R. Ex vivo and in vitro models in acetaminophen hepatotoxicity studies. Relationship between glutathione depletion, oxidative stress and disturbances in calcium homeostasis and energy metabolism. Arch. Toxicol. Suppl. 1995. V. 17. P. 209-214.

68. Titz N.U. // Clinical evaluation of laboratory tests. M .: Medicine, 1986.

69. Menshikov V.V. // Laboratory research methods in the clinic. M .: Medicine, 1987.

70. Warburg O. New manometric 2-vessel method. Hoppe Seylers Z. Physiol. Chem. 1967. V. 348. P. 1677-1682.

71. Pryce J.D. A modification of the Barker-Summerson method for the determination of lactic acid. Analyst. 1969. V. 94. P. 151-152.

72. Van Noorden C.J., Vogels I.M. Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium. Histochem. J. 1989. V. 21. P. 575-583.

73. H. Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning. Ed. IV, Baltimore, 1975, 427 p.

74. KK Sidorov On the classification of toxicity of poisons with parenteral routes of administration. Toxicology of new industrial chemicals. M. 1973, no. 13, 47-51.

75. Belosludtsev K.N. Belosludtseva N.V. Kondratyev M.S., Agafonov A.V., Purtov Yu.A. Interaction of phospholipase A of the E. coli outer membrane with the inhibitors of eukaryotic phospholipases A2 and their Effect on the Ca2 + -induced permeabilization of the bacterial membrane. J Membrane Biol (2014) 247: 281-288.

Claims (18)

1. Pharmaceutical composition with antioxidant activity, obtained from larvae of Galleria mellonella, including the following ingredients: thymogen from 0.5 to 6 μg / ml, thymosin-beta from 0.5 to 3 μg / ml, lysozyme from 0.1 to 2 μg / ml, superoxide dismutase from 1 to 15 μg / ml, aspartic acid 400-580 μg / ml, glutamic acid 620-840 μg / ml, glycine 160-440 μg / ml, alanine 660-1200 μg / ml, citric acid 600-1200 μg / ml, isolic acid 100-200 μg / ml, alpha-ketoglutaric acid 30-70 μg / ml. 2. A pharmaceutical composition according to claim 1, characterized in that its antioxidant activity, measured by the method of inhibition of adrenaline autooxidation, ranges from 150 to 500 units / ml. 3. The pharmaceutical composition according to claim 1, characterized in that its antioxidant activity, measured by the method of inhibiting the luminescence accompanying the thermal decomposition of α, α'-azodiisobutyroamidine dihydrochloride, is from 150 to 700 units / ml. 4. The pharmaceutical composition according to claim 1, additionally having a property normalizing the state of sympathetic hyperactivity and providing a protective effect in acute stress. 5. The pharmaceutical composition according to claim 1, additionally exhibiting a therapeutic effect in diseases selected from myocardial ischemia, tuberculosis, as well as stimulating the healing of infected wounds. 6. A pharmaceutical composition according to claim 1 in the form of a solution or in the form of a dry mixture. 7. The pharmaceutical composition according to claim 1, which is a powder, which additionally contains sorbitol.

Images