RU2801871C1 - Agent with the ability to increase lifespan, improve cognitive properties, motor control and coordination of animal movements - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to an agent with the ability to increase life expectancy, improve cognitive properties, motor control and coordination of animal movements. An agent that has the ability to increase life expectancy, improve cognitive properties, motor control and coordination of movements of animals, characterized by the following: it is an aqueous extract of the larvae of the darkling beetle Ulomoides dermestoides treated with antibacterial filtration containing a complex of antioxidant enzymes, heat shock proteins 70 kDa and 60 kDa. The product is obtained by thin dispersion of the biomass of larvae in water by the cavitation method in a cavitation disperser in a ratio of 1:4 until the temperature rises to 25–45°C, followed by extraction for a day with slow stirring, filtration of the extract and treatment with membrane filtration.

EFFECT: above invention allows to expand the arsenal of tools with the ability to increase life expectancy, improve cognitive properties, motor control and coordination of animal movements.

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Description

The invention relates to the pharmaceutical and feed industries, can be used in feed biotechnology to obtain a biologically active additive that can increase life expectancy, improve cognitive properties, motor control and coordination of animal movements, and find application as a product for healthy feeding.

The problem of active longevity attracts the attention of many researchers. The role of antioxidants is known to protect the brain in an imbalance of the prooxidant and antioxidant systems, which causes oxidative stress, which, according to some authors, is an important factor in aging. Kregel K., Zhang H. An integrated view of oxidative stress in aging: basic mechanisms, unctional effects, and pathological considerations. Am. J. Physiol Regul Integr Comp Physiol. 2007, v. 292(1), p. 18-36.

It is known to use the chemical compound 2-ethyl-6-methyl-3-oxypyridinium N-acetyl-L-glutaminate (EMOP-AG) as a neurotropic agent with antioxidant, antihypoxic, neuroprotective, antiamnesic, antiswaying activity and the ability to improve cognitive functions. EMAP-AG at a course application at doses of 10 and 20 mg/kg/day (daily for 5 days) has a pronounced antihypoxic effect in acute hypobaric hypoxia in rats with hemorrhagic stroke (HI). At the same time, EMAP-AG significantly increases the lifespan and survival of animals, prevents the occurrence of motor disorders that occur when climbing to a "height", and improves the general condition of animals. In rats with GI, EMOP-AG (20 mg/kg/day for 14 days) causes a significant decrease in neurological deficit, improves coordination of movements and reduces the death of animals by 2.3 times. Course administration of the agent restores cognitive functions impaired by hemorrhagic stroke in the CRPI test 7 and 14 days after surgery. EMAP-AG shows high activity in all indicators of cognitive function assessment. RU 2394816 C1, publ. 07/20/2010.

As you know, antioxidants, due to their ability to counteract free radicals and neutralize oxidants, are used to treat or alleviate the symptoms of diseases associated with reactive oxygen species, which include neurodegenerative diseases such as Parkinson's disease, sclerosis. Miller T.M., Pestronk A., David W. et al. An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: A phase 1, randomised, first-in-man study. Lancet Neurol., 2013, 12(5), p. 435-442.

In the practice of traditional medicine in China, Japan and Latin America, the darkling beetle Ulomoides dermestoides (Coleoptera, Tenebrionidae) is known, which is used against a wide range of diseases. Scientific studies have shown an antidiabetic effect. Jasso-Villagomez E.I., Garcia-Lorenzana M., Almanza-Perez J.C. et al. Beetle (Ulomoides dermestoides) fat improves diabetes: effect on liver and pancreatic architecture and on PPARg expression. Brazilian Journal of Medical and Biological Research, 2018, v. 51(6), e7238, http://dx.doi.org/10.1590/1414-431X20187238. Anti-inflammatory effect of an aqueous extract of the dark beetle Ulomoides dermestoides in laboratory experiments on a model of acute pleurisy in rats. Santos R.C., Lunardelli A., Caberlon E. et al. Antiinflammatory and immunomodulatory effects of Ulomoides dermestoides on induced pleurisy in rats and lymphoproliferation in vitro. inflation. 2010, 33, v. 3, p. 173-179.

The body's ability to produce antioxidants decreases with age, so supplementing with antioxidants is important. Insects are considered as a source of new biologically active substances and are promising objects for obtaining new substances for pharmaceutical use.

It is promising to find substances in insects that have a wide range of pharmacological effects, which can be assessed, for example, by their ability to act effectively in model systems to prolong the life of a nematode under conditions of oxidative stress, in tests to improve spatial orientation, motor control, and cognitive abilities to recognize objects with aging. mice, hamsters.

The technical task is to identify the ability to increase life expectancy, improve cognitive properties, motor control and coordination of animal movements with a new agent - an aqueous extract of the larvae of the darkling beetle Ulomoides dermestoides, containing a complex of antioxidant enzymes, heat shock proteins 70 kDa and 60 kDa.

The technical result achieved from the implementation of the invention is to expand the arsenal of tools that have the ability to increase life expectancy, improve cognitive properties, motor control and coordination of animal movements.

The technical result is achieved by an agent that has the ability to increase life expectancy, improve cognitive properties, motor control and coordination of movements of animals, according to the invention, which is an antibacterially treated aqueous extract of the larvae of the darkling beetle Ulimoides dermestoides, containing a complex of antioxidant enzymes, heat shock proteins 70 kDa and 60 kDa, obtained by fine dispersion of the biomass of larvae in water by the cavitation method in a ratio of 1:4 until the temperature rises to 25-45°C, followed by extraction for a day with slow stirring, filtration of the extract and treatment with membrane filtration.

The technical essence of the claimed invention is as follows.

The claimed remedy, which is an aqueous extract from the larvae of the black beetle Ulomoides dermestoides, is obtained as follows. The larvae are separated by sifting through a sieve with a mesh diameter of 1.5 mm and washed with water. Then they are placed in a cavitation disperser, in which a mixture of larvae and water in a ratio of 1:4 is subjected to grinding and homogenization until the temperature rises to 25-45°C (but not higher than 45°C). Next, extraction is carried out during the day with slow stirring. The extract is filtered first through nylon gauze for coarse cleaning of solid particles, and then through a Millipore membrane filter (Merck, Germany) with a pore diameter of 0.1 μm for antibacterial treatment.

The analysis made it possible to isolate the most significant proteins in the obtained extract, which can explain the biological activity of the proposed agent: a complex of antioxidant enzymes (superoxide reductase, catalase, glutathione S-transferase, peroxiredoxin, thioredoxin), heat shock proteins 70 kDa and 60 kDa.

The main attention was paid to the evaluation of the activity of antioxidant enzymes in the extract. The indicators of catalase and antioxidant activity (AOA) were determined.

Catalase activity was measured by the content of hydrogen peroxide using ammonium molybdate. AOA was assessed by the quenching of luminol chemiluminescence. The results obtained were compared with the action of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a water-soluble analogue of vitamin E (a natural antioxidant).

The results of measuring the AOA of the extracts according to Trolox: 1 mg/ml of the protein of the larval extract is equivalent in antioxidant activity to 1.36 mM Trolox.

The results of measuring the catalase activity of the extract of larvae -6.3±1.1 μmol H ₂ O ₂ /min/mg of protein.

Testing the biological effectiveness of the claimed agent (hereinafter referred to as the extract) was carried out in model experiments on a nematode under conditions of oxidative stress, aging mice, aging hamsters.

The effect of the extract on the lifespan of the free-living soil nematode Caenorhabditis elegans was tested under conditions of induced oxidative stress. Oxidative stress inducer - paraquat (Paraquat-dichlorid, Methyl viologen dichloride hydrate), SIGMA. An extract of larvae containing 1.2 mg protein/ml was tested on adult nematodes grown in a standard way to the Adl stage and washed from bacteria. Under oxidative stress (OS), an oxidant agent was introduced into 160 μl of S-medium and 20 μl of extract - 20 μl of a 0.5 M solution of paraquat PQ (concentration in the well 40 μmol). Determined NRM - median life expectancy.

Change in the relative number of nematodes in the presence of 40 μmol paraquat (conditions of massive oxidative stress): the extract extended the life expectancy of the nematode from 27 h to 39 h, which is more than 20%.

The obtained extract of larvae was tested for biological activity in behavioral tests on mice and hamsters. The study of cognitive abilities and motor skills was carried out on aging mice of the C57BL/6 line (age at testing 19 months out of 24 possible, i.e. 80% of the average life expectancy), 8 individuals in the experimental and control groups and the common hamster Cricetus cricetus (groups of 9 individuals).

The extract immobilized on a food carrier (sea buckthorn meal) in the form of an experimental dietary supplement was introduced into the feed of laboratory animals (C57 B1/6 mice and the common hamster Cricetus cricetus) for 3 months at a dose of 400 μg of the extract/kg of body weight twice a week. Then testing was carried out.

Tests:

Physical endurance, spatial orientation, cognitive abilities - tests on rotarod, vertical pole and object recognition in mice (cognitive abilities).

General indicators: life expectancy, body weight, general analysis and biochemistry of blood, cognitive abilities to recognize objects in hamsters.

Comparison of the maximum time (out of three attempts) spent by mice on a pole using the Craxkell-Wallis test showed significant differences between groups. The results are shown in table 1.

The time spent by mice on the rotarod drum and the number of revolutions per minute that the mice of the control and experimental groups withstood are shown in Table 2.

The results of the first two experiments showed that mice from the experimental group were able to stay longer on top of a vertical pole and stay longer on a rotating drum with an ever-increasing speed of rotation. This suggests that the mice in this group have stronger muscle tone and more efficient coordination because the pole is "uncomfortable" to hold onto for long periods of time. Indeed, during the entire time of the test, the mouse constantly changes its body position on the pole - the area of support at the upper end of the pole is small (the height of the pole is 50 cm, its diameter is 1 cm) and the mouse probably makes a lot of efforts to hold on and maintain balance, while without straining the muscles of the paws and body. It can be assumed that better motor control and coordination of movements allows mice from the experimental group to stay longer on the pole than mice from the control group. Higher muscle tone also allows mice from the experimental group to stay longer on the rotarod drum and withstand higher rotation speeds.

Test for the cognitive ability of mice to recognize objects.

The duration of examination of a new object by mice in the experimental group significantly exceeded that in the control group. Comparison of the total duration of the examination of the reference (old) and new object for 5 min of testing showed that mice from the experimental group examined the new object longer in test presentations, and they had a greater difference in the duration of the examination of the new and old objects. Thus, in the experimental group, the reaction to novelty is more pronounced, and in mice from this group, the mechanisms for recognizing new objects are more pronounced, the results are shown in Table 3.

Hamster tests. The results are shown in table 4.

Observations revealed that the change in body weight in both groups of hamsters (control and experimental) was seasonal: body weight increased in the autumn-winter period and decreased by spring, while the weight loss of overwintered animals (in March) in the experimental group that took the drug based on the claimed extract were significantly less than in the control. In both groups, in March, an increase in the average hemoglobin level was noted, and the latter in the experimental group was significant (hereinafter, Student's t-test, p≤0.05), in winter, on the contrary, there was a significant decrease in these indicators in both groups. Thus, the listed changes can have an adaptive value. In the experimental group, the content of leukocytes at the beginning of observations was higher compared to the control (the sample was random), and during the experiment this indicator decreased markedly. Despite the fact that all fluctuations occurred within the reference values, the decrease in the level of lymphocytes in experimental animals was significant, which is associated with a decrease in infection with helminths.

Helmintoovoscopy. The survey results are presented in Table 5.

During the initial examination in the spring of 2021, the infestation of animals of both groups with helminths was shown. 2 species of nematodes were found: Aspiculuris tetraptera (Oxyurata: Heteroxynematidae) and Capillaria spp. (Trichocehyalata Capillariidae).

A re-examination of stool samples in October revealed only capillariid nematodes: Capillaria spp. (Trichocehyalata Capillariidae) and Trichocehalus muris. The nematode Aspiculuris tetraptera, (Oxiurata), identified in the previous survey (spring) is absent. Another study conducted after overwintering (in March) when used in the diet of experimental animals on the basis of the claimed agent showed a general decrease in the extent of invasion in control animals to 33%, however, no helminths were found in experimental animals, and the total extent of invasion (IE) was 0 %. Mortality in the control group was higher than in the experimental group, and a higher mortality rate was noted among males. Out of 9 animals, 5 individuals (4 males and 1 female) died, which amounted to 55.6%. While in the experimental group only one female died, which amounted to 16.7%.

Test for cognitive abilities of hamsters Cricetus cricetus.

When comparing the average total duration of the examination of both the reference and new object, and the frequency of examination of the reference (old) and new object, it was shown that hamsters of both the control and experimental groups examined the new object longer. At the same time, there is a pronounced tendency to increased attention (total duration of sniffing) in experimental hamsters in relation to a new object compared to control ones, which indicates a better memorization of old objects and recognition of new ones in experimental animals. The results are shown in table 6.

Based on the conducted experimental studies, it can be concluded that the use of an antibacterially treated aqueous extract of the larvae of the darkling beetle Ulimoides dermestoides, containing a complex of antioxidant enzymes, heat shock proteins 70 kDa and 60 kDa, as a means that has the ability to increase life expectancy, improve cognitive properties, motor control and coordination of animal movements, allowed to expand the arsenal of means with the declared specific action.

Claims (1)

An agent that has the ability to increase life expectancy, improve cognitive properties, motor control and coordination of movements of animals, characterized in that it is an aqueous extract of the larvae of the darkling beetle Ulomoides dermestoides treated with antibacterial filtration, containing a complex of antioxidant enzymes, heat shock proteins 70 kDa and 60 kDa, obtained by fine dispersion of the biomass of larvae in water by the cavitation method in a cavitation disperser at a ratio of 1:4 until the temperature rises to 25-45°C, followed by extraction for a day with slow stirring, filtration of the extract and treatment with membrane filtration.