Characteristics of Biologically Active Properties and Therapeutic Use of Ulomoides Dermestoides Beetles

INTRODUCTION

The number of patients suffering from autoimmune and immunodeficiency diseases increases every year. In some economically underdeveloped countries, patients are forced to resort to various methods of treatment, one of the most common is insectotherapy. Most often, the black beetle Ulomoides dermestoides is used in insectotherapy. The use of these insects is becoming more and more popular every year, especially in Asia and South America [1, 2]. The effectiveness of their use in chemotherapy, bronchial asthma, oncology, psoriasis, and diabetes has been proven [3]. Substances contained in the feces of these insects can be used to treat stomach disorders, heart disease, myalgia, kidney diseases, and asthma, as well as against dermatitis, rheumatoid arthritis, hemorrhoids, inflammation, and pain in the liver and kidneys, Parkinson's disease, diabetes, various types of oncology [4-7].

Santos et al. [4] conducted a study aimed at evaluating in vitro and in vivo the anti-inflammatory effect of U. dermestoides. A model of acute inflammation by injection of carrageenan into the pleural cavity of rats was used. In experiments with an intrapleural injection of carrageenan-induced by an acute inflammatory reaction characterized by a noticeable accumulation of pleural, plasma exudation and intensive migration of polymorphisms of the pleural cavity. Treatment of animals with U. dermestoides extract weakened the inflammatory process: the total number of leukocytes, PMN cells, protein concentration, and volume of pleural exudation after treatment [8-11].

Tobon et al. [12] studied a neuropharmacological profile of biologically active substances extracted from U. dermestoides. The most significant effect was that the animals presented in this preliminary trial were sleepy and passive; during that time of the experiment, compared with the depressive effect of Diazepam and the stimulating effect of amphetamine on the central nervous system. Neuropharmacological tests performed showed the depressing effect of U. dermestoides extract: on memory, learning, and discrimination. Statistical analysis of the data shows that the extract of U. dermestoides produces a noticeable decrease in brain functions in these animals.

The purpose of the research work is to study the possibility of using U. dermestoides beetles as a source of raw materials for the development of an immunomodulatory agent.

MATERIALS AND METHODS

Despite the huge volume of biotechnological potencies, U. dermestoides beetles have not been used before in the preparation of drugs in the field of immunology, in this regard, there is no detailed description of the technological process of preparation of the drug based on the biomass of these beetles in the available literature. Relying on traditional methods and allowing our modifications, we have prepared an extract from U. dermestoides.

The technological process of preparing a biologically active substance from raw materials of animal origin is presented in the block diagram in Figure 1.

At the preparatory stage, beetles were grown in a laboratory, while part of the beetles was fed only banana peel (sample 1), part only sunflower meal (sample 2), and part only apple pulp (sample 3). Then the beetles were placed in a refrigerator for a day at a temperature of +4-6 ℃ for preservation. The cooled bodies of beetles improved the structural and mechanical properties and facilitated the subsequent homogenization process.

The need for homogenization (grinding) is since extraction is a mass transfer process, and its speed will depend on the contact area between the raw material and the extractant. After homogenization, extracts obtained from raw materials of animal origin U. dermestoides were placed in test tubes and subjected to centrifugation at a speed of 4000 revolutions per minute for five minutes, to separate into fractions according to density. Due to centrifugal forces, the primary juice is removed from the cellular material, a fresh extractant is supplied in its place, which is again removed from the material. The extractant circulates until saturation and then is replaced with a new one.

A saline solution was selected as an extractant. The production of the extract was carried out as follows: homogenate from beetle raw materials in a ratio of 1:10 was poured with saline solution. To prepare the extract taking into account the dry matter content in the homogenate, we took 20 g of homogenate and poured 350 ml of saline solution. The extract was infused for a week at room temperature 20-25 ℃, humidity 40-45%, and at normal atmospheric pressure. After the infusion of the extract, a two-time filtration was carried out through a sterile bandage folded in three layers. According to external characteristics, the extract is a viscous liquid of a light yellow color with a pungent odor.

In the final stage of extraction, packaging, and capping of semi-finished products in sterile bottles of 100 ml, as well as labeling, was carried out. Thus, when developing the technology for preparing the extract of the biologically active additive "Immutoron", based on the raw materials of U. dermestoides beetles, the most rational method of preparing the extract was used, allowing to preserve the immunomodulatory properties from the original raw materials, and also to develop several biologically active substances that improve the quality of the raw semi-finished product.

The rats are housed in cages mounted on metal racks. Experiments on Wistar rats (n = 40) were carried out following the rules for the protection of vertebrates used for scientific purposes.

After the animals were admitted to the laboratory, they were placed in clean, disinfected cages for quarantine for fourteen days. During the quarantine period, daily clinical observation and registration of the general condition of the animals were carried out. The animals were kept in rooms with a constant temperature of +20-22 ℃ and air humidity not exceeding 40-45%. The rats had free access to food and water. The rats received a standard vivarium diet recommended by the Research Institute of Nutrition of the Russian Academy of Medical Sciences for Animals, balanced by the main macronutrients and micronutrients, and chilled boiled water.

Four groups were formed, three experimental and one control group, 10 animals each. The duration of the experiment is 14 days. Rats of the experimental groups were administered orally daily dietary supplement "Immutoron" in a dose of 0.5 ml once. The rats of the control group remained without the introduction of the additive (Table 1).

Table 1. Distribution of experimental animals by groups

Body temperature was measured in all rats, as it is an important criterion for assessing the state of the body of warm-blooded animals.  The temperature of the animals was within the normal range from 37.0 ± 0.2 °C to 37.5 ± 0.3 °C. To evaluate the immunomodulating drug on the animal body, a cold model of the experiment developed by V.A. Dorovskikh was used [13].

The animals were monitored daily for 14 days. The general immunomodulatory process was evaluated: clinical condition, behavioral characteristics, intensity and nature of the motor activity, signs of intoxication, and possible mortality. It was found that in all tested doses it did not cause the death of rats and did not have a pronounced toxic effect on the body. When evaluating the effectiveness, body temperature, the physical and nutritional activity of animals, immune status indicators, and biochemical and hematological blood tests were taken into account.

RESULTS AND DISCUSSION

The study of blood biochemical parameters is of great diagnostic importance. Even an incomplete hematological and biochemical blood test helps a specialist to reliably determine the state of the animal's body, and periodic examination of the blood composition allows one to determine with high accuracy not only the general condition of the body but also to predict the outcome of the disease, adjust therapy, study the effect of certain medications. That is why the role of laboratory tests, as well as the range and number of studies required in preclinical studies, is constantly increasing.

A comprehensive analysis of the data obtained during the experiment allowed us to determine the effectiveness of the Immutoron dietary supplement. The effect of biologically active additives on hematological (number of erythrocytes, hemoglobin, leukocytes) and biochemical (total protein, cholesterol, glucose) indicators were studied. The negative influence of the studied "Immutoron" on the conduct and basic clinical parameters (temperature, pulse, respiration) of animals has not been established. Blood samples were taken before the start of the studies, as well as on the 7th and 14th days of the experiment.

Hematological and biochemical parameters in the second experimental group did not exceed the limits of the physiological norm during the entire study period.

On the 7th day of observation, an increase in the number of red blood cells was noted in the first experimental group – by 9.3%, in the second experimental group – by 8.9%, in the third – by 8.1% compared with the control group. The increase in hemoglobin content in the first experimental group was 11.3%, in the second experimental group – 13.8%, and in the third experimental group – 12.7% (Table 2). Biochemical blood parameters in experimental and control animals are shown in Table 3.

Table 2. Hematological studies

Table 3. Biochemical parameters of blood serum