RU2395289C1 - Method of manufacturing biogenic stimulator from larvae of drone brood of bees - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to veterinary. Method of manufacturing biogenic stimulator, which includes 50% of biologically active mass from drone larvae, 49.7% of sodium chloride solution and 0.3% of preservative, consists in the following: in order to obtain preparation comb with alive healthy 18-22 days old drone brood is kept in refrigerator at temperature 3-4°C during 5-6 days, after that comb with brood is opened and drone larvae of the same size, light-gray colour are selected, after which drone larvae are crushed in sterile laboratory mill, diluted with sterile sodium chloride solution with concentration 0.9% in ratio 1:1 and autoclaved at temperature inside boiler 120°C and steam pressure in jacket 1.5 atmosphere for one hour, after that the mass is filtered through two layers of gause into sterile measure vessel, brought to initial volume with sterile solution of sodium chloride with concentration 0.9%, preservative (phenol) is added, preparation is poured into prepared sterile vessels observing rules of asepsis, vessels are hermetically closed, autoclaved at 120°C and steam pressure in jacket 1.5 atmosphere for 20 minutes.

EFFECT: method allows to reduce considerably terms of preparation manufacturing, increase its safety, increase storage terms Biogenic stimulator obtained in said way contributes to improvement of animal growth and development, increase of offspring preservation, reduction of feed cost per unit of body weight increase, increase of general and specific resistance of organism.

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Description

FIELD OF THE INVENTION

The invention relates to veterinary medicine, in particular to preparations for increasing the overall resistance and productive qualities of farm animals.

State of the art

It is known that as a substrate used for the preparation of tissue preparations, most researchers recommend tissues with high growth activity and, consequently, metabolism. These are embryonic tissues, placenta, testes, blood, etc. [1, 3, 8].

A known method of preparing an extract from the placenta according to V.P. Filatov [3], including preservation of the placenta in the cold, washing off the blood by five immersion in distilled water, grinding, diluting with distilled water in a ratio of 1:10, heating in a water bath to 80-90 ° C for 30 minutes, cooling, filtering, boiling, packaging and sterilization by autoclaving at 120 ° C for 60 minutes

Known enzymatic hydrolysin and a method for its manufacture, characterized in that it is obtained by enzymatic hydrolysis using pancreatin and chloroform. Raw materials are fresh shaped blood elements of cattle [4].

Known dry biogenic stimulant made from animal embryos, wet tissues of which are kept under cooling according to the method of V. P. Filatov, and then crushed and dried in vacuum devices [3].

Known lysates (histolysates) - products of acidic or enzymatic hydrolysis of various organs and tissues of animals according to the method of academician MP Tushny [3, 6]. Get them in conditions of high temperature and high pressure.

Known ointment for treating open wounds based on beekeeping products, the preparation contains propolis, egg yolk, water and is characterized in that it contains butter or vegetable oil as a fat product (RU (11) 2082394 (13) C1, (51) 6 A61K 9 / 06, A61K 35/54, A61K 35/64) [9].

The disadvantage of this invention is exclusively the external use of this substance.

Known biostimulant - the placenta, the active principle of the placenta of women. The preparation is made according to a new technology, without heat treatment using the cavitation effect and newly discovered chemicals when sterilizing (see. A new generation of tissue preparations - PAN [8] // “Theoretical and practical aspects of the emergence and development of animal diseases and the protection of their health in modern conditions ". International Conference. Voronezh, October 3-4, 2002. Conference proceedings. - Volume 1. - Voronezh, 2000, - 295 p.).

The disadvantages of this method of preparing a biostimulant are the high cost, complexity and multi-stage preparation.

In recent years, organs and tissues of not only mammals, but also tissues of mollusks, fish, crabs and insects have been proposed as the initial substrate for the preparation of biogenic stimulants. We previously proposed a preparation in which the larvae of drone brood bees are used [7] (RU 2006 112147/13) and a method for its preparation, which includes selecting drones larvae, keeping the larvae in the cold, crushing them, diluting them with a 0.09% sodium chloride solution 1: 5, 5-fold heating at 58-59 ° С with daily intervals, filtration, application of honey (20%) and preservative (phenol or formalin - 0.3%), packing and again 5-fold heating at 58-59 ° С at daily intervals. The preparation prepared by the above method has high biological activity, but the process of its manufacture takes a lot of time - it only takes more than 10 days to heat treatment. The aim of this invention is to develop a new method of manufacturing a biogenic stimulant from drone brood larvae.

The implementation of the invention

Disclosure of invention

The aim of the invention is the creation of a biological stimulant based on the tissues of drone brood larvae and the development of methods for its manufacture. In this case, rational methods were used, aimed both at reducing the allergenic properties of the substrate, while preserving its initial qualities, and at reducing the production time of the drug.

The technical result that can be achieved using this invention is to obtain a new and affordable stimulant, the use of which helps to increase hormonal levels, increase the productive qualities of animals, preserve offspring, improve growth and development of animals. The achievement of the technical result is carried out by using the previously proposed new type of raw material - drone brood larvae and a new method (different from the method described in application RU 2006112147/13) for the preparation of the preparation. The method includes cooling the larvae in the cold at a temperature of 2-4 ° C for 4-6 days, grinding them, diluting with 0.9% sodium chloride solution (1: 1), heat treatment by autoclaving, filtration, packaging and final sterilization by autoclaving.

The essence of the proposed method of manufacturing the drug

To prepare the preparation, honeycombs with live healthy drone brood aged 18-22 days were kept in the refrigerator at a temperature of 3-4 ° C for 5-6 days, during this period there was a slow death of the brood in accordance with the teachings of V.P. Filatov. During this period, the formation of biologically active substances occurs in the body of larvae. Then the brood honeycombs were opened and drones larvae of the same size, light gray, were selected. After that, the drone larvae were crushed in a sterile laboratory mill, diluted with a sterile physiological solution of sodium chloride with a concentration of 0.9% in a 1: 1 ratio and autoclaved at a temperature inside the boiler of 120 ° C and a steam pressure in the jacket of 1.5 atmospheres for 1 hour. Then the mass was filtered through 2 layers of gauze in a sterile volumetric dish, adjusted to the initial volume with physiological saline, a preservative phenol or formalin (0.3% in the preparation) was added, poured into prepared sterile vials, following aseptic and antiseptic rules, and hermetically sealed. then kept in an autoclave at a temperature inside the boiler of 120 ° C and a steam pressure in the jacket of 1.5 atmospheres for 20 minutes.

Examples of specific performance of the method of manufacturing the proposed drug

Example 1. For the preparation of the preparation, the cells with live healthy drone brood of bees were kept in the refrigerator at a temperature of 3-4 ° C for 5-6 days. Then the brood honeycombs were opened and drones of the same size, light gray in color, were selected. Then the drone larvae were crushed in a sterile laboratory mill, diluted with a sterile physiological solution of sodium chloride with a concentration of 0.9% in a ratio of 1:10 and kept in a water bath at a temperature of 70-80 ° C for 60 minutes. Then the mass was filtered through 2 layers of gauze in a sterile volumetric container, adjusted to the initial volume with physiological saline, a preservative was added to its concentration in the substance of 0.3%, poured into prepared sterile vials, following aseptic and antiseptic rules, and hermetically sealed. When tested in experimental animals, we noticed numerous allergic reactions, which were manifested by the appearance of infiltrates at the injection site, a rash and, in some cases, Quincke's edema.

Example 2. Analogously to example 1, but the peculiarity of this modification is the dilution with a sterile physiological solution of sodium chloride with a concentration of 0.9% in a ratio of 1:10 and autoclaving at a temperature inside the boiler of 120 ° C and steam pressure in the jacket of 1.5 atmospheres for 1 hours. Then the mass was filtered through 2 layers of gauze in a sterile volumetric dish, adjusted to the initial volume with physiological saline, the preservative was added to its concentration in the substance of 0.3%, poured into prepared sterile vials, following aseptic and antiseptic rules, and hermetically sealed. Using this modification of the drug, we managed to avoid the manifestation of allergic reactions, but the effect of the use was insignificant, the shelf life of the drug was short.

Example 3. Analogously to example 1, but the peculiarity of this modification is the dilution with a sterile physiological solution of sodium chloride with a concentration of 0.9% in a ratio of 1: 1 and autoclaving at a temperature inside the boiler of 120 ° C and a vapor pressure in the jacket of 1.5 atmospheres for 1 hours. Then the mass was filtered through 2 layers of gauze in a sterile volumetric dish, adjusted to the initial volume with physiological saline, a preservative was added to its concentration in the substance of 0.3%, poured into prepared sterile bottles, observing aseptic and antiseptic rules and hermetically sealed, then kept in an autoclave at a temperature inside the boiler of 120 ° C and a steam pressure in the jacket of 1.5 atmospheres for 20 minutes. This modification is the most effective, because the concentration of active substances is optimal, the drug is safe for use in animals. The shelf life of the drug has increased significantly.

Example 4. Analogously to example 1, but the method consists in cooling the cells with live healthy drone brood of bees in the refrigerator at a temperature of 3-4 ° C for 5-6 days. Then the brood honeycombs were opened and drones of the same size, light gray in color, were selected. Then the drone larvae were crushed in a sterile laboratory mill, diluted with a sterile physiological solution of sodium chloride with a concentration of 0.9% in a 1: 1 ratio and autoclaved at a temperature inside the boiler of 120 ° C and a steam pressure in the jacket of 1.5 atmospheres for 1 hour. Then the mass was filtered through 2 layers of gauze in a sterile volumetric dish, adjusted to the initial volume with physiological saline, a preservative was added to its concentration in the substance of 0.3%, poured into prepared sterile bottles, observing aseptic and antiseptic rules and hermetically sealed, then kept in an autoclave at a temperature inside the boiler of 120 ° C and a vapor pressure in the jacket of 1.5 atmospheres for 40 minutes. With the additional time of the final autoclaving, a precipitate appeared in the substance and the caps were removed from many bottles.

Thus, the manufacturing method described in Example No. 3 is optimal. At the same time, the manufacturing time of the drug was reduced by more than 10 days; double autoclaving ensures reliable sterility of the drug and harmlessness, increases its shelf life.

Test results of a biogenic stimulator from drone brood in animals

Example 1. In the manufacture of an experimental batch of the drug (according to example No. 3 in the description of the manufacturing method), a biological sample was tested for its harmlessness. The test was carried out on laboratory mice and rabbits. When the drug was administered intramuscularly, subcutaneously and intraperitoneally, no acute toxic reaction was observed in animals, and no pyrogenic or allergic effect of the drug was established.

Example 2. For a more complete and objective assessment of the influence of the biogenic stimulant on the growth, development of sucking pigs and the reproductive qualities of sows, a scientific and economic experiment was conducted on pigs of the steppe type of early ripening meat breed (Table 1).

Table 1 The influence of biogenic stimulant on the growth and development of sucking pigs Age and gender groups Number of goals Dose, ml / kg Multiplicity of introduction Note Sucking pigs 40 0.1 4 times in 10 days A significant increase in the mass of the nest in 2 months. age. High live weight and average daily gains. Reduced feed costs per 1 kg of live weight gain. High safety of piglets both at the age of 21 days and at 60 days

After testing the drug on pigs, we obtained the following information:

- subcutaneous administration of the stimulant significantly affected the milk production of sows and the weight of the nest at two months of age;

- throughout the entire period of growing piglets showed a positive reaction to the introduction of a biological stimulant into the body, they were distinguished by better growth, development, had high live weight and average daily growths (average daily weight gain in piglets of the experimental groups was 40.7-98 higher, 9 g than control piglets);

- the use of a biostimulant contributed to an increase in the safety of sucking piglets by 5.5-10.4% compared with the control groups;

- the application has reduced the cost of feed per unit of increase in live weight.

Example 3. The study of the influence of biogenic stimulator from drone brood on the qualitative and quantitative indicators of sperm of boars-producers. The drug was administered three times at intervals of 10 days at a dose of 0.1 ml per 1 kg, subcutaneously. It was revealed that the drug contributes to an increase in ejaculate volume by an average of 72 ml, sperm concentration by 17 million per 1 ml, and the total number of sperm in the ejaculate by 35%.

Biochemical analysis of the drug is important as an indicator of its quality. According to many scientists, a number of compounds have high biological potential and can determine the main biostimulating function of the drug. Such substances, first of all, include amino acids. The amino acid composition of the drug is shown in table 2.

table 2 Amino acid composition of a biogenic stimulator from drone brood bee larvae Amino acids mg% Interchangeable Aspartic acid 16 Series 8 Glutamic acid 41 Alanya 12 Tyrazine 6 Total 83 Irreplaceable Threonine 7 Glycine 13 Valine 9 Methionine one Isoleucine eleven Leucine eleven Phenylalanine four Histidine 6 Lysine 10 Arginine 6 Total 78

To characterize the biological value of this drug, they use such a concept as the amino acid index, which determines the amino acid usefulness, it is equal to the ratio of essential amino acids to essential ones. In biologically complete substances, it varies within 1, the amino acid index of the samples of the drug (made according to example No. 3 in the description of the method) is 78/83 = 0.94.

The amino acids present in the preparation, such as methionine, histidine, and glutamic acid, are included in the pharmacopeia lists as independent immunostimulants and growth-promoting substances (Pharmacology, Mozgov I.E .; 1979).

In addition to amino acids, the drug contains another important group of biologically active substances - hormones.

Table 3 The hormone content in the biogenic stimulator from drone brood of bees The name of the hormones amount At to TG 0.09 FSH (follicle-stimulating hormone), mIU / ml 1.7 Free T4 (L-thyroxine), pmol / L 0.1 LH (luteinizing hormone), mIU / ml 2.5 Progesterone, nmol / L 9.5 Testosterone, nmol / L 14.5 Cortisol, nmol / L 0.01 Prolactin, mIU / ml 75.0 STH (growth hormone), mIU / l 12.0 DHEAc (dehydroepiandrosterone), μg / ml 0.1 ACTH (adrenocorticotropic hormone), pg / mg 1,0

Somatotropic hormone (STH) stimulates humoral and cellular immune responses, enhances protein synthesis in cells (D.N. Lazareva, E.K. Alekhin. Immunity stimulants. M: Medicine, 1985, - p.132-133). Cortisone and ACTH play a large role in providing adaptive trophic processes in the animal organism, which is especially important for young animals under the influence of constantly changing environmental conditions (Kostin A.P. Physiology of farm animals. M .: Kolos, 1974, p.249- 251).

The chemical composition of the nutrient stimulator from drone brood

Total moisture% Ash% Nitrogen,% Protein,% Fat% Ph 98.17 0.76 0.50 3,125 0.31 7.0

The quantitative composition of the drug

100 ml of the drug includes:

- 50 ml of biologically active mass from bee brood larvae;

- 49.7 ml of sodium chloride 0.9%;

- 0.3 ml of preservative.

The invention in comparison with the prototype and other known technical solutions has the following advantages:

- the substance used in the preparation of the preparation was not previously used in such a composition (50% instead of 20%);

- the biostimulator has the effect of tissue therapy, protein hydrolysatotherapy, organotherapy, non-specific protein therapy;

- reduction of the manufacturing time of the drug by 5 times.

The preparation method allows not only to obtain a drug with the effect of tissue and other types of therapy, but also allows you to save useful properties:

- high-quality amino acid and hormonal composition, high stimulating indicators;

- the use of a new type of raw material in a ratio of 1: 1;

- a significant reduction in the manufacturing time of the drug, increasing its safety, increasing the shelf life;

- a decrease in the volume (ml) of doses in 2 times and the frequency of injections.

Claims (1)

A method of preparing a biogenic stimulant comprising 50% of the biologically active mass from drones larvae, 49.7% sodium chloride solution and 0.3% preservative, characterized in that the cells are prepared in a refrigerator with live healthy drone brood aged 18-22 days in the refrigerator at a temperature of 3-4 ° C for 5-6 days, then the brood honeycombs are opened and drones of the same size, light gray in color, are selected, after which the drones are crushed in a sterile laboratory mill, diluted with a sterile chlorine solution yes sodium with a concentration of 0.9% in a ratio of 1: 1 and autoclave at a temperature inside the boiler of 120 ° C and steam pressure in a jacket of 1.5 atmospheres for 1 h, then the mass is filtered through 2 layers of gauze in a sterile volumetric dish, adjusted to sterile a solution of sodium chloride with a concentration of 0.9% to the original volume, add a preservative (phenol), pour, subject to the rules of asepsis and antiseptics, into prepared sterile bottles and seal them tightly, autoclave at 120 ° С and a vapor pressure in a jacket of 1.5 atmospheres in within 20 minutes