RU2698707C1 - Biogenic preparations production method - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a method for producing biogenic preparations. Method for producing a biogenic preparation includes using raw material from by-products and slaughter waste, holding raw materials in refrigerator conditions, raw materials milling, dilution thereof with an active solution of sodium hypochlorite during subsequent extraction of raw material in refrigerator conditions, wherein extraction is carried out in refrigerator conditions during subsequent dilution of extract with sodium hypochlorite and treatment of obtained extract with ultrasound, then produced extract is filtered, packed and sterilized in autoclave under certain conditions.

EFFECT: method described above allows to increase output of end product and increase its therapeutic effectiveness.

1 cl, 4 tbl, 1 ex

Description

The invention relates to veterinary medicine, in particular to a technology for producing tissue preparations, more specifically biogenic preparations from offal and slaughterhouse waste from farm animals.

There is a method of preparing an agarotissue preparation (ATP) in which the spleen of cattle from a healthy, freshly killed animal is used, which is placed in the refrigerator for 4-5 days at a temperature from 0 to + 4 ° C. Then it is ground in a sterile meat grinder and diluted with an isotonic sodium chloride solution in the ratio: 1 part of tissue and 2 parts of solution. The resulting mass is boiled for 1-1.5 hours, then left at room temperature for 2-3 hours. The boiled-up liquid is filled with sterile saline and sterile molten agar-agar is added at the rate of 0.1% by weight of the preparation. Agar-agar is added to give the tissue preparation prolonged properties. Then the liquid is filtered through a double gauze filter, poured into sterile vials and hermetically sealed. The drug is checked for sterility, safety and activity.

The disadvantage of this method should be considered the presence of a high temperature regime (boiling for 1.0-1.5 hours) during the extraction process, which leads to coagulation of proteins. In the process of manufacturing ATP, prolonged action requires agar-agar, due to its melting, the time and cost of the final product increases, the average price of 1 kg of agar-agar is 4586 rubles.

A known method of obtaining funds with adaptogenic and anti-radiation activity from the spleen by exposure to it with an aqueous extracting agent in the subsequent separation of the extract by dialysis and lyophilization. Exposure to an aqueous extractant is supplemented by simultaneous exposure to low-frequency ultrasound of 20-70 kHz, after which dialysis (purification) of the aqueous extractant is carried out, that is, the extractant is removed from the preparation by using membranes with a passage value for a molecular weight of 25000-50000. The weight ratio of spleen and extractant is spleen: extractant 1: 6-1: 3. The use of ultrasound in the extraction process allows to increase the yield of the drug (RF patent No. 2152219 A61K 35/28).

However, this method is characterized by the presence of the use of an extractant which must be disposed of at the end of the extraction in view of its harmful effects on the body. In addition, this method lengthens and complicates the process and complicates the organization of the production of biogenic drugs directly at agricultural enterprises.

A known method of obtaining biogenic medicinal stimulants (tissue preparations) according to V.P. Filatov. To prepare the extract, parenchymal organs or other tissues are taken from freshly killed healthy animals and placed in a refrigerator, where they are kept for 5-7 days at a temperature of + 2- + 4 ° С. Then the organs (tissues) are crushed in a sterile meat grinder and diluted with an isotonic sodium chloride solution at the rate of raw materials: 1: 2 solution. The mixture is boiled for 1-1.5 hours, insisted at room temperature for 2-3 hours, filtered, Packed in bottles, sterilized in an autoclave for an hour at a temperature of 120 ° C (I.E. Mozgov Pharmacology. - 8th ed., add. and revised .-- M .: Agropromizdat, 1985.S. 261-266).

The disadvantage of this method is the presence of a high-temperature regime (boiling for 1.5 hours) during the extraction process, which leads to coagulation of proteins, accompanied by a change in the most important properties of the protein: loss of biological activity (inactivation of enzymes), the ability of proteins to hydrate (when the conformation on the surface changes hydrophobic groups appear in the protein globule, while hydrophilic ones are blocked as a result of the formation of intramolecular bonds), increased aggregation of protein molecules. Outwardly, aggregation is expressed in different ways: in low-concentrated protein solutions, the formation of foam (flakes on the surface of the broths), in more concentrated protein solutions, the formation of a continuous gel during their simultaneous compaction and separation of the liquid into the environment (dehydration). This determines a low degree of extraction of biologically active substances.

A known method for the production of biogenic drugs including the use of raw materials from the slaughterhouse waste products, holding the raw materials in the refrigerator for 5-7 days at a temperature of + 2- + 4 ° C, grinding the raw materials with subsequent mixing with extractant, extraction, filtration, packaging and sterilization in an autoclave for an hour at a temperature of 120 ° C, an active solution of sodium hypochlorite at a concentration of 300.0-600.0 mg / l in the ratio of raw materials: sodium hypochlorite 1: 2-1: 3 is used as extractant, and the extraction is carried out at a temperature + 2- + 4 ° C for 18-24 hours. RF patent No. 2682641 A61K 35 / 12- (prototype).

The problem to which the claimed method is directed is to increase the yield of therapeutic efficacy of a biogenic preparation by optimizing technological regimes and the time of its production.

The specified technical result is achieved by the fact that in the method for the production of biogenic preparations, including the use of raw materials from offal and slaughterhouse waste, keeping the raw materials in the refrigerator for 5-7 days at a temperature of + 2 - + 4 ° C, grinding the raw material, diluting it with an active solution of hypochlorite sodium in the ratio of raw materials: sodium hypochlorite 1: 2-1: 3 during subsequent extraction of the raw materials in the refrigerator at a temperature of +2 - + 4 ° C, filtration, packaging and sterilization in an autoclave for an hour at a temperature of 120 ° C, according to the application According to the invention, extraction in the refrigerator is carried out for 8-10 hours, followed by dilution of the extract to a ratio of raw materials: sodium hypochlorite 1: 4-1: 5 and extraction of the solution under the influence of ultrasound at a frequency of 20 kHz for 2.5-3 hours at a temperature not higher than 50 ° C. An active solution of sodium hypochlorite is used at a concentration of 300.0-600.0 mg / L.

An aqueous solution of sodium hypochlorite (NaOCI) is a strong oxidizing agent and enters into numerous reactions, regardless of the acid-base nature of the medium, thereby showing its bactericidal activity. This agent kills microorganisms very quickly even at fairly low concentrations and is used in regulated dilutions of 300.0-600.0 mg / l (see the Manual on the use of an active solution of sodium hypochlorite, protocol No. 2 dated April 23, 1999, approved by the veterinary board of the Veterinary Department of the Ministry of Agriculture and Food RF). In the process of extraction of raw materials obtained from offal and slaughterhouse waste, the activity of sodium hypochlorite is lost due to its pharmacological properties and is finally destroyed when heated, therefore, after thermal sterilization (autoclaving), it turns into a normal saline solution, no toxic products are formed , therefore, there are no contraindications to use the biogenic preparation obtained by this method for subcutaneous and intramuscular administration to animals.

To develop the technical parameters of the claimed method, a number of studies were carried out. In the first experiment, the optimal moment of inclusion of ultrasonic action on the extracted substance into the technological chain was revealed. The experiment involved the raw materials: liver, spleen, lymph nodes, uterus with the fetus (2-3 months) and determined the dry matter content in extracts every 2 hours at a ratio of raw materials: sodium hypochlorite: 1: 1, 1: 2, 1: 3 , 1: 4, 1: 5 at a temperature of + 2- + 4 ° C in a refrigerator, i.e. according to the prototype with an extended degree of dilution. The results of the experiment are presented in table 1.

As studies have shown, the extraction process was significantly slowed down after 8-10 hours of extraction in a refrigerator (hundredths of a percent per hour), in contrast to the intensity of the process during the first eight hours, where the average increase was 0.17% per hour. It should also be noted the dynamics of the decrease in the yield of dry matter with increasing dilution (the minimum value of the product yield at a dilution of 1: 4-1: 5). Therefore, the introduction of ultrasonic action into the process chain from 8-10 hours of extraction, that is, starting from the moment the process intensity drops when the potential of the extractant is exhausted, should be considered an appropriate technique.

In the second experiment, the optimal time of exposure to ultrasound with a frequency of 20 kHz on the yield of dry matter was determined at the same dilution degrees (from 1: 1 to 1: 5) and the composition of the raw material. After 8 hours of extraction in a refrigerator, the extract was subjected to ultrasonic treatment (table. 2). The mechanical work of ultrasound enhances the diffusion of solvents into biological tissues. The physico-biological effect of ultrasound on biological objects is primarily associated with an increase in the permeability of cell membranes and diffuse processes change the concentration of hydrogen ions in tissues, causing splitting of high molecular weight compounds. Chemically, the decay products of ionized water molecules in tissues are extremely active, and their great activity is responsible for a number of general biological effects that manifest themselves under the influence of ultrasound. Cavitation leads to the breaking of molecular bonds in the biomaterial and the development of redox reactions.

Studies have shown that the optimal time of exposure to ultrasound on the course of the extraction process should be considered 2.5-3 hours while the yield of dry matter is at the level of 3.29-3.94%. In addition, it turned out that ultrasound more effectively affects the yield of the product in extracts with a dilution ratio of 1: 4-1: 5, which predetermined in the future, during the development of the method, additional dilution of the extract before exposure to ultrasound, since the extraction process is up to 8 -10 hours was carried out with a dilution degree of 1: 2-1: 3. In our opinion, this circumstance is due to the fact that by 8-10 hours of extraction, the extract becomes more saturated, which complicates the effect of ultrasound on cell structures. It should be noted that the effect of the ultrasonic field on the biomaterial allows to increase the percentage of extractable substance in the extract at a dilution of 1: 4-1: 5, thereby increasing its concentration (dry matter) and the amount (volume) of the extract, i.e. the yield of the product as a whole from a unit of raw materials.

With the propagation of ultrasound in biological media, it is absorbed and the conversion of acoustic energy into thermal energy. The biological effect of ultrasonic waves on the biomaterial is largely associated with the phenomenon of cavitation, that is, the process of formation of cavities in a liquid medium filled with vapor of the liquid itself, which arise under the action of large tensile stresses and collapse the next instant, accompanied by high pressures and local heating of the medium. Pressure pulses arising from the closure of cavitation cavities are capable of destroying not only solid and liquid bodies, but also many biological objects, in particular microorganisms, thereby minimizing the risk of undesirable consequences of banal microflora activity during production. In the inventive method, in order to prevent coagulation of proteins when the extract is heated under the influence of ultrasound, the extraction process is carried out at a temperature not exceeding + 50 ° C using a flowing water jacket for cooling during ultrasonic exposure.

The method is as follows:

Example. To prepare a biogenic preparation under aseptic conditions, a liver, a spleen, lymph nodes, a uterus with a fetus (2-3 months) from healthy animals were taken and the raw materials were placed in a refrigerator where they were kept for 7 days at a temperature of + 2- + 4 ° С. Then the organs were minced in a meat grinder and diluted with a solution of active sodium hypochlorite at a concentration of 370.0 mg / l in the ratio of 1 kg of biomaterial in 3 l of sodium hypochlorite (raw material: sodium hypochlorite ratio 1: 3), thoroughly mixed and placed in a household refrigerator for 8 hours at a temperature of + 2- + 4 ° C. After the specified time, the extract was diluted with one liter of sodium hypochlorite (raw material: hypochlorite ratio 1: 4) and transferred to an ultrasonic unit (ultrasonic processing apparatus of the Wave series UZTA-0.63 / 22nd) for 3 hours, cooling the contents of the vessel with extract to + 50 ° C using a water jacket. At the end of the indicated period, the obtained extract under aseptic conditions was filtered, Packed in sterile bottles, and sterilized in an autoclave for an hour at a temperature of 120 ° C.

To conduct production tests, experimental batches of tissue preparations were made according to the prototype and the claimed method. The test results are presented below.

Compared tissue preparations were administered 10-15 days before the expected calving by dry animals and the first day immediately after calving subcutaneously in the middle third of the neck at a dose of 20 ml. In the experimental group there were 90 cows, in the control (prototype) 80 animals. The results of comparative tests of the claimed method for producing biogenic preparations and the prototype method are shown in table 3.

It is considered the optimal norm if the separation of the placenta occurs within 2-6 hours after calving, sometimes the placenta leaves later, after 6-8 hours. Pathology is considered when the afterbirth is delayed for more than 10-12 hours. A long stay in the womb of a cow is associated with a risk of infection of the uterus and the birth canal with harmful microflora. In optimal terms, the separation of the placenta from 5.0-56.2% was noted after administration of a tissue preparation made according to the prototype, and by the claimed method, from 7.8-68.9%, respectively. Retention of the placenta over 12 hours was observed in 7 control cows that were injected with a tissue preparation prepared according to the prototype, which is 2.6 times higher than this indicator in the experimental group.

In the postpartum period, immediately after calving, a tissue preparation was injected prophylactically to retain the placenta at a dose of 20 ml subcutaneously. the middle third of the neck, and every 10 days 3-4 times in the same dose to reduce the time of uterine involution and time to restore sexual cyclicity. At the same time, the dates of coming to the hunt and the percentage of fruitful inseminations were taken into account, table 4.

The duration of the service period should normally not exceed 95 days. But you should strive for the optimal time for coming to hunting and fruitful insemination of 45-60 days, then the intensity of production does not suffer. According to the optimal timing for the restoration of sexual cyclicity, the claimed preparation exceeds the prototype by 8.0%, in the number of fruitful inseminations, respectively, by 6.5%.

Thus, the use of the claimed method for producing biogenic preparations can increase the yield and therapeutic efficacy of drugs while reducing the time of its production.

Claims (2)

1. Method for the production of a biogenic preparation, including the use of raw materials from offal and slaughterhouse waste, keeping the raw materials in the refrigerator for 5-7 days at a temperature of +2 - + 4 ° C, grinding the raw material, diluting it with an active solution of sodium hypochlorite in the ratio of raw materials: sodium hypochlorite 1: 2-1: 3 during subsequent extraction of raw materials in a refrigerator at a temperature of + 2 ... + 4 ° C, filtering, packing and sterilization in an autoclave for an hour at a temperature of 120 ° C, characterized in that the extraction in a refrigerator spend in t 8-10 hours during subsequent dilution of the extract to the ratio of raw materials: sodium hypochlorite 1: 4-1: 5 and extraction of the solution under the influence of ultrasound at a frequency of 20 kHz for 2.5-3 hours at a temperature not exceeding + 50 ° C. 2. The method according to p. 1 characterized in that the active solution of sodium hypochlorite is used at a concentration of 300.0-600.0 mg / L.