RU2386443C1 - Method for preparing biologically active concentrate of velvet antlers - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine and veterinary science, more exactly velvet antlers technology for making pharmacological and cosmetic preparations. There is offered the method which involves enzymatic hydrolysis of velvet antlers of drainage rate not exceeding 100 mcm with water and animal's pancreatic homogenate added at 100 g of pancreas per 1 kg of antler flour that is followed with water hydrolysate extraction by centrifugation and filtration, herewith enzymatic hydrolysis is performed with deer antler pancreas added in the weight ratio of antler flour: water 1:1.5, within 3-4 hours at temperature no more than 50°C, hydrolysate extraction is carried out in the weight ratio of hydrolysate: water 1:1.5, within 3 hours. Centrifugation and filtration is followed with drying the extract containing an end product at temperature 50°C and lower and pressure 0.9 atm.

EFFECT: invention provides preparing concentrate of deer antlers exhibiting high biological activity ensured by maximum content of hormones, growth factors, amino acids of macro- and microelements and vitamins.

1 ex, 4 tbl

Description

The invention relates to the production of pharmacological preparations with pronounced biological activity, in particular to preparations with a high content of hormones and growth factors, and can be used in the cosmetic industry, as well as in medicine and veterinary medicine.

A known method of producing pantolysate (liquid concentrate), including acid-enzymatic hydrolysis of crushed antlers in artificial gastric juice. Pantolizate contains in its composition a significant amount of macro- and microelements and essential amino acids. The fermentation process is carried out at a temperature of from 20 to 42 ° C. However, the use of acids necessitates the presence of a neutralization operation with subsequent removal of salts, as well as irreversible destruction of the protein by acid (see patent RU 2112396, MKI A23J 1/10, A23J 3/34, A61K 35/32).

A known method of preparing a biologically active preparation of antlers pantolysate (liquid concentrate), including enzymatic hydrolysis of crushed antlers and filtering the hydrolyzate. The degree of grinding of the antlers is not more than 100 μm, while before fermentation the flour is kneaded at room temperature in a double volume of distilled water, and after 1.5-2 hours the enzymatic complex of the pancreatic suspension is added at the rate of 10% by weight of flour. The mixture is stirred and hydrolysis is carried out for 6 hours at 38-40 ° C. The resulting hydrolysis is extracted with water in a ratio of 1: 5 for 6 hours and centrifuged before filtration (see patent RU 2268059, MKI AK61K 35/32 - prototype).

However, the liquid form of the drug has a limited shelf life, is inconvenient in transportation and use, so when introducing the drug into the product, it is necessary to take into account the presence of water in the preparation, which entails a number of unnecessary technological methods for eliminating this factor. In addition, the biological activity of antler drugs to date has been determined mainly by the presence of amino acids, macro- and microelements, vitamins, fatty acids and several hormones in them, while studies of the biochemical composition of antler products have revealed a sufficient number of previously non-hormones and growth factors Moreover, known production methods, as shown by studies, do not provide the maximum percentage of extraction of these substances from antler raw materials.

It is necessary to develop a method for producing a biologically active concentrate from antlers, convenient for use and providing due to the maximum extraction of hormones and growth factors (with a high content of amino acids, macro- and microelements, vitamins) high biological activity and expansion of the scope of use of the biologically active product from antlers.

The specified technical result is achieved in that in a method for producing a concentrate from antlers of deer, comprising enzymatic hydrolysis of antler flour with a grinding degree of not more than 100 μm in the presence of water and homogenate of the animal’s pancreas at the rate of 100 g of gland per 1 kg of antler flour followed by aqueous extraction of the hydrolyzate , centrifugation and filtration, enzymatic hydrolysis is carried out in the presence of a pancreas of antler deer at a weight ratio of antler meal: water 1: 1.5 for 3-4 hours at a temperature round, no more than 50 ° C. The hydrolyzate is extracted at a weight ratio of hydrolyzate: water of 1: 1.5 for 3 hours, and after centrifugation of the filtration, the extract containing the target product is dried at a temperature not exceeding 50 ° C and a pressure of 0.9 atm.

The essence of the method is as follows. Until recently, the optimality of the technological parameters of the process of production of biologically active preparations from antler products was assessed by the presence of mainly amino acids, vitamins, macro- and microelements and a number of hormones in them, and the influence of growth factors on the biological activity was ignored, since technological modes were aimed at the optimal yield of the above substances and, as studies have shown, growth factors were extracted in small quantities. At the same time, growth factors, like hormones, have a wide range of biological effects on many cells of the body - they stimulate or inhibit mitogenesis, chemotaxis and cell differentiation (maturation). So, cells divide under the influence of insulin-like (IGF) and epidermal (EGF) growth factors, and they mature using transforming (TGF) growth factor. Growth factors push back the “planned old age” of the cell, that is, they have a rejuvenating effect on the tissues and the whole organism: they restart the genes that are disconnected due to old age or due to illness, thereby prolonging the life of cells, increase the number of cell division, and stimulate metabolism in them. Growth factors stimulate the growth of the child, especially the development of the musculoskeletal system, which is especially important in adolescence. According to the latest clinical studies, growth factors facilitate the course of diseases of the digestive tract, and are most effectively used for acute intestinal diseases. The influence of growth factors is studied to prevent the development of hyperplastic processes (mastopathy, uterine fibroids, endometriosis) and reduce the progression of cancer. In particular, nerve growth factor (NGF) plays an important role in the processes of development and functioning of the nervous system, as well as in the regeneration of damaged neuronal structures. Transforming factor (TGF ₂ ) is an angiogenic factor, is involved in cell proliferation, as well as in the regulation of tumor cell growth. During the metabolism of dehydroepiandrosterone (DHEA), testosterone and dehydrotestosterone are formed in the peripheral tissues. Epidermal factor (EGF) controls and stimulates the proliferation of epidermal and epithelial cells, including fibroblasts, renal epithelium, glial cells, ovarian granulosa cells. Ciliary Neurotrophic Factor (CNTF) is seen as a key differentiation factor for developing neurons and glial cells. Bone morphogenetic protein (BMP-4) has a pronounced therapeutic effect in arthritis for bone tissue regeneration. Insulin-like growth factor (IGF) carries out endocrine, autocrine and paracrine regulation of the processes of growth, development and differentiation of cells and body tissues. It should be said that the action of growth factors must be considered in connection with other stimulants, primarily hormones, namely, hormones in biologically active preparations from antler products with the appropriate production technology (by the way, quite close in technological parameters, as studies have shown, to technologies for optimal output of growth factors) are in sufficient quantity.

Thus, the development of a method for the production of a biologically active product from antlers, which provides a high yield of growth factors, especially with the release of the product in the form of a dry concentrate, will expand the scope of the product with more convenient use.

When developing the method, studies were conducted to identify hormones and growth factors in whole antlers of deer, whole antlers of reindeer, the basis of antlers of reindeers, powder from skin and hair from canned antlers of reindeers. The content of hormones and growth factors was determined by the enzyme immunoassay using standard diagnostic kits of the German company JBL, American DSL, EGF, B&D Systems, CHEMICON, Austrian Bender Medsystems according to generally accepted methods. The research results are shown in table 1.

Table 1 Name Raw materials one 2 3 four 5 Antlers of the maral whole Whole reindeer antlers The basis of reindeer antlers Reindeer antlers skin and hair powder Reindeer antlers cake Testosterone (pg / g) 718 443 639 849 804 Dehydroepiandrosterone (DHEA), ng / g 14.8 6.1 3.6 12.7 33 Insulin-like Growth Factor 1 (IGF-1), ng / g 213 173 10,4 22 0 Insulin-like growth factor 2 (IGF 2), ng / g 314 148 40 48 34 Alpha transforming growth factor (TGF-α), pg / g 162 44 58.2 fourteen 11,2 Beta-transforming growth factor (TGF-β), ng / g 29.5 30,2 7.5 8.8 14.5 Epidermal growth factor (EGF), pg / g 26.8 46.3 43 32,0 46.0 Nerve Growth Factor (NGF), ng / g 33,4 47.0 38,0 158.0 33.0 Ciliary neurotrophic factor (NGF ₃ , CNTF), pg / g 134 23.5 26.9 42.3 65.3 Bone morphogenetic protein - 4 (BMP-4), pg / g 51.6 35,4 20.1 26,4 9.1

The initial stage in the development of the method was the search for an enzyme of animal origin, the most suitable for obtaining antler concentrate. Table 2 shows the biological activity of pantolysates made from reindeer antlers using enzyme complexes of cattle pancreas suspensions and reindeer pancreas suspensions as enzymes during their preparation according to the technological parameters of the prototype. Additionally, in this experiment involved a fermentation temperature of 50 ° C. Enzymatic complexes are pancreas (hereinafter referred to as homogenates) of cattle and antler milled on a homogenizer. The results of the experiment are shown in table 2.

table 2 Type of enzyme Dose, gram Duration of fermentation, hour The temperature of the fermentation process, ° C The duration of the extraction, hour The biological activity of pantolysate,% Enzymatic complex of the suspension of the pancreas of cattle (SRS cattle) one hundred 6 40 6 29.6 ± 2.1 fifty 31.4 ± 2.3 Enzymatic complex of antler deer pancreas suspension one hundred 6 40 6 33.6 ± 2.7 fifty 35.1 ± 1.4

Pancreatic homogenates contain a number of enzymes: trypsinogen (pretrypsin), trypsin A and B, chymotrypsin, carboxytrepsidase and elastase, which break down proteins into amino acids; aminopepsidase, which breaks down polypeptides, amylase (diastase), invertase and lactase, which turn complex carbohydrates into simple ones.

As can be seen from table 2, the biological activity of pantolysates using the antler pancreas homogenate during their preparation is 12.6% higher on average and 6.1, 4.5% higher when 50 ° C is used in the fermentation process.

In the process of identifying the optimal parameters of the method, providing a high yield of hormones and growth factors (with a high content of amino acids), 3 experiments were conducted on the preparation of concentrates from antlers of reindeer:

1-6 experiment - the dose of deer pancreas homogenate - 100 g per 1 kg of ground antlers (up to 100 microns);

- fermentation time 3, 4, 6 hours at a temperature of 50 ° C and a dilution of 1: 1.5 (for 1 kg of antlers 1.5 l of water). The weight of the hydrolyzate is 2.5 kg;

- the extraction time of 3 and 6 hours with a weight ratio of hydrolyzate: water 1: 1.5;

- drying pantolizate in a vacuum dryer at a temperature of 50 ° C and a pressure of 0.9 atm;

7 experience - a dose of homogenate of the pancreas of cattle 100 g per 1 kg of ground antlers;

- the time of fermentation, extraction and the degree of dilution in accordance with the optimal regime of the first six experiments (3 experiments);

- drying pantolizate according to the regime of 1-6 experiments;

8 experience (prototype) - the dose of the homogenate of the pancreas of cattle - 100 g per 1 kg of ground antlers;

- the time of fermentation, extraction and the degree of dilution in accordance with the technological regime of the prototype;

- drying pantolysate according to the mode of 1-6 experience.

The research results are shown in table 3.

As can be seen from table 3, with an increase in the duration of fermentation, a slight increase in the yield of total amino acids (on average by 3%) and free amino acids (on average by 5.9%) was observed, and despite the fact that the yield of dry matter also increased with increasing time fermentation, the release of hormones and growth factors was significantly reduced. The process of reducing the release of hormones and growth factors was also noted with an increase in the extraction time. Thus, the percentage reduction in testosterone ranged from 4.1% (with 3-hour fermentation) to 33.7% (with 6-hour fermentation), dehydroepiandrosterone from 7.6 to 41.2%, estradiol from 7 to 22.6% , insulin-like growth factor from 6.6 to 24.4%, epidermal factor from 15.3 to 17.6%, nerve growth factor from 10.0 to 15.7%, respectively (etc.). 3-4 hours fermentation should be considered the optimal technological regime providing a high yield of hormones and growth factors with a ratio (weight) of antlers: water 1: 1.5 and a temperature of not more than 50 ° C with 3-hour extraction and dilution of the hydrolyzate 1: 1 ,5. Thus, for 1 kg of ground antlers in the claimed method, the water flow rate is 6.25 liters, in contrast to 12 liters of water according to the prototype. A small amount of water is due to the need for drying in the production of concentrate. Table 3 shows a comparative analysis of the use of antler antler pancreas homogenate as an enzyme (experiment 3) and cattle (experiment 7). In the concentrate using the homogenate of the pancreas of cattle, the content of hormones and growth factors is lower by 10.6-25.5%, and in the concentrate prepared according to the prototype (experiment 8), the decrease in these indicators is even higher (from 26.5 to 72 , 2%).

Table 4 shows comparative data on the content of nutrients in pantolizate and concentrate.

Table 4 Indicators Product type Total amino acids (mg / g) pantolysate 14.0 concentrate 676.2 Free amino acids (mg / g) pantolysate 3,7 concentrate 179.8 Collagen (mg / g) pantolysate 9.6 concentrate 462.5 Vitamins (mg / 100 g) pantolysate 14.6 concentrate 708.9 Macronutrients (mg / g) pantolysate 384.1 concentrate 18600 Trace elements (mg / g) pantolysate 1,1 concentrate 52.08 Testosterone (pg / g) pantolysate 32 concentrate 1559 Dehydroepiandrosterone (DHEA), ng / g pantolysate 0.1 concentrate 4.28 Estradiol, pg / g pantolysate 1.8 concentrate 89.0 Insulin-like Growth Factor 1 (IGF-1), ng / g pantolysate 1,2 concentrate 57.2 Epidermal growth factor (EGF), pg / g pantolysate 0.4 concentrate 19.8 Nerve Growth Factor (NGF), ng / g pantolysate 1.8 concentrate 86.8 Alpha transforming growth factor (TGF-α), pg / g pantolysate 3.8 concentrate 186.0 Beta-transforming growth factor (TGF-β), ng / g pantolysate 1.27 concentrate 61.5 Ciliary neutrophic growth factor (NGF ₃ , CNTF), pg / g pantolysate 2.0 concentrate 94.8 Bone morphogenetic protein (BMP-4), pg / g pantolysate 4.9 concentrate 237.1

As can be seen from table 4, the saturation of the concentrate with useful components is 48 times higher than the pantolysate, and since the biological activity of the drug is its ability to change the functional capabilities of the components of the body (in vitro or in vivo), the claimed method of producing a biologically active concentrate taking into account the greater the release of growth factors provides (based on the healing properties of these factors) high biological activity.

Example. The method is as follows. Antler meal (milled reindeer antlers) with a grinding degree of not more than 100 microns) is mixed with distilled water at the rate of 1 kg of milled antlers with 1.5 liters of water containing 100 g of deer pancreas homogenate, i.e. when using a fresh or frozen pancreas, it is ground to a homogeneous state.

After thorough mixing, the substrate is placed in a thermostat for 3-4 hours at a temperature of not more than 50 ° C. At the end of the fermentation period, water is added to the hydrolyzate in the ratio hydrolyzate: water 1: 1.5 and extracted for 3 hours on a PK5M unit, followed by centrifugation and filtration. The volume of pantolysate is 4.6 liters. Pantolizate is dried in a vacuum dryer at a temperature not exceeding 50 ° C (i.e., at a temperature of fermentation) and a pressure of 0.9 atm to 6% humidity. The concentrate is packaged in plastic bags or other sterile sealed containers.

In reindeer, iron is collected from isolated internal organs, when the carcass is internalized. During the period of mass slaughter, the gland is harvested in the form of a powder, the homogeneous mass of the gland is dried in a vacuum dryer at a temperature not exceeding 45 ° C to 6% humidity and stored in a closed sterile container.

Thus, the claimed method of obtaining a biologically active concentrate from antlers provides due to the high content of hormones and growth factors, their therapeutic properties (with a sufficient content of other useful components), high biological activity, which will expand the scope of the concentrate. The concentrate is convenient in transportation, storage and use.

Claims (1)

A method of obtaining a biologically active concentrate from antlers of deer, including enzymatic hydrolysis of antlers with a degree of grinding no more than 100 microns in the presence of water and homogenate of the animal’s pancreas at the rate of 100 g of iron per 1 kg of antler flour, followed by aqueous extraction of the hydrolyzate, centrifugation and filtration, characterized in that enzymatic hydrolysis is carried out in the presence of a pancreas of antler deer at a weight ratio of antler meal: water 1: 1.5 for 3-4 hours at a temperature of not more than 50 ° C, ex RAC hydrolyzate is carried out at a weight ratio of hydrolyzate: water 1: 1.5 for 3 hours, and after centrifugation and filtration, the extract containing the desired product are dried at a temperature not higher than 50 ° C and a pressure of 0.9 atm.