RU2738478C1 - Method and device for producing hormonal concentrate from urine - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: group of inventions refers to pharmaceutical industry. Device and a method for producing a hormonal concentrate from urine of a human or mammal are disclosed. Device includes housing, cover, connector with horizontal outlets for fluid inlet and outlet, cooling element, heating element, concentrator for collection of hormonal concentrate, photo-optic unit, level sensor and temperature sensor. Method includes urine cooling directed from periphery to centre to −10 °C with replacement of ice concentrate containing hormonal concentrate with liquid concentrate to concentrator, draining the hormonal concentrate into an external accumulator having a precipitant and a preserving agent, mixing and holding the hormonal concentrate at room temperature, followed by separation of the residue.

EFFECT: inventions provide higher quality of urine pre-treatment, as well as quality and efficiency of technological process of obtaining hormonal concentrate therefrom.

12 cl, 2 ex, 6 tbl, 3 dwg

Description

The present invention relates to a method and device for producing a hormone concentrate from biological raw materials as a starting material suitable for creating pharmaceutical preparations that contain a natural mixture of conjugated hormones in the form of a biological active component.

Applied technologies occupy an important place among the main areas of biotechnology and are directly related to the results of the implementation of advanced solutions in various sectors of the national economy, including medicine and veterinary medicine, the cosmetic and food industries, etc.

One of the most relevant biotechnological topics is hormonal, which includes the optimization of already known technologies and the development of fundamentally new solutions for the production, purification and use of hormonal preparations from raw materials of plant or biological origin.

In this regard, the development of new approaches based on the introduction of new technologies, leading to a significant improvement in the qualitative and quantitative characteristics of the final product, which can be further used to obtain individual hormones, contributes to progress in this direction.

The proposed invention is associated with a fundamentally new approach to the isolation of hormonal substances through the predominant use of physical methods of concentration and purification of hormonal mixtures for subsequent use in the isolation of individual drugs.

The aim of the present invention is to significantly improve the quality of preliminary purification of the biological initial product - urine, as well as to significantly improve the quality and efficiency of the technological process of obtaining hormonal concentrate from it for subsequent use.

The purpose of the present invention is achieved through the use of the developed method and device for obtaining hormone concentrate by using physical principles for the allocation of hormone concentrate from urine.

From the current state of the art, the urine content of an extremely wide spectrum of biologically active substances is known. Some of them are successfully used in medicine and cosmetology, primarily as hormonal drugs.

Most of the known methods for producing hormones are based on the use of animal or human urine as the most biologically compatible starting product.

Almost all methods of hormone release require preliminary purification of raw materials and their concentration.

The overwhelming majority of known methods for the isolation of hormones use technical means, most of which are various chromatographic ones using gels or adsorbents, as well as ultrafiltration, centrifugation, etc.

Depending on the goals set for the release of hormonal substances, the priority of using one or another technical means remains the choice of the contractor, however, the general tendency to use them in the technological process has common criteria, first of all, such as: safety and purity of the final product, maximum possible productivity and minimization stages of the technological process, the availability of technical means and components, as well as the economic efficiency and cost of the final product.

Most of the known methods have been developed taking into account the release of predominantly steroid hormones, as the most popular in modern medicine and cosmetology, as well as due to the rather large possibilities of obtaining the initial product, including urine.

The classification of steroid hormones distinguishes two main groups that differ in their source of origin: hormones of the adrenal cortex - corticosteroids and hormones of the sex glands - gonadosteroids. In turn, corticosteroids are also divided into two subgroups. One of them is represented by glucocorticoids, such as: cortisone, cortisol, deoxycortisol.

The second subgroup is made up of mineralocorticoids, such as deoxycorticosterone, corticosterone, aldosterone.

Gonadosteroids are represented by three subgroups, among which progestogens are distinguished, such as pregnenolone, progesterone, allopregnenolone. The second subgroup is made up of androgens: androstenediol, testosterone, dehydrotestosterone. The third subgroup is represented by estrogens: estrone, estradiol, estriol. In addition, steroids include herbal compounds with valuable pharmacological properties: steroid alkaloids, digitalis glycosides (cardiac glycosides), and steroid saponins (24).

The source of raw materials for the isolation of steroids are numerous and diverse products of biological and plant origin. Most often, steroids are isolated from the spinal cord and bile of cattle, animal fats, from the alkaline hydrolyzate of yeast, vegetable oils and wastes from the pulp and paper industry (8).

Despite the fact that a complete synthesis of the main steroid hormones has now been developed, the demand for natural hormones remains extremely high. Intensive searches for ways to optimize the production of natural hormones from plant and biological raw materials are associated and determined with this. From this perspective, urine is the ideal, most readily available and constantly renewable source of virtually all hormones of animal origin.

The urine contains a natural mixture of hormones, in particular steroid hormones, which is presented mainly in a conjugated form, for example, in the form of the sodium salt of a half-ester of sulfuric acid. The content of conjugated hormones (calculated for estrogen sulfate) can be between 0.3 and 1% by weight, based on dry matter. Known methods for the isolation of steroid hormones from plant materials. One of them is known as a method for producing ecdysteroids (RF Patent No. 2063763) (8), which consists in extracting raw materials with 70% ethanol, removing accompanying substances from the primary extract by a series of sequential liquid-phase extractions, purifying the resulting product by column adsorption chromatography on aluminum oxide, and its subsequent crystallization. The disadvantages of this method are the use of significant volumes of ethanol in the first stage of extraction, large volumes of organic solvents in subsequent multiple liquid-liquid extractions, multistage and duration of the process.

There is a known method of obtaining α-ecdysone described in patent RU 2246966 (18) by extracting plant raw materials of the Serratulacoronata species, which consists in squeezing and filtering the juice from freshly harvested leaves of the Serratulacoronata plant in the budding phase, concentrating and extracting it with ethyl acetate, the extract is evaporated, cooled the mixture of crystals of 20-hydroxyecdysone and α-ecdysone is filtered, followed by dissolution in acetone and addition of phosphoric-molybdic acid, the resulting mixture of 2,3: 20,22-diacetonide 20-hydroxyecdysone and 2,3-acetonide α-ecdysone is separated by column chromatography, the isolated 2,3-acetonide α-ecdysone is hydrolyzed, the reaction mixture is extracted, the extract is evaporated, and crystals of α-ecdysone are isolated by column chromatography. The disadvantages of this method are the use of 12 operations, including operations of liquid-liquid extraction, which require multiple repetitions and at least three times the volume of the extractant to the volume to be extracted at each extraction operation, the duration, laboriousness and multistage nature of the process, as well as significant energy consumption of the method due to three operations evaporation and duration of operation of the vacuum circulation unit.

It is known to isolate from urine an immunologically active substance from the urine of mammals, including humans, which has the biological activity of serum thymic factor (RF Patent 2139065) (12). According to the description, urine containing serum thymic factor is processed using various technical means, in particular chromatographic columns, freeze drying, for which it is successively chromatographed on a 2.5 × 30 cm column with Toyoperl HW-40 gel equilibrated with 0.2 M NaCl, material in the region of 80-100 ml is eluatalyophilized, chromatographed on a column 1 × 100 cm with Toyoperl HW-40 gel equilibrated with 0.2 M NaCl, the material in the region of 35-40 ml is eluted and desalted on Sephadex G-15, taking the final product into area mol. m 6000-1000 Daltons.

There is a known method of obtaining raw chorionic gonadotropin, RF patent 2071334 (14), according to which the urine of pregnant women with a gestational age of 8-12 weeks is clarified by passing through microfiltration membranes with a pore size of 0.22-0.5 microns. The clarified raw material is concentrated on a hollow fiber ultrafiltration module with a cutoff limit of 10-70 kDaltons. The resulting concentrate is washed from urea and salts with distilled water at pH 5.8-7.0. In the concentrate, the pH is adjusted to 3.8-4.2 and a precipitate containing chorionic gonadotropin is isolated by adding an excess of acetone. For reprecipitation of an active precipitate containing chorionic gonadotropin, the precipitate is dissolved in 5-10% ammonium acetate buffer containing 50-70% ethyl alcohol. Precipitation of the target product is carried out by increasing the ethanol concentration to 90-92%. The activity of the obtained product is 1500-2000 U / mg, the yield is 85.7-90%.

In the technological process of the above method, clarification procedures on microfiltration membranes and concentration on an ultrafiltration module are used, followed by step-by-step sequential precipitation with acetone and ethyl alcohol.

Disadvantages of the method: low productivity during microfiltration and concentration on the ultrafiltration module, which significantly limits the possibilities of industrial production. In this case, clarification does not lead to the removal of urea and urine salts, which leads to the need for an additional procedure for washing the concentrate with distilled water.

A group of patents is known that describe similar methods for the isolation of estrogens based on the use of liquid-liquid extraction methods (patent RU 2308955 (11), patent RU 2329818 (6), patent RU 2351607 (10).

As an example of one of them, we can cite the "Method of isolating a natural mixture of conjugated equine estrogens depleted in unconjugated lipophilic compounds". Patent RU 2329818 (6).

For this, the initial aqueous phase is selected from the group including: an aqueous solution of a natural mixture of conjugated estrogens from the urine of pregnant mares, already depleted in phenolic components of urine; an aqueous concentrate of a natural mixture of conjugated estrogens from the urine of pregnant mares, already depleted in phenolic components of urine; concentrate of liquid urine obtained by thickening; and concentrated urine retentate. Any of these aqueous phases are subjected to liquid-liquid extraction. Obtained according to the description of the method, the pre-purified urine concentrate had a concentration ratio of up to 1:10. In particular, it is indicated that the concentrates can also be subjected to further concentration by a known method, for example by distillation, before being processed by the process according to the invention, in order to thereby obtain a concentrate practically free of organic solvent. Also in the description, in particular, it is indicated that the use of technical means, in particular, a membrane filter with pore sizes lying in the nanometer range, ensures the production of a retentate of urine of pregnant mares with an increased concentration of estrogens contained practically without their loss, while simultaneously removing up to 50 wt.% Low molecular weight components.

It will be appreciated that the above known liquid-liquid extraction methods have to deal with many problems, such as intense foaming, sludge formation, emulsion formation and poor phase separation. Usually, extraction is required in several stages, which leads to the loss of estrogenic components and only their incomplete excretion from urine. In addition, these extraction methods require large volumes of solvents that are partly harmful to health. In addition, the above patent publications do not contain any information on the content of unconjugated lipophilic components or unconjugated, primarily androstane and pregnane steroids in the resulting products, nor on the separation of these components. The descriptions of these methods do not allow making a conclusion about their real effectiveness in relation to the yield or purity of the obtained extracts and the effect of undesirable from a toxicological point of view solvents.

An analogue of the invention proposed by us is the method and device described in patent RU 2277241 C2 (1) for accumulation (concentration) and stabilization of conjugated estrogens from mare's urine. The author proposes a method and a device for the accumulation and stabilization of conjugated estrogens from the urine of pregnant mares on solid carriers in order to obtain a starting material suitable for the production of pharmaceutical preparations, which contain a natural mixture of conjugated estrogens as an active ingredient.

As a device, the proposed cartridge is filled with an adsorbent permanently in a liquid, suitable for adsorption of a mixture of conjugated estrogens contained in urine. According to the description, urine, from which mucus and solids are optionally removed, or a thickened concentrate of this urine or a thickened retentate obtained by filtering this urine through a membrane filter (a substance that has not passed through a membrane filter), is brought into contact with sufficient for adsorption contained in urine the mixture of conjugated estrogens is separated from the rest of the urine by the amount of the semipolar polymeric adsorbent resin and the semipolar polymeric adsorbent resin saturated with the mixture of conjugated estrogens;

b) saturated with a mixture of conjugated estrogens, the semipolar polymeric adsorbent resin is washed with a washing liquid with a pH value set at least 12.0, especially 12.5-14.0;

c) the washed adsorbent resin is contacted with a sufficient amount of eluent to desorb the adsorbed mixture of conjugated estrogens, which is a water miscible organic solvent from the group consisting of water miscible ethers, lower alkanols and lower aliphatic ketones, or a mixture of water miscible organic solvent water of an organic solvent and optionally alkaline water, and the eluate containing the natural mixture of conjugated estrogens is separated from the adsorbent resin.

The disadvantages of this patent are that, based on the known properties of the adsorbents used in the invention, we can confidently assume that in the cartridge for accumulation and storage equipped with one or another adsorbent, non-selective accumulation (concentration) of a mixture of most hormones present in urine occurs, including and estrogens. At the same time, in order to isolate them from the cartridge, it is necessary to apply at least 5-fold volumes of the eluent (the ratio of the adsorbent volume / eluent volume), which ultimately leads to the fact that instead of the concentrate at the outlet from the cartridge, a diluted solution of a mixture of hormones is actually obtained, which is necessary in further concentrate again.

It is important to note that all the analogs listed above generalize and apply already known technological solutions and technical means, such as ultrafiltration, the use of ion exchange resins and adsorbents, precipitation with various precipitants, for example ketones, alcohols, etc., which are not selective in relation to a specific hormone, and the final product is a mixture of almost all urine hormones, in varying proportions, depending on the method used.

At the same time, without diminishing the importance of attempts to improve the qualitative and quantitative characteristics of the final product, regardless of the name of the method, how the authors want to present it (for example, a method for producing estrogens, or a method for producing gonadrotropin, etc.) in all cases of using the above non-selective methods for isolating hormonal preparations, it should be understood that we are talking about a concentrated solution or concentrate of a mixture of hormones, at best, with a predominance of one or another hormone.

The currently existing chemical and thermal methods for concentrating bioorganic products have a number of disadvantages that contribute to the destruction of unstable compounds, condensation and polymerization of substances, etc. In this regard, the development of alternative methods of concentration that do not lead to a change in the qualitative and quantitative composition of the final product is relevant. One of the promising directions is the method of concentration by freezing, which is based on the fact that when water freezes to form ice - a solid phase, the dissolved components remain and concentrate in the liquid phase (5, 19, 23).

The successful development of technical means of cooling made it expedient to use them in the food industry for concentrating fruit juices, beer, various extracts (9), as well as in biotechnological industries, as a preliminary stage for concentrating biologically active substances for their subsequent individual isolation.

The physicochemical principles of freeze concentration are based on crystallization, during which the main part of the water in solution turns into ice upon cooling, and the remaining concentrate, which has a higher specific gravity, is separated under the action of the formed ice. It was found that in the process of crystallization, areas of special micro-formations are formed, in the center of which there are mainly molecules of pure water, and at the periphery there is a saturated solution of the components present. Since during the cooling process, pure water first freezes, the resulting ice crystals displace and thereby concentrate the dissolved components (20, 21, 30).

With a slow and smooth process of crystallization, the force vector will also smoothly repel the concentrate from itself, however, during rapid freezing, the crystallization process takes on a chaotic nature, which leads to the disappearance of the direction of the force vector and the formation of numerous autonomous centers of crystallization inside the ice, making it impossible to selectively remove the concentrate from it. Thus, an obvious condition for obtaining and separating the concentrate from ice is the maximum possible combination of the optimal rate and temperature of cooling of the initial solution, as well as the creation of directionality and the preservation of the force vector throughout the entire technological process of its preparation (16). It has been established that the conditions for the formation and growth of crystals depend on several factors, such as: the degree of supercooling of water below its crystallization temperature, the number of crystallization centers, the dynamic state of water, its purity and heat removal during crystallization (26). Practical conclusions are that the higher the degree of supercooling of water, and the more centers of crystallization appear, the more intense the process of freezing and concentration is. (twenty)

In (9), the results of studies of the effect of cooling on the production of juice concentrate and wine material are presented. Freezing the juice yields a concentrate with a high density and a pronounced varietal aroma. It is shown that after freezing, the concentration of sugars in the juice increases by more than three times, along with an increase in the concentration of titratable acids, both in the juice and in the wine material. The process of freezing wine material is accompanied by a threefold increase in the content of ethanol, methanol and acetic acid. The proportion of ether-soluble compounds increases from 0.56% to 1.2%. However, as in freezing the juice, part of the extractive substances of the wine material remains in the ice. Similar conclusions are given in publication (7), where it is shown that during freezing of concentrated solutions, a part of the substance is captured by ice, as evidenced by turbid, opaque ice.

When studying the factors affecting the degree of extraction of organic compounds by freezing, the dependence of the value of the final volume of the concentrate was established, which, to fulfill the experimental conditions, could not be less than 30 ml, regardless of the value of the initial volume, varying from 100 to 400 ml, which, according to the author, is explained by the mechanical factor associated with the deterioration of stirring of the solution (31).

It is known that aqueous solutions upon freezing form two main phases. Studying the conditions for the formation of such phases is of great importance for evaluating their interactions in solutions upon cooling (28). It is known that, depending on the ratio between the cooling rate of the solution and the rate of water crystallization in the presence of various components, ice crystals, an amorphous phase, or a mixture of them can form during freezing (24). Factors directly affecting the freezing process include: temperature conditions depending on the cooling rate, concentration of the initial solution, final volume of concentrate, concentration of inorganic ions in solution, pH of an aqueous solution (27, 29). Experimentally confirmed and recommended preferred temperatures for freezing for concentration purposes are in the range of -10 ° C to -12 ° C. In addition to this, we carried out experimental work, which made it possible to find the optimal sizes of devices for freezing aqueous solutions in order to isolate an organic concentrate, as well as the optimal ratios of the internal working volume with their height and internal radius.

From the technical literature available to us, we know a number of patents related to the production of concentrate by freezing.

Thus, patent RU 2060774 C1 (13) is known, intended for use for removing water from solutions by freezing, preferably in the production of concentrated juices. The essence of the invention: a liquid with a boiling point at atmospheric pressure below 0 ° C, selected from the group of inert gases, nitrogen, nitrous oxide and mixtures thereof, is introduced into an aqueous solution. In this case, the liquid evaporates and ice crystals are formed, separated from the concentrate.

Disadvantages: the method is not intended for use in the pharmaceutical industry.

A known method of automation of technological processes in the freezing of liquid products by automatic control of the process of freezing moisture in a two-stage freezing unit, which provides for a change in the temperature and flow rate of the refrigerant in the crystallizers of the installation, the flow rate, temperature and residence time of the initial product in the crystallizers, the flow rate of cooling water, the power of the adjustable drives of refrigeration compressors machines and drives of crystallizer drums by acting on the drum rotation frequency and on the consumption of coolant, cooling water, initial product and water obtained by melting frozen ice, the initial product is preliminarily supplied for cooling to a recuperative heat exchanger washed with fresh water formed during melting of frozen ice, and then into the recuperative cooler, washed by the concentrated product, the flow rate and dry matter content in the initial product are measured at the inlet to the first crystallizer, the current value consumption and dry matter content in the concentrated product at the outlet of each crystallizer, the residence time of the product in each crystallizer, and according to these data, the boiling point of the refrigerant in the inner cavity of the crystallizer drum and its rotation frequency are corrected by changing the capacities of the variable drives of the compressors and drives of the crystallizer drums in depending on the electrical conductivity of frozen ice, as well as changes in the flow rate of water supplied to the additional condenser, the amount of which depends on the current values of the temperature and flow rate of fresh water at the outlet of the melters, as well as on the measured values of the dry matter content in the original product and the residence time in the freezing unit and the amount of the frozen layer of ice, in accordance with which the rotational speed of the drum of the crystallizer is set, while the pre-cooling of the initial product is carried out due to the recuperation of the heat of fresh water obtained as a result at the melting of ice and due to the recovery of the heat of the concentrated product, and the melting of frozen ice is carried out due to the heat of the refrigerant pumped by the compressor of the refrigeration machine while simultaneously cooling and condensation of the refrigerant in the melting zone, and the boiling point of the refrigerant in the inner cavity of the crystallizer drums is reduced when the content of dry substances deviates in the concentrated product at the outlet of the crystallizers from the set value towards the increase and increase the boiling point of the refrigerant when the content of dry substances in the concentrated product deviates from the set value towards the decrease (Patent 2235581) (2).

The method is intended for automatic control in a two-stage freezing unit with a technological process suitable for use in the chemical, microbiological, food industries, as well as at the enterprises of the agro-industrial complex. The technical parameters of this process do not provide for its use in biotechnological production.

Known patent RU 2294644 C1 (15), which describes the use of granular carbon dioxide for the crystallization of water in a passing solution, which serves as a starting point for obtaining a concentrate. In this case, the dispersed product crystallizes into carbon dioxide granules having a temperature of -78 ° C. To separate ice crystals, the authors suggest using centrifugation, presses, and washing columns.

The disadvantages of the proposed method are the complexity and energy consumption of the technological process, as well as the loss of concentrate remaining in ice crystals on granules. All this complicates the technological process and, ultimately, reduces the efficiency of obtaining the final product. For the effective implementation of the method, large amounts of the initial product, in particular plant raw materials, are needed, which is hardly applicable for significantly smaller amounts of a product of biological origin. The method does not provide for its use in the pharmaceutical industry for the isolation of biologically active substances. At the same time, the method does not provide for the further use of the liquid after the crystals are thawed and washed off the granules, which further reduces its economic component.

Closest to the claimed invention is patent RU 2491976 (22), adopted by us as a prototype, which describes a method for concentrating solutions by freezing, including crystallization of a solution followed by separation of the concentrate from solvent crystals. In a freeze-out solution having a temperature not lower than the liquidus temperature, the direction of crystallization is carried out: if the density of the concentrated solution is less than the density of the initial solution, then it is predominantly opposite to the force of gravity; if the density of the concentrated solution is greater than the density of the initial solution, then it is predominantly in the direction of the force of gravity. The device for concentration of solutions by freezing contains an absorber for the heat of crystallization of a solution, a heat insulator that prevents the environment from affecting the freezing of the solution, devices for compensating for the thermal expansion of substances and removing the concentrated solution. In the device for concentration of solutions by freezing, the absorber of the heat of crystallization of solutions is installed relative to the solution to be frozen out in space in accordance with the method of concentration of solutions.

The disadvantage of this method is its dependence on the composition of the initial solution, which manifests itself in the need to change the direction of crystallization, which in turn requires the use of two structurally different devices, which significantly reduces its economic advantages. In addition, the design of the device itself is quite complex in operation. Another disadvantage of the above method is that it does not provide for dynamic control of the technological process by technical means, as a result of which it is difficult to determine the end of the freezing cycle and the degree of concentration of the final product, and the technological process itself is performed manually.

Based on the well-known theoretically and experimentally proven technical solutions that determine the main parameters of the process of concentration freezing of aqueous solutions of organic and inorganic substances, taking into account the factors affecting this process, as well as the results of our research, we have developed the proposed method and device for obtaining hormonal concentrate from human urine and mammals. From these positions, the inventive method and device for obtaining a hormonal concentrate from urine is based on the use of physical principles that are significantly superior to the established counterparts in most of the main indicators, without carrying their inherent disadvantages.

The method for producing a hormonal concentrate is developed on the basis of concentration of solutions by removing the frozen solvent from them. The principle of the method of obtaining a hormonal concentrate and the operation of the device is that with gradual cooling of urine, the resulting ice pushes organic and inorganic components of urine out of its structure, which form their concentrate. With directed cooling, for example, from the periphery to the center, the concentrate is formed in one predetermined place, specifically in the center, from where it can be selectively removed.

The method is as follows. As a starting solution containing a mixture of natural hormones, human or mammalian urine is used, which is pre-purified from the bulk of urinary salts, denatured proteins, mucus, macromolecular formations according to the method described in patent RU 2621311 (4). This method is based on the effect of cryoaggregation, which manifests itself when urine is cooled to a preferred temperature of -2 ° C, without ice formation. At this temperature, the maximum possible formation of cryo-aggregates is observed, consisting of uric salts, proteins, which also involve large macromolecular formations, including mucus, bacteria, and uniform elements. As a result of this process, the urine is largely purified from the above-described impurities, which increases the efficiency of the subsequent stages of purification and obtaining directly the hormonal concentrate.

In accordance with the inventive method, urine, previously purified by cryoaggregation and cooled to -2 ° C, to which microparticles of activated carbon with a size of preferably 500 microns in an amount of preferably 200 mg / l are also added, enters the inventive device equipped with a cooling element, in which it is cooled to a preferred temperature of -10 ° C. Cooling of urine is carried out in the horizontal direction and from the periphery to the center in a constantly controlled mode, not exceeding the set temperature of -10 ° C. The ice crystals formed during the cooling process displace organic and inorganic liquid components also from the periphery to the center, which enter and accumulate in a concentrator having a tubular cylindrical shape installed in the center of the container of the device. In the microprocessor of the control unit, restrictions are preliminarily introduced on: the temperature of the coolant, the heating temperature of the wall of the device case, the initial level of the liquid inside the case, the optical density of the hormone concentrate, as well as the control of switching on and off the solenoid valves. The accumulation of the dark brown concentrate is controlled by a photo-optical unit installed in the upper part of the concentrate, which, after reaching the set value of the optical density of the hormone concentrate, sends a signal to an externally installed control unit to terminate the working cycle. The automatically activated solenoid valve opens a horizontal outlet of the device, through which the hormone concentrate is removed from the concentrator into an externally mounted accumulator, which is a container that exceeds the volume of the concentrate in a ratio of 4: 1 to 6: 1, preferably 5: 1. For additional purification of the hormonal concentrate from accompanying impurities, powdered sodium hydroxide is introduced into the storage device before feeding the hormonal concentrate in a ratio of 0.5 g per 100 ml of the volume of the hormonal concentrate.

We have surprisingly found that when sodium hydroxide is added to the hormone concentrate in the preferred amounts described above, a rapid precipitation and precipitation is observed, the composition studies of which have shown the presence of predominantly glycosaminoglycans and proteoglycans. These studies are consistent with the data (RF patent 2401838) (17) and our studies (RF patent 2621311) (4), which describe the use of sodium hydroxide for the isolation of glycosaminoglycans and proteoglycans in the process of obtaining drugs from them.

According to the claimed method, the use of sodium hydroxide serves two purposes. One of them is as follows. The above references indicate the use of sodium hydroxide for the precipitation of glycosaminoglycans and proteoglycans from raw materials of animal origin in order to obtain highly purified preparations of these substances. However, we have not found technical information on the use of sodium hydroxide for the purification of a hormonal concentrate obtained from urine or other biologically active solutions in order to purify them from impurities of glycosaminoglycans and proteoglycans. These high molecular weight substances are undesirable components, since their presence significantly reduces the efficiency of using the hormonal concentrate to obtain highly purified hormonal preparations and in any case requires additional purification methods. The author of the proposed method found that using an aqueous solution of 0.5M sodium hydroxide, it is possible to successfully remove proteoglycans and glycosaminoglycans from the hormonal concentrate obtained from urine for the purpose of additional purification.

We have experimentally proven the presence of glycosaminoglycans and proteoglycans in the hormonal concentrate in a concentration exceeding their level in the original urine, on average, 2 times. So, with the initial values of 0.120 ± 0.01 mg / ml of the initial urine, determined by the carbazole method, the content of total glycosaminoglycans and proteoglycans in the hormone concentrate was 0.256 ± 0.012 mg / ml (p <0.05, n = 10). After the application of sodium hydroxide, proteoglycans and glycosaminoglycans in the hormonal concentrate were not determined.

The technical result is achieved by the fact that after adding the hormonal concentrate, the powdered sodium hydroxide in the storage device dissolves within 20-30 seconds, and at the same time there is an increasing turbidity of the solution, followed by the formation of a coarse precipitate, consisting mainly of glycosaminoglycans and proteoglycans, falling out within 5 -6 minutes to sediment. In the next 5-6 minutes, the sediment is compacted and when the ratio of the volume of the concentrate / volume of the sediment is preferably 10: 1, the hormonal concentrate is separated from the sediment and is used as intended.

We have studied the weight of the dry residue in the hormone concentrate before and after the application of sodium hydroxide to remove glycosaminoglycans and proteoglycans. To prepare the dry residue, the hormonal concentrate obtained on the inventive device was divided into two samples, one of them is a control sample without the use of sodium hydroxide, and the second sample was mixed with powdered sodium hydroxide in a ratio of 0.5 g per 100 ml of the hormonal concentrate volume. After stirring for 10 seconds, the hormone concentrate solution was left for 10 minutes at room temperature until complete precipitation and compaction. The supernatant, which is a hormonal concentrate without proteoglycan and glycosaminoglycan, was separated from the sediment. Both samples were dried in an oven at 100 ° C for 18 h to completely remove moisture. Fine weighing of the remaining dry residue was carried out on a digital balance (GF-400, A&D Corp., Taiwan). As a result, from 100 ml of hormonal concentrate of the control sample after drying, 1.246 g of dry matter was obtained, which is 1.246% in percentage. The final weighing of the dry residue from 100 ml of hormone concentrate after treatment with sodium hydroxide and subsequent removal of proteoglycans and glycosaminoglycans established the dry residue weight equal to 0.788 g, which is 0.788% in percentage. Comparison of the results of weighing the dry residue and their interpretation indicates a significant reduction in the weight of the dry residue, by almost 36%, which is convincing evidence of the effectiveness of sodium hydroxide for additional purification of the hormonal concentrate from undesirable components, in particular from proteoglycans and glycosaminoglycans.

The study of hormonal activity, using the example of estradiol and cortisol, in the hormonal concentrate after the application of sodium hydroxide (with the previous study of the neutralization of sodium hydroxide with an equivalent amount of citric acid) did not reveal statistically significant differences both in the first hours after the separation of the hormonal concentrate from the sediment, and in the subsequent period observation, which has already exceeded 2 years.

The second purpose of using sodium hydroxide is that sodium hydroxide has a powerful bactericidal effect on almost the entire bacterial flora, as well as viruses, including: viral hepatitis, HIV, etc. In our bacteriological studies to study the effectiveness of sodium hydroxide as a preservative, in no case was the presence of bacterial microflora detected both in the first hours after the separation of the hormonal concentrate from the sediment, and in the subsequent observation period, which had already exceeded 2 years. Thus, the use of sodium hydroxide for sterilization of the final product, as well as a preservative for long-term storage, has proven technically and economically justified effectiveness due to the simplicity and reliability of its use, as well as the low cost and availability of sodium hydroxide itself.

The technical result of the implementation of the method is achieved by the fact that pre-purified and cooled to -2 ° C urine, to which also microparticles of activated carbon with a size of preferably 500 microns in an amount of preferably 200 mg / l, after opening the electromagnetic valve installed on the inlet horizontal outlet, enters through the connector, which is installed on the outer side of the bottom of the body, into the container of the body and is poured to the required volume, limited by the setting of the level sensor. After the level sensor is triggered, the signal from it is sent to an externally installed control unit, from which a signal is sent to turn off (close) the solenoid valve on the input horizontal branch. At the same time, a photo-optical unit is switched on, sending a signal to an externally installed control unit, the values of which are taken as initial ones for monitoring the state of the liquid inside the concentrator. Also, at the same time, an externally installed cooling unit is switched on, which, after switching on, starts the cooling process, which consists in the fact that a coolant with a preferred temperature of -10 ° C begins to flow into the tubular spiral cooling element in a flow-through mode. This makes it possible to achieve the desired effect by smooth cooling of the liquid directed towards the center, accompanied by the directed formation of ice from the periphery to the center. In this case, the formed hormonal concentrate is gradually pushed out by the formed ice from the periphery to the center and, ultimately, passes through the drainage holes into the concentrator, from where, after the photo-optical unit is triggered and the electromagnetic valve on the horizontal outlet is opened, it enters an externally installed storage device for additional purification of the hormonal concentrate from impurities of proteoglycans and glycosaminoglycans. The process of additional purification consists in the fact that the hormonal concentrate enters an externally installed accumulator, which is a container, the volume of which exceeds the volume of the concentrate in a ratio of 4: 1 to 6: 1, preferably 5: 1, where, before feeding the hormonal concentrate, as a precipitant and a preservative is preferentially introduced, preferably, powdered sodium hydroxide in a ratio of 0.5 g: 100 ml of the volume of the hormonal concentrate, after which the hormone concentrate is poured into the reservoir in the above volume ratio, which is gently stirred for preferably no more than 10 seconds, and kept at room temperature temperature for 8-12 minutes, preferably 10 minutes, followed by the separation of the sediment, consisting mainly of glycosaminoglycans and proteoglycans, from the hormone concentrate, which is used as a final product for subsequent use.

The essential distinguishing features of the proposed method for producing a hormonal concentrate from urine from known analogs and the prototype are as follows.

1. An essential distinguishing feature of the proposed method is the use of preliminary cooling of the original urine to a temperature of -2 ° C for its purification based on the cryoaggregation method, which allows to remove the bulk of urine salts, denatured proteins, mucus, macromolecular formations such as formed elements, bacteria and macroaggregates ... This method has not previously been used as a preliminary purification of urine to obtain a hormonal concentrate from it, and its use in the claimed method makes it possible to significantly increase the efficiency of the subsequent freezing process to obtain a hormonal concentrate.

2. An essential feature of the proposed method is the use of activated carbon microparticles with a preferred size of 500 microns and a preferred concentration of 200 mg / l of urine to stabilize the process of directional crystallization of water when urine is cooled at -10 ° C, which prevents the chaotic formation of dentrites. The use of microparticles of activated carbon for these purposes has not been previously described, and their use contributes to a significant increase in the stability of the parameters of the directed concentration process during cooling, which makes it possible to significantly increase the efficiency of freezing to obtain a hormonal concentrate.

3. An essential distinguishing feature of the proposed method is the proven effect of purification of the hormonal concentrate from a significant part of the unwanted urine components, which accompanies the ice crystallization process during freezing, which can significantly improve the quality and degree of concentration of the hormonal concentrate at the outlet of the device.

4. An essential distinguishing feature of the proposed method is the use of sodium hydroxide as a precipitant for additional purification of the hormonal concentrate from the group of unwanted high molecular weight components of urine, such as proteoglycans and glycosaminoglycans, which are almost completely removed. In the claimed method, for the first time, the possibility of using sodium hydroxide for purification of a hormonal concentrate obtained from urine or other biologically active solutions is described for the purpose of purification from impurities of glycosaminoglycans and proteoglycans. This makes it possible to significantly improve the quality of the final product and to avoid additional purification procedures from these substances at the subsequent stages of individual hormone isolation.

5. An essential distinguishing feature of the proposed method is the use of sodium hydroxide as a preservative, since it has not previously been used to sterilize the hormonal concentrate, however, its use makes it possible to completely eliminate the development of bacterial cantamination during storage or transportation, and at the same time preserve the biological activity of individual hormones in the hormonal concentrate. These conclusions have proven technically and economically justified efficiency and because of the simplicity and reliability of its application, as well as the low cost and availability of sodium hydroxide itself.

Composition of a device for producing a hormone concentrate from urine A device for producing a hormone concentrate includes a body 1, cylindrical, with an internal volume of at least 500 ml and not more than 1500 ml, preferably 1000 ml, preferably made of stainless steel, closed with an external lid 2 with external thermal insulation, with a cone-shaped bottom 3, in the center of which, on the outside, there is a connector 4 with at least two horizontal outlets, one of which, an inlet horizontal outlet 5, is designed to introduce liquid into the internal volume of the housing 1, and the second , the outlet horizontal outlet 6 is intended for the withdrawal of the hormonal concentrate, and at the end of the horizontal inlet 5 there is a normally closed solenoid valve 7, and at the end of the horizontal outlet 6 there is a normally closed solenoid valve 8, and in addition, on the outer side of the housing wall cooling tubular element 9 of a spiral shape, connected to an externally mounted autonomous cooler equipped with a microcircuit for regulating cooling parameters, and between the spiral of the tubular element 9 on the outer wall of the housing 1, as well as on the horizontal branches 5 and 6, a plate-shaped heating element 10 is installed, connected to the externally located a power supply equipped with a microcircuit to regulate the temperature parameters of heating the wall of the housing 1, and in the center of the housing 1 a freely movable tube-shaped concentrator 11 is installed parallel to its vertical axis, designed to collect the hormonal concentrate, made, preferably of stainless steel, on the walls of which throughout there are through drainage holes 12, and on the concentrator 11 there are installed at least four guide plates 13 arranged symmetrically to each other, vertically and parallel to the axis of the concentrator so that the inner edge of each guide plate the entire length is firmly connected to the outer wall of the concentrator 11, without closing the lumen of the drainage holes 12 of the concentrator 11, and the outer opposite edge of each guide plate 13 freely adjoins the inner wall of the housing 1 throughout its entire length, in addition to this, on the lower part of the concentrator 11 there is a support plate 14 of an annular shape, located perpendicular to its vertical axis, also provided throughout the entire length of through holes 15, while in the center of the upper third of the concentrator 11, a photo-optical unit 16 is installed, equipped with a light source and a photocell, hermetically protected from liquid, and additionally to this, a level sensor 17 is installed in the upper part of the concentrator 11, and a temperature sensor 18 is installed below on the outer side of the bottom of the housing 1. The drawing of the claimed device is shown in FIG. 3.

The specific technical result of the implementation of the work of the claimed device is disclosed by the fact that at the preliminary stage urine undergoes a cryoaggregation process described in the RF patent No. 2621311 (4), which consists in the fact that when it is cooled to -2 ° C, urine salts and large molecular formations fall , bacteria, mucus, shaped elements. The urine purified in this way, after opening the solenoid valve 7 installed on the horizontal inlet 5, enters through the nozzle 3, which is installed outside on the bottom 2 of the housing 1, previously closed with an outer cover 2, into the container of the housing 1 and is poured to the required volume, limited by the sensor setting level 16. In the microprocessor of an externally installed control unit, restrictions are preliminarily introduced on: the temperature of the coolant, the temperature of the wall of the device housing, the initial level of the liquid inside the housing, the optical density of the hormonal concentrate. After the level sensor is triggered, a signal from it is fed to an externally installed control unit, from which a signal is sent to turn off (close) the solenoid valve 7 on the input horizontal branch 5. At the same time, the photo-optical unit 15 is turned on, sending a signal to the externally installed control unit, values which are taken as the initial ones for monitoring the state of the liquid inside the concentrator 11. Also, at the same time, an externally installed cooling unit is turned on, which, after switching on, starts the cooling process, which consists in the fact that the cooling liquid begins to flow into the tubular spiral cooling element 9 in a flow-through mode at a speed of 3 liters / min and with a preferred temperature limited to a range of not less than -8 ° C and not more than -12 ° C, the most preferred temperature is -10 ° C.

It has been experimentally established that at a coolant temperature of less than -8 ° C, the urine cooling rate is low, which affects an increase in the production cycle time and a decrease in efficiency. On the contrary, an increase in the cooling rate by lowering the temperature to 12 ° C and below leads to an undesirable rapid freezing of the liquid throughout the entire volume of the housing. At the most preferred temperature of -10 ° C, the required effect of smooth and controlled cooling of the liquid directed from the periphery to the center is achieved, which is also accompanied by ice formation directed horizontally and perpendicular to the vertical axis of the housing and the concentrator. In this case, the formed hormonal concentrate is gradually pushed out by the formed ice from the periphery to the center and, ultimately, passes through the drainage holes 12 into the concentrator 11.

It has been experimentally established that the optimal one-time urine volume for obtaining the hormone concentrate is in the range from 500 ml to 1500 ml, preferably 1000 ml. This is due to the fact that, in practice, a single volume of individual urine in animals is, on average, 1 liter. In a person, a one-time volume of individual urine is, on average, 300 ml, allowing mixing with one or two additional volumes of his own individual urine in a time range limited to no more than 6 hours. These limitations are associated with the need to minimize the effect of microbial cantamination during urine exposure.

It has also been experimentally established that a rapid decrease in the cooling temperature leads to a violation of the direction of cooling, as a result of which the concentration gradient is blurred and a liquid with a higher inorganic and organic composition does not separate and freezes over the entire volume of the container without a focused concentration.

It has been experimentally established that the optimal ratio of the internal working volume of the housing 1 and the internal working volume of the concentrator 11 is in the range from 40: 1 to 60: 1, preferably 50: 1. This is due to experimental observations, which found that with the selected internal volume of the housing 1 of 1000 ml, in the internal working volume, the indicated parameters are preferable due to the need for a smooth and gradual decrease in the temperature of the liquid inside the container in order to cool it directed from the periphery to the center. Thus, the most preferred temperature for cooling is -10 ° C, at which the required effect of directed towards the center of cooling of the liquid is achieved, accompanied also by directional formation of ice from the periphery to the center. In this case, the formed concentrate is gradually pushed out by the formed ice from the periphery to the center and, ultimately, passes through the drainage holes 12 into the concentrator 11.

With the internal working volume of the concentrator 11 over 20 ml, the concentration of the liquid decreases, and with the internal working volume of the concentrator 11 less than 20 ml, the hormonal concentrate formed outside the collector remains outside of it, due to the discrepancy and limited volume of the concentrator 11.

In the process of experimental selection of cooling parameters, it was found that the rate of ice formation progressively decreases with distance from the cooled walls of the housing, and therefore, with the selected internal volume of housing 1 of 1000 ml, the preferred values of the inner radius of housing 1 are in the range of at least 40 mm and not more than 60 mm, however, the most preferred is an inner radius of 50 mm.

The height of the device body has a lower threshold, limited to a minimum value of 127 mm, which is associated with the possibility of overflow from the body when using more than one liter of urine with an internal working volume of the body container designed for 1 liter. The upper limit of the height is not limited, but is preferably not more than 150 mm, but 140 mm is most preferable.

If it is necessary to use more than one liter of urine for one time, the height of the wall of the body is increased by 80 mm in multiples for each liter of urine. The preferred maximum height of the wall of the body is not more than 1000 mm. In this case, the body of the device is a column, with a top cover and a bottom bottom. The made and tested prototype of the column with such a height showed that the large dimensions of the column height over 1000 mm complicate its technological maintenance, primarily due to the inconvenience of removing the concentrator with ice after the completion of the cooling cycle.

With an increase in the height of the body over 140 mm, for each subsequent 80 mm, additional spiral tubular cooling elements are installed from the outer wall of the device body, in an amount that is a multiple of each added urine volume. This is due to the fact that the experimentally calculated cooling area of the outer wall of the device body is in direct proportion to the volume and speed of the coolant. With the declared optimal cooling temperature down to -10C, with a preferred body height of 140 mm, the temperature of the coolant at the inlet and outlet differs by 2.3C -2.6C. It was found that the indicated temperature differences associated with heat loss during cooling of 1 liter of urine are constant and on their basis the time of one cooling cycle was calculated. With an increase in the volume of cooled urine, it is required to significantly increase the rate of passage of the coolant and simultaneously lower its temperature, which is technically less profitable than adding an additional cooling element with a controlled optimum temperature of -10C. This technical solution becomes even more preferable with an increase in the one-time processed urine volume over 2 liters, but not more than 10 liters.

As the concentrated liquid accumulates inside the concentrator, its color changes from light yellow to dark brown, and when the required values of the optical density of the liquid, established experimentally, are reached, the photo-optical unit gives a signal to complete its cooling, which is accompanied by turning off the cooling unit, turning on (opening) solenoid valve on the horizontal outlet, and the discharge of the hormonal concentrate into an external storage for further use. After draining the hormone concentrate for 30 seconds, which are experimentally established and required for its complete draining, a heating element is turned on, heating the housing wall and horizontal branches, in the range of 20 ° C-30 ° C, preferably 25 ° C. The indicated values of the heating interval were selected experimentally and are due to the fact that at lower values of the body heating, the process time increases, and an increase in temperature above 30 ° C leads to overheating of the liquid, which is impractical due to the fact that this liquid is used for a parallel technological process. Therefore, a temperature of 25 ° C is most preferred for this stage. The heating process of the case is controlled by a temperature sensor installed on the outer side of the bottom, which gives a signal to turn off the heating when the required temperature is reached. Short-term heating is used only so that the wall ice formed during cooling thaws to a liquid, which allows the remaining compact volume of frozen urine in the form of ice to be removed from the container for the next cycle with a new portion of urine. When extracting frozen urine, the base plate installed in the bottom of the concentrator acts as a piston, contributes to the complete removal of ice from the body, while through the holes on the base plate the remainder of the hormone concentrate passes to the bottom of the body and is subsequently removed or mixed with a new portion of urine in next cycle.

The average time of one treatment cycle for 1000 ml of urine is, on average, 90 minutes, and with the output, on average, 18 ml of hormonal concentrate.

The automation of the hormonal concentrate production process is carried out due to the fact that:

1. Apply externally mounted microprocessor control unit.

2. Applied installed on the device: a sensor of the initial level of liquid for freezing inside the body, a temperature sensor for heating the wall of the body of the device, a sensor for monitoring the optical density of the hormonal concentrate.

3. Applied solenoid valves installed on the horizontal branches of the device, one of which is designed to supply the original urine, and the second for the output of the resulting hormonal concentrate.

4. The microprocessor of the externally installed control unit is preliminarily introduced with restrictions on: the coolant temperature, the heating temperature of the wall of the device body, the initial level of the liquid inside the body, the optical density of the hormonal concentrate.

5. In the program of the externally installed control unit, the function of turning off the cooling has been introduced in the absence of changes in the increase in the signal from the photo-optical unit for 60 seconds.

6. Tubular spiral cooling element of the device is connected to an externally mounted cooling unit, which provides control of the supply of liquid cooled to -10 ° C (based on polyethylene glycol mw 10,000), preferably at a rate of 3 liters / min. and control of the set cooling parameters.

Using the device described above, from 1 liter of the original product (urine after the previous treatment in the previous stages), 19 ml of the hormone concentrate was obtained, which is determined by the internal volume of the concentrator. The ratio of the internal volume of the concentrator to the internal volume of the housing is 100: 1.9. The values of the ratio of the internal working volumes of the housing and the concentrator were selected experimentally and realized on the basis that the internal working volume of the housing in 1000 ml is most suitable for working with portions of urine also in 1000 ml. This is primarily due to the requirements for the safety of raw materials, since the rejection of one portion of raw materials cannot affect the quality of the entire batch. An important point is that when the inner volume of the concentrator decreases below that given in the claimed device, it leads to the loss of the hormone concentrate outside of it from the outside, and a larger volume of the concentrate helps to reduce the concentration of the hormonal concentrate itself.

The conducted studies of the composition and properties of the hormonal concentrate and liquid after the withdrawal of the hormone concentrate (HPIGC) are as follows.

Materials and research methods.

The urine of 10 apparently healthy women aged 17-30 years was examined. Urine was taken in the morning, on an empty stomach, in the middle of the second phase of the menstrual cycle.

The studies were carried out on a working prototype of the claimed device in compliance with all the declared parameters of its operation.

In samples of LPIGC and the hormonal concentrate itself, the following indicators were investigated: color, optical density, specific gravity, dry residue. The dry residue was determined by its weight, the content of ash and protein components, total lipids and carbohydrates.

In addition, the content of high-polymer urine components, such as proteoglycans and glycosaminoglycans, was analyzed using the carbazole method. Studies of the main, quantitatively prevailing hormones were carried out on an ELISA analyzer Multiskan FC, "Thermo Scientific".

The concentration of estradiol (E2) was determined using the set "Estradiol-ELISA", "Hema-Hema-Medica". The result was expressed in pg / ml.

The concentration of progesterone, testosterone and cortisol was determined using the diagnostic systems kits, N. Novgorod, RF. The results of the study of progesterone and testosterone were expressed in nmol / l, cortisol in μg / day.

The obtained indicators were processed by the method of variation statistics with the determination of the arithmetic mean (M) and standard error of the arithmetic mean (m). The means were compared using the Mann-Whitney U-test and the Wilcoxon W-test. The differences between the two compared values were considered significant if the probability of their identity was less than 5% (p <0.05). (3)

The study of the dynamics of cooling showed that the process of directional gradual gradual cooling of urine leads to the appearance of a hormonal concentrate, which is clearly visible and determined by technical means. FIG. 1 shows samples of hormone concentrate (1.1) and LPIGC (1.2). As you can see, the color of the hormone concentrate is dark brown, while the LPIGC is light, slightly cloudy.

The optical density of the hormone concentrate (in% of the initial) increased to 92%. The results of measuring the optical density of the hormonal concentrate in the concentrator of the inventive device when cooled to -10 ° C are presented in table 1 and FIG. 2.

As can be seen from the presented data, in the first 30 minutes after the start of cooling, the changes in optical density are insignificant, however, starting from 50 minutes up to 70 minutes, the optical density values increase sharply, and also sharply slow down after 70 minutes. The revealed patterns in the dynamics of changes in the optical density of the hormonal concentrate are due to its accumulation in the concentrator. At the same time, the decrease in accumulation after 70 minutes indicates the completion of ice formation in the entire volume of the device body. Based on these results, the optimum cooling time was determined to be 90 minutes. Further continuation of cooling is impractical due to the completion of the freezing process of the entire mass of urine and, accordingly, the formation of the maximum possible final volume of the hormonal concentrate.

The study of the specific gravity of the samples of the hormonal concentrate and LPIGC showed that the specific gravity of the hormonal concentrate is, on average, 1033.8 ± 3.7, with the values of the initial urine, on average, 1010.9 ± 4.8 (p <0.05). The specific gravity of LPIGC was, on average, 1001.5 ± 2.3 and was significantly lower than both the initial urine and the hormonal concentrate (n = 10, p <0.05).

To obtain a dry residue obtained on the claimed device, the hormonal concentrate and the original urine were dried in an oven at 100 ° C for 18 hours to completely remove moisture. Fine weighing of the remaining dry residue was carried out on a digital scale (GF-400, A&D. Taiwan). As a result, 1.251 g was obtained from 100 ml of the hormonal concentrate after drying. dry matter, which is a percentage of 1.25%. The final weighing of the dry residue from 100 ml of the original urine was 1.116 g. dry residue, which is 1.2% as a percentage. Comparison of the results of weighing the dry residue and their interpretation proves the following. Proceeding from the fact that to obtain 100 ml of the hormonal concentrate requires 5 liters of initial urine, then the total dry residue must also be taken into account per 5 liters of initial urine. Thus, in terms of comparable values of 1 liter, the weight of the dry residue in the hormonal concentrate is not 1.2%, but 5 times less, i.e. corresponds to 0.24 gr. In this case, when compared with the weight of the dry residue of the original urine, also in terms of 1 liter, the weight of its dry residue is 11 g, which is more than 45.8 times more than the weight of the dry residue of the hormonal concentrate obtained from 1 liter of urine. Ultimately, the calculations show that the freezing process in the claimed device leads to the formation of a hormonal concentrate with a simultaneous significant, almost 46-fold decrease in the proportion of substances that make up the components of the dry residue. Those. the freezing process in the claimed device performs an additional function of cleaning the hormonal concentrate from accompanying undesirable components, increasing the quality and degree of its purification.

The results of the study of the weight and composition of the dry residue in the original urine, which has passed the preliminary freezing process (after preliminary cryoaggregation) and the hormonal concentrate obtained using the proposed method and device are summarized and presented in Table 2.

Studies have shown that during the freezing process (cryoconcentration), two main fractions are formed. One of them, in the form of frozen liquid, after defrosting is LPIGC, prevails in volume, weakly colored, practically free of the main steroid hormones, in particular, cortisol and estrogens. The second fraction is liquid - hormonal concentrate.

The main amount of hormonal substances, accounting for 80% to 90% of their total content in the original urine, is in the hormonal concentrate, which differs in color, in particular, from yellow to dark brown shades and its saturation. Studies have shown that in humans, the ratio - the volume of the hormonal concentrate / the volume of LPIGC is in the range 1: 4-1: 60, averaging 1:50. The conducted studies of the content of 4 different steroid hormones in the original urine, as well as in the hormonal concentrate and LPIGC showed significant differences. The results are shown in Table 3.

As can be seen from the data presented in Table 3, the level of estradol in the hormone concentrate (obtained from normal urine) exceeded 90,000 pg / ml and, with initial values of 5805.1 ± 324.4 pg / ml, was almost 15 times higher than in the initial urine and 42 times higher than in ZhPIGK.

Similarly, from the data presented in Table 3, it can be seen that the level of cortisol at initial values of 430.9 ± 26.4 μg / day, in the hormonal concentrate exceeded 8000 μg / day and was almost 60 times higher than the values of LPIGC.

The results of the study of the content of other hormones, such as testosterone and progesterone showed that their concentration in the hormonal concentrate is also significantly higher than that determined in the original urine and LPIGC, and these changes occur after the application of the claimed method and the claimed device.

Additional information proving the effectiveness of the proposed device is presented in table 4.

The conducted studies of the content of various steroid hormones confirm the significant advantages of the proposed method and device for producing a hormonal concentrate over the already known analogues, since high values of the concentration coefficient and hormone content in the final product are unattainable when using previously known methods.

The device works as follows

The pretreated urine, after opening the solenoid valve 7 installed on the horizontal outlet 5, flows through the connector 4 installed on the bottom 3 of the body 1 into the container of the body 1 after the preliminary closing of the body 1 with the outer cover 2, and is poured to the required volume, limited by the setting of the level sensor 16. After the level sensor 16 is triggered, the signal from it is fed to an externally installed control unit, from which a signal is sent to turn off (close) the solenoid valve 7 on the horizontal branch 5. At the same time, the photo-optical unit 15 is turned on, sending a signal to the externally installed control unit , the values of which are taken as the initial ones for monitoring the state of the liquid inside the concentrator 11. Also, at the same time, an externally installed cooling unit is turned on, which, after switching on, starts the cooling process, which consists in the fact that the tubular spiral cooling element 9 begins to flow into the flow m mode coolant with a temperature of -10 ° C. The forming hormone concentrate passes through the drainage holes 12 into the concentrator 11. As it accumulates inside the concentrator 11, the photo-optical unit 15 gives a signal to complete the cooling, which is accompanied by the shutdown of the cooling unit, the switching on (opening) of the electromagnetic valve 8 on the horizontal outlet 6, and the discharge of the hormonal concentrate into a storage tank for further use. After draining the hormone concentrate, the heating element 10 is switched on, which briefly heats the wall of the housing 1, preferably up to 25 ° C to defrost the wall ice. The heating process of the housing 1 is controlled by a temperature sensor 17 installed on the outer side of the bottom 3, which gives a signal to turn off the heating when the required temperature is reached. When extracting frozen urine, the support plate 13, installed in the lower part of the freely movable concentrator 11, contributes to the complete removal of ice from the housing 1, while through the holes 14, on the support plate, the remainder of the hormone concentrate passes to the bottom 3 of the housing 1 and is subsequently removed or mixed with a new portion of urine in the next cycle.

The essential distinguishing features of the proposed device, from the known analogs and prototype, allowing to solve the tasks, are as follows.

1. An essential feature of the claimed device is the use of a spiral tubular cooling element installed on the outside of the housing, which creates a directed horizontally and perpendicularly from the periphery to the center, controlled cooling of the original product to a preferred temperature of -10 ° C for smooth and uniform ice formation.

2. An essential distinguishing feature of the claimed device is the use of a heating element installed on the outside of the housing and on the horizontal outlets, which makes it possible to briefly warm up the housing wall and horizontal outlets for the unhindered removal of ice from the device after each cycle, as well as for defrosting the ice formed during the cooling process in horizontal ice outlet for the smooth removal of the hormone concentrate from the device after the end of the cycle.

3. An essential distinguishing feature of the claimed device is the use of a concentrator in the center of the body of the device for the accumulation of hormonal concentrate, which makes it possible to immediately obtain a hormonal concentrate.

4. An essential distinguishing feature of the claimed device is the use of guide plates installed in the concentrator, which make it possible to create at least four independent concentration sectors in the direction from the periphery to the center, which increases the uniformity of cooling, its directionality, and has a positive effect on stability, reproducibility and the effectiveness of the method for producing the hormonal concentrate.

5. An essential distinguishing feature of the claimed device is the automation of its operation through the use of technical means for monitoring and control of the hormonal concentrate production process, such as an externally installed control unit with a microprocessor, electromagnetic valves, temperature sensors, liquid level, photo-optical unit, which allow fully automating a cycle for obtaining a hormone concentrate, and in the complex they represent a device of a modular type, which, if necessary, can be integrated into a single technological line with several similar devices, and also functionally expanded for one-time processing up to 10 times the volume of the original urine.

The advantages of the claimed device

1. Does not require the use of expensive chromatographic equipment, gel filtration, ultrafiltration, centrifugation, ion-exchange resins or other adsorbents, which are significantly expensive, on the one hand, and significantly smaller volumes of the final product, on the other hand.

2. To obtain the hormone concentrate, only 1 stage is required.

3. Ability to work continuously for 24 hours.

4. Simplicity of equipment and reliability of regulation of the parameters of the hormone concentrate production process, as well as the reproducibility of the process.

5. Substantially better rates / volume / quality.

6. Full automation of the production cycle.

7. Possibility of using several devices at the same time, integrated into one technological line in a quantity that meets the needs of production.

8. Industrial technology for producing hormonal concentrate.

9. No need to use chemical reagents at the stage of concentration and at the previous stages of urine processing.

10. Possibility of using the device for laboratory and analytical purposes.

11. No need to use imported equipment,

components, consumables.

12. The possibility of using the method and device for other technological processes, on the contrary, requiring preliminary purification of the starting material from undesirable components, in particular hormonal components.

Examples of implementation of the method and operation of the device

Example 1.

The urine of a practically healthy woman D., 18 years old, was examined and processed. Urine was taken in the morning, on an empty stomach (320 ml received), and also twice in the afternoon (400 ml received). Morning urine was stored at 1 ° C prior to use. A total of 720 ml of urine was obtained. The woman was in the middle of the second phase of her menstrual cycle.

The studies were carried out on a working prototype of the claimed device in compliance with all the declared parameters of its operation.

In the samples of the initial urine, LPIGC and the hormonal concentrate itself, the following parameters were investigated: color, optical density, specific gravity. In addition, the content of hormones was analyzed, namely: estradiol, cortisol, progesterone and testosterone.

The study of the dynamics of cooling showed that the process of directional gradual gradual cooling of urine leads to the appearance of a hormonal concentrate of dark brown color, while the LPIGC is light and transparent.

The optical density of the hormonal concentrate (in% of the original) was 98.5%. The study of the specific gravity of the samples of the hormonal concentrate and LPIGC showed that the specific gravity of the hormonal concentrate was 1030 at the values of the initial urine of 1010. The specific gravity of the LPIGC was 1001 and was significantly lower than both the initial urine and the hormonal concentrate.

The results of determining the content of steroid hormones are presented in table 5.

As can be seen from the data in Table 5, the content of all the studied hormones without exception in the hormonal concentrate was significantly higher compared to the original urine.

Example 2.

The urine of a practically healthy woman I., 30 years old, was examined and processed. Urine was taken in the morning, on an empty stomach (340 ml received), and also three times in the afternoon (590 ml received). Morning urine was stored at 1 ° C prior to use. A total of 930 ml of urine was obtained. The woman was in the middle of the second phase of her menstrual cycle.

The studies were carried out on a working prototype of the claimed device in compliance with all the declared parameters of its operation.

In the samples of the initial urine, LPIGC and the hormonal concentrate itself, the following parameters were investigated: color, optical density, specific gravity. In addition, the content of hormones was analyzed, namely: estradiol, cortisol, progesterone and testosterone.

The study of samples before and after the operation of the device showed that the process of directed gradual gradual cooling of urine leads to the appearance of a hormonal concentrate of a dark brown color, while the LPIGC is light and transparent. The optical density of the hormonal concentrate (in% of the initial) was 98.3%.

The study of the specific gravity of the samples of the hormonal concentrate of the initial urine and LPIGC showed that the specific gravity of the hormonal concentrate was 1036, with the values of the initial urine being 1012. The specific gravity of the LPIGC was 1002 and was significantly lower than both the initial urine and the hormonal concentrate.

The results of determining the content of steroid hormones are presented in table 6.

As can be seen from the data in Table 6, the content of all studied hormones, without exception, in the hormonal concentrate was significantly higher than in the original urine.

Literature

1. Yu. Anzorge "Method and device for accumulation and stabilization of conjugated estrogens from mare's urine". Patent RU 2277241 C2, priority from 2001.07.26.

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Claims (12)

1. A device for obtaining a hormone concentrate from human or mammalian urine, including a housing, a lid, characterized in that the housing is made of stainless steel, has a cylindrical shape, a cone-shaped bottom and an internal volume from 500 ml to 1500 ml, while it is closed with an outer cover with external thermal insulation, in the center of the bottom of the case on the outside there is a connector with at least two horizontal outlets, one of which is intended for injecting liquid into the inner volume of the body, and the second for withdrawing the hormonal concentrate, and at the ends of the inlet and of the outlet horizontal branches, normally closed solenoid valves are installed, a spiral-shaped cooling tubular element is installed on the outer side of the housing wall, connected to an externally mounted autonomous cooler equipped with a microcircuit for regulating cooling parameters, and between the spiral of the tubular element on the outer wall of the housing There is a plate-shaped heating element connected to an externally located power supply equipped with a microcircuit for regulating parameters that provide a temperature range for heating the housing wall, and in the center of the housing, a freely movable concentrator is installed parallel to the vertical axis of the housing, designed to collect the hormone concentrate, made of stainless steel and tubular, on the walls of which there are through drainage holes along the entire length, and on the lower part of the concentrator there is an annular support plate located perpendicular to its vertical axis, also equipped with through holes throughout, while a photo-optical unit is installed in the center of the upper third of the concentrator, equipped with a light source and a photocell, which are hermetically protected from liquid, and a level sensor is installed in the upper part of the collector and a temperature sensor is installed on the bottom outer side of the bottom. 2. The device according to claim 1, characterized in that the internal volume of the housing is 1000 ml. 3. A device according to any one of claims 1, 2, characterized in that the autonomous cooler provides smooth and uniform cooling from all sides of the housing wall and maintains the required parameters of temperature, direction and rate of urine cooling inside the housing. 4. A device according to any one of claims 1 to 3, characterized in that the ratio of the internal working volume of the housing and the internal working volume of the concentrator is from 40: 1 to 60: 1. 5. The device according to claim 4, characterized in that the ratio of the internal working volume of the housing and the internal working volume of the concentrator is 50: 1. 6. The device according to claim 2, characterized in that the size of the inner radius of the body is from 40 mm to 60 mm. 7. The device according to claim 6, characterized in that the inner radius of the body is 50 mm. 8. The device according to any one of claims 1-7, characterized in that the cycle of the device is fully automated by using an externally mounted control unit with a microprocessor and temperature sensors, liquid level, optical density, electromagnetic valves installed on the body. 9. A method of obtaining a hormone concentrate from human or mammalian urine by freezing using a device according to any one of claims 1 to 8, characterized in that urine cooled to 2 ℃ and previously purified from urine salts, denatured proteins, mucus and macromolecular aggregates, to which also added microparticles of activated carbon with a size of 500 microns in an amount of 200 mg / l, poured into the device case, where it is additionally cooled in a constant mode to a temperature of -10 ℃, and the cooling process is directed horizontally and from the periphery to the center, and crystals formed during the cooling process ice gradually displace the liquid concentrate containing the hormonal mixture to the center of the device, which enters the concentrator installed in the center of the device, vertically and parallel to its axis, from where, after the end of the freezing cycle, the hormonal concentrate is drained into an externally installed storage device, which is a container whose volume exceeds the volume concentrate rat in a ratio from 4: 1 to 6: 1, where, before feeding the hormonal concentrate, powdered sodium hydroxide is preliminarily introduced as a precipitant and preservative in a ratio of 0.5 g: 100 ml of the volume of the hormonal concentrate or concentrated sodium hydroxide solution in a ratio of 0.5: 100 to the hormonal concentrate, after which the hormonal concentrate is poured into the storage device in the above volume ratio, which is stirred for no more than 10 seconds and kept at room temperature for 8-12 minutes, followed by separation of the sediment , consisting mainly of glycosaminoglycans and proteoglycans, from a hormone concentrate that is used as a final product for subsequent use. 10. A method according to claim 9, characterized in that the volume of the storage tank exceeds the volume of the concentrate in a ratio of 5: 1. 11. A method according to any one of claims 9, 10, characterized in that the hormonal concentrate is kept in the reservoir for 10 minutes. 12. The method according to any one of claims 9-11, characterized in that before using the hormone concentrate, sodium hydroxide is neutralized with an equimolar amount of citric acid.

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