RU2189961C2 - Method of immobilization of physiologically active compounds - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention proposes a method of immobilization of physiologically active compounds of trace elements comprising the composition of a mixture of these compounds with addition of immobilizing agent and the following treatment of mixture and granulation of the ready product. Method is based on the use phosphates and their mixtures with molybdates, carbonates, sulfates and nitrates including trace elements as a immobilizing agent, the following mixture melting, preparing vitreous active micrononhomogeneous amorphous mass and its granulation. EFFECT: improved method of immobilization. 2 tbl, 2 ex

Description

 The invention relates to the chemistry of physiologically active compounds based on phosphates with the addition of any other necessary components and can be used in pharmacology, medicine, veterinary medicine and in agriculture.

In pharmacology, sustained-release drugs or oral drugs that are absorbed in a given section of the digestive tract are constantly being developed. For this purpose, slowly destroyed organic substances, for example, in the form of shells, are most often used.
This task is no less relevant in agriculture. The annual application of fertilizers is not only wasteful, but also environmentally harmful, since at least 50% of the fertilizer goes into groundwater and erodes. Attempts to coat fertilizers with polymer shells improved the situation by only 10% by reducing weathering. Another disadvantage of this type of encapsulation is the washing out by the enzymes of an animal or plant of useful components during the first damage to the membrane, which leads to excessive saturation of these components and causes damage to the object. In this case, plants or the animal organism accumulate an excessive amount of nitrates, selenium, thallium, lead and other elements, which sometimes leads to damage to the parenchyma of the body. The introduction of hormonal drugs that accelerate the development of animals and plants causes international protests by anti-globalists against beef and a number of other products of Western countries. Thus, there is a need for the creation of physiologically active substances in an assimilable form with an adjustable rate of assimilation, maintaining a predetermined balance of potassium, magnesium, phosphorus, boron and trace elements in animal and plant organisms without compromising the quality of food products.

 A known method of immobilization of biological components (a.a. USSR 1594410, G 01 M, 09/23/90, Bull. 35), comprising preparing a mixture of compounds with the addition of an immobilizer in the form of gelatin, physical processing of the mixture and shaping it in the form of a coating of enzyme electrodes.

 The disadvantage of this method is the low stability of the product, the uncertainty of measurements over time due to changes in its properties and short service life due to inhibition of surface-active centers.

The RF certificate for utility model 9840 dated May 16, 1999, adopted for the prototype, is known, which includes the "execution of a two-layer granule" obtained from a mixture of oxides and phosphoric acid "by heat treatment in a temperature gradient of 250-1400 ^o С, followed by processing in a mode that provides replacement in the outer layer with a thickness of 5-50 microns 5-15% of metal oxides of 1-2 groups of elements of the periodic table of DI Mendeleev for water molecules. " The prototype "ensures the transformation of a homogeneous internal material of a two-layer granule into a surface layer within 1-4 years as a result of a layer-by-layer, simultaneous release of all components into a solution."

 However, the prototype does not provide for the immobilization of trace elements (catalysts) and enzymes on the surface of the granules and the formation of biochemically active complexes on the surface of microinhomogeneous granules in the process of their dissolution, and does not provide for the introduction of carbonates, sulfates, molybdates, nitrates and other compounds that affect the microinhomogeneous structure of glassy phosphates .

The objective of the present invention is to provide a method for producing a physiologically active substance of prolonged action with auto-controlled action in the process of uniform dissolution in time
The method provides long-term immobilization of bioactive compounds of trace elements through the use of a chemically inhomogeneous immobilizer. The method provides reliable and long-term prolongation of the physiologically active micro-inhomogeneous glassy granules.

 The problem is achieved in that in the method of immobilization, including the preparation of a mixture of physiologically active compounds, adding an immobilizing component to it, physical processing, forming a mixture in an assimilable form, in accordance with the present invention, phosphates and their mixtures with carbonates, sulfates are used as an immobilizing substance , silicates, molybdates, and nitrates, the physical processing of the mixture is carried out by the method of its melting, to obtain an amorphous glassy mass, which is shaped granulation, crushing or drawing into fibers with a diameter of up to 2 mm.

The rejection of an organic or inorganic immobilizer with a crystalline structure and its replacement with an inorganic immobilizer with an amorphous structure provides not only how pleasingly prolonged the dissolution process, but also allows the uniformity of time-dependent action of immobilized trace elements, which is very important in all of the above directions for the use of physiologically active compounds
Among the trace elements necessary for the development of any biological systems that ensure the creation of physiologically active substances and vitamin complexes in the food chain: soil microorganisms - plants - animals - people include: copper, cobalt, zinc, vanadium, molybdenum, iron, sulfur, selenium, iodine , boron, chromium, nickel, silicon, etc.

 The study of ash residues shows that the concentration of trace elements in plants and animals ranges from 0.0005 to 0.2 wt.%. Moreover, different types of microorganisms, plants, animals and even their individual organs accumulate various trace elements. From this it was concluded that they act as catalysts for biochemical processes.

 The most important condition for the action of trace elements is their availability, i.e. presence in the form of active complexes. All leading manufacturers of fertilizers, pharmaceuticals and food additives introduce trace elements, as a rule, in the form of salt additives - sulfates, nitrates, chlorides, hellates, etc. In the practice of animal husbandry and poultry farming, the use of polycrystalline feed phosphates is widely known.

 In this case, both undesirable shock loads on living objects and the rapid leaching of trace elements during dissolution without their assimilation are possible.

The principal difference of the proposed method is: the preparation of a given mixture of trace elements in the ratios determined by the type of biological object. The mixture is composed of silicates, nitrates, sulfates and molybdates of iron, manganese, zinc, cobalt, copper, etc. The resulting mixture of physiologically active trace elements in a total amount of up to 5 wt.% Is introduced into the melt of potassium, magnesium, calcium meta- and pyrophosphates, which acts as an immobilizing component - an amorphous micro-inhomogeneous glassy mass - component of 90 wt.%. All trace elements in the melting process (at a temperature of 1000-1350 ^o C) due to thermal destruction of the starting compounds are concentrated in areas of microinhomogeneities. Inhomogeneous structure is an important feature of materials. Due to the heterogeneous structure, the local concentration of trace elements significantly exceeds the average composition in the synthesis - the segregation of activators. Introduction to the melt of the immobilizing component along with trace elements carbonates, silicates, borates, aluminates and molybdates in a total concentration of up to 5 wt. % allows you to influence the dissolution rate of the material and its microinhomogeneous structure.

 A mixture of physiologically active compounds is composed of inorganic salts of cations of trace elements, including cobalt, zinc, manganese, copper, iron, boron, selenium, molybdenum, vanadium, nickel, etc. These trace elements form stable chemical salt compounds in the form of carbonates, nitrates, silicates, sulfates , molybdates, the concentration of each of these trace elements in the mixture is 0.1-1.0%. The total amount of trace elements is 2-5 wt.% In relation to the immobilizing component of potassium, calcium and magnesium meta- and pyrophosphates. A mixture of physically active compounds is most often prepared in a nitric acid aqueous solution at a pH of less than –3, followed by the addition of carbonates and silicates that increase the pH to more than 7, which leads to the formation of an insoluble precipitate. The resulting filtered and dried product is a mixture of physiologically active compounds, which is introduced into interaction with the immobilizing component in the process of high-temperature synthesis.

 The proposed method is intended to obtain a glassy immobilizer of micronutrients of prolonged action. Purpose and scope: as a feed additive for animals, including macro- and microelements in a balanced ratio, as a fertilizer for plants, providing autocatalytic action in soil biochemical processes and improving the quality of agricultural products, starting from animal feed products to human food .

The presence in the immobilizing component of the active compounds of trace elements and their concentration are selectively controlled by spectroscopic analysis of plane-parallel samples on standard spectrophotometers according to the characteristic known absorption bands of activator cations. The yield of a trace element mixture in aqueous solutions is also controlled by sampling solutions from the absorption spectra of ions hydrated with trace elements
The dissolution of the immobilizing component is controlled by the weight loss of the granules. The inhomogeneous structure of glassy meta- and pyrophosphates is determined by the methods of small-angle X-ray scattering, Rayleigh and Mandelstam-Brillouin scattering. The development of an inhomogeneous structure occurs in the melt; at the same time, a thermodynamic equilibrium is established by the distribution of physiologically active compounds in the regions of heterogeneity.

 The time to establish equilibrium depends on temperature and pressure and is monitored visually when the homogeneity of the melt is achieved, as well as selectively by X-ray diffraction analysis to achieve the X-ray amorphous state. Areas of heterogeneity enriched with trace elements are characterized by dispersion in size and structure and therefore cannot be characterized as individual chemical compounds.

The size of the chain phosphates entering the solution is quantified by paper chromatography. It was shown that with an increase in silica concentration to 5 wt.%, The average degree of polymerization of phosphates in solutions decreases from 30 to 10 [PO ₄ ] tetrahedra in the chain.

The physical and chemical characteristics of the final product should include: refractive index - n _d in the range of 1.480-1.495, density - ρ in the range of 2.5-2.8 g / cm ³ , X-ray fluorescence analysis data confirming the absence of crystalline inclusions and the presence of trace elements in predetermined concentrations and experimentally controlled kinetics of dissolution of the weight of the granules. These methods allow you to uniquely control the completion of the claimed process.

The preparation of inorganic polymers of variable composition based on meta- and pyrophosphates of the elements of the first and second groups of the periodic system and transition metals was carried out from the melt at 800–1300 ^o С with the introduction of silicates, nitrates, molybdates, sulfates, borates, aluminates in concentrations up to 5 wt. .%, which provided a change in the dissolution time of inhomogeneity regions, including trace elements, according to the exponential law with a change in the dissolution rate constant up to 5 times. Phosphoric acid with a concentration of 35-85% of the basic substance, carbonates and nitrates of salts of the elements of the first and second groups of the periodic system, as well as nitrates, silicates, borates, molybdates and sulfates of transition elements were used as starting materials.

Example 1
The synthesis of a physiological active mineral compound of variable composition having a micro-heterogeneous, low molecular weight structure while maintaining a macroscopically disordered structure and X-ray amorphism was carried out in the following sequence. The reaction mixture, including phosphoric acid H ₃ PO _{4 with a} concentration of 85-35 wt.%, Was mixed with potash - K ₂ CO ₃ at a concentration of 15 wt.%, Apatite concentrate 23 wt.%, Magnesium carbonate 6 wt. %, nitrates, sulfates and molybdates of zinc, manganese, cobalt, iron, copper - the total concentration of 1% of the composition by weight.

The reaction mixture was kept for 5 hours in paper bags and then subjected to heat treatment at 350 ^o C, which led to briquetting in 2 hours. Briquettes were placed in a high-temperature chamber at a temperature of 900 ^o C for 1 hour. After which the temperature rose to 1250 ^o C and the resulting melt was maintained at this temperature for about 3 hours until a disordered inhomogeneous structure was achieved. After the specified time, the melt was granulated or molded, inertially cooled to room temperature and crushed into granules of 1-5 mm.

Tests have shown that the obtained product is X-ray amorphous, characterized by the kinetics of dissolution in water, obeying the law In p ₀ / p = kt, where P ₀ is the initial weight, and P is the current weight of the granules, k is the dissolution rate constant that exponentially depends on temperature, t - time. The dissolution time at various temperatures is given in table. 1.

In this case, a layer enriched with microelements forming active centers forms on the surface of the granules. The layer thickness increases during dissolution and reaches 1–2 mm in 1 year due to the selective leaching of low molecular weight groups. Microprobe analysis shows that the concentration of trace elements in areas of microinhomogeneities is 3-5 times higher than the average volume
The product has passed toxicological examination and certification on dissolution kinetics (certificate "Mechanobr-Analit" G / 13 dated 02.02.94).

 Agrochemical and microbiological tests on sod-podzolic soil samples using a composition product (Table 2) (according to X-ray fluorescence analysis carried out in the Mechanobr Analit), wt.%, Revealed the following patterns.

 A strong stimulating effect on almost all groups of soil microorganisms, there was an increase in the number of nitrogen-fixing microorganisms, amylolytic and protolytic bacteria, an increase in the number of fungi, accompanied by an increase in species diversity.

 The presence of Trihoderma Viridae fungi involved in the decomposition of organic residues was detected in the soil.

 Control and testing data for the second year of testing confirms the prolonged action of the product.

Example 2
Potassium metaphosphate (potassium monophosphate) 32 wt.% Was mixed with phosphoric acid 85% - 37 wt.%, Apatite concentrate 14 wt.% With dolomite flour 14 wt. %, sulfates of iron, copper, zinc, manganese, molybdenum, cobalt with a total content of 2 wt.%.

 The resulting mixture was divided into 3 parts, in one of which - A was added 3% sodium disilicate, in the second - 3% sodium aluminate, and in the third - 3% borax.

Further, all three mixtures were placed in quartz crucibles and loaded into a furnace at a temperature of 900 ^o C. Then the temperature rose to 1300 ^o C. After keeping at this temperature for 3 hours, the microinhomogeneous melt formed was cast. Tests on the kinetics of dissolution showed that mixture A led to the production of an X-ray amorphous product with a dissolution rate of two times that of product B, and the dissolution rate of product B was 5 times less than product A, due to the difference in microinhomogeneous structure and segregation activators. Polythermal studies of the samples by combined scattering, Mandel'shtam-Brillouin spectroscopy, and small-angle X-ray scattering showed that inorganic polymers obtained by high-temperature synthesis are characterized by disordered structure and heterogeneity of the structure due to the freezing of supramolecular groups existing in high-temperature melt. The substance obtained in this way has excess free energy compared with crystals and is in a nonequilibrium state.

 Catalysis by complex compounds is similar to the mechanism of action of enzymes. In contrast to immobilized enzymes artificially associated with water-insoluble carriers — actually heterogeneous catalysts — the authors propose active meta- and pyrophosphates that immobilize trace elements in areas of microinhomogeneity.

 The obtained compounds were used with a prolonged effect both to increase the biomass of microorganisms and accelerate their growth, and for plants as fertilizers with a controlled dissolution time from 1 month to 5 years. A toxicological examination carried out at the Institute of Toxicology of the Russian Academy of Sciences (St. Petersburg) showed that the products obtained by the proposed method are non-toxic and can be used as feed additives and fertilizers.

Claims (1)

 A method of immobilizing physiologically active compounds, including composing a mixture of inorganic compounds, adding an immobilizing component to it, physical processing and forming a mixture in forms assimilable by microorganisms and plants, characterized in that meta- and pyrophosphates or mixtures thereof containing molybdates are used as immobilizing substances , carbonates, sulfates and alkaline nitrates, the physical processing of the mixture is carried out by its melting method to obtain an amorphous micro heterogeneous glassy mass, which is shaped by granulation, crushing or drawing.

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