RU2736186C1 - Method for producing medicinal agents - nitrogen-containing betaines of hydrochlorides - based on post-distilled corn silage - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a substance and a dosage form containing nitrogen-containing betaines hydrochlorides based on a liquid phase of a post-alcohol corn mackerel, intended for use as digestive, fermental medicinal agents (LS), as well as to methods for preparing them. Method of producing a drug substance consists in the fact that from the liquid phase separated from the solid phase of the post-alcohol corn brew by filtering, reducing sugars are removed after pre-hydrolysis of maltose by crystallisation, filtration and drying of glucose, concentration and cooling of the filtrate, the proteins contained in the filtrate and the peptides are hydrolysed with concentrated HCl, cooled, filtered, contained in the amino acid filtrate, methylated, treated with concentrated HCl, concentrated, cooled, filtered precipitate A, the filtrate is concentrated, crystallized, the residue B is separated, the combined (A and B) sediment B is dissolved in water, purified with activated carbon, filtered, the filtrate is concentrated, purified precipitate B is separated, the filtrates formed after separation and cleaning precipitate B, diluted with water, treated with activated carbon, cooled, filtered, concentrated, cooled, treated with concentrated HCl, crystallized, crystals (precipitate D) filtered, re-precipitated from aqueous solution with concentrated HCl, filtered and dried purified precipitate G, filtrates formed after separation and purification of the residue G, evaporated and dried to dry residue (precipitate D), precipitation B, D, D are combined and mixed with pharmacopoeial pepsin (weight ratio 4:1). Pharmaceutical substance is a homogeneous crystalline powder of white colour with an acidic odour, with composition of nitrogen-containing betaines of hydrochlorides: glycine (sediment B, 36.5 wt. % to substance), glutamic acid (sediment G, 32.3 wt. % to substance), arginine, alanine, aspartic acid, tyrosine, histidine, serine, valine, leucine, lysine, threonine (precipitate D, 11.2 wt. % to substance) and pharmacopoeial pepsin (20.0 wt. % to substance), with quantitative content of betaine hydrochlorides: 99.8 % (residue B), 99.6 % (precipitate D), 99.5 % (precipitate D), pepsin 100 ± 7.5 %. Method of producing a dosage form (tablet) is that a pharmaceutical substance of weight 250.0 mg is dried to residual moisture content of not more than 3 %, is mixed with 29.9 mg of a sliding substance of aerosol, 15.0 mg of colitisone CL lemonisant, moisturised with 1.0–1.5 ml of ethyl alcohol of 96 %, ground, strained through a sieve, "powdered" with 3.0 mg of calcium lubricant of stearate, obtained dry granulate is pressed to obtain tablets. Dosage form represents a tablet for oral administration with weight 296.4–303.6 mg, diameter 9 mm, height 3 mm, round shape, with biconvex surfaces, solid edges, without risks and chamfers, with smooth and uniform surface, white colour, with acidic odour, with composition of biologically active compounds in 1 tablet : 200.0 mg of nitrogen-containing betain hydrochlorides, 50.0 mg of pepsin, with excipients in 1 tablet : 29.9 mg of aerosil, 15.0 mg of collidone CL, 3.0 mg of calcium stearate, with 2 years shelf life of tablets at storage temperature of 10 °C.

EFFECT: disclosed is a method for preparing medicinal agents - nitrogen-containing betaines of hydrochlorides - based on a post-alcohol corn duff.

4 cl, 17 tbl

Description

The invention relates to the pharmaceutical industry and relates to a method for producing a substance and tablets of nitrogen-containing betaines hydrochlorides based on the liquid phase of post-alcohol corn stillage, intended for use as digestive, enzymatic drugs (MP).

A known method of obtaining from the liquid phase post-alcoholic grain (including corn) stillage of pharmaceutical substance "Biobardin", containing, along with other biologically active compounds, 54.0% of proteins and amino acids. The method consists in separating and concentrating the liquid phase of vinasse to a density of 1.480 g / ml, treating it with ethyl alcohol 95% (by volume 1: 3), filtering and drying (60 ° C) the precipitated substance "Biobardin" (yield 3.3% to liquid phase) (Kaisheva N.Sh., Kaishev A.Sh. Antioxidant drug based on grain distillery stillage. RF Patent 2404766, IPC A61K 31/375, publ. 10.09.10). This method for producing drugs is close to the claimed method in terms of the raw materials used (post-alcohol grain stillage), the target product (amino acids), specific pharmacological activity (drugs of digestive action), and is selected as a prototype. The disadvantages of this method are:

- loss of proteins and amino acids due to precipitation with alcohol 95%;

- loss of betaines, soluble in alcohol, when processing the concentrate with 95% alcohol (Poludek-Fabini R., Beirich T. Organic analysis. Guidelines for the analysis of organic compounds, including medicinal substances. Translated from German, Leningrad, Chemistry, 1981 , 624 p.);

- no stage of purification of target products from accompanying substances (oligogalacturonides, biogenic elements);

- low technological yield of target products.

To obtain tablets "Biobardin" from lactose (filler) and polyplasdone (binder) (1: 1), a granulate is prepared, into which a 50% solution of the substance is introduced, the mixture is granulated, "powdered" with calcium stearate (lubricant) and flat-cylindrical tablets are obtained by pressing ... Composition of 1 tablet: 0.250 g of the "Biobardin" substance (contains 0.135 g of proteins and amino acids), 0.300 g of lactose, 0.300 g of Polyplasdone XL-10, 0.001 g of calcium stearate. The tablets are practically non-toxic, intended for use in gastroenterology (Kaisheva N.Sh., Kaishev A.Sh., Shevchenko AM, Mikelov VA Pharmaceutical tablets "Biobardin", obtained on the basis of the liquid phase of post-alcohol grain stillage and intended for gastroenterology. Pat RF 2662064, IPC A61K 31/70). The target product exhibits specific pharmacological activity not due to all components, but only due to the partial presence of amino acid betaines. Apparently, the targeted production of nitrogen-containing betaines of hydrochlorides from vinasse and the introduction of a digestive enzyme can enhance the specific pharmacological effect.

Close to the claimed tablets are tablets "Acidin-pepsin" (Registration Certificate (RU) LS001355241011: order of the Ministry of Health of the Republic of Belarus No. 843 dated 08.24.2015). One tablet with an average weight of 252 mg contains as active substances: 200 mg of betaine hydrochloride (betaine glycine hydrochloride) and 0.5 mg of the proteolytic enzyme of porcine pepsin (in terms of 10% pepsin); as auxiliary substances: 12 mg of povidone-K25 (polyvinylpyrrolidone) (binder), 5.2 mg of aerosil (colloidal anhydrous silicon dioxide) (substance that improves wettability; glidant), 2.5 mg of calcium stearate (antifriction substance), 31.8 mg sorbitol (bulking agent, thinner, glidant). Tablets are round, flat, scored and beveled, white or white with a slightly yellowish sheen, with a specific odor, sour taste. According to the pharmacological properties, the tablets are classified as digestive, enzymatic drugs (ND 42-9712-05 "Acidin-pepsin", Republic of Belarus), while the acid must comply with the requirements of the US State Pharmacopoeia (United States Pharmacopoeia, 2019, USP 42-NF 37), pork pepsin - enterprise standard (STP 55-98). There is no description of the technology of tablets "Acidin-Pepsin" in the literature. The disadvantages of this composition of tablets are:

- underestimated content of pepsin (0.5 mg); to provide digestive, enzymatic action in tablets "Acidin-pepsin" should be 200 mg of acidin and 50 mg of pepsin (4: 1) (Mashkovsky M.D. Medicines. - 16th ed., revised and additional - M .: New Wave, 2016 .-- 1216 p.);

- the quality of pepsin should be regulated by pharmacopoeial monographs, and not by standards intended for food quality control;

- a relatively high proportion of excipients (20.4% of the tablet weight);

- the use of povidone-K25, a low-molecular excipient, does not provide satisfactory disintegration of tablets that meet pharmacopoeial requirements (State Pharmacopoeia of the Russian Federation (GF RF): in 4 vol. XIV ed., M, 2018);

- tablets, due to their high water absorption, even before the expiration date “spread”, sticking to each other;

- short shelf life (1 year 6 months) (Instructions (information for specialists) on the medical use of Acidin-Pepsin drugs: order of the Ministry of Health of the Republic of Belarus No. 843 dated 08.24.2015).

The purpose of the invention is to develop a method for producing the amount of nitrogen-containing betaines hydrochlorides (substances) based on the liquid phase of post-alcohol corn stillage, as well as the composition and technology of tablets with optimal pharmaceutical and technological parameters, long shelf life, suitable for use as digestive, enzymatic drugs.

This goal is achieved by the fact that after the separation of post-alcohol corn stillage into liquid and solid phases by filtration, first, reducing sugars (maltose, glucose) are removed from the liquid phase by hydrolysis of maltose with anhydrous acetic acid (mass ratio of maltose and acid 1: 1, 100 ± 2 ° C, 0.5 hour; Morrison R., Boyd R. Organic Chemistry.Translated from English by V.M.Demyanovich and V.A.Smith, under the editorship of I.K.Korobitsyna, M., Mir, 1974, 1133 s.) Until the formation of glucose, treatment of the hydrolyzate with anhydrous acetic acid (total mass-volume ratio of glucose and acid 1: 2), crystallization (0-5 ° С, 1 day), filtration and drying (70 ± 2 ° С) of crystals glucose, concentration of the filtrate (55 ± 2 ° С, up to 1/25 volume) and its cooling (up to 20 ± 2 ° С). The proteins and peptides contained in the concentrated filtrate are hydrolyzed to the formation of amino acids by treating the filtrate with concentrated hydrochloric acid (density 1.097 g / ml, concentration 6.0 mol / l, 19.98 wt%) (by volume 1: 1, 110 ± 2 ° C, 72 hours), slow cooling of the mixture (up to 20 ± 2 ° C), removal of ballast sediment by filtration, treatment of the filtrate with activated carbon (0.3% by volume of the mixture, 55 ± 2 ° C, 0.5 hour), filtration and washing the precipitate with water (the precipitate is discarded), combining the filtrate with the washing waters. The products of hydrolysis - amino acids (in the filtrate) are subjected to exhaustive methylation at the nitrogen atom to form betaines (trimethylamino acids) by the Hoffmann method (Hauptmann Z., Grefe Y., Remane X. Organic chemistry. Transl. From German., M. Chemistry, 1979, p. 502) using methyl iodide (molar ratio of nitrogen in the composition of amino acids and reagent 1: 4) in the presence of sodium carbonate (molar ratio of nitrogen in the composition of amino acids and reagent 2: 1) (42 ± 2 ° C, 30 hours). To obtain glycine betaine hydrochloride, concentrated hydrochloric acid (by volume 1: 1) is added to the mixture, concentrated (55 ± 2 ° C, to 1/3 of the volume), cooled (to 20 ± 2 ° C), filtered and the resulting precipitate is washed ( precipitate A) with concentrated hydrochloric acid (by volume 1:10). The filtrate combined with the washings is concentrated (55 ± 2 ° C, up to 1/3 of the volume), crystallized (0-5 ° C, 8 hours), the precipitate (precipitate B) of betaine hydrochloride glycine is separated by decantation; precipitates A and B are combined (precipitate C). Sediment B is purified by dissolving in water (55 ± 2 ° C, 1: 2), treating the solution with activated carbon (0.3% by weight of the solution, 40 ± 2 ° C, 0.5 hour), filtration, concentration of the filtrate (55 ± 2 ° С, up to 1/3 of the volume), crystallization (0-5 ° С, 8 hours), filtration and drying (20 ± 2 ° С). Further, glutamic acid betaine hydrochloride is isolated from the mother liquors formed after the separation and purification of the glycine betaine hydrochloride precipitate by diluting the solution with water (1: 1), clarifying it with activated carbon (0.3% by weight of the solution, 55 ± 2 ° C, 0 , 5 hours), cooling (up to 40 ± 2 ° C), filtration, concentration of the filtrate (55 ± 2 ° C, up to 1/3 of the volume), cooling (up to 20 ± 2 ° C), treatment with concentrated hydrochloric acid (density 1.097 g / ml, concentration 6.0 mol / l, 19.98%) wt.) (1: 1), crystallization (0-5 ° C, 5 days), filtration of crystals. The crystals obtained are purified by reprecipitation from an aqueous solution (1:10) with concentrated hydrochloric acid (density 1.097 g / ml, concentration 6.0 mol / L, 19.98 wt%) (1: 1) followed by filtration, drying (20 ± 2 ° C) precipitate of glutamic acid betaine hydrochloride (precipitate D). From the mother liquors formed after the isolation and purification of precipitate D by evaporation under vacuum (55 ± 2 ° C) and drying in a vacuum desiccator, the remainder of betaines of other amino acids hydrochlorides (precipitate D) is obtained. The obtained target products (precipitates C, D, D weighing 93.6229 g, 82.8289 g, 28.8863 g, respectively) are mixed until a homogeneous white powder (weight 205.3381 g) is formed, which, after preliminary dry granulation, is subjected to tableting ... Per 1 tablet, a mixture of 200.0 mg (66.7%) of the substance and 50.0 mg (16.7%) of the state standard sample (SSS) of pepsin (corresponding to the requirements of the pharmacopoeial monograph FS 42-0077-01) (pharmaceutical substance) is dried (40 ± 2 ° C) to a residual moisture content of not more than 3%. Then add 29.9 mg (10%) Aerosil (antifriction agent), 15.0 mg (5.0%) Kollidon CL (baking powder), moisten with 1.0-1.5 ml of ethyl alcohol 96%, grind in a mortar. The resulting dry granulate is rubbed through a No. 15 sieve (hole diameter 1.5 mm), "powdered" with 3.0 mg (1.0%) calcium stearate (lubricant) and pressing to obtain round tablets, with biconvex surfaces, solid edges, without risks and chamfers, white, with a specific odor. The composition of one tablet with a diameter of 9 mm, a height of 3 mm, an average weight of 300.0 mg: substances of betaines hydrochlorides 200.0 mg, pepsin 50.0 mg, aerosil 29.9 mg, collidone CL 15.0 mg, calcium stearate 3.0 mg.

Post-alcohol corn stillage is preferable for obtaining amino acids (precursors of nitrogen-containing betaines), the content of which in stillage is weighty: 28.8-28.9% in terms of dry residue (Kaishev A.Sh., Kaisheva N.Sh. Scientific bases of pharmaceutical use of raw materials alcohol production. Volgograd, Volgograd State Medical University, 2013, 156 p.). The object of isolation of the target products was the liquid phase of post-alcohol corn stillage, containing (28.8-36.0%) 13 times more amino acids and peptides than the solid phase (2.5%) (Kaishev A.Sh., 2013).

When processing corn, starch is extracted, subjected to enzymatic hydrolysis to the formation of glucose, which is subjected to alcoholic fermentation (Production technological regulations (MTP) for the production of alcohol from starchy raw materials for the enterprise Suvorovsky LLC: at 2 hours. Approved by the Ministry of Agriculture of the Russian Federation, M., 2004 ). In this regard, secondary raw materials may contain residual amounts of carbohydrates: in corn distillery no more than 0.5% (PTR, 2004). To control the residual content of carbohydrates, the method of high performance liquid chromatography (HPLC) was used, based on the separation of carbohydrates, their registration with a refractometric detector and quantitative determination by the method of external standards (GOST 32167-2013 Method for the determination of sugars). The determination was carried out on an UltiMate 3000 liquid chromatograph (Dionex, USA) with columns 250 mm in length, 4.6 mm in inner diameter, filled with amine-modified silicon dioxide (Eclipse XDB-C18, 5 μm). Data collection and processing was carried out using the system "Chromeleon, version 7" ("Dionex", USA). To prepare a standard solution, exact weights of fructose about 2.0 g, glucose about 1.5 g, sucrose about 0.25 g, maltose about 0.15 g are dissolved in 30 ml of water in one volumetric flask with a capacity of 100 ml, add 25 ml of methanol, volume the solution is brought up to the mark with water, filtered through a nylon filter. To prepare the test solutions, an accurate weighed portion of molasses about 5.0 g is added to a 100 ml volumetric flask, 20 ml of water is added; in another, the same flask, add 5 ml of the liquid phase of corn stillage; 30 ml of water are poured into both flasks. 25 ml of methanol, dilute the solution with water to the mark, filter through a nylon filter. The standard and test solutions are successively chromatographed under the following conditions: column temperature 30.0 ± 0.1 ° С, flow rate 1.3 ml / min, mobile phase (eluent): acetonitrile and water (80:20), volume of injected solution 0.020 ml, refractometric detector. The retention times of carbohydrates on HPLC chromatograms are shown in Table 1. By comparing the retention times on HPLC chromatograms of the standard and test solutions, fructose, glucose, sucrose were identified in molasses, and glucose, maltose were identified in post-alcohol corn stillage.

The quantitative content of carbohydrates (HPLC, external standards method, GOST 32167-2013) was determined from the peak areas of the determined carbohydrates in the chromatograms of the test solutions in relation to those of the standard solution. The experimental values for calculating the quantitative content are shown in Table 2. The quantitative content of carbohydrates (X,%) is calculated by the formula (1) (GOST 32167-2013):

where: S ₁ and S ₂ - the area of the peak of the carbohydrate in the chromatogram of the test and standard solutions, respectively, mm ² ;

a ₁ and a ₂ - weight of a sample of vinasse (molasses) and a standard sample of carbohydrate, respectively, g;

V ₁ and V ₂ - the total volume of the test and standard solutions, respectively, ml.

The data obtained indicate the content of 17.5% of reducing sugars (3.5% maltose, 14.0% glucose) in the liquid phase of post-alcohol corn stillage.

Unlike the prototype, the claimed method provides for the removal of reducing sugars from raw materials after preliminary acid hydrolysis of maltose with the formation of glucose (Morrison R., 1974) and subsequent crystallization of glucose from anhydrous acetic acid (Guben I. Methods of organic chemistry. Vol. 3, no. 1, M.-L., 1934, p. 274). Most amino acids, peptides are soluble in anhydrous acetic acid (Borovlev I.V. Organic chemistry: terms and basic reactions. - M .: BINOM. LZ. 2018. - 359 p.), So they remain in the filtrate after crystallization of glucose.

In the claimed method, almost complete hydrolysis of proteins and peptides to the formation of amino acids is ensured: within 72 hours with concentrated hydrochloric acid (density 1.097 g / ml, concentration 6.0 mol / l, 19.98% by weight), which is recommended by the RF State Pharmacopoeia ( GF RF, XIV, 2018). A slight excess of concentrated hydrochloric acid in relation to the filtrate (1: 1) is due to the partial loss of acid due to its volatility and the ease of its removal after isolation of the target products.

The ratio of the hydrolyzate of proteins, peptides and activated carbon, as well as the duration of the treatment of the hydrolyzate with coal, was selected experimentally by visual determination of the color of the solutions (tables 3, 4). As can be seen from the data presented, the optimal concentration of activated carbon in relation to the volume of the hydrolyzate is 0.3%, the optimal duration of the treatment of the hydrolyzate with activated carbon is 0.5 hour.

The resulting hydrolyzate was tested for the presence of amino acids in comparison with standard samples (CO) of α-amino acids (at a concentration of 2.5 mol / l) by two-dimensional chromatography in a thin layer of sorbent on Sorbfil PTSKh-P-A plates. For chromatography in the first direction, a solvent system was used: n-butanol - acetic acid - water (4: 1: 5). in the second direction - the system: ethanol 95% - water (95: 5). The detection of adsorption zones was carried out with a 1% alcohol solution of ninhydrin (Yakubke Kh.D .. Eshkait Kh. Amino acids. Peptides. Proteins. Translated from German, edited by Yu.V. Mitin, M., Mir, 1985, 82 p. .). The spots were identified by the mobility coefficients (Rf) and relative mobility coefficients (Rs) (relative to the CO of the corresponding amino acids) (Table 5). The data obtained indicate the presence of 14 α-amino acids in corn distillery hydrolyzate.

The quantitative determination of amino acids (in terms of glutamic acid) in the hydrolyzate of corn stillage was carried out by spectrophotometry by the reaction of interaction with ninhydrin at a wavelength of 566 nm and a layer thickness of 10 mm (Gakkel V.A. Candidate dissertation of pharmaceutical sciences, Moscow, 2008, 21 p.). The calculation of the quantitative content of amino acids was carried out according to the specific absorption index (A ^1% _{1 cm} = 0.673) of the product of the interaction of glutamic acid CO with ninhydrin at 566 (Kaishev A. Sh., 2013). The optical density (A) of the product of the interaction of the hydrolyzate (50 ml) with ninhydrin was 0.639. The quantitative content of amino acids (in terms of glutamic acid) (X,%) in the stillage hydrolyzate is calculated by the formula (2) (Kharitonov Yu.Ya. Analytical chemistry (analytics): in 2 books. M., VS, 2005):

where: V is the volume of the test vinasse hydrolyzate, ml.

Thus, the amino acid content was 1.90% to the liquid phase of vinasse.

Sediments of ballast substances formed as a result of acidic hydrolysis of proteins and peptides did not give positive results for reactions with ninhydrin, a biuret reagent (there were no blue-violet colors of solutions), which indicates the absence of proteins or amino acids in the sediments (Kharitonov Yu.Ya., 2005) ...

Exhaustive methylation of α-amino acids at the nitrogen atom to the formation of betaines (trimethylamino acids) was carried out by the Hoffmann method using methyl iodide in the presence of sodium carbonate (Hauptmann Z., 1979). The method is based on the following reaction equations:

According to the results of the quantitative determination of amino acids, their content in 3600 ml of the liquid phase of vinasse is 68.4 g (1.90%). It is known that in most amino acids the nitrogen content is approximately the same and can be taken equal to 16% (GF RF. XIV, 2018), therefore, 68.4 g of amino acids contain 10.944 g or 0.782 mol of a nitrogen atom. In accordance with the above equations, the molar ratio of nitrogen in the composition of amino acids and methyl iodide is 1: 3. However, an excess of methylating reagent is always required (usually 1: 4), therefore, for 0.782 mol of a nitrogen atom, we consumed 3.128 mol of methyl iodide (or 443.86 g, taking into account its molar mass of 141.9 g / mol). In accordance with the stoichiometric coefficients of the reaction equations, the neutralization of methylation products with the participation of an excess (0.782 mol) of methyl iodide requires 0.391 mol (2: 1) of sodium carbonate (or 41.446 g, taking into account its molar mass of 106 g / mol).

In the claimed method, nitrogen-containing betaines, after hydrochlorination with concentrated hydrochloric acid, are isolated by crystallization from an acidic solution, which ensures their isolation from related substances. Another feature of the method is the crystallization temperature: with a decrease in temperature, crystallization proceeds more actively due to the lower solubility of the target product, therefore, in the claimed method, the crystallization temperature is 0-5 ° C. It is practically established that at a temperature of 0-5 ° C, the crystallization of betaines is completed after 8 hours (Lazurevsky G.V., Terentyeva I.V., Shamshurin A.A. Practical work on the chemistry of natural compounds. 2nd ed., M., VS , 1966, 335 p.).

Identification of target products was carried out by qualitative color, precipitation and thermal reactions (Luttseva T.Yu., Sadchikova N.P., Arzamastsev A.P. Methods of analysis and standardization of medicinal quaternary ammonium bases. Questions of biological, medical and pharmaceutical chemistry, 2002, No. 3.p. 17-20; GF RF, XIV, 2018) (table 6). The presented results indicate that all three target products (precipitates C, D, E) contain a quaternary nitrogen atom (betaine), as well as a chloride ion; neither primary, nor secondary, nor tertiary amino groups were found anywhere.

In addition, the identification of precipitates C and D was carried out by the melting point by an open capillary method (State Foundation of the Russian Federation. XIV, 2018). The melting point of sediment B was 227.5 ± 0.2 ° C (with decomposition), which corresponds to betaine glycine hydrochloride (Lazurevsky G.V., 1966). The melting point of the precipitate G was 134 ± 0.2 ° C, which corresponds to betaine of glutamic acid hydrochloride (Lazuryevsky G.V., 1966).

The analysis of the qualitative composition of precipitation components C, D, D was carried out by HPLC using an unconventional chromatography mode: with dynamically induced phase separation (DIRP) (Saprykin L.V., Serdan A.A., Saprykina L.V. Direct analysis of betaines in biological liquids by HPLC. Internet-journal "Chemical Analysis", 2005). The determination was carried out on an UltiMate 3000 liquid chromatograph (Dionex, USA) equipped with a UV detector (model 115), with columns 250 mm long. 4.6 mm inner diameter filled with unmodified silica gel (Separon SGX, 5 μm). The eluent used was an aqueous acetonitrile solution of potassium dihydrogen phosphate containing 70% acetonitrile in a 0.1 mol / L phosphate buffer solution with pH 6.2; elution was carried out in 250 μl with the study of each fraction on a chromatograph; the value 198 nm was selected as the operating wavelength of UV detection. Aqueous solutions of precipitates C, D, D (0.1 mg / ml) and a model aqueous mixture of working standard samples (RSS) of betaines of amino acid hydrochlorides (0.1 mg / ml each) obtained by methylation and hydrochlorination of CO of amino acids according to described techniques. Data collection and processing was carried out using the system "Chromeleon, version 7" ("Dionex", USA). By comparing the retention times on the HPLC chromatograms of the tested solutions and the model mixture of PCO (Table 7), precipitate B was identified rather reliably by the chromatograph detector as glycine betaine hydrochloride, precipitate G - glutamic acid betaine hydrochloride, precipitate D - a mixture of betaines hydrochlorides of arginine, alanine aspartic acid, tyrosine, histidine, serine, valine, leucine, lysine, threonine.

The quantitative determination of the target products was carried out according to the content of hydrochlorides by the method of direct alkalimetry by titration of solutions obtained by dissolving about 0.20 g (exact weight) of powders of crushed sediments C, D, D in 30 ml of water, 0.1 mol / L sodium hydroxide solution 42-9712-05) with fixation of the pH value on the pH-150MI device (indicator electrode - glass, reference electrode - silver chloride) (solution temperature 23 ± 2 ° С). The volume of the titrant at the equivalence point is determined by the titration curve built in the graphic coordinate system “(ΔpH / ΔV) = f (V + 1 / 2ΔV)” (Kharitonov Yu.Ya., 2005). The curves of potentiometric alkalimetric titration of the three target products are of the same type. The quantitative content of the target products (X,%) is calculated by the formula (3) (Kharitonov Yu.Ya., 2005):

where a is a sample of a substance for analysis, g;

V is the volume of the titrant at the equivalence point, ml;

K - correction factor to the concentration of the titrant solution;

T is the titer of the titrant for the substance to be determined, mg / ml;

P is the total mass of the analyzed substance.

The T value for sediment B is 15.36 mg / ml (based on glycine betaine hydrochloride), for sediment G - 22.56 mg / ml (based on glutamic acid betaine hydrochloride), for sediment D - 25.27 mg / ml (based on the total nitrogen-containing betaines hydrochlorides in terms of arginine betaine hydrochloride).

Experimental data for the quantitative determination of target products by the method of alkalimetry with potentiometric fixation of the equivalence point are shown in Table 8. The results obtained indicate the satisfactory quality of the target products, as pharmaceutical substances, according to the "Quantification" indicator: equally high values of average results and deviations from average results no more than ± 5% (GF RF, XIV, 2018).

To provide a digestive, enzymatic action, the optimal content of pepsin in one tablet is a dose of 50 mg, i.e. the mass ratio of betaines and pepsin should be 4: 1 (200 mg: 50 mg) (Mashkovsky M.D., 2016. - 1216 p .; Kryzhanovsky S.A., Vititnova M.B.Modern medicines: More than 10,000 names. - M: Ripol-Kl., 2016 .-- 640 p.). Therefore, in the proposed method in the specified ratio, the obtained precipitates of betaines (precipitates C, D, D) and pepsin (hereinafter "pharmaceutical substance") are mixed.

The assessment of the pharmaceutical and technological properties of the obtained pharmaceutical substance to determine the possibility of obtaining tablets (known dosage forms of betaines (acidin)) was carried out according to the following indicators and methods (State Foundation of the Russian Federation, XIV, 2018; Industrial technology of drugs.Edited by V.I. Chueshov, Kharkov : NFAU, 2002, v. 2, 716 p.):

- organoleptic properties.

- the flowability of the powder (the ability to pour out under its own gravity and evenly fill the matrix channel of the tabletting machine) - the time (sec) for 100 g of powder to pass through the outlet 15 mm in diameter of the "VP-12A" vibration device (average value from 3 determinations):

- angle of repose of the powder (in angular degrees) - a three-dimensional angle relative to the horizontal surface, formed by a cone-shaped pyramid of bulk material, estimated on a vibration device "VP-12A" using a protractor (average value from 3 determinations);

- powder compressibility - the ability of particles to cohesion under pressure, determined by evaluating 10 tablets obtained on a rotary tablet machine "RTM-12" in a matrix with a punch diameter of 13 mm at a pressure of 120 MPa, according to the average compression ratio (the ratio of the tablet mass to its height , g / mm) and average crushing strength of tablets (Newtons. N) in the Erveka device;

- bulk (bulk) density (g / cm ³ ) - mass per unit volume of powder, determined on the device "545R-AK-3" (average value from 3 determinations);

- pressure (force) of expulsion (MPa) of the obtained tablets from the matrix was determined according to the readings of the press pressure gauge on a rotary tablet machine "RTM-12" in a matrix with a punch diameter of 9 mm at a pressure of 120 MPa (average value from 3 determinations).

The average results obtained (table 9) indicate unsatisfactory pharmaceutical and technological indicators of the pharmaceutical substance, which is complemented by the hygroscopicity and caking of the substance, the low crushing strength of the tablets. The presented data necessitated the selection of excipients capable of optimizing the pharmaceutical and technological parameters of the substance.

In order to correct the technological parameters of a pharmaceutical substance, labile to external factors (moisture, high temperature, light (ND 42-9712-05)). for the manufacture of tablets, dry rather than wet granulation is preferred. In this regard, the use in the known method (registration certificate LS001355241011) of the low molecular weight excipient povidone-K25 is not acceptable, since it is used for wet granulation, not providing the proper tablet strength during dry granulation (Egoshina Yu.A., Potselueva L.A. Modern excipients in tablet production. Successes of modern natural science, 2009, No. 10, pp. 30-33). Disintegrants (disintegrants) are used to improve the disintegration of tablets in the gastrointestinal tract and the release of drugs with the necessary therapeutic effect. Such a disintegrant in the known tablets "Acidin-Pepsin" is a low molecular weight polymer - povidone K-25, which does not provide satisfactory disintegration of the tablets: the tablets already spread in air, and upon contact with water they disintegrate within 5 seconds. Therefore, we have selected the optimal disintegrant that ensures satisfactory disintegration of tablets in water at a temperature of 37 ± 2 ° C (according to the Russian Federation State Pharmacopoeia, XIV, 2018: within 15 minutes) (table 10, average values from 3 determinations). The obtained data testify to the provision of satisfactory disintegration of tablets (within 15 minutes) when povidone K-25, copovidone, and collidone CL are introduced into them as disintegrants. However, the use of the first two disintegrants, in contrast to Kollidon CL, causes a very rapid disintegration of the tablets (during a time insufficient for their entry into the gastrointestinal tract) and the spreading of the tablets in the air, therefore, Kollidon CL was chosen as the optimal disintegrant.

To ensure good loosening properties, the recommended amount of Kollidone CL is 2-5% of the tablet weight (Egoshina Yu.A., 2009), the experimentally selected dose of Kollidone CL was 5% of the average tablet weight (Table 11, average values from 3 determinations). The data obtained show that in the studied range of the mass fraction of Kollidone CL from the tablet weight, the optimal disintegration time of the tablets in water is observed, however, only at a disintegrant concentration of 5% is satisfactory (71-72 N) tablet crushing strength provided.

Another auxiliary substance in the well-known tablets "Acidin-pepsin" is sorbitol - a diluent, a sliding substance (Yegoshina Yu.A., 2009). It is known that diluents are used in the case of small amounts of active ingredients to ensure the required tablet weight (Grossman V.A. Pharmaceutical technology. M .: GEOTAR-Media, 2014, 320 p.), Which does not apply to known tablets, where the content of active ingredients (betaine and pepsin) high: 200.5 mg, in the proposed tablets even more - 250.0 mg. On the other hand, the composition of the known tablets "Acidin-Pepsin" contains aerosil, which acts as a sliding substance, therefore it is necessary to introduce another sliding substance - sorbitol, moreover, in a high concentration (31.8 mg or 12.62% by weight tablets) with a relatively high proportion of excipients (20.4% by weight of the tablet), is impractical. In this regard, in the proposed method sorbitol is excluded from the composition of the tablets and the content of aerosil is increased until the optimum pressure for pushing the tablets out of the matrix of the tablet machine is achieved (table 12, average values from three determinations). The data presented indicate that at the maximum permissible concentration of aerosil in the composition of tablets of 10% (no more than 10% according to the Russian Federation State Fund. XIV. 2018), the sliding of the tableted mass becomes optimal: the minimum pressure of ejection of the tablets from the matrix is achieved. In this case, the proportion of glidants decreases from 14.68% to the weight of the tablet (2.06% Aerosil and 12.62% sorbitol) in the known tablets to 10% in the proposed tablets.

The content of calcium stearate in the proposed tablets (1% by weight of the tablet) is similar to the known tablets.

To moisten a granulate prepared from a pharmaceutical substance capable of forming a sticky, flowing, poorly granulated mass with water, ethyl alcohol 96% is usually used (Egoshina Yu.A., 2009). in this connection, this particular humidifier is used in the proposed method.

Thus, the comparative composition of tablets in the known and proposed methods differs in the content of both active and auxiliary substances (table 13). The content of active substances in the proposed tablets was 83.4%, in the known tablets 79.6%, the content of excipients in the proposed tablets was 16.6%, in the known tablets 20.4%.

Pharmaceutical and technological characteristics of the proposed tablets are shown in Table 14. The obtained data fully meet the requirements of the RF GF (RF GF XIV, 2018) for uncoated tablets.

Quality control of the claimed tablets without a shell with a diameter of 9 mm, a height of 3 mm, an average weight of one tablet of 300 g, was carried out according to the following indicators:

- description (GF RF, XIV, 2018),

- authenticity:

- nitrogen-containing betaines hydrochlorides (Lutseva T.Yu., 2002; GF RF. XIV, 2018).

- pepsin (ND 42-9712-05),

- uniformity of mass of dosage forms (GF RF, XIV, 2018),

- abrasion strength of tablets (GF RF, XIV, 2018),

- disintegration of tablets in water at a temperature of 37 ± 1 ° С (State Pharmaceutical Foundation of the Russian Federation, XIV, 2018),

- dissolution of a tablet in a 0.01 mol / L solution of hydrochloric acid at a temperature of 37 ± 1 ° C (State Pharmaceutical Foundation of the Russian Federation, XIV, 2018),

- excipients (aerosil, calcium stearate) (GF RF. XIV, 2018),

- quantitative determination (nitrogen-containing betaine hydrochlorides, pepsin) (ND 42-9712-05).

The results of testing the quality of the claimed tablets in comparison with the norms are shown in Table 15. In terms of authenticity, physicochemical characteristics and quantitative content, the claimed tablets comply with pharmacopoeial standards.

The stability of the claimed tablets during storage was studied using the accelerated storage method (accelerated aging) (GF RF, XIV, 2018). For this, 3 series of tablets (6 pieces each) were stored in thermostats at temperatures of 40, 50, and 60 ° C, respectively, for 114, 57 and 29 days. The quality parameters of the tablets shown in Table 15 were monitored after 11 (at 40 ° C), 6 (at 50 ° C) and 2.9 (at 60 ° C) days. During the experimental shelf life, no series of tablets deviated from the indicators shown in Table 10. Based on the experimental shelf life, using the mathematical expression of the Van't Hoff rule, the shelf life of the claimed tablets at a temperature of 10 ± 2 ° C was calculated, which was 2 years.

The method of obtaining the claimed drugs based on nitrogen-containing betaines hydrochlorides: pharmaceutical substances and tablets made from the liquid phase of post-alcohol corn stillage and suitable for use as digestive, enzymatic drugs, is illustrated by the following specific example.

Example 1.

For separation into liquid and solid phases, post-alcohol corn stillage (volume 5000 ml) is filtered through a cloth filter and cotton wool, the solid phase is discarded. The liquid phase of vinasse (yellow liquid with a specific yeast odor, volume 3600 ml), containing 3.5% maltose and 14.0% glucose (table 2), is freed from reducing sugars. To do this, first hydrolyze maltose: to the liquid phase with a volume of 3600 ml, containing 126 g (3.5%) of maltose, add 120 ml (126 g) of anhydrous acetic acid (density 1.052 g / ml, concentration 99.8%) (wt. ratio of maltose and acid 1: 1) and heated for 0.5 hour in a boiling water bath in a flask equipped with a reflux water ball condenser.

The total content of reducing sugars in the liquid phase of vinasse is 17.5%, which means that 3600 ml of vinasse contains 630 g of glucose (free and formed after hydrolysis of maltose). Since glucose crystallization requires a double volume (1260 ml) of anhydrous acetic acid (Guben I., 1934), taking into account the already added volume of acid (120 ml), it is necessary to add 1140 ml (1260-120 ml) of acid. Therefore, 1140 ml of cold (5 ° C) anhydrous acetic acid are added to the cooled hydrolyzate, crystallized with stirring (0-5 ° C, 1 day). Glucose crystals are filtered on a Buchner funnel with a glass filter, dried (70 ± 2 ° C); the yield of glucose is 470 g or 74.6% to the content of reducing sugars, 13.1%) to the liquid phase of vinasse. The resulting filtrate is concentrated in a water bath under a water-jet pump vacuum (55 ± 2 ° C) to 1/25 of the original volume, cooled (to 20 ± 2 ° C).

Next, acid hydrolysis of proteins and peptides is carried out. contained in the concentrated filtrate, for which the filtrate (volume 180 ml) in a flask equipped with a reflux water ball condenser is treated with 180 ml of concentrated hydrochloric acid (density 1.097 g / ml, concentration 6.0 mol / l, 19.98 wt% .) in a water bath at a temperature of 110 ± 2 ° С (72 hours). During hydrolysis, ballast sediment precipitates. At the end of hydrolysis, the reaction mixture is slowly cooled (to 20 ± 2 ° C), additionally separating the precipitate of ballast substances, which is filtered on a Buchner funnel with a glass filter. The filtrate is treated with 1 g of activated carbon (55 ± 2 ° C, 0.5 hour), filtered on a Buchner funnel with a glass filter, washing the precipitate with 10 ml of hot water; the filtrate is combined with washing water, the precipitate is discarded.

The α-amino acids contained in the filtrate are subjected to exhaustive methylation at the nitrogen atom a with methyl iodide in the presence of sodium carbonate until betaines are formed by the known Hoffmann method (Hauptman Z., 1979). To this end, 443.860 g of methyl iodide and 41.446 g of sodium carbonate are added to the resulting filtrate, placed in a flask equipped with a reflux water ball condenser. The mixture is heated in a water bath to the boiling point (42 ± 2 ° C) and boiled until the evolution of carbon dioxide stops (30 hours).

To obtain glycine betaine hydrochloride, concentrated hydrochloric acid (1: 1 by volume) is added to the mixture, concentrated in a water bath under a water-jet pump vacuum (55 ± 2 ° C, up to 1/3 of the volume). The mixture is cooled to a temperature of 20 ± 2 ° C, while a copious precipitate of "crude acidin salt" precipitates, which is filtered on a Buchner funnel with a glass filter; the precipitate (precipitate A) on the filter is washed with 100 ml of concentrated hydrochloric acid. The filtrate with washings is concentrated in a water bath under vacuum of a water-jet pump (55 ± 2 ° C, up to 1/3 of the volume), cooled, transferred to a crystallizer (0-5 ° C, 8 hours). This precipitates a glassy precipitate of glycine betaine hydrochloride (precipitate B), which is decanted and squeezed between sheets of filter paper. Both precipitates A and B are combined (glycine betaine hydrochloride, precipitate C).

Next, precipitate B is purified by dissolving in warm water (55 ± 2 ° C, 1: 2), processing a solution of 0.35 g of activated carbon, heating in a flask equipped with a reflux water ball condenser on a water bath (40 ± 2 ° C, 0.5 hour), filtration on a Buchner funnel with a glass filter (the precipitate is discarded). The filtrate (clear, colorless liquid) is concentrated in a water bath under vacuum of a water-jet pump (55 ± 2 ° C, up to 1/3 of the volume), crystallized (0-5 ° C, 8 hours); The precipitate separated from the Buchner funnel is squeezed between sheets of filter paper, dried in air (20 ± 2 ° C) and dried in a vacuum desiccator. The precipitate of purified glycine betaine hydrochloride is a white powder; its yield is 93.6229 g or 2.60% to the liquid phase of vinasse.

The mother liquors formed after the separation and purification of the glycine betaine hydrochloride precipitate are combined and used to isolate glutamic acid betaine hydrochloride For this, the solution is diluted with water (1: 1), clarified with activated carbon (0.3% by weight of the solution, 55 ± 2 ° C , 0.5 hour), cooled (to 40 ± 2 ° С), filtered on a Buchner funnel with a glass filter, the precipitate is discarded, the filtrate is concentrated in a water bath under vacuum of a water-jet pump (55 ± 2 ° С, up to 1/3 volume) , cooled in an ice bath (up to 20 ± 2 ° C), treated with concentrated hydrochloric acid (density 1.097 g / ml, concentration 6.0 mol / l, 19.98 wt%) (by volume 1: 1), crystallized ( 0-5 ° C, 5 days), the formed crystals of glutamic acid betaine hydrochloride are separated on a Buchner funnel with a glass filter. The crystals are purified by dissolving the precipitate in water (1:10), treating the solution with concentrated hydrochloric acid (density 1.097 g / ml, concentration 6.0 mol / L, 19.98 wt%) (by volume 1: 1), filtration on Buchner funnel with glass filter. The precipitate is wrung out between sheets of filter paper, dried in air (20 ± 2 ° C) and dried in a vacuum desiccator; the mass of the precipitate of purified glutamic acid betaine hydrochloride (precipitate D) 82.8289 g or 2.30% to liquid stillage gas. The mother solutions formed after the separation and purification of the precipitate D are combined, evaporated in a water bath under the vacuum of a water-jet pump (55 ± 2 ° C) almost dry, dried in a vacuum desiccator; the resulting residue contains betaines of other amino acids, hydrochlorides (precipitate D); the yield of precipitate D is 28.8863 g or 0.80% to the liquid phase of vinasse.

The quantitative content of betaines hydrochlorides in the pharmaceutical substance: 99.8% (sediment C), 99.6% (sediment D), 99.5% (sediment D).

The obtained purified target products (precipitates C, D, D with a total weight of 205.3381 g) are placed in a mortar, where they are mixed and ground until a homogeneous white powder (pharmaceutical substance) is formed. Next, tablets are made (all numerical data are given per 1 tablet), for which 200.0 mg of the obtained substance is placed in a mortar, 50.0 mg of pepsin GSO ("Meito Sangyo Co., LTD", Japan, quantitative content 100 ± 7.5%), mixed, dried in a vacuum drying oven (40 ± 2 ° C) to a residual moisture content of not more than 3%. To the resulting mixture, for the preparation of dry granulate, add 29.9 mg of Aerosil (antifriction, sliding, substance that improves wettability), 15.0 mg of Kollidone CL (disintegrant, swelling substance), moisten with 1.0-1.5 ml of ethyl alcohol 96%, mix. The granulate is rubbed through a No. 15 sieve (hole diameter 1.5 mm), the granules are "powdered" with 3.0 mg of calcium stearate (lubricant), pressed on a rotary tablet machine "RTM-12" in a matrix with a punch diameter of 9 mm at a pressure of 120 MPa. The tablets are packed in boxes with lids with granular silica gel (desiccant). The target product is round tablets, with biconvex surfaces, solid edges, without risks and chamfers, with a smooth and uniform surface, 9 mm in diameter, 3 mm in height, uniformly white, with a specific (acidic) odor. The composition of one tablet with an average weight of 300.0 mg: betaine hydrochlorides 200.0 mg, pepsin 50.0 mg, aerosil 29.9 mg, collidone CL 15.0 mg, calcium stearate 3.0 mg.

Pharmaceutical technological characteristics of tablets

Indicators of granulate: powder flowability 11.5 g / s, angle of repose 28 °, bulk density 0.95 g / cm ³ . Indicators of tablets: compression ratio 0.1 g / mm, crushing strength of tablets 71 N, pressure of extrusion of tablets from the matrix 11.1 MPa.

The average weight of one tablet out of 20 tablets tested was in the range of 296.4 mg-303.6 mg; the deviation of the weight of each tablet from the average weight of 20 tablets did not exceed ± 1.2%. Loss in weight of 10 tablets during abrasion was ± 0.7% of the initial weight of the tablets (no more than 3%). Within 12 minutes in water at a temperature of 37 ° C, all 6 test tablets disintegrated. When tested according to the "dissolution" test of one tablet in the "Rotating basket" device at a basket rotation speed of 100 rpm in a 0.01 mol / L hydrochloric acid solution (pH 2.0 ± .01) with a temperature of 37 ± 1 ° С and a volume of 0.9 l for 45 min by the method of alkalimetry with potentiometric indication of the equivalence point, 199.6 mg (99.8%) of betaines hydrochlorides were found. The quantitative content of excipients aerosil and calcium stearate, determined by the method of gravimetry by the mass of ash (GF RF, XIV, 2018), was 9.9% and 1.0%), respectively. The quantitative content of betaines hydrochlorides, determined by alkalimetry, was 199.6 mg per tablet. The quantitative content of pepsin, determined by biochemical and spectrophotometric methods, was 47.5 mg per tablet.

The shelf life of the claimed tablets, assessed using the accelerated storage method (accelerated aging) in terms of authenticity, physicochemical characteristics and quantitative content of betaines hydrochlorides, pepsin, aerosil, at a temperature of 10 ± 2 ° C was 2 years.

Biological tests of the claimed tablets were carried out according to the tests: acute toxicity, hyperacid and proteolytic action in experiments on animals, each group of which included 6 individuals. The test results were processed by the method of multiple statistics (GF RF, XIV, 2018) using the parametric Student's test, determining the arithmetic mean and deviation from it, the probability of differences in the results of the compared groups of animals.

The acute toxicity of the claimed tablets was determined by the Kerber method (Sidorov K.K. Methods for determining the acute toxicity and hazard of chemicals (toxicometry). M., Medicine, 1970, 117 pp.) By single oral administration to mice weighing 20 g tablets in doses (mg of substance / kg of mouse weight): 100, 250, 500, 1000, 5000 in 5 ml of solution. During the observation period (14 days) for the state of the animals, their death was not noted. Autopsy of the animals, 14 days after the introduction of the tablets, no changes in the liver, kidneys, spleen were found. The results of studying the acute toxicity of the claimed tablets (table 16) indicate that even the maximum administered dose (5000 mg / kg) did not cause toxicity, therefore it was not possible to calculate the LD ₅₀ , obviously, LD ₅₀ > 5000 mg / kg. In this regard, according to the classification of toxic substances (Sidorov K.K., 1970), the claimed tablets are classified as practically non-toxic substances.

The hyperacidal effect of the claimed tablets was assessed by the content of acidic components of gastric juice in Wistar rats weighing 180-220 g. Three groups of animals were used, 6 animals in each group, on a stationary diet: group No. 1 - intact animals that received isotonic solution (control ); group No. 2 - animals that received the known tablets "Acidin-Pepsin" (reference drug); group No. 3 - animals that received the claimed tablets. The dose of administration of the claimed tablets to animals was calculated taking into account the dose of the known tablets "Acidin-Pepsin": 4 tablets or 1600 mg of acidin per day (Instruction, Order No. 843), which is 23 mg / kg of adult weight. In terms of the weight of an adult by the weight of an animal (coefficient 5.9). daily doses of betaines and pepsin are 23–5.9 = 136 mg and 5.75–5.9 = 34 mg per 1 kg of animal weight, respectively, or 170 mg of substance per 1 kg of animal weight. The calculated dose of the substance corresponds to 0.68 of the claimed tablet. Thus, for 7 days, the animals were daily orally (after meals) injected with the claimed tablets in a daily dose of 0.68 tablets or 170 mg of the substance per 1 kg of animal weight (in four doses). On the next day after the end of the experiment, the animals were sacrificed, the stomach was removed, opened along the lesser curvature, the stomach contents were taken, in which the concentration of hydrochloric acid (free, total, associated with proteins) and total acidity were determined by the method of alkalimetry (Stroyev E.A., Makarova VG Workshop on biological chemistry. M., VSh. 1986, 231 p.). The determination of acidic components of gastric juice is based on their titration with sodium hydroxide solution using two indicators: p-dimethylaminoazobenzene (pH 2.3-4.2) and phenolphthalein (pH 8.2-10.0). Free hydrochloric acid was determined by changing the color of p-dimethylaminoazobenzene (from red to orange); by the color transition of phenolphthalein (from colorless to pink) - the total acidity of gastric juice.

Assessment of the proteolytic function of the stomach by the content of proteins was carried out by the method of photometry by reaction with a biuret reagent (Stroyev E.A., 1986).

The results of studying the effect of the claimed tablets on the content of acidic components, the total acidity of gastric juice and the proteolytic function of the stomach of rats are shown in Table 17. According to the data obtained, the claimed tablets, like the known tablets "Acidin-Pepsin", in comparison with the control, do not cause changes in concentration free hydrochloric acid in the stomach of rats, but significantly increase the content of total hydrochloric acid (1.94 times), hydrochloric acid associated with proteins (2.4 times), total acidity (1.90 times). The comparison drug acts in a similar way: an increase in indicators by 2.10 times, 2.62 times, 2.00 times, respectively, in relation to the control. A significant decrease in the concentration of proteins in the stomach, both of the claimed and known tablets, was established in comparison with the control: by 1.22 and 1.31 times, respectively. There are no significant differences between the claimed and known tablets, both in hyperacid and proteolytic activity (P ₂ > 0.05). Thus, the claimed tablets are not inferior to the comparison drug in terms of hyperacid and proteolytic action, increasing the content of all acid components, except for free hydrochloric acid, and reducing the concentration of proteins in the stomach.

Thus, the proposed method for producing medicines - the substance of nitrogen-containing betaines hydrochlorides and tablets containing betaine hydrochlorides and pepsin as active components - provides the following positive effect:

1. Obtaining 12 nitrogen-containing betaines of hydrochlorides (glycine, glutamic acid, arginine, alanine, aspartic acid, tyrosine, histidine, serine, valine, leucine, lysine, threonine) due to the high concentration of amino acids (28.8- 28.9%).

2. Increase in the technological yield of the target product: 5.70% to the liquid fraction (in the claimed method) versus 3.3% to the liquid fraction (in the method taken as a prototype). This is due to the optimization of the technological process: almost complete acid hydrolysis of proteins and peptides (72 hours in the claimed method), semisynthesis of target products by methylation and hydrochlorination of amino acids, “sparing” temperature regimes that prevent the volatilization of hydrochlorides, excluding the stage of treatment with ethyl alcohol, leading to loss of proteins , peptides, amino acids.

3. High degree of purity of the obtained target product, confirmed by the quantitative content (99.5-99.8%), which is due to the stage of removing carbohydrates and optimization of technological conditions.

4. Optimal for the provision of hyperacid and enzymatic (digestive) action, the mass ratio of betaines and pepsin (200 mg: 50 mg, 4: 1), recommended by pharmacologists, in contrast to the known substance (200 mg: 0.5 mg, 400: 1) ...

5. Introduction of pepsin with pharmacopoeial quality standards (pharmacopoeial article FS 42-0077-01), in particular, with a quantitative content of 100 ± 7.5% in the composition of a pharmaceutical substance.

6. Optimization of pharmaceutical and technological properties of granules and tablets:

- for granules: decrease in flowability (11.5 sec), angle of repose (28.0 °), bulk (bulk) density (0.95 g / cm ³ );

- for tablets: an increase in the compressibility factor (0.10 g / mm), the crushing strength of the tablets (71 N), a decrease in the pressure (force) of the tablet pushing out (11.1 MPa).

The specified optimal properties are achieved by correcting the tablet technology and the composition of the excipients: the use of dry granulation; replacing the low molecular weight disintegrant povidone K-25 (known drug) with the high molecular weight disintegrant Kollidone CL (claimed drug); reducing the proportion of glidants (12.62% sorbitol and 2.06% aerosil in known tablets) to 10.0% aerosil (in the claimed tablets); reducing the proportion of excipients from 16.6% (claimed drug) to 20.4% (known drug).

7. Use of technological equipment available for any pharmaceutical production (dryers, rotary tablet machine, etc.), auxiliary substances.

8. Compliance of the claimed tablets with pharmacopoeial standards in all respects: description, authenticity, physicochemical characteristics (mass homogeneity, abrasion strength, disintegration in water, dissolution), quantitative content of active and auxiliary substances.

9. Providing an accurate dose of drugs when using tablets, in contrast to powder, the use of which is associated with significant fluctuations in the concentration of active substances.

10. Optimal consumer properties of the claimed tablets: no smell, readiness for use.

11. Increasing the shelf life of tablets at a storage temperature of 10 ° C: from 1.5 years to 2 years.

12. The claimed tablets are practically non-toxic: they have a pronounced hyperacid effect (increase the content of total hydrochloric acid by 1.94 times, hydrochloric acid associated with proteins by 2.4 times, total acidity by 1.90 times), pronounced proteolytic effect (reduce the concentration proteins in the stomach by 1.22 times). In terms of specific activity, the claimed tablets are at the level of known tablets "Acidin-pepsin".

Claims (4)

1. A method of obtaining a pharmaceutical substance containing nitrogen-containing betaines hydrochlorides, which consists in the fact that from the liquid phase separated from the solid phase of post-alcohol corn stillage by filtration, reducing sugars are removed after preliminary hydrolysis of maltose (anhydrous acetic acid, wt. Ratio of maltose and acid 1: 1, 100 ° С, 0.5 h) by crystallization (0-5 ° С, 1 day), filtration and drying (70 ° С) of glucose, concentration (55 ° С, up to 1/25 volume) and cooling (up to 20 ° C) of the filtrate, proteins and peptides contained in the filtrate are hydrolyzed with concentrated hydrochloric acid (density 1.097 g / ml, volumetric ratio of filtrate to acid 1: 1, 110 ° C, 72 h), cooled (to 20 ° C), ballast substances, treat the filtrate with activated carbon (0.3% by volume of the mixture, 55 ° C, 0.5 h), filter the precipitate after washing, the amino acids contained in the filtrate are exhaustively methylated with methyl iodide (the molar ratio of nitrogen in the composition amino acids and methyl iodide 1: 4) in the presence of sodium carbonate (molar ratio of nitrogen in the composition of amino acids and sodium carbonate 2: 1) (42 ° C, 30 h), the resulting mixture is treated with concentrated hydrochloric acid (volume ratio of the mixture and acid 1: 1 ), concentrated (55 ° С, up to 1/3 of the volume), cooled (up to 20 ° С), after washing with the same acid (volume ratio of precipitate A and acid 1:10), precipitate A is filtered, the filtrate is concentrated (55 ° С, up to 1/3 of the volume), crystallize (0-5 ° C, 8 h), separate precipitate B, the combined (A and B) precipitate C is dissolved in water (55 ° C, 1: 2), purified with activated carbon (0, 3% by weight of the solution, 40 ° C, 0.5 h), filtered, the filtrate is concentrated (55 ° C, up to 1/3 volume), isolated from it by crystallization (0-5 ° C, 8 h), filtration and drying (20 ° С) purified precipitate B, the filtrates formed after isolation and purification of precipitate B, diluted with water (1: 1), treated with activated carbon (0.3% by weight of the solution, 55 ° C, 0.5 h), cooled (up to 40 ° C), filter They are concentrated, concentrated (55 ° С, up to 1/3 of the volume), cooled (up to 20 ° С), treated with concentrated hydrochloric acid (volumetric ratio of filtrate and acid 1: 1), crystallized (0-5 ° С, 5 days), crystals (precipitate D) are filtered, reprecipitated from an aqueous solution (1:10) with concentrated hydrochloric acid (volume ratio of solution to acid 1: 1), filtered and dried (20 ° C) purified precipitate D, filtrates formed after isolation and purification of the precipitate D, evaporated (55 ° C) and dried in a vacuum desiccator to dry residue (precipitate E), precipitates C, D, E are combined and mixed with pharmacopoeial pepsin (mass ratio 4: 1). 2. The pharmaceutical substance of the digestive (enzymatic, hyperacid) action, obtained by the method according to claim 1, is a homogeneous crystalline powder of white color with an acidic odor, with the composition of nitrogen-containing betaines hydrochlorides: glycine (precipitate B, 36.5 wt.% To the substance) , glutamic acid (sediment G, 32.3 wt.% to the substance), arginine, alanine, aspartic acid, tyrosine, histidine, serine, valine, leucine, lysine, threonine (sediment D, 11.2 wt.% to the substance) and pharmacopoeial pepsin (20.0 wt.% to the substance), with a quantitative content of betaines hydrochlorides: 99.8% (sediment C), 99.6% (sediment D), 99.5% (sediment D), pepsin 100 ± 7.5%. 3. A method of obtaining a dosage form (tablet), which consists in the fact that the pharmaceutical substance obtained by the method according to claim 1, weighing 250.0 mg (all data per 1 tablet) is dried in a vacuum drying oven (40 ° C) to a residual humidity not more than 3%, mixed in a mortar with 29.9 mg of aerosil sliding substance, 15.0 mg of baking powder Kollidon CL, moistened with 1.0-1.5 ml of ethyl alcohol 96%, rubbed, rubbed through a sieve No. 15 with a hole diameter 1.5 mm, 3.0 mg of a lubricant calcium stearate is "powdered", the obtained dry granulate with a flowability of 11.5 g / s, an angle of repose of 28 °, a bulk density of 0.95 g / cm ³ is compressed to obtain tablets with a coefficient compressibility 0.1 g / mm, crushing strength 71 N, ejection pressure from the matrix 11.1 MPa. 4. Dosage form of digestive (enzymatic, hyperacid) action, obtained by the method according to claim 3, which is a tablet for oral administration weighing 296.4-303.6 mg, 9 mm in diameter, 3 mm in height, round, with biconvex surfaces, solid edges, without risks and chamfers, with a smooth and uniform surface, white, with an acidic odor, with loss in weight by abrasion 10 tablets ± 0.7%, with disintegration of 6 tablets in water at 37 ° C for 12 minutes , with the release of 199.6 mg or 99.8% of nitrogen-containing betaines hydrochlorides when 1 tablet is dissolved in a 0.01 mol / L hydrochloric acid solution (pH 2.0 ± 0.1) with a temperature of 37 ° C and a volume of 0.9 L for 45 minutes at a basket rotation speed of 100 rpm, with the composition of biologically active compounds in 1 tablet: 200.0 mg of nitrogen-containing betaines hydrochlorides, 50.0 mg of pepsin, with the content of excipients in 1 tablet: 29.9 mg of Aerosil, 15.0 mg Kollidone CL, 3.0 mg calcium stearate, with a shelf life of tablets at a storage temperature of 10 ° C for 2 years.