Improved method of purification of pravastatin
SUBSTANCE: purification of pravastatin is carried out from stereoisomer 6-epipravastatin. The culture liquid is centrifuged with a content of 6-epipravastatin 7% by weight of pravastatin for separation of mycelium. The native solution is prepared having pH 6.6. Purification of the native solution is carried out by filtration through a layer of basic alumina with pH 10, having activity corresponding to 8% of moisture. Alumina is taken in an amount of 25:1 relative to the amount of pravastatin in the native solution. Pravastatin is extracted by the organic solvent. Final purification of pravastatin is carried out through obtaining of intermediate ammonium salt using 25% aqueous ammonia solution and subsequent conversion to its sodium salt.
EFFECT: invention enables to carry out purification of pravastatin according to the pharmaceutical requirements to quality.
2 cl, 2 ex
The invention relates to chemical-pharmaceutical industry, namely the processes of biosynthesis, and relates to a method of purification of the drug pravastatin.
Pravastatin has expressed antihyperlipidemic effect: it reduces the level of cholesterol low density lipoprotein, in varying degrees increases the level of high density lipoprotein. Used in hyperlipidemia without coronary heart disease (risk reduction of myocardial infarction, atherosclerosis and ischemic heart disease, including myocardial infarction (to slow the progression of atherosclerosis and reduce the likelihood of a second heart attack).
Pravastatin-(3R,5R) - for 3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-2-methyl-8-[[(2S)-2-methylbutanoyl]-oxy]-1,2,6,7,8,8A-hexahydronaphthalen-yl]-heptane acid (sodium salt)
Pravastatin. Gross formula C23H36O7(Na) is the biosynthetic representative of the first generation statins, has become firmly rooted in medical practice.
1. Pravastatin is produced by bioconversion of compactin, first isolated in 1976 by a group of Japanese researchers. (Endo A., Kuroda, M. and Y. Tsujita ML - 236B, and ML - 236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. // J. Antiiot. - 1976. - 29. - P.1346-1348, Endo a, Kuroda m, Terahara, A., Tsujita Y., Tamura C. Physiologically active substances and fermentative process for the same. // United States Patent No. 4,049,495; September, 20, 1977.
Pravastatin get the two stages:
first - biosynthesis compactin using the fungus Penicillium citrinum (CAN 2185598, KP 20040052335 A).
second - microbiological hydroxylation sodium salt compactin using Streptomyces carbophilus) (EN 2235780 C2) (Scheme 2).
Describes several methods for the isolation and purification of pravastatin.
The most common of them is the extraction of pravastatin in the organic solvent from the filtrate of the culture fluid and its subsequent re-extraction into water. This operation is usually repeated several times to remove most of the impurities. Then the organic solvent is evaporated in vacuum, and technical pravastatin cleaned using the method of column chromatography on silica gel, eluate evaporated in vacuo, and the obtained product is recrystallized several times from a mixture of organic solvents (for example, US 4.346227, EN 2265665 C2, WO 2009/121870).
The second popular method of allocation is based on the use of information that pravastatin is culturally fluid in the two equilibrium forms - acid and lactone.
After the bioconversion pravastatin extracted from the culture fluid or filtrate obtained after removal of the mycelium.
The mycelium can be removed from the culture fluid by filtration or centrifugation. Before extraction of the culture liquid is or its filtrate is acidified with mineral acid to a pH of 3.5-3.7, then the extraction is carried out pravastatin esters of acetic acid. The extract is dried, and complete the formation of the lactone by adding to the extract of catalytic amounts of triperoxonane acid. After completion of the process of formation of the lactone sequentially washed with the reaction mass 5% aqueous sodium bicarbonate solution and then water, dried and evaporated in vacuum to dryness. Received a technical product (pravastatin lactone) is cleaned by the method of column chromatography on silica gel, eluate combined and evaporated in vacuum. The pravastatin lactone is dissolved in acetone and hydrolyzing with an equivalent amount of sodium hydroxide. After completion of the process of formation of the sodium salt of pravastatin planted her from a solution with excess acetone, filtered off, dried and recrystallized from a mixture of organic solvents (for example, US 4.346227, EN 2235780).
Great interest is also the method of isolation and purification of pravastatin through an intermediate product of its salt with secondary amines containing alkyl, cycloalkyl, arylalkyl - substituents. For this purpose non-toxic amines, such as dioctylamine, dicyclohexylamine, dibenzylamine and other
After the bioconversion pravastatin extracted from the culture fluid or filtrate organic dissolve elem, then add the amine in the amount of 1.5 mole per mole of pravastatin contained in the extract, and concentrating the extract under vacuum to a volume of approximately 5% from the original. To the concentrate is added an additional amount of amine (0,2 mol), forming a crystalline salt of pravastatin, which is filtered off and purified (recrystallization, the clarification of its solutions activated carbon and others). The purified amine salt of pravastatin transform in pravastatin sodium salt by treatment with sodium hydroxide or sodium alkoxide, preferably by ethoxide sodium(US 6.444452, WO 2009/068556).
One of the variants of this method is the use of gaseous ammonia to obtain the ammonium salt of pravastatin (US 6.444452).
From a review of the prior art shows that the biosynthesis of pravastatin flows is ambiguous. The result is a large number of by-products (6-approvalstatus, 3-and-pravastatin etc), which seacadets together with the target product. Receiving pravastatin, suitable for use as a medicine, calls for a multi-step cleaning, which inevitably entails a significant loss of yield of the target compounds (up to 30-40%), depending on the selected method, and also increases the time pravastatin is m, which is highly undesirable, given its high reaktsionnosposobnykh.
The most difficult problem of purification of pravastatin from impurities is the separation of pravastatin from its stereoisomer of 6-epiprawastatina, the content of which is strictly regulated by the international quality requirements of the drug. (European Pharmacopoeia No. 7 is not more than 0.3%). Biosynthesis and compactin (predecessor of pravastatin and pravastatin is not stereoselective and therefore in the culture fluid may contain from 4 to 12% of 6-epiprawastatina.
Known methods of obtaining and purification of pravastatin from its stereoisomer (US 7425644 B2).
Two options are described:
1. Cleaning compactin from apicoectomy and its subsequent use for the biosynthesis of pravastatin.
2. Isolation and purification of the sodium salt of pravastatin.
Cleaning compactin performed using chromatography or by recrystallization and then use the purified product in the biosynthesis of pravastatin. The result is a product of high purity from impurity 6-epiprawastatina.
Sodium salt of pravastatin is also purified using chromatographic methods, thus obtaining a high-purity product.
The described method is the closest to the proposed invention.
However, in the prototype, unfortunately, naukasana other indicators of the quality of the final product (the content of the basic substance, the amount of impurities and so on), without which the substance cannot be considered to be pharmaceutically pure. Not specified the output in relation to the number of pravastatin in the culture fluid, how many episoder contains source compactin prior to extraction from the culture fluid. The authors describe the cleanup has already been allocated and, apparently, a purified or partially purified preparation - i.e. its purification. In addition, the use of chromatographic methods of cleaning is undesirable for industrial methods. When creating inventions following objectives were set:
- to create an industrial purification method of the drug pravastatin;
- to improve the quality of the target product while maintaining high output;
to facilitate the process.
This goal is achieved by:
- filtering the native solution of pravastatin through the layer of aluminum oxide that allows you not only to remove the remnants of ballast substances that interfere with the extraction, but also, unexpectedly, almost completely freed from impurities 6-epiprawastatina. Filtration through a layer of aluminum oxide allows not only clean pravastatin, but also significantly reduce the time to process, and thereby reduce the likelihood of accumulation of the degradation products of the target compounds under the action of light and atmospheric oxygen. In addition to t the th, this technique is much simpler in practice than, for example, column chromatography.
by using when getting the target of connection of the intermediate product ammonium salts, obtained by using 25% ammonia solution, with further transfer of its sodium salt.
To achieve this goal we have studied in detail the working conditions with pravastatin at each stage of purification. Experimental by the pH values of solutions which can minimize the appearance of the degradation products.
At the first stage of the treatment is extraction of pravastatin from native solution (formed after centrifugation of the culture liquid) organic solvent, the process remains difficult proteins in the filtrate. For exemption from them, before carrying out the extraction process, you can use the ultrafiltration, but we decided to simplify and cheapen the process by filtering a native solution through a layer of aluminum oxide. Used for this purpose aluminum oxide basic (pH 10), having an activity corresponding to 8% moisture. The result was not only to remove the remnants of ballast substances, preventing extraction, but, unexpectedly, almost completely free from impurities 6-epiprawastatina. Moreover, it should be noted that the use is of any other type oxide with another activity has the opposite effect, i.e. it can even lead to the increase in the number of 6-epiprawastatina.
To obtain the substance pharmaceutical quality we experimentally tested a number of methods of purification of pravastatin from inorganic salts and concluded that the best results are obtained by using the method of purification through ammonium salt, with subsequent translation into sodium salt. The method allows the most complete free from by-products of bioconversion and metabolicheskikh the products contained in the culture fluid and switched to native solution. We spent clearing pravastatin through ammonium salt, which was obtained using an aqueous solution of ammonia (25%), which allowed to considerably reduce the cost of the process and at the same time to simplify, as using ammonia solution, we eliminate the need to clean the product from the inorganic salts. After transfer of ammonium salts in sodium we consistently received target pharmaceutical product quality with high yield.
The proposed method is as follows.
1. Separating the mycelium from the culture fluid by centrifugation to obtain native solution (the content of 6-epiprawastatina - 7% by HPLC).
2. The native filtering the solution through a layer of aluminum oxide (contents 6-epiprawastatina (0,01%).
3. Extraction of pravastatin is islote from native solution.
4. Receipt and allocation of the ammonium salt of pravastatin.
5. Translation ammonium salt of sodium with a preliminary bleaching solution ammonium salt of pravastatin.
The invention is illustrated by the following examples.
500 ml of the culture fluid with a concentration of pravastatin 7,4 g/l and a content of 6-epiprawastatina 7% by weight pravastatin centrifuged to separate the mycelium and receive native solution. Then native solution having a pH of 6.6, filtered through a layer of aluminum oxide (92,5 g of basic aluminum oxide with a pH of 10 and a humidity 8%) and get a native solution containing 0.01% 6 epiprawastatina.
Received native solution is acidified to pH of 3.5-3.7 with 5% hydrochloric acid, then poured 200 ml ethyl acetate and extracted with pravastatin acid. The extract was washed with 60 ml of water, dried with anhydrous sodium sulfate and evaporated in vacuo to a volume of 50 ml To the obtained concentrate, add 3 ml of 25% aqueous ammonia solution, thoroughly mixed and divided layers. First reextract containing the ammonium salt of pravastatin, is separated, by remaining in a separating funnel to ethyl acetate is added 2 ml of water containing 0.05 ml of ammonia solution (pH 9-10), stirred for 10 minutes, the layers are separated and the lower aqueous layer (second reextract) attached to the first. To the United reex is rectum pravastatin ammonium salt with a volume of 5-6 ml gradually, within 10 minutes, with stirring, add 60 ml of acetone, the resulting suspension is cooled to 10-15°C and continue stirring for 2 hours. The crystalline precipitate is filtered off, washed on the filter with cold acetone, dried, and get 2,98 g of pravastatin ammonium salt in the form of a white to yellow crystalline powder. The basic substance content of 99.2% (norm-not less than 97%),the content of 6-epiprawastatina 0.01% (a rate of not more than 0.3%), the amount of impurities 0.5% (norm, not more than 0.6%).
2,98 g of pravastatin ammonium salt is dissolved in 35 ml of 70% aqueous isopropyl alcohol solution add 0.05 ml of 25% ammonia and 1.5 g of activated carbon and stirred for 30 minutes. The charcoal is filtered off, washed with 70% isopropanol. Isopropanolic the solution is evaporated in vacuum to dryness, the resulting residue is dissolved in 6 ml of a solution of 0.14 g of sodium hydroxide and add with stirring 50 ml of acetone. Stirring is continued for 30 minutes, then the precipitate is filtered off and dried, yielding 2.8 g of pravastatin sodium salt in the form of white crystalline powder. The basic substance content of 99.3%, (norm-not less than 97%), the content of 6-epiprawastatina 0.01%) (rate of not more than 0.3%), the amount of impurities of 0.4% (norm, not more than 0.6%).
500 ml of the culture fluid with a concentration of pravastatin 7,4 g/l and a content of 6-epiprawastatina 7% by weight of the PRA is of astatine centrifuged to separate the mycelium and receive native solution. Then native solution having a pH of 6.6, filtered through a layer of aluminum oxide (111 g of basic aluminum oxide with a pH of 10 and a humidity 8%) and obtain a solution containing 0.01% of 6-epiprawastatina.
Next, the process is conducted as described in example 1.
Obtain 2.6 g of sodium salt of pravastatin in the form of white crystalline powder. The content of the basic substance 99.8%, (norm-not less than 97%), the content of 6-epiprawastatina 0.01% (a rate of not more than 0.3%), the amount of impurities 0.35% (normal is less than 0.6%).
1. The method of purification of pravastatin from 6-epiprawastatina, including the production of native solution by centrifugation of the culture liquid, clean, natural solution, extraction of pravastatin organic solvent, the purification of pravastatin through the intermediate ammonium salt with the subsequent transfer of its sodium salt, characterized in that the clearing of native solution lead by filtering through a bed of basic alumina with a pH of 10, having an activity corresponding to 8% moisture content, and the purification of the product is carried out by receiving ammonium salt using 25% aqueous ammonia solution.
2. The method of purification of pravastatin on p. 1, characterized in that the aluminium oxide charge in the amount of 25:1 in relation to the number of pravastatin, which is the native solution.
FIELD: chemistry; biochemistry.
SUBSTANCE: invention pertains to bioengineering. Penicillium citrinum VKM F-4043 D fungus is cultured in a mineral medium of the following composition (g/l): sucrose - 50.0-100, urea - 2.14-4.28, KH2PO4 -1.00-2.00, MgSO4·7H2O - 0.30-0.60, ZnSO4·7H2O -0.0044-0.0088 and distilled water - up to 1l. After separation of the biomass, epoxyagroclavine-1 is separated from the alkaline extract of the culture fluid through extraction with chloroform at acidic and alkaline pH values with subsequent dehydration and concentration. The obtained alkaline extract is dissolved in acetone and while stirring, a solution of tartaric acid in methanol is gradually added. Quinocitrinines are separated from the acidic extract of the culture fluid through column chromatography (with silica gel, ratio of the height to the diametre of the column equal to 1:1.8-2.0), with chloroform first and then with a mixture of chloroform and methanol in ratio 9:1.
EFFECT: method increases content of epoxyagroclavine-1 in the culture fluid, simplification of methods of purifying epoxyagroclavine-1 and quinocitrinines, reduced cost of the medium, longer period of storing epoxyagroclavine-1.
SUBSTANCE: group of inventions relates to biotechnology. Claimed is group of inventions: method of obtaining chemical product and apparatus for obtaining chemical product by claimed method. Microorganisms or culture cells are cultivated in fermentation reservoir. Culture liquid is transferred from fermentation reservoir into reservoir of membrane separation for filtration of culture liquid through separation membrane. Fermentation product is collected from liquid obtained after filtration as chemical product. Reverse discharge of unfiltered culture liquid into fermentation reservoir for combination with culture liquid, which did not pass through membrane separation reservoir, is provided. One part of culture liquid is directed to bypass membrane separation reservoir back into fermentation reservoir. Volume of culture liquid flow is regulated in such a way that manometric pressure of culture liquid from the side of flow outlet into membrane separation reservoir constitutes 1 MPa or less. Apparatus includes fermentation reservoir, membrane separation reservoir, circulation pipeline, which connects fermentation reservoir with membrane separation reservoir, means for transfer of culture liquid, installed into circulation pipeline, bypass pipeline for membrane separation reservoir, means for registration of flow pressure from the side of flow inlet into membrane separation reservoir, means for regulation of flow volume, installed into bypass pipeline.
EFFECT: inventions ensure increased output of end product.
13 cl, 23 dwg, 6 tbl, 11 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: presented group of inventions refers to medicine. What is presented is a method for preventing and treating the diabetes mellitus complications associated with the developing degenerative processes of the nervous tissue, comprising administering a medicine containing a peptide of general formula Pro-Gly-Pro, or a pharmaceutically acceptable salt thereof in effective amounts. There are presented pharmaceutical composition comprising said peptide for the neuroprotective therapy of the complications of diabetes mellitus and the method for preparing it.
EFFECT: group of inventions enables the more effective prevention and treatment of the diabetes mellitus complications associated with the developing degenerative processes of the nervous tissue by the use of the peptide Pro-Gly-Pro, or a pharmaceutically acceptable salt thereof.
9 cl, 2 dwg, 4 tbl, 7 ex
SUBSTANCE: fish roe is homogenised. Fish roe hydrolysis is carried out with a ferment preparation "Collagenase" in presence of an inhibitor for 10-12 hours. Chitosan is added to the produced hydrolysate at the ratio of 0.5-1.0:1.0. Components are mixed.
EFFECT: invention makes it possible to accelerate process of chitosan-nucleic complex production.
1 dwg, 1 tbl, 3 ex
SUBSTANCE: method is proposed to produce a foaming agent. The method includes cultivation of a microorganism in a fermentative medium, the cells of which produce a foaming agent extracellularly. At the same time the fermentative medium contains a defoaming agent, which has opacity temperature. Then the defoaming agent is removed at the temperature of the fermentative medium of at least 10°C higher than the opacity temperature of the defoaming agent.
EFFECT: method makes it possible to simplify removal of a defoaming agent from a fermentative medium.
15 cl, 2 dwg, 6 tbl, 4 ex
FIELD: oil and gas industry.
SUBSTANCE: invention relates to the method to produce oil fuel, in which mixing is carried out and reaction of hydrolysis is done with water containing a ferment, which a hydrocarbon oil product, besides, water containing a ferment, is produced by means of mixing of a natural vegetable ferment, containing, at least lipase, in water. The natural vegetable ferment may additionally contain cellulase. The invention also relates to a device for production of oil fuel.
EFFECT: increased efficiency of fuel, which is stable, and also suppression of hazardous substances formation.
10 cl, 11 dwg, 1 ex
SUBSTANCE: phytoplankton is cultivated in electromagnetic bioaccelerators. Oxygen and biomass, consisting of lipids, hydrocarbons and sugars are obtained. Biomass is oxidised. Thermodynamic energy is obtained. Carbon dioxide and NOx are returned to electromagnetic bioaccelerators.
EFFECT: invention makes it possible to obtain biofuel and reuse CO2 and NOx.
9 cl, 10 dwg
SUBSTANCE: method involves preparation of material for enzymatic hydrolysis. Alkaline hydrolysis is carried out with proteolytic enzyme preparations with neutralisation of the obtained solution to pH=7. A salt is added to the obtained enzymatic hydrolysate to a value of not less than 0.1 mol/l. Successive ultrafiltration is carried out, first on a membrane with maximum retention of 50 kD with separation of high-molecular weight impurities, and then on a membrane with maximum retention of 5 kD with separation of low-molecular weight substances. The chondroitin sulphate solution retained at the membrane is washed on the same membrane with distilled water until complete removal of salts. Final washing with distilled water is carried out on a membrane with maxim retention of 50 kD.
EFFECT: invention enables to obtain a chondroitin sulphate preparation with weight ratio of the basic substance.
SUBSTANCE: disclosed is an enzymatic method of producing electroconductive polymers, specifically polyaniline, polypyrrole, polythiophene and substituted derivatives thereof. The method involves use of a salt of a transition metal complex as an enzymatic reaction accelerator, having redox potential in the range of 0.35-0.95 V. The salt is selected from a group comprising cyanide complexes of molybdenum, osmium, ruthenium, tungsten and iron. The process is carried out at pH 2.5-5.5 and temperature 0-30°C and the oxidant used is molecular oxygen.
EFFECT: method enables to carry out high-speed oxidative polymerisation using low concentration of enzymatic reaction accelerator and an acid dopant.
3 dwg, 12 ex
SUBSTANCE: method involves preparation of plant oil with heating to 80°C, alkaline ethanolysis using potassium hydroxide in ethanol with molar concentration of 2 mol/dm3 to obtain an ether-glycerine mixture which is separated to form two fractions - glycerine and a mixture of ethers. The mixture of ethers (biodiesel fuel) undergoes filtration, sorption purification and dehydration. The obtained biodiesel fuel is stored. Preparation of the plant oil is carried out such that before heating, the plant oil is mixed with 1% aqueous solution of an Ecofriend enzyme-probiotic preparation and the obtained mixture is held for 24 hours at temperature 23-27°C. After holding for 24 hours, the mixture of plant oil and this preparation is heated to said heating temperature. The products are taken in the following ratio, pts.wt: plant oil 5; 1% aqueous solution of Ecofriend enzyme-probiotic preparation 5; potassium hydroxide in ethanol 5.
EFFECT: method increases the amount of the obtained biodiesel fuel.
2 tbl, 3 ex
FIELD: food industry.
SUBSTANCE: fermented food product based on linseed is produced by defatting linseed, its crushing and milling, blending crushed and milled linseed with water, probably with addition of other cereals or plant seeds or linseed fraction at a concentration of nearly 3 - nearly 8 wt % to produce a suspension. The suspension is fermented with a starter, i.e. Bifidobacterium lactis Bb12 strain, enriched and stabilised to produce a viscous or fermented drinkable snack product.
EFFECT: invention allows to produce a product enriched with probiotic bacteria and having low fat content.
15 cl, 15 tbl, 11 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention relates to methods of synthesis and to intermediate products of compounds of formula (XVII) and salts thereof.
EFFECT: preparing the hepatitis C virus protease inhibitor.
32 cl, 13 ex
SUBSTANCE: invention relates to a stereoselective method of producing norbornadiene-based stressed skeleton carbocyclic compounds. The method is characterised by that it involves codimerisation of norbornadiene with acrylic esters. The catalyst used is a bis(allyl)nickel-based single-component system.
EFFECT: use of the present method increases stereoselectivity of the codimerisation reaction and increases product output.
1 cl, 5 ex, 1 tbl