SU624562A3 - Method of producing substance exhibiting hormonal action - Google Patents

Description

Trichloroacetic acid and sulfosalicylic acid are used as precipitating proteins. Proteins can be removed with solvents and salting out.

The separation of proteins, polypeptides and other fractions is carried out and using gel filtration, the substance is obtained with high purity. The biologically active substance concentrate is purified by other methods, for example, chromatography, dialysis, extraction, precipitation, adsorption, or a combination of these methods.

To destroy calcium-active zarahormone, they are treated with substances that easily react with protein substances or with polypeptides. For this purpose, nitrous acid, ethylene oxide, formalin is used.

The content of biologically active substances in preparations may be within wide limits and depends on a number of conditions. All these preparations are prepared by known methods from solid or liquid concentrates or mixtures in the form of tablets, suppositories, solutions, ointments, powder mixtures or formulations for spraying. Tablets with a content of a biologically active substance of 0.05-3 mg are made by wet granulation methods, but dry methods are also used. Inactive substances are used as fillers and auxiliaries.

Injection solutions are obtained from aqueous solutions with a concentration of 0.05-5 mg / ml, with the removal of protein substances. Prepare the injection solution by sterile filtration. Ointments are prepared with a concentration of biologically active substances of 0.01 - 1 g / kg. For this purpose, hydrophilic and hydrophobic ointment bases are used. Candles are also prepared for which cocoa butter is used or, for synthetic ones, carbowax. The concentration of the active ingredient in the candle is 0.05-5 mg, depending on the purity of the concentrate.

Cosmetic preparations are also prepared which, as a biologically active substance, contain a substance having a torus montal action. These cosmetic preparations are made from solid or liquid concentrates of a substance with known solvents, additives, fillers, diluents, and excipients in the form of lubricants, solutions, creams, emulsions; powdery mixtures, spray formulations.

The content of biologically active substance is 0.01-0.5 mg / g.

The resulting substance with a hormonal action m does not affect the level of calcium in the blood serum, but increases the silicon content and changes

concentrations of other trace elements such as C, F and others.

In clinical therapy, the substance has a beneficial effect on the syndrome

Sjogren, on Rhino-laringo-pharingitis sicea, scleroderma, skin fungus, erythema radiation and other diseases.

Example 1. 10 g of a lyophilized and milled gland powder is defatted in a known manner. The powder is transferred from 25 ml of acetone at-about: at {natal temperature and filtered on a glass filter. The well-squeezed gland powder is then shifted from 25 ml of chloroform at room temperature and filtered on a glass filter. The operation is repeated. The squeezed material is then suspended in 25 ml of absolute acetone at room temperature and sucked off on a glass filter. The resulting gland powder is dried under vacuum at room temperature. Dry weight 7-9 g, depending on the fat content of the starting material. Then 10 g of a defatted, anhydrous gland powder is extracted three times in 100 ml of distilled water at room temperature for one hour. Between the extraction stage, the gland powder is separated from the extract by centrifugation.

Extracts before processing are stored in a nitrogen atmosphere at a temperature of 3.5 ° C. Aqueous extracts have a positive reaction with respect to ninhydrin, biuret, and trichloroacetic acid. Then the aqueous extracts are collected and instilled with an aqueous 50 ° / o-th solution of trichloroacetic acid with constant agitation until the final concentration of trichloroacetic acid is 15%. After a one-hour aging at 3-5 ° C, the precipitate of three. Chloroacetic acid is centrifuged.,

D. The upper layer is a clear, pale yellow aqueous solution, which gives a negative reaction to biuret and trichloroacetic acid, and a positive reaction to n-pyhydrin.

Excess trichloroacetic acid is removed by sequential extraction at room temperature with an aqueous ethereal solution. Extraction is carried out 17-18 times to completely remove trichloroacetic acid. After the last extraction, the aqueous phase should have a pH of 4.5-5. The aqueous phase is freed from the ester using a water jet pump at a temperature not higher than 30 ° C for 6 hours. The release from the ether is controlled by the absence of odor. Thereafter, the aqueous solution is lyophilized and stored in a closed vessel. The drug is hygroscopic. The yield, based on the total weight of the gland powder (before degreasing), is 10-15% (depending on the fat content). Example 2. In Example 1, an extract that does not contain protein and precipitant is prepared from an aqueous extract of a defatted parathyroid gland. 5 g of lyophilisate inert to trichloroacetic acid is subjected to gel filtration on a Sephadex G-15 column. The mass of the column is 4.3 x X 144 cm. Water is eluent. 6 ml / h flow rate Fraction size 15 ml. The main fraction of the number of tubes 46-83 with a content of 913 mg is rich in a substance that has hormonal action. This substance is contained in the first two upper portions of the gel filtration. The separation of fractions is carried out as follows. Spectrophotometric absorption measurement at 280 nm, then at 234 and 360 nm, then the electrical conductivity is measured, then a qualitative study on the chloride ion with a reagent - silver nitrate. The pH of the combined fractions belonging to the individual vertices is measured. Then, a ninhydrin reagent study is performed and the naked eye is examined for opalescence and staining. The yield of the main fraction of the number of tubes 46-83 in terms of dry, non-fat, parathyroid powder is approximately 2-2.5%. Example 3. The procedure of Example 2, with the difference that both first peaks are captured in separate main fractions. In this way, 302 mg of the main fraction 1 and 555 mg of the main fraction 11 are obtained from 5 g of an aqueous extract of the protein-free extract. Then the main fraction 11 is again subjected to gel filtration. 456 mg of substance are applied to the column, water is used as eluent. The column size was 4.3 X 144 cm, the flow rate was 50 ml / h, the fraction size was 10 ml, Sephadex G was 15. In this way, the main fraction 11 was divided into three other upper parts. Fractions related to the middle part are combined. The weight of the latter after lyophilization is 237.5 mg. The yield in terms of non-fatty powder of the parathyroid gland is 0.6-0.8 / o. It is a yellow hygroscopic substance containing CHNOS or P. Characteristic groups SH-COOH, -OH. At research by a mass spectrometer, the following characteristic fragments are obtained: C / Hi iN hSP; CsHgNgOg and C4HbNO. The fraction contains other structures. This fraction has a physiological or therapeutic effect already at a dose of I nm / kg body weight. The product is purified by chromatography or other methods. The output of the trial is 0.3-0.4% in terms of the dry parathyroid gland. Example 4. Obtained in example 1, the lyophilized final product is subjected to dialysis. 700 .mg substances dissolved in 33 ml of distilled water are dialyzed. The external aqueous phase is replaced every 3.5 hours. For the next 3 nights, the aqueous phase is replaced every 16 hours. Dialysis is carried out at 5 ° C. The dry matter content after lyophilization is 35 mg. The external phase, concentrated in vacuo at 30 ° C and then subjected to lyophilization, gives 662 mg of dry matter. Example 5. 20 g of the gland powder (lyophilized, non-fat) of Example 1 is dissolved in water. The concentration of the aqueous solution freed from protein and ether is reduced to 1/3 and extracted by shaking 3 times with 1: 1 volume of chloroform, and the aqueous portion is freed from chloroform in vacuo. The chloroform-free aqueous solution is concentrated in vacuo to 20 ml and 5 volumes of acetone are added at room temperature. The solution was left overnight on ice. From the almost colorless acetone phase, a yellow oily portion is precipitated. The acetone solution is settled and the yellow oily substance is present in vacuum to constant weight. 1.1 g of a resinous yellow concentrate are obtained. The acetone solution is evaporated to dryness in vacuo. Get 1 g of resinous concentrate. Example 6. 50 g of a lyophilized gland powder is defatted as in Example 1. The resulting gallstone-free powder (38 g) is mixed with 40 ml of a 10% / urea solution. After one hour of exposure, the mixture is stirred in 400 ml of glacial acetic acid and 400 ml of acetone. It is aged for 1 hour, then the remaining 1200 ml of acetone and 11.2 ml of normal sodium hydroxide solution are added to the solution; After deposition, the gas is filtered through a bed. To the urea and acetone containing extract was added 2 liters of ether. The mixture is left overnight and then decanted. The suspension is centrifuged, washed 2 times with 50 ml in a mixture of acetone and ether (1: 1). The resulting precipitate is mixed. With 200 ml of a 10% acetic acid solution, the precipitate is dissolved. Then 10 g of sodium chloride is added, and a precipitate is formed from the solution, which is absorbed in 50 ml of water. Dialysis is performed 6 times with 2 liters of water, dialysis water is drained and the residue is lyophilized. 732 g of solid concentrate are obtained. The substance (197 ml) that remains in solution after precipitation with sodium chloride and centrifugation is treated with 40 ml of 45% trichloroacetic acid and then centrifuged. The upper phase is dialyzed 4 times with 3 liters of water, evaporated in vacuo and lyophilized. Get 86 g of concentrate.

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Claims (2)

The claims give proteins and polypeptides of trichloroacetic acid from it, then the excess 1 is removed. -5 the target product is subjected to purification, the soothing ones are dried, crushed, 2. The method according to claim I, characterized in that it is extracted with water, the resulting water, that the aqueous extraction is carried out at a temperature extract separated from the solid particles, precipitation is 20-60 ° C, 624562