RU2091073C1 - Method of immunostimulating agent preparing - Google Patents

Abstract

FIELD: medicine, biotechnology. SUBSTANCE: preliminary frozen salmon fish milts were milled and extracted with 3% acetic acid solution in the presence of zinc chloride for 24-48 h. Extract is cleared by centrifugation and filtration. Ultra- and diafiltration of extract were carried out in 0.9% sodium chloride solution at pH = 6.0-6.5 through porous material at separation limit 5000 Da. Preparation is sterilized, poured into vials and dried by lyophilization. The yield is 0.12% of the parent milt. Immunomodulating activity of the obtained preparation is evident at the dose 0.01-0.1 mg/1 kg mass and can be used as immunomodulating agent. EFFECT: simplified method, high activity, improved ecology. 2 tbl

Description

 The invention relates to the field of medicine and the medical industry, in particular to methods for producing immunostimulants from milk from salmon processing waste.

 The drug obtained by the proposed method can be used to stimulate the immune response, for example, in animal producers, farm animals and humans, as well as in the production of immune serums.

 The prior art is as follows. A known method for producing DNA from sturgeon milk is that the milk is homogenized and extracted in a saline solution containing citrate and sodium chloride (ed. St. USSR N 1410494, class C07M21 / 04, 1986).

 Methods of obtaining immunostimulants from fish milk are unknown.

 The closest analogue is a method for producing an immunostimulant, including freezing animals treated with acetone lymph nodes, grinding them, extracting with a 3% solution of acetic acid with the addition of zinc chloride, treating n-sufficient liquid with acetone, separating the precipitate and isolating the target product (ed. St. USSR N 1187824, CL A61K35 / 30, 1985).

 The disadvantage of this method is the laborious technology using acetone, which is an environmentally harmful substance.

 The invention consists in the following.

 The invention is aimed at eliminating from the technology the step of precipitating the target product with acetone by producing an immunostimulator by ultrafiltration through a porous material with a separation limit of 5000 D and using salmon fish milk as waste from the processing of sea products as a raw material.

 The technical result is to simplify the process of obtaining an immunostimulant using environmentally friendly technology with higher activity.

 The essential features characterizing the invention are as follows.

 Limiting signs: freezing of raw materials, grinding, extraction with a 3% solution of acetic acid with the addition of zinc chloride, filtration, separation of insufficient liquid and isolation of the target product.

 Distinctive features: salmon milk is used as raw material, extraction is carried out for 24-48 hours, followed by ultra- and diafiltration of the extract in a solution of 0.9% sodium chloride with a pH of 6.0-6.5 through a porous material with a separation limit of 5000 D.

 The use of salmon milk as a raw material makes it possible to utilize biologically valuable raw materials from fish processing waste.

 Extraction for 24-48 hours allows you to increase the yield of the biologically active fraction of the drug. Ultrafiltration replaces the precipitation of the target product with acetone. Diafiltration of the extract in a solution of 0.9% sodium chloride with a pH of 6.0-6.5 through a porous material with a separation limit of 5000 D contributes to a more complete removal of low molecular weight impurities from the centrifugate, as well as the isolation of the biologically active fraction.

 Example. 5 kg of frozen salmon milk is ground in a meat grinder and 30 l of pyrogen-free distilled water containing 90 g of zinc chloride is poured and mixed. Extraction is carried out by adding 900 ml of acetic acid for 24-48 hours with periodic stirring at room temperature. Then carry out the purification of the extract. The extract is centrifuged at 3000 rpm for 40 minutes. The sediment is thrown away. The centrifuge is filtered through membrane filters with a pore size of 0.8; 0.45; 0.22 μm in series.

 Obtain 32 l of a purified sterile filter, to which 130 l of a 0.9% sodium chloride solution pH 6.0-6.5 is added and dia and ultrafiltration of the diluted extract on hollow fibers with a separation limit of 5000 D is carried out. The purified extract is concentrated to a content of 0 5 mg of protein in 1 ml. The resulting 1.5 l of the semi-finished product is subjected to sterilizing filtration on membrane filters. The sterile preparation is poured into vials or ampoules and subjected to freeze drying. The result is a non-toxic, sterile, pyrogen-free drug for injection. The yield of the target product 6 g (0.12%) from the feedstock.

 The preparation, obtained from salmon milk, is a white powder, contains a complex of polypeptides and proteins, with a molecular weight of 5000 20,000 D. Absorption spectrum 280 ± 2 nm. It has immunomodulatory activity in doses of 0.01-0.1 mg per 1 kg of mass. The specific activity of an immunostimulant obtained from salmon milk was evaluated in comparison with an immunostimulant from animal lymph nodes (prototype) by the effect on immunodeficiency and T-lymphocytes.

An immunodeficiency model was created by transferring mice thymocytes (6 x 10 ⁶ ) adjuvant arthritic rats. The degree of suppression of the immune system was judged by the severity of the immune response on the 5th day after intraperitoneal immunization of the same mice with sheep erythrocytes, determining the level of AOK, rosette-forming blood lymphocytes (ROCK), and hemagglutinin titer (Table 1).

 The proposed immunostimulant from salmon milk and an immunostimulant (prototype) were administered subcutaneously daily, starting immediately after the injection of sheep erythrocytes. Control mice were injected with isotonic sodium chloride solution.

 Evaluation of the effect of the proposed preparation from salmon milk and the preparation from animal lymph nodes (prototype) on T-lymphocytes was carried out in the reaction of “active” rosette formation (Ea ROCK), treated with trypsin of guinea pig thymocytes with rabbit erythrocytes (Table 2).

 As can be seen from the data table. 1 and 2, the drug obtained from salmon milk, in a number of indicators (AOK, late ROCK, Ea ROCK) surpasses the immunostimulant obtained from the lymph nodes of animals (prototype) and can be used as an immunostimulant.

Claims (1)

 A method of obtaining an immunostimulant, including freezing raw materials, grinding, extraction with a 3% solution of acetic acid with the addition of zinc chloride, filtering, separating the supernatant and isolating the target product, characterized in that salmon milk is used as a raw material, extraction is carried out for 24 48 h followed by ultra- and diafiltration of the extract in a solution of 0.9% sodium chloride with a pH of 6.0 - 6.5 through a porous material with a separation limit of 5000 D.

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