CS256635B1 - A method for extracting a polyvalent protease inhibitor from animal glands - Google Patents

Abstract

The method of extracting a polyvalent protease inhibitor from animal glands, for example from bovine lungs, with an aqueous solution of organic solvents, for example ethanol, with the addition of soluble inorganic salts at elevated temperature consists in that the animal tissue is extracted in two phases, whereby in the first phase the animal tissue is extracted with stirring in a high turbulence area for 3 to 8 hours, preferably 5 hours, at a pH of 5.5 to 6.7, preferably 6.5, at a temperature of 40 to 45 °C, with an aqueous solution of ethanol with a mass concentration of 70 to 80 % and the addition of 1 to 3 % by weight. calcium chloride, in the second phase, the animal tissue is extracted with stirring in a laminar flow area while diluting the mixture with water to 10 to 90% of the original ethanol concentration, preferably to 60% of the original concentration, for a period of 1 to 18 hours, preferably 16 hours at pH 5.5 to 6.5 at a temperature of 18 to 40°C, and the solid phase is separated from the obtained suspension and the extract is processed in a known manner.

Description

DURDIL PETR ing. CSc., REVAJNEG STANISLAV, PRAGUE

A method of extracting a polyvalent protease inhibitor from animal glands

A method for extracting a polyvalent protease inhibitor from animal glands, e.g., bovine lungs, with an aqueous solution of organic solvents, e.g. ethanol, with the addition of soluble inorganic salts at elevated temperature is to extract the animal tissue in two phases, the first phase the animal tissue is extracted with stirring in a region of high turbulence for 3 to 8 hours, preferably 5 liters at a pH of 5.5 to 6.7 preferably 6.5 at a temperature of 40 to 45 ° C with an aqueous ethanol solution of 70 to 80% by weight % by addition of 1 to 3 wt. calcium chloride, in the second phase the animal tissue is extracted with stirring in the laminar region while diluting the mixture with water to 10 to 90% of the original ethanol concentration, preferably to 60% of the initial concentration for 1 to 18 hours, preferably 16 hours at pH 5, 5 to 6.5 at a temperature of 18 to 40 ° C, the solid phase is separated from the suspension obtained and the extract is worked up in a manner known per se.

236635 (51) l6 c6

B 11/00

- 1,256,635

The invention relates to a method of extracting a polyvalent protease inhibitor from animal glands.

The procedure for obtaining a polyvalent protease inhibitor (Kallikrein inactivator, Aprotinin, Antilysin) from animal glands is elaborated in a number of works. The inhibitor is obtained, for example, from pancreas, liver, spleen, lung and other material.

In known methods of preparing the inhibitor, aqueous or water solutions with organic solvents / alcohols, organic acids, ketones and the like / in the presence of alkaline earth metal salts, alkali metals, transition metals and the like are used to extract the crushed glands. See U.S. Patent Nos. 1,014,288, 1,011,576, U.S. Pat. U.S. Pat. 2,840,986 /. Extraction temperatures range from 20 to 40 ° C. Extractions are carried out with stirring from a few minutes to 5 hours.

According to the Czechoslovak invention AO No. 155 891, the extraction procedure is as follows: the lungs, fresh or thawed, are milled to pieces of the order of millimeters, the extraction medium consists of an aqueous solution of ethanol with calcium chloride, Extraction is carried out with stirring for 2 hours, followed by separation of the solid from the extract on a centrifuge. In this way, part of the inhibitor is recovered from the glands. The yield is far from optimal in terms of the actual utilization of the feedstock, since it is attained a state that is very far from the equilibrium between the extracted feedstock and the extraction medium.

- 2,256,635

The above-mentioned drawbacks are eliminated by the method of extracting the polyvalent protease inhibitor from animal glands according to the invention, which consists in the extraction of animal tissue in two phases, wherein in the first phase the extraction takes place under agitation in high turbulence for 3 to 8 hours, preferably 5 hours at pH 5.5 to 6.7, preferably at a temperature of 40 to 45 ° 0 with an aqueous solution of ethanol having a concentration of 70 to 80% by weight, with the addition of 1 to 3% by weight of ethanol; calcium chloride, in the second stage of extraction, the mixing takes place in the laminar zone while the mixture is diluted with water to 10 to 90% of the original ethanol concentration, preferably to 60% of the initial concentration for 1 to 18 hours, preferably 16 hours at pH 5.5 to 6 The solid phase is separated from the suspension obtained and the extract is worked up in a manner known per se.

The process according to the invention achieves an inhibitor yield of 350,000 to 400,000 TU / kg of lung, which is about 30% more than in the prior art.

The actual extraction procedure is demonstrated.

The example kg of lungs is disintegrated into particles of maximum size of 3 nm. The mass is immediately transferred to an extraction apparatus in which the ethanol / water mixture is 75% by weight ethanol. with addition of 3 wt. calcium chloride. The mixture is subjected to vigorous stirring at a temperature of 40 to 42 ° C in the high turbulence region for 5 hours while maintaining the pH continuously

6.5 by addition of mineral acid (HCl). After this operation, the stirring is changed and the mixture is further stirred slowly in the laminar flow area for 16 hours, reducing the ethanol concentration to 40% by weight. and the temperature is spontaneously lowered to 20 ° C at pH 6. Next, the mixture is centrifuged, the solid phase being waste. The inhibitor contained in the solution precipitates after several ballast removal operations. The crude product obtained is first purified by fractionation, followed by a gel molecular sieve purification phase. The yield of the inhibitor was 0.75 g with an activity of 5,200 TU / mg.

Claims (1)

SUBJECT OF THE INVENTION 256 635 A method of extracting a polyvalent protease inhibitor from animal glands, e.g., bovine lungs, with an aqueous solution of organic solvents, e.g. ethanol, with the addition of soluble inorganic salts at elevated temperature, characterized in that the animal tissue is extracted in two phases, wherein the tissue is extracted with stirring in a high turbulence region for 3 to 8 hours, preferably 5 hours at a pH of 5.5 to 6.7 preferably 6.5 at a temperature of 40 to 45 ° C with an aqueous ethanol solution of 70 to 80% by weight 1 to 3 wt. calcium chloride, in the second phase the animal tissue is extracted with stirring in the laminar region while diluting the mixture with water to 10 to 90% of the original ethanol concentration, preferably to 60% of the initial concentration for 1 to 18 hours, preferably 16 hours at pH 5, 5 to 6.5 at 18 to 40 [deg.] C. and from the suspension obtained, the solid phase is separated and the extract is worked up in a manner known per se.