RU2195296C2 - Method of preparing gangliosides - Google Patents

Abstract

FIELD: pharmaceutical chemistry. SUBSTANCE: phospholipid-free gangliosides that can be used as drugs are prepared by homogenization and acetone-assisted dehydration of gray matter followed by extraction with chloroform-acetone mixture and subsequent acetone (5:1)- mediated isolation of desired product, supernatant being preliminarily purified by acetone at ratio (1.5-2.0): 1 and filtered before isolation pf product. EFFECT: enabled removal of phospholipids without losses in specific biological activity of gangliosides. 4 ex

Description

 The invention relates to medicine, namely to medicines containing materials of the brain of mammalian animals, and can be used to obtain phospholipid-free impurities of gangliosides, which are tissue receptors of a number of biologically active substances and toxins.

 The urgency of the task of cleaning gangliosides from phospholipid impurities is due to the fact that recently studies of the therapeutic effect and anti-infective activity of gangliosides are actively developing [Efremenko V. I. Gangliosides - cell receptors for bacterial enterotoxins. ZhMEI, 4, 1985, p. 96-100; Shvets V.I., Krasnopolsky Yu.M. Lipids in drugs. West. Acad. honey. Sciences of the USSR, 6, 1990, p. 22-24].

 Known methods for the isolation of gangliosides from a common chloroform methanol extract of lipids by dialysis or gel filtration do not exclude non-lipid and phospholipid impurities from entering the ganglioside fraction [A.S. USSR 1022357, A 61 K 9/10, A 61 K 35/12, op. 12/30/83, bull. 48; Preparative biochemistry of lipids. M .: Nauka, 1981, p. 259].

 Closest to the claimed is a method for producing gangliosides from the brain tissue of mammalian animals [A.S. USSR 1113743, G 01 N 33/48, op. 09/15/84, Bull. 34].

 The method is as follows. Separate the gray matter of the brain from white. The gray matter is homogenized with acetone at the rate of 4 ml per 1 g of aqueous tissue. Then it is continuously stirred for 30 minutes and the suspension is filtered on a Buchner funnel.

The resulting precipitate was extracted twice with a mixture of chloroform-ethanol (1: 1) at the rate of 2 ml per 1 g of the original tissue. Shake for 1 hour on a shunt and centrifuge. Supernatants are collected and the pellet is again extracted with a mixture of chloroform-ethanol (1: 1) at the rate of 1 ml per 1 g of the starting tissue. This suspension is heated in a water bath for 30 minutes. at 50 ^o C and centrifuged hot. Supernatants combine.

 5 volumes of acetone are added to the obtained extract. A mixture of gangliosides precipitates. After the complete formation of the precipitate (after 15-20 minutes), the supernatant is sucked off by a siphon, and the ganglioside mixture is collected.

 The disadvantage of the prototype is the presence in the sediment of gangliosides of impurities of phospholipids, which reduces their specific biological activity.

 The objective of the invention is the purification of the target product from impurities of phospholipids. The technical result of the invention is manifested in a decrease in the yield of the target product due to its purification from phospholipids and the preservation of a sufficient specific biological activity of gangliosides.

 The specified technical result is achieved by the fact that the method of producing gangliosides is carried out by homogenizing the gray matter of the brain tissue and dehydrating it with acetone, extracting it with a mixture of chloroform and ethanol, separating the extract, followed by purification with acetone, first in a ratio of 1.5-2.0: 1 with separation supernatant, and then in a 5: 1 ratio.

 In relation to the prototype of the claimed method is characterized in that before the precipitation of the target product is an additional purification of the extract with acetone in a ratio of 1.5-2.0: 1.

 In the process of additional purification, phospholipid fractions precipitate and purified gangliosides remain in the supernatant, while the yield of the target product decreases and its specific biological activity increases, which is confirmed by examples of its specific implementation.

 Example 1

A cattle brain is used as a source of ganglioside production. Separate the gray matter of the brain from white. The gray matter in an amount of 1000 g is homogenized in 4000 ml of acetone, then continuously stirred for 12 hours at 20 ^o C and the suspension is filtered on a Buchner funnel.

The resulting precipitate was extracted with 2000 ml of a mixture of chloroform-ethanol (1: 1) by shaking on the shutter for 1 h at 20 ^o C. the Supernatant is collected, and the precipitate is again extracted with 1000 ml of a mixture of chloroform-ethanol (1: 1) at 50 ^o C for 30 minutes and centrifuged at this temperature.

 The resulting supernatants are combined and 1.5 volumes (4500 ml) of chilled acetone are added. After the formation of a precipitate (after 15-20 minutes), it is separated by filtration, and up to 5 volumes (15000 ml) of cooled acetone are added to the supernatant and a precipitate is formed again, which is a mixture of gangliosides, which are finally freed from the supernatant by centrifugation.

 The output of the ganglioside preparation was 3.8 g (0.38% of the weight of the feedstock). The preparation contains 5433400 ± 42300 toxin-neutralizing units identified in the neutralization test of the biological effect of cholerogen in the skin sample according to the Graig method.

 Example 2

 It is carried out analogously to example 1, except that during the preliminary purification of the extract add 1 volume (3000 ml) of chilled acetone.

 The output of the ganglioside preparation was 4.6 g (0.46% of the weight of the feedstock). The preparation contains 5866700 ± 32400 toxin-neutralizing units identified in the neutralization test of the biological effect of cholerogen in the skin sample according to the Graig method.

 Example 3

 It is carried out analogously to example 1, except that when pre-cleaning the extract add 2 volumes (6000 ml) of chilled acetone.

 The output of the ganglioside preparation was 1.5 g (0.15% of the weight of the feedstock). The preparation contains 5011500 ± 52200 toxin-neutralizing units detected in the neutralization test of the biological effect of cholerogen in the skin sample according to the Graig method.

 Example 4

 It is carried out analogously to example 1, excluding additional purification of 1.5 volumes of chilled acetone.

 The output of the ganglioside preparation was 16 g (1.6% of the weight of the feedstock). The preparation contains 6122500 ± 51500 toxin-neutralizing units identified in the neutralization test of the biological effect of cholerogen in the skin sample according to the Graig method.

 As the experimental results show, the optimal solution to the task of cleaning the target product is additional purification of the extract with 1.5-2.0 volumes of chilled acetone, since when using 1 volume of chilled acetone (example 2) phospholipidic impurities are not completely removed, as evidenced by the absence of changes the yield of the product compared to the prototype and the decrease in specific biological activity compared to example 1. An increase in the amount of acetone to 2 volumes (example 3) practically does not change the results Reamer 1.

 Thus, the claimed method has advantages over known similar solutions in terms of ease of implementation and purity of the target product.

Claims (1)

 The method of producing gangliosides by homogenization and dehydration of the gray matter of the brain with acetone, extraction with a mixture of chloroform and ethanol, separation of the supernatant, followed by isolation of the target product with acetone in the ratio of acetone: supernatant 5: 1, characterized in that the supernatant is pre-purified with acetone in a ratio of 1.5- 2.0: 1, filtered, and the selection of the target product is carried out from the resulting supernatant.