RU1836092C - Method of obtaining of "phosphotim" lipin complex from animal raw material - Google Patents

Abstract

The invention relates to the pharmaceutical industry, the resulting lipid complex can be used in medicine as an immunocorrective agent. SUMMARY OF THE INVENTION: the thymus gland of cattle is used as a raw material, which is crushed, homogenized in physiological saline, centrifuged from the floating lipid fraction of the suspension by extraction with yzopropanol in a ratio of 1: {5-10). a lipid complex is isolated, centrifuged, from the supernatant after reprecipitation with acetone in a ratio of 1: (2-10), the desired product is obtained.

Description

The invention relates to the pharmaceutical and perfumery and cosmetic industries, and the resulting lipid complex can be used in medicine as an immunocorrective agent, in cosmetics as a component for the regeneration of skin cells.

In contrast to the known nature of the proposed method is as follows:

 - as a raw material, the thymus gland of cattle is used, which is crushed and homogenized in physiological saline;

| - the liquid phase is separated (centrifuged), and a precipitate and suspension are formed with a floating lipid fraction;

- a lipid complex is extracted from the floating fraction with izopropanol;

- separating the liquid phase (decantation and centrifugation);

- precipitate with acetone a complex of biologically active lipids;

-concentrate (distillation of acetone) and dry the final product.

The resulting complex contains: glycerides 37-55%, cholesterol 4-13% (in free and esterified form), free fatty acids 6-12% with a mass ratio of saturated fatty acids to unsaturated 1: 1. 14–40% plasmalogens, phospholipids 2–13% (phosphatidylcholine, phosphati. Didethanolamine, sphingomyelik, phosphatidylinositol in a ratio of 1: 1: 0.7: 0.3).

Despite the variety of generally accepted sources of raw materials for producing lipids, thymus iron is known to those skilled in the art as a source of raw materials for the production of lipids.

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laziness of immunospecific proteins has never been used for this purpose. Currently, exclusively protein preparations with immunocorrective properties are isolated from this gland, for example: vilosen (7), thymalin (8), taktivin (9). In this case, the raw materials are not used economically enough, since the yield of known preparations is only 5-7% of the weight of the feedstock.

The proposed invention, not excluding the isolation of these protein preparations from the BILTIC gland, allows biologically active lipid complexes to be obtained in parallel due to the optimal utilization of fractions of homogenized raw materials after centrifugation: the supernatant is used to isolate known protein preparations (7, 8, 9), and the float fraction for isolating a new complex of biologically active lipids.

Studies of the composition of the resulting complex reveal a significant immunocorrective effect.

The possibility of implementing the new method, as well as evidence of the biological activity of the lipid complex, are illustrated by examples of the specific implementation of the invention and testing the biological effect of the final product.

Example I. 10 kg of the thymus gland are crushed on a chopper with a die diameter of 2 mm, homogenized in 2 volumes of physiological saline, centrifuged for 30 min at 3000 rpm and a temperature of 4 ° C in a refrigerator centrifuge. Preparations such as vilosen are isolated from the supernatant. The floating supernatant fraction was collected and extracted in the reactor with 7 volumes of isopropanol for 3 hours with stirring and subsequent sedimentation of the extract for 16 hours and centrifugation (3000 rpm 30 min, -4 ° C). The suspension is decanted and concentrated by vacuum distillation of the solvent. The desired product is obtained by reprecipitation with 2 volumes of acetone at 4 ° C. Filtration and drying. The yield of the final product is 4% of the weight of the feedstock and 20% of the wet weight of the floating lipid fraction.

The composition of the lipid complex: cholesterol 6% in free and esterified form: free fatty acids 12% with a mass ratio of saturated fatty acids to unsaturated 1: 1, phospholipids 2% (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylinositol

with a ratio of 1: 1: 0.7: 0.3), glycerides 45%, plasmalogens 35%. .

Example 2. 10 kg of the thymus gland are treated analogously to example 1 and get a floating fraction of the supernatant, which is selected and extracted in the reactor with 5 volumes of isopropalone for 3 hours with stirring and subsequent sedimentation of the extract.

0 The supernatant is decanted and centrifuged at 0 ° C for 20 min at 1500 rpm. The supernatant was decanted and the solvent was removed by vacuum distillation. Target Product Receive

5 reprecipitation of lipids in 5 volumes of acetone at 0 ° C, filtration and drying. The yield of the final product is 6% of the weight of the feedstock and 30% of the wet weight of the float fraction.

0 Composition of the complex,%: glycerides 55, cholesterol 13 in free and esterified form, free fatty acids 8 with a mass ratio of saturated fatty acids to unsaturated 1: 1, phospholipid 5 dya 10 (phosphatidylcholine,

phosphatidylethanolamine, sphingomyelin. .. phosphatidylnositol in a ratio of 1: 1: 0.7: 0.3), plasmalogens 14.

PRI me R 3. 10 kg of the thymus gland

0 is treated analogously to Example 1 and a flotation fraction of the supernatant is obtained, which is collected and extracted in the reactor with 10 volumes of isopropanol for 3 hours with stirring and subsequent sedimentation of the extract. The supernatant was decanted and the solvent was removed by vacuum distillation. The target product is obtained by reprecipitation of lipids in 10 volumes of acetone at 0 ° C,

0 filtration and drying. The yield of the final product is 7% of the weight of the feedstock and 35% of the wet weight of the float fraction.

The composition of the complexes,%: glycerides 37, cholesterol 4 in free and esterified form, free fatty acids 6 with a mass ratio of saturated fatty acids to unsaturated 1: 1, phospholipids 13 (phosphatidylcholine, phosphatidylethanolamine,

0 sphingomyelin, phosphatidylinositol in a ratio of 1: 1: 0.7: 0.3). plasmalogens 40.

Example 4. The activity of the obtained phosphotime lipid complex was determined in an in vitro experiment in parallel with the control drug Wilosen (7). The thymus lymphocytes of mice are isolated by spreading tissue with forceps in RPM1-1640 medium (Komissarenko S.V. Umansky V.Yu., Dmitrenko N.P. Activity of adenosine deaminase and AMP metabolic enzymes in

ConA-stimulated rat thymocytes. Immunologists, 1982, No. 2. S. 16-18). The medium contains NEPEC (20 mM), MANSOz (1.5 mg / ml), penicillin (50 U / ml), streptomycin (50 μg / ml) and fetal blood serum (5%), the pH of the medium is adjusted to 7.3-7.5 % sodium bicarbonate. The cell suspension is filtered through a nylon sieve and centrifuged twice for 10 min at 8000 d. Lymphocyte viability is determined by vital staining with 0.1% trypan blue in isotonic NaCI solution. The resulting suspension contains at least 99% of living cells. Thymocytes are cultured in a culture medium at 37 ° C. the concentration was 1x10 cells in 1 ml. Activation of thymocytes is caused by pre-incubation of the cells for 2 hours with the obtained ipid complex (0.05-0.3 mg / ml) and visas preparation (control) at the same concentrations, followed by washing with incubation red by centrifugation for O min, at 800 d, after which concavalin A was introduced into the culture medium of the incubating cells of the experimental ti control group, about a final concentration of 5 μg / ml. Labeling of DNA is carried out over a 2-hour period with H-thymidine (6-H-thymidine, 4 N kM). Label incorporation was determined 72 hours after the start of culture.

The incorporation of labeled precursor b, the DNA of the thymocytes under study was determined by the radioactivity of the acid-insoluble cell pellet after they were deposited on Whatman paper filters - 3MM and washed with 6% TCA, H20 and ethyl alcohol, dried with ether. Radioactive filters are counted in scintillation liquid ZhS-1.

The inclusion of labeled thymidine in the DNA of cells preincubated with 0.05 mg / ml of the new lipid complex is 3 times higher than in thymocytes preincubated with 0.05 vilosen (control),

EXAMPLE 5 Immunological tests of the resulting phosphatime lipid complex were carried out in vitro on blood lymphocytes of 20 patients (10 rheumatoid arthritis, 10 complicated spinal cord injuries).

In vitro experiments evaluate the effect of preincubation of a new complex with peripheral blood lymphocytes isolated in the Ficoll Verography density gradient on the processes of total spontaneous rose formation (TT-PO) of diseased lymphocytes of sheep with ram red blood cells, which are a marker of T lymphocytes (M. Gondal and

et al. 1972) and active spontaneous rosette formation (Ta-PO), which is a marker of the differentiating sub-population of T-lymphocytes (H. MIne. N. Kenichl 1978, G. Bertogllo 1976). For research, lymphocytes of individuals whose Ta-PO content in the peripheral blood is less than 20%, i.e. having a defect in the process of maturation of lymphocytes. Pre-incubation is carried out for 2 hours, followed by washing of the lymphocytes with 10 ml of medium 199.

The TT-PO reaction was carried out according to the method of MJendal (1972) in the modification of Petrov (1978), and Ta-PO was carried out according to the method of J. Wybran, N.N. Fundenberg (1973). The final concentration in the incubation medium of the new complex is 0.05 mg / ml.

The number of lymphocytes forming Ta-PO increases after their preincubation with the new lipid complex in the group of patients with rheumatoid arthritis by 82% and is observed in all 10 examined individuals of this group. A more distinctly stimulating effect (by 12% - p 0.01) in the group of patients with spinal cord injury complicated by spinal cord injury.

Similar trends, although less pronounced, are observed under the influence of the new lipid complex on TT-PO. The new complex increases by 5.6% the ability of lymphocytes in patients with rheumatoid arthritis in people to form total outlets. In lymphocytes of patients with spinal injury complicated by spinal cord injury, the activation of the new complex is more significant and amounts to 7.8% (p 0.01).

The fact that the new complex has a more significant effect on the Ta-PO process compared with TT-PO suggests an effect, primarily on cell precursors, of the T-lymphocyte population. surface markers of which are detected in the Ta-PO reaction. those. cells at the stage of differentiation. :

Thus, the new complex, obviously, activates the maturation of T-lymphocytes, especially in conditions of their deficiency in complex pathologies such as rheumatoid arthritis and spinal injury, complicated by damage to the spinal cord.

Claims (1)

SUMMARY OF THE INVENTION A method for producing a complex of phosphatime lipids from animal feed, including grinding the feed, extraction with an organic solvent, separating the supernatant containing the desired product, centrifuging, followed by concentration, distillation, heating and h and the fact that as a raw material the extract is extracted, the thymus gland is used with coarse meat in a ratio of 1: 5-10, centrifuged cattle again, after grinding, the raw material is centrifuged, and the target product after distillation homogenized in physiological saline-treated with acetone in the ratio re, centrifuged, floating fraction-5 1: 2-10,