RU2362573C1 - Way of spinochrome reception and protein of sea urchins interacting with polyhydroxynaphtoquinone - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: way of spinochrome A reception and the sea urchins protein interacting with polyhydroxynaphtoquinone is offered, characterised that raw materials are wash with water, extracted using ethyl alcohol, demineralised with 20% phosphoric acid; then the water-acid admixture is filtered, centrifugated, further the received solution is placed on polychrome 1, admixtures are washed out with 3% phosphoric acid and the distilled water, and the complex of protein with polyhydroxynaphtoquinone is eluted by 10% ethyl alcohol, then 40% ethyl alcohol, fractions are aggregated; further ethyl alcohol is removed, the water residue is freeze dried; received lyophilic powder is dissolved in the water, a non-dissolved deposit is separated, and ethyl alcohol is added to an aqueous solution at a parity 1:9 and maintained within 18-24 hours at 4°C, the dropped out protein is separated, ethyl alcohol is washed out and chromatographed on the sephacryl S-200 and freeze dried; then spirituous solutions is aggregated, concentrated to dryness, the dry rest is dissolved in ethyl alcohol and chromatographed using sephadex LH-20, then the violet fraction of spinochrome A is separated, boiled to dryness, the dry residue is dissolved in acetone and crystallised.

EFFECT: expansion of a spectrum of biologically active substances received from sea urchins.

1 ex, 3 dwg

Description

The invention relates to a technology for processing natural objects and relates to a method for producing polyhydroxynaphthoquinone - spinochrom A and a protein interacting with it from the shell and needles of the commercial species of the sea urchin Strongylocentrotus nudus.

Naphthoquinone pigments are known to have antioxidant properties. These include echinochrome A and spinochromes, the related hydroxylated derivatives of 5,8-dihydroxy-1,4-naphthoquinones. This class of antioxidants is distinguished by the presence of a labile quinoid structure susceptible to redox transformations, and the hydroxyl substituents of the naphthoquinone cycle determine the antioxidant properties of polyhydroxy-1,4-naphthoquinones (PHNH) [Lebedev A.V., Levitskaya E.L., Tikhonova E.V. . et al. Antioxidant properties, autooxidation and mutagenic activity of echinochrome A in comparison with its structural analogues. // Biochemistry. 2001, vol. 66, p. 885-893].

The antioxidant properties of spinochromes were studied on the models of initiated oxidation of alkylbenzenes, thermal oxidation of methylene oleate and oxidation of mineral and vegetable oils. The antiradical activity of spinochromes is characterized by the constant of interaction with peroxide radicals, comparable with the constant of ionol [E.A. Koltsova. The dissertation for the degree of candidate of chemical sciences. Study of the chemical structure and physiological function of the quinoid pigments of sea urchins. Vladivostok, 1983, p. 84; Lebedev A.V., Ivanova M.V., Krasnovid N.M. et al. Acidic properties and interaction with superoxide anion radical of echinochrome A and its structural analogues // Issues of Medical Chemistry. 1999, vol. 45, p.123-130].

Known and successfully used in medicine, natural polyhydroxynaphthoquinone - echinochrome A, which has anti-ischemic and anti-infarction activity. Its activity is due to the ability to improve the supply of peripheral tissue with oxygen as a result of interaction with both cells and individual enzyme systems [N.P. Mishchenko, S.A. Fedoreev, V.L. Bagirova. New original domestic drug Histochrome. // Chem.-farm. Journal 2003. Volume 37, no. 8, p. 49-53; A.V. Shvilkin, L.I. Serebryakova, O.V. Tskitishvili. The effect of echinochrome on experimental reperfusion damage to the myocardium. // Cardiology. 1991. Volume 31, No. 11, pp. 79-81].

Other polyhydroxine naphthoquinones, which are part of the shell cells of the shells and needles of sea urchins, which are less studied than echinochrome A, in particular spinochrome A [C.W. J.Ch., J.C. Moore, can exhibit similar activity. Pigments from Some Marine Speciments. Journal of Chemical Education, vol.50, p.102, 1973; E.A. Koltsova, O.B. Maksimov, N.K. Utkina, A.P. Shchedrin. Quinoid echinoderm pigments. // Manuscript dep. VINITI, No. 106, p. 74, 1974].

Of particular interest are the proteins of the shells and needles of echinoderms, interacting with naphthoquinone pigments, in particular DT-diaphorase, which is an integral part of another enzyme, nitric oxide synthase (NOS).

As you know, NO synthase in the body produces endothelial vascular relaxation factor (NO, EGF), which prevents coronary heart disease by improving the supply of peripheral tissue with oxygen, which is transported throughout the circulatory system by red blood cells. In addition, NO prevents the adhesion of leukocytes and platelets to the vascular endothelium and platelet aggregation. This can be of great importance at the stages of the development of blood clots, not only in the genesis of atherosclerotic lesions of the vessel wall, but also be a factor in the activation of the antioxidant and immune systems of the body.

Naphthoquinones in living systems act as substrates of various enzymes. When interacting with enzymes (DT-diaphorase and NO synthase), they participate in the synthesis of H ₂ O ₂ , NO and electron transfer in redox reactions with the direct use of NADP (H ⁺ ) and flavin coenzymes. This helps to improve the supply of peripheral tissue with oxygen, reducing the risk of cardiovascular disease, heart attack and stroke.

A known method of producing quinoid pigments (spinochromes) by dissolving the calcium skeleton of sea urchins in concentrated hydrochloric acid, neutralizing the acid solution with sodium bicarbonate, extraction of spinochroms with sulfuric acid, acidification with hydrochloric acid to restore them in sodium form, dehydration of the ether extract with anhydrous sodium sulfate, uparium solution spinochromes in vacuo and chromatographic purification on acid-treated silica gel using 5% methanol in chloroform as ve eluent. After concentration, the resulting eluate was redissolved in ethyl acetate and evaporated in vacuo [C.W. J.Chang, J.C. Moore. Pigments from Some Marine Speciments. Journal of Chemical Education, vol.50, p.102, 1973].

There is a method of producing quinoid echinoderm pigments by dissolving a calcium skeleton in concentrated or 6 N hydrochloric acid, extraction with sulfuric ether, separation of echinochrome in the form of water-soluble sodium derivatives, repeated extraction with sulfuric ether or benzene and subsequent chromatographic purification of echinochrome on silica gel or cellulose-chloromethane system : petroleum ether: hexane [E.A. Koltsova, O.B. Maksimov, N.K. Utkina, A.P. Shchedrin. Quinoid echinoderm pigments. Manuscript dep. VINITI, No. 106, p. 74, 1974].

A known method for producing echinochrome A from sea urchins by processing raw materials with a 18% solution of hydrochloric acid in water, liquid extraction with n-butyl alcohol, subsequent chromatographic purification of the target product on polytetrafluoroethylene (polychrome-1) using 40-50% ethyl alcohol as an eluting agent and crystallization of echinochrome A from aqueous ethyl alcohol [SU 1233443 A1, 08/02/1986].

A known method of producing protein - hemagglutinin from the coelomic fluid of sea urchins Anthocidaris crassispina by ion exchange chromatography on DEAE cellulose and subsequent affinity chromatography using shadows of human red blood cells bound by glutaraldehyde, eluting with 10 mM EDTA. Using SDS electrophoresis, the molecular weight of the obtained protein was determined [Giga Y., Suutoh K., Ikai A. A new Multimeric Hemagglutinin from the Coelomic Fluid of the Sea Urchin Anthocidaris crassispina].

A known method for the isolation and purification of a flavoprotein having DT-diaphoretic activity from a raw beef kidney extract by treating the extract with ammonium sulfate, dialysis, chromatography on carboxymethyl cellulose in phosphate buffer with elution with a solution of ammonium sulfate, precipitation of the protein with ethanol at -2 ° C in the ratio of 1: 1,2, by chromatography on cellulose in a phosphate buffer, eluting with the same buffer, treating the eluate with calcium phosphate gel in a ratio of 1: 2 at 0 ° C, centrifuging, treating with an ammonium sulfate solution Ia and dialysis [Liao S., Dulaney J.T., Williams-Ashman H.G. Purification and Properties of Flavoprotei Catalyzing the Oxidation of Reduced Ribosyl Nicotinamide. The Journal of Biological Chemistry, v. 237, No. 9, 1962].

Methods for the simultaneous production of podihydroxinaphthoquinone - spinochrome A and a protein that interacts with polyhydroxynaphthoquinone from sea urchins have not been found in available patent and other scientific and technical literature.

Sea urchins of the species Strongylocentrotus nudus are a commercial species. After removing the eggs, the shells with needles fall into the waste. However, they can serve as a source of biologically active substances - polyhydroxynaphthoquinones and their natural complexes with proteins.

The objective of the invention is to develop a method for producing a complex of spinochrome A with protein from wastes of industrial processing of hedgehogs (shells and needles of sea urchins of the species Strongylocentrotus nudus) and obtaining individual preparations of spinochrome A and protein from it.

The technical result provided by the invention is to expand the range of biologically active substances obtained from the waste from industrial processing of sea urchins.

As a result of the invention, three products are obtained.

1. A complex of spinochrome A with the protein of the shell and needles of sea urchins, which can serve as the basis for the development of new dietary supplements that reduce the risk of cardiovascular diseases, heart attacks and strokes, while being a nutritious dietary product in the prevention of excess weight and associated diseases (arthritis , arthrosis, diabetes and others).

2. Spinochrome A is an effective antioxidant (an analog of echinochrome A), it can be used for transformations in organic synthesis;

3. The protein of the shell and needles of sea urchins, capable of binding and enzymatic transformation of polyhydroxynaphthoquinones, which is of interest as a potential antisclerotic and immunostimulating agent.

The essence of the proposed method is as follows.

Shells with needles of commercial sea urchins, remaining after the extraction of eggs, fresh or defrosted, are washed with water to remove mechanical impurities and the remains of viscera. The lipid substances contained in the shell are removed by extraction with ethanol three times, followed by ethanol regeneration. The fat-free shell of sea urchins is subjected to demineralization with a solution of mineral acid until the carbonate skeleton of the urchins is completely decomposed.

The resulting aqueous acidic extraction mixture is filtered to remove undissolved residues of the feed and, if necessary, centrifuged to remove microparticles. The obtained supernatant (aqueous acid extract of sea urchin pigments) is chromatographed on a hydrophobic sorbent Polychrome-1.

A solution of colored compounds is applied to a chromatographic column, the adsorbed components are washed away from impurities with a weak solution of mineral acid and distilled water. The elution of pigment complexes with polychrome-1 is carried out by a gradient gradient of ethyl alcohol (10% and 40% aqueous solutions of ethanol). The fractions containing pigments are combined, ethanol is removed by concentration in vacuo. The concentrate is lyophilized. A dark red lyophilic powder is obtained, which is a complex of polyhydroxynaphthoquinones with the protein of the shell and needles of sea urchins. The product yield is 0.2-0.3% of the weight of the feedstock.

The resulting lyophilic powder is dissolved in distilled water. Insoluble precipitate is separated by centrifugation. Ethanol was added to an aqueous solution of the resulting complex in a ratio of 1: 9. To form a protein precipitate, the alcohol solution is placed for 18 hours in a refrigerator (4 ° C). The precipitated protein is separated by centrifugation or filtration and washed with ethanol. The protein collected by centrifugation is dissolved in distilled water and chromatographed on Sephacryl S-200 in distilled water. Protein stained peak is collected and lyophilized. The resulting protein is analyzed by SDS electrophoresis in 13% polyacrylamide gel. The isolated protein has a molecular weight of about 28-29 kDa, which coincides with the molecular weight of DT-diaphorase (27.3-31.0 kDa). DT-diaphorase has a huge affinity for naphthoquinones and uses them as substrates when working in cell structures.

The combined alcohol solutions are concentrated in vacuo, dissolved in a minimal volume of ethanol and chromatographed on Sephadex LH-20 in ethanol. The escaping lilac-violet fraction of the pigment was evaporated to dryness from ethanol. An individual compound is obtained - spinochrome-A.

Figure 1 shows the absorption spectrum of spinochrome A obtained from the shell and needles of sea urchins. Spectral maxima λ _max : 251, 317, 514 nm.

Figure 2 presents the absorption spectrum of the protein obtained from the shell and needles of sea urchins. The spectral maximum is λ 270 nm.

Figure 3 presents SDS-electrophoresis of a protein obtained from the shell and needles of sea urchins in 13% polyacrylamide gel.

The invention is illustrated by the following example.

EXAMPLE

The shells with needles of sea urchins remaining after removing eggs in the amount of 18 kg are crushed and washed with water to remove internal soft tissues. Get 15 kg of feedstock.

The feed is loaded into the reactor-extractor and extracted three times with 96 ° ethanol (3 × 15 kg) until complete removal of lipid substances. Ethyl alcohol is regenerated. Fat-free shell with needles of sea urchins is treated with 15 l of 20% phosphoric acid.

After decomposition of the carbonate backbone of the hedgehogs, the resulting aqueous acidic extraction mixture is filtered through a woven filter and centrifuged. The resulting pigment solution is applied to the prepared column with 5 kg of polychrome-1 in water. Adsorbed pigments are washed from impurities with 15 liters of a 3% solution of phosphoric acid in water, and then 20 liters of distilled water. Pigments with polychrome-1 were eluted with 15 liters of a 10% solution of ethyl alcohol, and then with 15 liters of a 40% solution of ethyl alcohol.

Fractions containing hedgehog pigments are combined. Ethyl alcohol was removed by concentration in vacuo at 60 ° C. The aqueous residue is lyophilized. Get a dark red lyophilic powder - an enzyme complex of polyhydroxynaphthoquinones. The product yield is 42 g.

The resulting lyophilic powder is dissolved in 500 ml of distilled water. Insoluble precipitate is separated by centrifugation. To 400 ml of an aqueous solution of the resulting complex add 3600 ml of 96 ° ethanol (1: 9 ratio). The water-alcohol solution is placed for 18-24 hours in a refrigerator (4 ° C) to form a protein precipitate. The precipitated protein is separated by centrifugation and washed with 500 ml of 96 ° ethanol. The protein obtained after precipitation and centrifugation is dissolved in 100 ml of distilled water and chromatographed on Sephacryl S-200 in distilled water. The stained protein peak is collected and lyophilized. 2.45 g of protein powder is obtained.

Alcohol solutions are combined and concentrated in vacuo to dryness at a temperature of 50-60 ° C. A dry residue in an amount of 10 g is dissolved in 500 ml of 96 ° ethanol and chromatographed on a Sephadex LH-20 column in ethanol. The violet fraction of the pigment is separated off and evaporated to dryness in vacuo at a temperature of 50-60 ° C, dissolved in dry acetone and crystallized. Obtain 0.3 g of a purple powder, which is an individual compound - Spinochrome-A.

Claims (1)

A method for producing spinochrom A and sea urchin protein interacting with polyhydroxine naphthoquinone, characterized in that the feed is washed with water, extracted three times with 96% ethanol, demineralized with 20% phosphoric acid, then the aqueous acid mixture is filtered, centrifuged, then the resulting solution applied to polychrome 1, impurities are washed with 3% phosphoric acid and distilled water, and the protein complex with polyhydroxynaphthoquinone is eluted with 10% ethyl alcohol, then with 40% ethyl alcohol, fractions of about unite; then ethanol is removed by concentration in vacuo, the aqueous residue is lyophilized; then the obtained lyophilic powder is dissolved in water, the insoluble precipitate is separated by centrifugation, and ethanol is added to the aqueous solution at a ratio of 1: 9 and kept for 18-24 hours at 4 ° С to form a protein precipitate, then the precipitated protein is separated by centrifugation and washed with 96 % ethanol, then the resulting protein is dissolved in water, chromatographed on Sephacryl S-200 and lyophilized; then the alcohol solutions are combined and concentrated in vacuo to dryness, then the dry residue is dissolved in 96% ethanol and chromatographed on Sephadex LH-20, then the violet fraction of spinochrome A is separated, evaporated in vacuo to dryness, then the dry residue is dissolved in acetone and crystallized.

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