RU2568604C1 - Method for producing 2,3,7-trioxyjuglone (spinochrome b) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: raw material is sea urchin's (Strongylocentrotus intermedius) shell and spines, which are defrosted or separated from a preserving agent by decantation, washed in water, defatted by ethanol, dried, ground, demineralised by concentrated phosphoric acid and added with distilled water, extracted three times in ethanol; the produced extract is passed through a chitosan column; the column effluent is boiled off; the residue is dissolved in distilled water, filtered, passed through a polychrome-1 column; the column is washed with distilled water; the end product is eluted with an aqueous solution of ethanol, evaporated to dryness, dissolved in ethyl acetate; the end product is deposited by hexane, filtered and dried.

EFFECT: extending the range of biologically active substances produced by industrial processing of commercial sea urchins (S intermedius) that makes it possible to produce an individual compound - spinochrome B, which is an effective antioxidant and exhibits the antiallergic properties.

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Description

The invention relates to a technology for processing natural objects and relates to a method for producing spinochrome B pigment from wastes from commercial sea urchins of the species Strongylocentrotus intermedius.

The structure of spinochrome B was first established by Kuroda [Kuroda S, Koyasu K. // Proceedings of the Imperial Academy. 1944. V. 20. N. 1. P. 23-25.] And confirmed by Anderson [Anderson H.A. et al. // Comp.Biochem. Physiol. 1969. V. 28. P. 333-345]. It was shown that the dominant pigment of the commercial sea urchin S. intermedius is Spinochrome B [Li D. - M. et al. // Eur. Food Res. Technol. DOI 10.1007 / s00217-013-1996-8].

Pigments of sea urchins are known to be effective antioxidants. The antioxidant properties of spinochromes have been studied in models of initiated oxidation of alkylbenzenes, thermal oxidation of methylene oleate and oxidation of mineral and vegetable oils. The interaction constant of spinochromes with peroxide radicals significantly exceeds the effect of ionol and the natural antioxidant tocopherol [Koltsova EA and other Quinoid pigments of echinoderms // Manuscript dep. VINITI. 1974. No. 106. S. 74; Karpov R.S., Dudko V.A. Atherosclerosis: pathogenesis, clinical features, functional diagnostics, treatment // Tomsk .: Ed. STT, 1998.672 pp.]. In addition, naphthoquinones exhibit cytotoxic activity against various tumor cell lines [Thomson R.N. Naturally Occurring guinones // London N-Y: Academic Press, 1971.2 ed. p 257-276].

Along with the well-known antioxidants of sea urchins, such as echinochrome A, spinochrome A, spinochrome E [RU 2283298 C1, 09/10/2006; Lebedev A.V. et al. // Questions of medical chemistry. 1999. T: 45 (2). S. 123-130; RU 2362573 C1, 07.27.2009, RU 2411939 C1, 02.20.2011], spinochrome B exhibits antioxidant activity [Li D. - M. et al. // Eur Food Res Technol DOI 10.1007 / s00217-013-1996-8; Kuwahara R. et al. // Food Science and Technology. 2010. V. 43. P. 1185-1190; Pozharitskaya O.N. et al. // Planta Med. 2013.V. 79 (18). P.1698-1704].

In addition to antioxidant activity, spinochrome B exhibits pronounced antiallergenic properties. Currently, allergies of various etiologies are widespread diseases. Spinochrome B has proven effective in treating allergic conjunctivitis and local skin irritations [Pozharitskaya O.N. et al. // Planta Med. 2013.V. 79 (18). R. 1698-1704].

A known method for producing pigments from the shells of red sea urchins Strongylocentrotus franciscanus [Amarowicz R. et al. // Food Chemistry. 1994. V. 51 P. 227-229]. The frozen raw materials are thawed, the internal organs are removed, the shells are washed with cold water, dried in air at 20 ° C for 24 hours, and the dried shells are crushed in a blender. The milled shells are dissolved in 10% HCl at room temperature, then extracted with diethyl ether (1: 1 by volume), the ether layer is washed with water, dried over anhydrous sodium sulfate. The solvent is removed by a stream of nitrogen. The precipitate was dissolved in methanol, placed on a Sephadex LH-20 column and eluted with methanol. The identification of the composition is carried out on a spectrophotometer. The fraction containing spinochrome B was determined by the absorption maximum at 270, 324 and 464 nm.

There is a method of obtaining the sum of quinoid pigments from sea urchins Strongylocentrotus droebachiensis by defrosting frozen raw materials, removing entrails, washing with water, drying, grinding, dissolving in concentrated hydrochloric acid, extraction with sulfuric ether, washing the ether layer with 10% NaCl, evaporation, dissolving in ethyl alcohol, concentration and separation by thin layer chromatography (TLC) on glass plates with silica gel in the system: chloroform: methanol: acetic acid: water, drying, elution methanol and identification by high performance liquid chromatography (HPLC). [Shikov A.N. et al. // Journal of Chromatography A. 2011. V 1218. P. 9111-9114].

However, the known methods do not provide individual pigment - Spinochrome B, they use highly toxic and flammable solvents - diethyl ether and methanol. They relate to laboratory analytical methods for obtaining the amount of quinoid pigments and are not suitable for technological purposes.

As a prototype, a method for producing spinochrome B from the needles of the sea urchin Strongylocentrotus intermedius [Li D. - M. et al. // Eur Food Res Technol DOI 10.1007 / s00217-013-1996-8]. The collected needles are washed with cold water, lyophilized and ground to a powder state. The powder is dissolved in an aqueous solution of inorganic acid, centrifuged, neutralized and centrifuged again. Chromatography of the sample is carried out in a static mode on a polar macroporous resin, the ratio of sorbent to sample 1:10. Desorption is carried out with ethanol, the eluate is evaporated on a rotary evaporator until ethanol is removed.

Separation of spinochromes and identification of the composition is carried out by HPLC mass spectrometry at 475 nm. A sample is obtained containing predominantly spinochrome B of undetermined concentration.

However, this method relates to laboratory analytical methods. Single-stage chromatography in static mode does not make it possible to separate the sum of pigments into individual components, therefore, the prototype method does not provide the production of spinochrome In a high degree of purification. Spinochrome B was isolated as an individual substance only by HPLC to identify it, which is unacceptable on an industrial scale. As raw materials, only the needles of sea urchins are used, and not the entire biomineral carapace containing an identical set of spinochromes.

Sea urchins of the species S. intermedius are the main commercial species of the Japan Sea coast due to significant biomass reserves and high commodity indices of food gonads [Levin B.C., Korobkov V.A. Sea urchins of Russia. Biology, industry, use // St. Petersburg: Publishing House. DORN, 2003.256 s.]. The shells and needles of sea urchins are industrial waste.

The technical result provided by the invention is to expand the spectrum of biologically active substances obtained from industrial wastes from commercial sea urchins S. intermedius, which ensures the manufacturability of the proposed method.

The technical result is also to increase the degree of purification of the target product. As a result of the implementation of the proposed method, an individual compound is obtained - spinochrome B, which is an effective antioxidant and exhibits anti-allergic properties.

The inventive method for producing 2,3,7-trioxydigone (spinochrome B) is as follows.

Shells with needles of commercial sea urchins S. intermedius, remaining after gonad extraction, are fresh, defrosted or preserved, washed with water to remove entrails, solids and sea salts. When using canned raw materials, the preservative is first separated by decantation. Substances of a lipid nature contained in the feed are removed by extraction washing with 96% ethanol followed by ethanol recovery. Fat-free raw materials are dried in air or in an oven with forced ventilation at a temperature of 30-40 ° C. Dried raw materials are crushed.

Then, the prepared raw material is demineralized with a stoichiometric amount of concentrated phosphoric acid (feed: acid ratio 5: 1), followed by the addition of distilled water at an acid: water ratio of 2: (1-0.5) to improve the removal of carbon dioxide formed during the process.

The resulting product was extracted with 96% ethyl alcohol three times at a ratio of product: ethanol (1: 1.0-1.2) for 10-14 hours. Then the intense red alcohol extract was separated by filtration, followed by centrifugation. It contains the total pigment complex of S. intermedius sea urchins and has a pH of 3-3.5.

Next, the total alcohol complex of spinochromes is cleaned of impurities on chitosan. For this, the alcoholic extracts are combined and passed through a chromatographic column filled with chitosan. The residual acidity of the ethanol extract does not allow polyhydroxynaphthoquinones to be adsorbed on chitosan. Spinochromes pass the chitosan column without delay. At the same time, the impurity compounds contained in the extract are fixed on chitosan. In particular, in this way, the separation of naphthoquinones and binaphthoquinones occurs.

Then the ethanol solution of naphthoquinones is evaporated to dryness, the residue is dissolved in distilled water. Spinochrome B is isolated from the pigment complex on the hydrophobic sorbent polychrome-1. The aqueous solution of the pigment complex obtained in the previous step is filtered and passed through a polychrome-1 chromatographic column. Adsorbed components are washed from the acid with distilled water. Elution of spinochrome B is carried out with a 5% aqueous solution of ethyl alcohol. The eluate was evaporated to dryness in vacuo. Then the dry residue is dissolved in ethyl acetate and spinochrome B is planted with hexane. The resulting microcrystalline precipitate is filtered off, washed with hexane and dried in a vacuum oven at a temperature not exceeding 60 ° C.

The manufacturability of the proposed method provide the following procedures.

The use of demineralization of waste from the industrial processing of aquatic organisms (shells and needles of sea urchins), rather than a laborious and not industrially acceptable procedure for collecting and grinding needles of sea urchins to a powder state, as in the prototype method.

The use of purification of the extract containing the total pigment complex from impurity compounds on chitosan and column chromatography on polychrome-1, which allows selective extraction of spinochrome B from the sum of pigments, rather than chromatography on a macroporous resin in static mode, as in the prototype method that does not have sufficient resolution for the selective extraction of individual compounds from the sum of the pigments.

The precipitation of spinochrome B with hexane from ethyl acetate, which provides an individual compound - spinochrome B, which does not provide for the prototype method.

To confirm the purity of the product, spinochrome B samples were analyzed by high performance liquid chromatography (HPLC).

HPLC was performed on a LaChrom chromatograph (Merck Hitachi) equipped with an L-7400 UV detector, an L-7100 pump, an L-7300 thermostat, a D-7500 integrator and an Agilent Technologies Zorbax Eclipse XDB-C18 column, 5 μm (75 mm × 4.6 mm) with Hypersil ODS protection column, 5 µm (4.0 mm × 4.0 mm). The column was thermostated at 30 ° C. The impurities were separated by a mixture of solvents: A (water + 1% glacial acetic acid) and B (acetonitrile + 1% glacial acetic acid) in the following mode: 0-5 min - isocratic, 90% A, 10% B; 5-35 min gradient, 90-10% A, 10-90% B. The feed rate of solvents is 1 ml / min. Detection was carried out at 270 nm.

In FIG. Figure 1 shows the HPLC sum of S. intermedius spinochromes (eluate from chitosan).

In FIG. Figure 2 shows the HPLC of a mixture of S. intermedius spinochromes obtained by hydrophobic chromatography on polychrome-1.

In FIG. Figure 3 shows the HPLC of a microcrystalline precipitate of spinochrome B.

The invention is illustrated by the following example.

Example

The shells with needles of Strongylocentrotus intermedius sea urchins remaining after gonad extraction, fresh or defrosted, are washed in the amount of 10 kg with drinking water to remove mechanical impurities and sea salts. Then the raw materials are degreased with 10 l of 96% ethanol. Ethyl alcohol is regenerated. The raw materials are dried in the dark and crushed. The crushed raw material is loaded into the reactor-extractor, 2.2 L of concentrated phosphoric acid are added, and then 1 L of distilled water is added sequentially, portionwise.

After decomposition of the calcium skeleton and the cessation of gas formation, the resulting product was extracted three times with 96% ethanol (3 × 11 L). The extraction is carried out for 12 hours at room temperature. Then the extracts containing the naphthoquinone complex are combined, filtered through a tissue filter and centrifuged. The resulting alcoholic extract has a pH of 3.5.

Next, an alcoholic extract in the amount of 33 l is passed through a column with 0.5 kg of crushed chitosan, balanced with 96% ethyl alcohol. Impurity compounds are adsorbed on chitosan. Column regeneration is carried out with 2 l of 96% ethanol.

Then, the resulting eluate containing a complex of spinochromes is evaporated to dryness in vacuo. The dry residue is dissolved in 0.5 l of distilled water. The solution is filtered and passed through a column of 0.5 kg of polychrome-1, balanced with distilled water. In this case, the pigments are adsorbed on polychrome-1. Adsorbed pigments are washed from the acid with 2 L of distilled water. Spinochrome B is then eluted from a 2 liter column of a 5% aqueous solution of ethyl alcohol. The resulting eluate was evaporated to dryness. The dry residue is dissolved in 0.2 l of ethyl acetate and precipitated with spinochrom 0.5 l of hexane, the precipitate is filtered off, washed with hexane and dried in a vacuum oven at a temperature not exceeding 60 ° C.

The product yield is 2.72 g (0.0272% of the weight of the feedstock) with a purity of 80-90%.

Claims (1)

A method for producing 2,3,7-trioxy diamine (spinochrome B) from Strongylocentrotus intermedius sea urchins, including extraction and isolation of the target product, characterized in that the shell and sea urchin needles are used, which are defrosted or separated from the preservative by decantation, washed with water, degreased 96 % ethanol, dried, crushed, demineralized with concentrated phosphoric acid, followed by the addition of distilled water at a ratio of raw materials - acid 5: 1, acid - water 2: (1-0.5), then extracted three times with 96% ethyl alcohol volume in the ratio of raw materials - ethanol 1: (1-1.2) for 10-14 hours, the obtained extract is passed through a column with chitosan, the eluate is evaporated, the residue is dissolved in distilled water, filtered, passed through a column with polychrome-1, washed column with distilled water, the target product is eluted with a 5% aqueous solution of ethyl alcohol, evaporated to dryness, dissolved in ethyl acetate, the target product is precipitated with hexane, filtered and dried.

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