RU2697197C1 - Method of producing water-soluble salt form of echinochrome a, suitable for use in pharmacological and food industry - Google Patents

Abstract

FIELD: technological processes.SUBSTANCE: invention relates to a method of producing water-soluble monosodium salt of echinochrome A from sand dollar suitable for use in pharmacological and food industry. Method is characterized by that the freshly caught or defrosted raw material is preliminarily washed with drinking water, dried, poured with 96 % ethyl alcohol with addition of ascorbic acid at ratio of raw materials : ethyl alcohol : ascorbic acid 1:(1.0–1.2):(0.003–0.005) (kg : l : kg) and extracted at room temperature for 22–26 hours, then obtained extract is drained and evaporated, then the concentrated residue is diluted with distilled water and passed through the column with polychrome 1, then the pigment fractions are eluted with 40 % ethyl alcohol, the eluate is evaporated to dryness, then defatted raw material is subjected to double extraction with 96 % ethyl alcohol with addition of phosphoric acid at room temperature in ratio of raw materials : ethyl alcohol : phosphoric acid 1:(1.0–1.2):(0.002–0.005) (kg : l : kg) for 22–26 hours, then obtained extracts are drained, combined and evaporated, and the dry residue obtained at the extraction step is combined with the eluate concentrate and washed with distilled water. Further, the concentrate is dissolved in 1.0 % aqueous solution of sodium bicarbonate in ratio of concentrated residue : soda solution 1:(1.5–2.0) (kg : l), then obtained solution is centrifuged, residue is discarded, and obtained aqueous solution containing echinochrome A monosodium salt is dried on lyophilic or spray drier to produce target product.EFFECT: disclosed method enables to obtain echinochrome A monosodium salt which is well soluble in water without loss of functional properties inherent in echinochrome A.1 cl, 4 dwg, 2 ex

Description

The invention relates to a method for producing a water-soluble salt form of echinochrome A from flat sea urchins, specifically to its monosodium salt, suitable for use in the pharmacological and food industry as an active ingredient for the production of biologically active food additives (BAA) and functional food products (FPP) .

Echinochrome A (ECA) is used as a substance for the production of drugs of the Histochrome series, used in cardiology and ophthalmology, as well as for the production of dietary supplements for food Timarin, Hitochrom, and Golden Horn. They are designed to prevent atherosclerosis, coronary heart disease, improve blood lipid status, provide antioxidant protection of the body [RU 2134107, RU 2137472, RU 2137400, RU 2340216, RU 2337696, RU 2360683, RU 2359686].

ECA activity in the body is due to an improvement in the supply of peripheral tissues with oxygen due to biological reactions, both with the cells themselves and with individual enzyme systems.

The raw materials for the production of ECA are flat sea urchins of the species Scaphechinus mirabilis, which are widespread along the Far Eastern coast of the Sea of Japan.

ECA production is a technological process using organic solvents, such as chloroform, hexane, acetone, dioxane, which are not approved for use in the food industry. To remove toxic solvents, vacuum sublimation is used at a temperature of 220-222 ° C, labor-intensive and energy-intensive. Such techniques are due to the need to obtain a highly purified individual substance used as a substance for the preparation of drugs for injection. In addition, echinochrome A is soluble in alcohol and insoluble in water, which limits its scope in the production of dietary supplements and FPP.

A known method for producing 2,3,5,7,8-pentahydroxy-6-ethyl-1,4-naphthoquinone (echinochrome A) by extraction of sea urchins with a solution of an inorganic acid in an organic solvent, liquid extraction with chloroform and recrystallization from an organic solvent, for example dioxane, followed by vacuum sublimation at 220 ° C [RU 2283298 C1, 05.22.2006].

The disadvantages of this method are the use of chloroform and dioxane, not approved for use in the food industry, as well as the energy-intensive and time-consuming process of sublimation of the target product. The method is designed to obtain ECA - the substance of the series of drugs "Histochrome".

As a prototype, a method was selected for producing 2,3,5,7,8-pentahydroxy-6-ethyl-1,4-naphthoquinone from canned sea urchins, including decantation of a preservative, extraction of raw materials with ethyl alcohol with the addition of inorganic acid, and chromatography of the extract on a column with chitosan, elution of the target product with 96% ethanol with the addition of hydrochloric acid, neutralization with baking soda, evaporation followed by re-dissolution in chloroform or acetone and recrystallization from ethanol [RU 2352554 C1, 04/20/2009].

The disadvantage of this method is the chromatographic purification of the target product on chitosan. The process is time consuming, lengthy and requires the evaporation of large volumes of solvents. ECA obtained by this method is unsuitable for use as an ingredient in dietary supplements for food and FPP, as insoluble in water and obtained using toxic solvents. The method uses additional purification of the target product by its re-dissolution in chloroform and recrystallization. In this case, a highly purified expensive substance for drugs is obtained, the use of which in the food industry is not economically feasible.

Since dietary supplements and FPP are used as nutrient forms, in this regard, there was a need to develop a method for producing a water-soluble form of ECA while maintaining its functional properties using extraction solvents and technological materials approved for use in the food industry. ECA monosodium salt meets these requirements. When it enters the gastrointestinal tract in an acidic environment, the monosodium salt of ECA goes into the active form, suitable for absorption.

The technical result provided by the invention is the development of a method for producing a water-soluble form of echinochrome A from flat sea urchins in the form of its monosodium salt, without losing the functional properties inherent in echinochrome A, suitable for use in the pharmacological and food industries as a dietary supplement and FPP.

The inventive method is less time consuming, because it does not require evaporation of large volumes of toxic solvents; in the production process only materials and solvents approved for food production are used, such as drinking water, ethyl alcohol, ascorbic acid, phosphoric acid, baking soda. The essence of the developed method is as follows.

Freshly harvested or defrosted flat sea urchins of the species Scaphechinus mirabilis are washed with drinking water to remove impurities (sea salts and sand) and dried in air. Then the raw material is extracted with 96% ethyl alcohol with the addition of ascorbic acid at a ratio of raw materials: ethyl alcohol: ascorbic acid 1: (1.0-1.2) :( 0.003-0.005) (kg: l: kg) for 22-26 hours at room temperature. The extraction of freshly caught or defrosted raw materials with ethyl alcohol with ascorbic acid is due to the need for degreasing the raw materials in order to remove lipids. The addition of ascorbic acid prevents the oxidation of ECA and, consequently, the loss of the target product. The extract is drained, ethyl alcohol is distilled off and sent for regeneration. The concentrated residue containing a small amount of pigments is diluted with distilled water and passed through a column with a hydrophobic sorbent Polychrome 1 to avoid loss of ECA.

The prepared raw materials are extracted with 96% ethyl alcohol with the addition of phosphoric acid in the ratio of raw materials: ethyl alcohol: phosphoric acid 1: (1.0-1.2) :( 0.002-0.005) (kg: l: kg). The extraction is carried out twice at room temperature for 22-26 hours. The resulting extracts are poured, combined and evaporated. Alcohol distillation is sent for regeneration and reuse. The dry residue is washed with distilled water. Pigment fractions from polychrome 1 elute with 40% ethanol. The eluate is evaporated to dryness, the alcohol is sent for regeneration.

The dry residue obtained in the extraction step is combined with the eluate concentrate and washed with distilled water. The washed concentrated residue containing echinochrome A is dissolved in a 1.0% aqueous sodium bicarbonate solution in the ratio of concentrated residue: soda solution 1: (1.5-2.0) (kg: l).

The resulting solution is centrifuged. The sediment containing lipids is discarded. An aqueous solution containing the monosodium salt of echinochrome A is dried on a freeze dryer or spray dryer. A reddish-brown loose powder is obtained containing 83-85% of the monosodium salt of echinochrome A, readily soluble in water, without loss of functional properties inherent in ECA.

The content of ECA monosodium salt in the target product was analyzed by high performance liquid chromatography (HPLC) on a LaChrom chromatograph (Hitachi Merck) equipped with a UV detector L-7400, thermostat L-7300, pump L-7100, integrator D-7500. For analysis, a ZORBAX Eclipse XDB-C18 (Agilent) column 4.6 × 75 mm, 3.5 μm was used. Detection was carried out at λ = 210 nm, the feed rate of the solvents was 0.9 ml / min, and the thermostat temperature was 30 ° C. The elution was carried out with a mixture of solvents A (acetonitrile + 1% CH ₃ COOH), B (water + 1% CH ₃ COOH); gradient (10% A + 90% B) → (90% A + 10% B). When acidifying the solution of the monosodium salt of ECA, sodium acetate and ECA are formed.

To determine the authenticity of the target product, a comparative analysis of the absorption spectra of the monosodium salt of ECA and pharmacopoeial ECA was carried out. The analysis was carried out by spectrophotometry in the UV and visible ranges on a spectrophotometer SF-200 (Design Bureau "Spectrum", St. Petersburg, Russia). We used quartz cuvettes with a layer thickness of 1 cm. Pharmacopoeia echinochrome A with a purity of 98% was used as a reference substance.

In FIG. 1 shows an HPLC chromatogram of an ECA monosodium salt with a purity of 85%. The obtained HPLC spectrum has an absorption band at λ 270 nm corresponding to a retention time of 15.55 min, which is typical for ECA in this solvent system.

In FIG. 2 shows an HPLC chromatogram of an acidified alcohol solution of pharmacopeia ECA with a purity of 98%, used as a standard sample. The absorption band of the pharmacopoeial ECA at λ 270 nm corresponds to a retention time of 15.55 min (for this solvent system).

In FIG. Figure 3 shows the UV-visible absorption spectrum of an alcoholic solution of the obtained ECA monosodium salt, acidified with hydrochloric acid to convert ECA monosodium salt to echinochrome A (the abscissa axis is the wavelength λ (nm), the ordinate axis is the optical density in relative units).

In FIG. Figure 4 shows the UV-visible absorption spectrum of an acidified alcohol solution of pharmacopoeial echinochrome A used as a standard (the abscissa axis is the wavelength λ (nm), the ordinate axis is the optical density in relative units).

The UV-visible absorption spectrum of the alcohol solution of the obtained ECA monosodium salt (FIG. 3) has absorption maxima identical to the standard sample shown in FIG. 4. The presence of well-defined absorption maxima indicates a high degree of purity of the sample of the drug.

The invention is illustrated by examples.

Example 1

Raw materials - 100 kg of flat sea urchins of the species Scaphechinus mirabilis, freshly caught or defrosted, washed with drinking water to remove sea salts, sand and other impurities. The washed raw materials are dried in air, placed in a reactor and filled with 100 l of 96% ethanol with the addition of 0.3 kg of ascorbic acid “under the mirror”. The infusion time is 22 hours at room temperature. The resulting extract was drained and evaporated. Ethyl alcohol is sent for regeneration.

The concentrated residue containing a small amount of pigment is diluted with 5 L of distilled water and passed through a chromatographic column with polychrome 1. The pigment fraction is eluted with a 40% aqueous solution of ethyl alcohol. The eluate is evaporated to dryness. Ethyl alcohol is sent for regeneration.

The raw materials are dried in air and extracted with 100 l of 96% ethanol with the addition of 0.2 kg of phosphoric acid. The extraction is carried out twice at room temperature for 22 hours. The resulting extracts are poured, combined and evaporated. Distillation of ethyl alcohol is sent for regeneration. The dry residue is washed with distilled water. Pigment fractions from polychrome 1 elute with 40% ethanol. The eluate is evaporated to dryness.

The dry residue obtained in the extraction step in an amount of 1 kg is combined with an eluate concentrate in an amount of 0.01 kg and washed with distilled water.

The washed concentrated residue is dissolved in 1.5 L of a 1.0% aqueous sodium bicarbonate solution. The resulting solution is centrifuged. The sediment containing lipids is discarded. An aqueous solution of the monosodium salt of echinochrome A is dried on a freeze dryer. Obtain 0.058 kg of a reddish-brown loose powder containing 83% of the monosodium salt of echinochrome A.

Example 2

Raw materials - 100 kg of flat sea urchins of the species Scaphechinus mirabilis, freshly caught or defrosted, washed with drinking water to remove sea salts, sand and other impurities. The washed raw materials are dried in air, placed in a reactor and filled with 100 l of 96% ethanol with the addition of 0.5 kg of ascorbic acid “under the mirror”. The infusion time is 26 hours at room temperature. The resulting extract was drained and evaporated. Ethyl alcohol is sent for regeneration.

The concentrated residue containing a small amount of pigment is diluted with 5 L of distilled water and passed through a chromatographic column with polychrome 1. The pigment fraction is eluted with a 40% aqueous solution of ethyl alcohol. The eluate is evaporated to dryness. Ethyl alcohol is sent for regeneration.

The raw materials are dried in air and extracted with 100 l of 96% ethanol with the addition of 0.5 kg of phosphoric acid. The extraction was carried out twice at room temperature for 26 hours. The resulting extracts were poured, combined and evaporated. Distillation of ethyl alcohol is sent for regeneration. The dry residue is washed with distilled water. Pigment fractions from polychrome 1 elute with 40% ethanol. The eluate is evaporated to dryness.

The dry residue obtained in the extraction step in an amount of 1 kg is combined with an eluate concentrate in an amount of 0.01 kg and washed with distilled water.

The washed concentrated residue is dissolved in 2.0 L of a 1.0% aqueous sodium bicarbonate solution. The resulting solution is centrifuged. The sediment containing lipids is discarded. An aqueous solution of the monosodium salt of echinochrome A is dried on a freeze dryer. Obtain 0.060 kg of reddish-brown loose powder containing 85% monosodium salt of echinochrome A.

Claims (1)

A method of obtaining a water-soluble monosodium salt of echinochrome A from flat sea urchins suitable for use in the pharmacological and food industries, characterized in that the freshly harvested or defrosted raw materials are pre-washed with drinking water, dried, filled with 96% ethyl alcohol with ascorbic acid in the ratio of raw materials: ethyl alcohol: ascorbic acid 1: (1.0-1.2) :( 0.003-0.005) (kg: l: kg) and extracted at room temperature for 22-26 hours, then the resulting extract is drained and evaporated melt, then the concentrated residue is diluted with distilled water and passed through a column of polychrome 1, then the pigment fractions are eluted with 40% ethyl alcohol, the eluate is evaporated to dryness, then the fat-free raw materials are subjected to double extraction with 96% ethyl alcohol with the addition of phosphoric acid at room temperature in the ratio of raw materials: ethyl alcohol: phosphoric acid 1: (1.0-1.2) :( 0.002-0.005) (kg: l: kg) for 22-26 hours, then the obtained extracts are poured, combined and evaporated, and the dry residue obtained at the extraction stage, volume tinned with the eluate concentrate and washed with distilled water, then the concentrate is dissolved in a 1.0% aqueous sodium bicarbonate solution in the ratio of concentrated residue: soda solution 1: (1.5-2.0) (kg: l), then the resulting solution is centrifuged, the precipitate is discarded, and the resulting aqueous solution containing the monosodium salt of echinochrome A is dried on a lyophilic or spray dryer to obtain the desired product.

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