RU2645077C1 - Method for naphthoquinones obtaining from sea urchins - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: method for naphthoquinones obtaining from sea urchin includes sea urchins coelomic fluid taking as raw material, before coelomic fluid taking, sea urchins are exposed to stress by conditioning hypoxia, followed by isolation of the desired product.

EFFECT: above method is effective for naphthoquinones obtaining, moreover, it allows multiple usage of a sea urchin for coelomic fluid extraction, then the sea urchins can either be released into their natural habitat, or repeatedly used to obtain the coelomic fluid.

2 cl, 5 dwg, 4 tbl, 6 ex

Description

The invention relates to biotechnology, pharmaceutical and food industries, to the production of biologically active compounds, in particular naphthoquinones, and can be used to obtain a water-soluble form of spinochromes, which can be used in the correction of pathological disorders of lipid, carbohydrate metabolism and antioxidant status of the body.

Marine invertebrates are one of the promising objects of world fishing: they account for up to 8.5% of the catch. About 800 species of edible marine invertebrates are known, which are widely used for the preparation of food, technical, feed products, as well as for therapeutic and prophylactic purposes [Handbook on the chemical composition and technological properties of algae, invertebrates and marine mammals / Ed. V.P. Bykova. - M.: VNIRO Publishing House, 1999. - 262 s].

Sea urchins attract steady attention due to high environmental adaptability and significant regenerative potential.

It is known that naphthoquinone pigments synthesized by sea urchins have antioxidant and antibacterial properties. These include echinochrome A and spinochromes, the related hydroxylated derivatives of 5,8-dihydroxy-1,4-naphthoquinones.

In medicine, natural naphthoquinone - echinochrome A, which has anti-ischemic and anti-infarction activity, is successfully used. 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 injury of the myocardium // Cardiology. 1991. Volume 31, No. 11, p. 79-81].

Other naphthoquinones, which are part of the shell cells of shells and needles of sea urchins, which are less studied than echinochrome A, in particular, spinochrome A, can exhibit similar activity [C.W.J. Chang, J.C. Moore Pigments from Some Marine Speciments. Journal of Chemical Education, vol. 50, p. 102, 1973].

A known method for the extraction of naphthoquinones from canned flat sea urchins with a solution of inorganic acid (hydrochloric, phosphoric) in alcohol, followed by chromatographic purification of the extract on a column with chitosan in —OH form, eluting echinochrome A with acidified ethyl alcohol, neutralizing the eluate, removing the solvent, and re-dissolving the residue in chlorine with redissolved solvent or acetone, recrystallization from ethyl alcohol and drying the product [RU 2352554 C1, IPC C07C 50/32, C07C 46/10, publ. 04/20/2007].

A known method of producing naphthoquinones (quinoid pigments) from the shell of a sea urchin, which includes the following steps: cleaning and drying the shell of a sea urchin, after removing gonads and internal organs, grinding to a particle size of not more than 0.1 mm; abrasion of crushed shell with dry citric acid or monosubstituted sodium phosphate for 60 minutes; soaking and extraction in an acid solution (15 volumes) at room temperature for 5 hours, filtration and concentration in vacuo to obtain a mother liquor. The mother liquor is extracted with an aqueous solution of an acid in an organic solvent, where formic or hydrochloric acid may be used as the acid; and as an organic solvent, methanol, ethyl alcohol or acetone. The mixture is placed in the extraction vessel of a supercritical carbon dioxide extraction apparatus. The separation is carried out at 30-60 ° C at a pressure of from 20 to 50 MPa. The extracted product is frozen and dried. For further purification, chromatography is performed on a column with macroporous resin, and then on a column with reverse phase [CN 101812242 V, IPC A23L 1/275; С09В 61/00; C09B 67/04; С09В 67/10; С09В 67/54, publ. 2012.09.05].

A known method of processing the shell of a sea urchin, cleaned from the remnants of the insides and dried to obtain the pigment complex (naphthoquines). To achieve this goal, the shells are crushed, demineralized at a temperature of 40-90 ° C with a small stoichiometric excess of weak organic acid, using a 10-80% aqueous acid solution. The resulting mixture was centrifuged, the organic acid salt was precipitated by the addition of an organic solvent miscible with water, and filtered. The filtrate is concentrated, neutralized to pH 4-7 by the addition of water-soluble salts of sodium, potassium or the like, concentrated in vacuo at a temperature not exceeding 60 ° C, freeze-dried, the antioxidant pigment complex (naphthoquine mixture) is redissolved from an organic solvent [RU 2441661 C1, A61K 35 / 56, B01D 11/02, publ. 02/10/2012].

The disadvantages of the above methods are the duration and multi-stage process, the low yield of the pigment complex and the high content of organic impurities in the target product (lipids, resinous substances), the use of complex, expensive equipment for chromatography. Since the calcium and magnesium complexes of the shells and needles of sea urchins, which contain naphthoquinones, are stable compounds, this requires the use of highly active solvents to isolate them. Corrosive substances such as hydrochloric acid and sulfuric acid are used, as well as expensive reagents, including toxic substances (methanol, dioxane), which requires subsequent multi-stage purification of extracts from toxins with the inevitable loss of the final product.

Also a disadvantage of the above methods is the irrational use of biological raw materials.

A known method of complex processing of sea urchins (RU No. 2432956 C1, IPC A61R 35/56, B01D 11/02, publ. 10.11.2011), selected as a prototype. A method of complex processing of sea urchins, in which sea urchins are preliminarily divided into four fractions, caviar, coelomic fluid, viscera and carapace are obtained, then the target products are extracted: peptide-amino acid complex from coelomic fluid; gangliosides and phospholipid concentrate from caviar; the amount of quinoid pigments, calcium and magnesium from the shell; enzymes from the insides.

In this method, the amount of quinoid pigments, including echinochrome A, is obtained from the shell with needles, and coelomic fluid is used only to obtain substances of a protein nature: a peptide-amino acid complex and a protein concentrate. To do this, 1.5-3 volumes of ethyl alcohol (preferably 2-2.5 volumes) are added to the coelomic liquid, left overnight at a temperature of 3-15 ° C (preferably 8-12 ° C) and then the precipitate formed is filtered off (protein concentrate ) The extract obtained is concentrated to 0.2-0.5 volumes, the lipid layer is decanted, frozen and freeze-dried. A peptide-amino acid complex is obtained. The protein concentrate is dried.

Unfortunately, the concentration of coelomocytes and biologically active substances in the coelomic fluid is low, which does not allow making the processing of coelomic fluid economically viable.

Thus, one of the important tissues of the sea urchin, which is currently not used for the production of polyhydroxynaphthoquinones, is a coelomic fluid that fills the internal cavities and washes the internal organs, the salt composition of which is close to the composition of sea water (Barrington, 1979), but contains increased concentration of potassium chloride, lipids, proteins, sugars and wholeocytes (Smith, 1981; Chia, Xing, 1996).

The problem solved by the invention is the development of a simple method for obtaining naphthoquinones from sea urchins in vivo.

The technical result achieved by the claimed invention coincides with the task - the development of a simple method for producing naphthoquinones from sea urchins in vivo.

The developed method for producing naphthoquines from sea urchins consists in the use of coelomic fluid of sea urchins as a raw material, before taking coelomic fluid of sea urchins, they are exposed to stress by keeping them under hypoxia, followed by isolation of the target product from it.

For the method, various types of sea urchins can be used, in particular the green sea urchin Strongylocentrotus droebachiensis, the black sea urchin Mesocentrotus nudus, the sea urchins of the Strongylocentrotidae family - the red sea urchin Mesocentrotus franciscanus; Echinarachnidae family of order Clypeasteroida - flat shield-shaped sea urchin Echinarachnius parma; Arbaciidae families of the Arbacioida order: Arbacia lixula and Arb. Punctulata.

Isolation of the target product - natoquinones - from the coelomic fluid is carried out by known methods, for example, sublimation of the coelomic fluid, after its preliminary filtration or centrifugation.

The use of coelomic fluid from sea urchins as raw materials for the production of naphthoquinones does not require the use of expensive equipment and various toxic substances, and thereby ensures the development of not only a simple method for producing naphthoquinones, but also a gentle, rational one, since the same individuals of sea urchins are used to extract coelomic fluid repeatedly.

The content of sea urchins before removing coelomic fluid from them increases the number of producer cells and the concentration of naphthoquinones in the coelomic fluid, which, in the final result, allows us to offer coelomic fluid as a raw material for the production of naphthoquinones.

Stress conditions are created, in particular, by the content of sea urchins in sea water, covering the shells of sea urchins at a level of 1/3 to 2/3, at a temperature of 4-15 ° C, exposure to air at room temperature, the content of sea urchins in sea water covering the shells of sea urchins at a level of 1/3 to 2/3, at room temperature.

Under stressful conditions, sea urchins should be kept for no more than 6 days, since being in such conditions for more than 6 days leads to the death of sea urchins.

The method is illustrated by the following drawings.

FIG. 1 - the color of coelomic fluid collected from the sea urchin Strongylocentrotus droebachiensis: (a) control; (b) the sea urchin was exposed at 15 ° C for 72 hours in cuvettes with sea water, covering animals by 1/3; (c) the sea urchin was exposed to air at 4 ° C for 144 hours.

FIG. 2: (A) - coelomic fluid of the intact sea urchin Mesocentrotus nudus; (B) - coelomic fluid of the sea urchin after incubation for 96 hours under hypoxia.

FIG. 3 - ¹ N (300 MHz) NMR spectrum of the coelomic fluid of the black sea urchin Mesocentrotus (= Strongylocentrotus) nudus. 1. The upper spectrum is an intact sea urchin. 2. The average spectrum is the sea urchin after exposure for 96 hours under hypoxia. 3. The lower spectrum is the control preparation Cardio-Histochrome containing echinochrome A.

FIG. 4 - technology for the production of naphthoquinones from the coelomic fluid of sea urchins.

FIG. 5 - coelomic fluid of sea urchins Strongylocentrotus intermedius: 1 - hedgehogs after aging under stress, 2 - coelomic fluid of control animals.

Table 1 - average pH, concentration of lactic acid, solid particles and turbidity of coelomic fluid from green sea urchins, which were contained in water at 15 ° C for 72 hours; at 4 ° С for 144 hours in water or in air (lactic acid concentration is given in mmol / l, turbidity values are given in NTU (nephelometric turbidity units), total number of coelomocytes in millions of cells, naphthoquinone content in (μg / ml) )

Table 2 - signal intensity (469 and 260, 5 nm) corresponding to the naphthoquinone group.

Table 3 - the average value of the intensity of the red, green, and blue colors of the coelomic fluids of the intact and hypoxic sea urchins Mesocentrotus nudus, determined in the ColorPix program.

Table 4 - the maximums in the UV spectra in the range from 190 to 550 nm. Echinochrome (Cardio-Histochrome preparation containing echinochrome A) coelomic fluid of an intact sea urchin, coelomic fluid of a sea urchin after aging for 144 hours under conditions of hypoxia.

The method is as follows.

All experiments were carried out with two species of sea urchins - a green sea urchin (Strongylocentrotus droebachiensis) and a black sea urchin (Mesocentrotus nudus), caught from June to October 2015.

Sea urchins were delivered to the laboratory live within 24 hours after capture.

Conventional cooler bags were used to transport sea urchins. A layer of ice prepared from sea water is placed at the bottom of the bag, then sea urchins are placed (in an amount occupying no more than 2/3 of the bag’s volume when the package is loose), the hedgehogs are poured with cold sea water, another layer of sea ice is placed on top and the bag is closed.

After transportation, sea urchins were kept in aquariums with continuous aeration. Under these conditions, at a water temperature of 2-6 ° C, it was possible to keep sea urchins in a working condition for several weeks (up to 4 weeks).

Example 1

The study used 6 control individuals Strongylocentrotus droebachiensis and 18 experimental animals. Animals were caught in Aniva Bay (Sakhalin Island).

Control animals were kept in aerated aquariums with running seawater. Experimental animals were divided into three groups.

Before extracting coelomic fluid, the first group of 6 individuals was placed in cuvettes with seawater, covering the animals by 1/3, and kept at a temperature of 15 ° C for 72 hours.

The second experimental group (6 individuals) was placed in cuvettes with sea water, covering sea urchins by 2/3 of the shell, and kept at a temperature of 4 ° C for 144 hours.

The third experimental group (6 individuals) was exposed in the refrigerator at a temperature of 4 ° C for 144 hours.

Upon completion of exposure under stressful conditions caused by a lack of oxygen (hypoxia), a selection of coelomic (cavity) fluid from experimental animals was performed. Control animals also received coelomic fluid. After sampling the coelomic fluid, sea urchins were returned to aerated aquariums. They remained viable and after 10-14 days could be reused for taking coelomic fluid.

Samples of the cavity fluid were obtained using a sterile 26 gauge needle (Pst 5) (outer diameter 0.45 mm, length 13 mm, with a cone-shaped welded tip and a side outlet, designed to better protect against clogging when piercing materials), using disposable syringe. So that the needle does not enter the cavity of the Aristotelian lantern, it should be inserted into the peristomal membrane at an angle to the axis of the body.

The appearance of samples of coelomic fluid collected from different groups of Strongylocentrotus droebachiensis sea urchin is shown in FIG. 1. From the presented figure 1 it is clearly seen that the coelomic fluid of experimental animals in comparison with the control has an intense color.

The effect of exposure of S. droebachiensis hedgehogs in air or in water at 4 or 15 ° С to the color and pH of a coelomic fluid, its lactic acid content, turbidity, percent solids, naphthoquinone content, and the number of coelomocytes was studied.

The measurement of lactic acid as D-lactate was performed using a Lactate Pro ™ meter (Arkray Inc, Kyoto, Japan).

The turbidity of the coelomic fluid was measured after centrifugation (1.5 ml) of the sample at 10,000 g for 10 minutes, and the supernatant was absorbed at 600 nm using an SF-256UVI spectrophotometer (OJSC LOMO, St. Petersburg, Russia).

The percentage of solids present in the solution was measured with a hand-held refractometer (Atago Co., Ltd, Itabashi, Japan).

The pH of the coelomic fluid was determined using an Orion SA 720 pH meter with a glass electrode at 0 ° C.

Calculation of the number of coelocytes in the intracavitary fluid was carried out using a light microscope according to a standard technique using a hemocytometer.

Analysis of naphthoquinones in the abdominal fluid was carried out according to the following procedure.

Coelomic fluid (5 ml) immediately after leaving the hedgehog was placed in a chilled tube, centrifuged for 10 min at 2000 g and the supernatant was discarded. Echinochrome A was extracted from a cell pellet with 5 ml of a mixture of 50% acetone, 45% ethanol and 5% 0.1 N HCl. After stirring for 30 minutes, the tube was centrifuged for 10 minutes at 2000 g. The supernatant was used for spectrophotometry at 475 nm. The extractant was used as a control, and a solution containing 20 μg / ml of a mixture of naphthoquinones was used as a reference sample.

To obtain ¹ H nuclear magnetic resonance spectra, a Bruker spectrometer with a resonant proton frequency of 300 MHz was used. To suppress the water signal, the pre-saturation method was used and the zgpr pulse sequence was used. To adjust the uniformity of the field, 10% deuterated water (D ₂ O) was added to the sample.

Data on the change in the parameters of the light measurement of the samples were obtained using a UV-1600 spectrophotometer. Samples were tested in pure form without the addition of any solvents.

The analysis results are presented in table 1. From the presented table it follows that when the hedgehogs are kept under stressful conditions, the number of naphthoquinones increases 1.9–5.4 times.

Example 2. They took 10 control individuals of the Japanese sea urchin Mesocentrotus nudus and 10 experimental individuals. Animals were collected in the East Bay of Peter the Great Bay of the Sea of Japan.

The experimental animals were placed in cuvettes with sea water, covering the shells of sea urchins not completely at a temperature of 22 ° C. Control animals were kept in aerated aquariums with running seawater.

A day later, coelomic fluid was taken from sea urchins, while it was found that visually the coelomic fluid of the experimental animals did not visually differ from the coelomic fluid of the control animals. However, in the UV spectra of the coelomic fluid of the sea urchin, which is under hypoxic conditions for 24 hours, an absorption region appears at a wavelength of 469.0 nm. A similar absorption region is also observed in the analysis of the control preparation Cardio-Histochrome, which contains a water-soluble form of echinochrome A. Table 2 shows the signal intensities (469 and 260, 5 nm) corresponding to the group of naphthoquinones in both control and experimental animals.

As can be seen from table 2, the method allows to stimulate the synthesis of naphthoquinones in coelomic fluid without the use of any additional equipment.

Example 3

10 control specimens of the Japanese sea urchin Mesocentrotus nudus and 10 experimental specimens were taken. The experimental animals were placed in cuvettes with sea water that did not completely cover the shell of the sea urchins and kept for 4 days at a temperature of 4 ° C to slow their metabolism and better survival under stress. The control animals were kept in aerated aquariums with running sea water. Four days later, coelomic fluid is extracted from sea urchins. The difference in the color of the coelomic fluid of the experimental and control animals becomes visually distinguishable (see Fig. 2, where A is the coelomic fluid of the intact sea urchin Mesocentrotus nudus; B is the coelomic fluid of the sea urchin after incubation for 96 hours under conditions of hypoxia). As follows from Table 3, which presents the average intensity value of the red, green, and blue colors of the coelomic fluids of the intact and hypoxic sea urchins Mesocentrotus nudus, determined in the ColorPix program, the most noticeable change is observed in the red range by 19 units, which indicates the appearance in coelomic fluid quinoid pigments (naphthoquinones).

In the UV spectra of the coelomic fluid of sea urchins under hypoxic conditions for 96 hours (the coelomic fluid for analysis was taken from animals aged under the conditions of Example 3), in contrast to the coelomic fluid of intact hedgehogs, an absorption region appears at a wavelength of 469.0 nm, which is also in the spectrum of the Cardio-Histochrome preparation containing echinochrome A. Table 4 shows the maxima in the UV spectra in the range from 190 to 550 nm. Echinochrome (Cardio-Histochrome preparation containing echinochrome A) of the coelomic fluid of the intact sea urchin, coelomic fluid of the sea urchin after standing for 96 hours under hypoxic conditions.

The UV spectra presented also confirm the presence of naphthoquinones in the coelomic fluid of the hedgehog subjected to stress caused by hypoxia.

In intact sea urchins in physiologically comfortable conditions, in tissues, in particular in coelomic fluid, the concentration of quinoid pigments is lower than the sensitivity level of most analytical instruments, i.e. lower than noise level. After keeping sea urchins under stress caused by a lack of oxygen dissolved in water, the concentration of naphthoquinones becomes comparable to natural metabolites and can exceed 0.1 mmol / l, which makes it available for isolation. By NMR spectroscopy, signals from the ethyl and methyl groups of naphthoquinones in the coelomic fluid of stressed sea urchins are clearly detected. In FIG. 3 shows - ¹ N (300 MHz) NMR spectrum of the coelomic fluid of the black sea urchin Mesocentrotus (= Strongylocentrotus) nudus, where 1. The upper spectrum is the intact sea urchin; 2. The average range is the sea urchin after exposure for 144 hours under hypoxia; 3. The lower spectrum is the control preparation Cardio-Histochrome containing echinochrome A.

Example 4

A schematic diagram of the preparation of a mixture of biologically active naphthoquinones is given in FIG. four.

Take 50 individuals of the green sea urchin S. droebachiensis and expose in air at 4 ° C for 144 hours. At the end of the exposure, coelomic fluid is removed from the individuals. For this, the peristomal region of the sea urchin is washed with sterile sea water and an incision is made in the peristomal membrane. Coelomic fluid is collected in a clean plastic container without the addition of an anticoagulant. The volume of collected coelomic fluid was about 2.5 liters. To it add the same amount of extracting mixture containing 50% acetone, 45% ethanol and 5% 0.1 N HCl. The resulting liquid was stirred with a mechanical stirrer at room temperature for 30 minutes. The mixture is then centrifuged at 3000 g for 15 minutes. The supernatant is neutralized with baking soda to a pH of 4.0-4.5, evaporated to completely remove organic solvents and lyophilized. The lyophilisate is dissolved in 96% ethanol, filtered and evaporated until the solvent is completely removed.

The dry residue was dissolved in acetone, filtered and evaporated again. The resulting fine powder is crystallized from ethyl alcohol and dried in a vacuum oven to constant weight. The result was 263 mg of naphthoquinone.

Example 5

10 control specimens of the Japanese Sea-urchin Strongylocentrotus intermedius and 10 experimental specimens were taken. Experimental animals were placed in cuvettes with sea water so that the water covered up to 1/3 of the carapace. More than half of the animal was in the air. Control animals were kept in aerated aquariums with running seawater. The vitality of the hedgehogs was determined by their ability to move with the help of ambulacral legs and needles.

The experimental animals were kept for 4 days at a temperature of 4 ° C to slow their metabolism and better survival of animals under stress. After four days, the difference in color of the coelomic fluid of the experimental and control animals becomes visually distinguishable (see Fig. 5). The most noticeable change is observed in the red range.

Example 6

The study also used 10 control individuals of the flat sea urchin Scaphechinus mirabilis and 10 experimental individuals. The experimental animals were placed in cuvettes with sea water. Control animals were kept in aerated aquariums with running seawater.

Experimental animals were kept for 4 days at a temperature of 6 ° C to slow their metabolism and better survival of animals under stress conditions. After four days, the difference in color of the coelomic fluid of the experimental and control animals becomes visually distinguishable (Fig. 5). The most noticeable change is observed in the red range.

The claimed method offers for the production of naphthoquinones a new raw material, which until today has not been used for these purposes. Naphthoquinones of sea urchins are obtained from coelomic fluid, which is contained in the body cavity of sea urchins, limited by their biomineral carapace, internally coated with coelomic epithelium. In intact individuals, coelomic fluid is transparent and contains almost no pigments. Pigments appear in the coelomic fluid of sea urchins subjected to stress when kept under hypoxia. This is a water-soluble form of naphthoquinones, which can be distinguished, for example, by sublimation of coelomic fluid after its preliminary filtration or centrifugation. The method is simple to implement, does not require expensive equipment and is rational and resource-saving, as individuals of sea urchins after extracting coelomic fluid from them can either be released into the natural habitat or reused to produce coelomic fluid.

Claims (2)

1. The method of producing naphthoquinones from sea urchins, which consists in the fact that the coelomic fluid of sea urchins is taken as a raw material, before taking coelomic fluid of sea urchins, it is subjected to stress by exposure to hypoxia and subsequent isolation of the target product from it. 2. The method according to p. 1, characterized in that under conditions of hypoxia of sea urchins can withstand no more than 6 days.

Images