RU2481119C1 - Peptide, amino acid and phospholipid rich agent - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, biotechnology, as well as to pharmaceutical, food and cosmetic industries, and concerns an agent made of sea urchin viscera being waste after edible gonads have been separated. The agent with anti-inflammatory and immunotropic activity is characterised by the fact that it represents an alcoholic extract prepared of sea urchin viscera after gonad separation, containing 10-15% of peptides, 35-45% of amino acids, 4-8% of phospholipids, trace substances, and has the following characteristics: molecular weight of the peptide fractions as determined by polyacrylamide gel electrophoresis is 19-15 kDa, 6-3,5 kDa and less than 3.5 kDa. The agent is prepared by separating gonads from sea urchins, taking the viscera, extraction in ethanol in ratio 1:1-3 at temperature 0 to plus 10°C for 2 h to 10 days, separation of a liquid portion to produce a water-alcohol extract, alcohol distillation, and drying.

EFFECT: invention provides preparing the safe agent with the evident therapeutic effect.

3 cl, 7 ex, 1 tbl

Description

Invertebrates are one of the promising objects of world fishing: they account for 8-8.5% 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 preventive purposes [1].

Sea urchins are typical representatives of invertebrates and a source of a number of interesting biologically active substances [2, 3].

There is 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, chitosan, calcium from the shell; enzymes from the guts [4]. The disadvantage of this invention is the loss of part of the biologically active substances contained in the viscera, for example peptides, amino acids, minerals.

A known method for producing therapeutic and prophylactic agents, food and feed additives, culture media for the cultivation of microorganisms and eukaryotic cells. The technical result in the present invention is achieved by creating a method for producing an enzymatic hydrolyzate based on fish proteins, in which, according to the invention, a protein-containing mass is obtained from the intestines of fish or by mixing the entrails and carcasses of the fish, and the hydrolysis of the mass is carried out directly or by mixing it with water, as the agent uses calcium hydroxide, and acid - phosphoric acid. The final purification of the product (removal of pigments, specific smell and taste of fish) is carried out by chromatography on media such as Amberlight XAD. The invention provides a final product with a low content of ash (salts); reducing the duration of technological modes of the process; improving the taste properties of the resulting product by eliminating a specific smell [5]. The disadvantage of this invention is the use of hydrolysis technology, leading to a large amount of waste and wastewater requiring treatment.

A known method of obtaining a feed additive for animals from holothuria: holothuria (trepang and cucumaria) is cut, the entrails, corollas and intraperitoneal film are removed and boiled in sea, fresh or salted water for 5-15 minutes at a ratio of 1: 1 or 1: 2 ( by weight). Cooking in the same cooking medium of new portions of raw materials is carried out from 3 to 5 times, obtaining a semi-finished product for the manufacture of food products, selecting the cooking frequency so that the amount of solids in the cooking water is from 4 to 10%, and the heat-treated cucumaria is dried at a temperature of 10 to 65 ° C to a water content of 5-12% in the product, 4-8% of minerals to produce a semi-finished product for the production of a food product. The internals of holothuria, corollas and the intraperitoneal film are subjected to drainage, light pressing, freezing, grinding and drying at a temperature of 10-75 ° C, to a residual water content of 5-15%. It is cooled to ambient temperature, packaged in waterproof bags and stored at a temperature not exceeding 20 ° C for 8 months. The obtained biologically active feed additive is used as a means of increasing the resistance of animals to infectious diseases in the form of a feed additive [6]. The disadvantages of this technology are repeated evaporation at high temperatures, the presence of lipids in the final product, leading to rapid spoilage of the final product, as well as the limited use as an animal feed additive.

A known method of complex processing of bivalve burrowing mollusks to obtain gourmet products, biologically active substances and mineral-protein nutrition for animals and birds. As raw materials, shellfish of any size are used. The method includes sorting, washing mollusks, extracting mollusks and viscera from the shell of raw muscle tissue, cutting muscle tissue with separation of the motor muscle - legs and mantles with an adductor, washing and hydrothermal treatment of the latter for 5-25 minutes at a temperature of 40-55 ° C. Then the motor muscle - leg and mantle with an adductor is treated with a salted mixture or a salted mixture with an enzyme preparation, which is used as a proteolytic enzyme from king crab liver in the amount of 0.5-1.5% by weight of the raw material, washed with water and kept in an acetic solution acid pH 3.2 ± 0.2 for 20-25 minutes. The resulting raw materials are used either to obtain preserves or paste. As raw materials for the production of biologically active substances and mineral-protein nutrition for animals and birds, insides and mollusk shells are used. When cutting mollusks, the insides and shells are taken separately. The insides are crushed, dehydrated and divided into protein and fat fractions. The shells are boiled in water for 40-60 minutes and then crushed to obtain mineral flour. The protein fraction of the entrails is mixed with mineral flour and a mineral-protein feed additive for animals and birds is obtained [7]. The disadvantage of this invention is the use of enzymatic hydrolysis, resulting in a large amount of wastewater requiring treatment, and the lack of confirmed biological activity of the product obtained from the insides with the subsequent addition of this fraction to the feed additive.

Closest to the claimed method is a method of complex processing of the internals of holothuria with the production of biologically active food additives and biologically active food additives "TINGOL-2" and "EROGOL" [8], characterized in that the interiors of holothuria are crushed to particles of no more than 0, 5 mm, extracted with ethyl alcohol in a ratio of 1: 1 to 1: 3 at a temperature of minus 5 to plus 60 ° C for 5 hours to 10 days, the liquid part is separated to obtain an aqueous-alcoholic extract, then the remainder of the holothurium internals with soder by water, from 55 to 75% are dried at 60-75 ° C for 2.5-4 hours, crushed to a powder state, tabletted or encapsulated. As a result of processing the entrails of holothuria according to this method, two biologically active food supplements (BAA) are obtained: dietary supplement TINGOL-2, which is a water-alcohol extract from the entrails of holothuria containing triterpene glycosides in an amount of 700-3500 μg / ml containing alcohol 10-50% and lipids 0.5-4% and dietary supplement "Erogol", which is a dry powder containing 3-12% water, triterpene glycosides in an amount of 800-4500 μg / ml, protein 35-45%, manganese 13, 5-24.5 mg / kg, zinc 44.0-82.0 mg / kg, iron 481.2-893.5 mg / kg, copper 5.6-10.4 mg / kg, nickel 2.4- 4.2 mg / kg; and the sum of the elements of calcium, magnesium, potassium 6500-12000 mg / kg. The holothurium used as raw material is rich in triterpene glycosides.

From literature data it is known that echinoderms, in particular coelomic fluid of sea urchins, are a source of peptides [9]. However, other biologically active substances are also contained in the hedgehog's insides.

The objective of the invention is to expand the arsenal of funds with anti-inflammatory and immunotropic activity.

The problem is solved by creating a complex of biologically active substances containing peptides, amino acids, phospholipids and mineral substances of natural origin, obtained from the insides of the sea urchin, which has anti-inflammatory and immunotropic activity.

The objective of the invention is solved in that in the known method, the internals of holothuria are crushed to particles no larger than 0.5 mm, extracted with ethyl alcohol in a ratio of 1: 1 to 1: 3 at a temperature of minus 5 to plus 60 ° C for 5 hours up to 10 days, the liquid part is separated to obtain a water-alcohol extract, the remainder of the internals of holothuria with a water content of 55 to 75% is dried at 60-75 ° C for 2.5-4 hours, ground to a powder state, tabletted or encapsulated, according to the invention after separation of the gonads from the hedgehogs are extracted inside rynnosti, extracted with ethyl alcohol in a ratio of 1: 1-3 at a temperature of from 0 to plus 10 ° C for 2 hours to 10 days, the liquid part is separated to obtain a water-alcohol extract, the alcohol is distilled off, dried, used as an agent, possessing anti-inflammatory and immunotropic activity.

Moreover, the raw materials used are the insides of sea urchins in fresh, ice cream or dry form.

A distinctive feature of the present invention is:

- the use of the entrails of commercial sea urchins of any species, in particular green sea urchins;

- obtaining in the end a product that is a complex of biologically active substances containing peptides, amino acids, phospholipids and minerals, which has anti-inflammatory activity.

Fundamentally new in the present invention is the use of a complex of biologically active substances obtained from the insides of the sea urchin, which are selective inhibitors of cyclooxygenase-2 (COX-2), has an anti-inflammatory and immunotropic effect.

A new property of a complex of biologically active substances containing peptides, amino acids, phospholipids and mineral substances isolated from the entrails of a sea urchin was established by the authors for the first time experimentally.

A new property of a complex of biologically active substances containing peptides, amino acids, phospholipids and minerals isolated from the insides of the sea urchin does not explicitly follow from the prior art and its description was not found by the authors in the patent and medical literature.

As an anti-inflammatory and immunotropic agent, a complex of biologically active substances containing peptides, amino acids, phospholipids and minerals secreted from the insides of the sea urchin can be used for the prevention and treatment of various diseases, including inflammatory and allergic processes on the skin and mucous membranes: dermatitis of various etiologies; skin manifestations of diseases of internal organs, acute diseases of the joints, diseases of the nasopharynx (acute rhinosinusitis, etc.).

The technical result of the invention is to obtain a safe tool in the form of various finished dosage forms with a pronounced therapeutic effect for the treatment of inflammations of various etiologies.

Our studies have shown that fresh, frozen or dried insides of sea urchins, which are waste after the extraction of edible gonads, are not required to be crushed. The insides are filled with alcohol at a ratio of raw materials: extractant 1: 1-1: 3. The suspension is incubated at a temperature of from 0 to plus 10 ° C for 2 hours to 10 days. Heating above 10 ° C can lead to the decomposition of biologically active substances. Incubation at low temperatures requires additional cooling and does not increase the yield of the target components. It is advisable to separate the precipitate on an industrial separator or continuous flow centrifuge. After filtration and distillation of the alcohol, the solid residue is dried in one way or another, with the exception of high-temperature evaporation.

The final product is a light brown substance with a brackish taste and no unpleasant odor, does not contain toxic elements in amounts exceeding the MPC, and pathogenic microflora, i.e. is safe.

The product contains peptides 10-15%, amino acids 35-45%, phospholipids 4-8%, trace elements. When conducting polyacrylamide gel electrophoresis, it was found that peptide fractions with a molecular weight of 19-15 kDa, 6-3.5 kDa and less than 3.5 kDa remained in the final product. In the peptide substance there are no high molecular weight protein compounds (according to the qualitative reaction to the protein by the precipitation method with trichloroacetic acid).

Example 1

2.5 l of 95% ethyl alcohol are added to 1 kg of the insides of sea urchins, extracted at 5 ° C for 7 days, filtered. The resulting liquid portion was evaporated to remove the alcohol and dried. The result is 18 g of dry biologically active complex.

The final product is a light brown powder, non-hygroscopic, with a brackish taste and no unpleasant odor, does not contain toxic elements in quantities exceeding the MPC, and pathogenic microflora, i.e. complies with safety indicators.

Example 2

Solution for inhalation use (in g per 100 g)

Biologically active complex - 0.01

Preservatives - q.s.

Water for injection - up to 100

Example 3

Solution for parenteral use (in g per 100 g)

Biologically active complex - 0.1

Preservatives - q.s.

Water for injection - up to 100

Example 4. Evaluation of anti-inflammatory effects on a model of experimental acute rhinosinusitis in rats induced by intranasal administration of formalin.

The agent prepared according to example 2 was administered intranasally at doses of 0.025; 0.050; 0.100 ml / rat nasal passage for 7 days.

It was found that the agent prepared according to pr. 2 had a pronounced dose-dependent anti-inflammatory effect. The number of goblet cells significantly differed from those in the intact and control groups of animals. At the maximum dose, the agent statistically significantly reduced the severity of infiltration by mononuclear cells and leukocytes in the own plate of the mucous membrane and leukocyte infiltration in the submucosal layer. This effect is associated with the earlier disappearance of blood cells that appeared in the focus earlier (neutrophilic granulocytes) upon completion of the inflammatory process, and the later elimination of mononuclear cells with less chemotoxic sensitivity. These facts indicate recovery and reflect the pronounced anti-inflammatory effect of the composition based on a complex of biologically active substances, superior in comparison drugs.

Example 5. Evaluation of the immunotropic effect

To assess the effect of the composition on the cellular immune response in accordance with the guidelines for experimental (preclinical) study of new pharmacological substances, a delayed-type hypersensitivity reaction (HRT) was used.

In order to identify the pharmacological activity of the agent with respect to the T-cell immune response, the mice were injected with the agent prepared according to Example 3 at a dose of 40 μg / kg of mouse weight for 6 days (delayed-type hypersensitivity reaction development period). The results of the study are presented in table 1.

It was found that the agent prepared according to pr. 3 has an effect on the development of a delayed type reaction, in particular, it reduces it by almost 2 times. The data obtained indicate that a six-day administration of the drug has an immunosuppressive effect.

Table 1 Delayed hypersensitivity reaction in inbred mice with intramuscular injection (M ± m) Group The number of animals in the group Reaction index Intact 10 0.4 ± 0.1 The control 10 6.9 ± 0.9 ^* The tool according to example 3 10 3.3 ± 1.3 ^* _** * - the differences are statistically significant compared with the intact group at p <0.05, ** - the differences are statistically significant compared with the control at p <0.05.

Example 6. The effect on the enzymatic activity of COX-1 and COX-2

The influence of the agent according to pr.1 on the enzymatic activity of COX-1 and COX-2 was carried out using a commercial COX inhibitor screening assay kit (Cayman Chemical, USA). The COX-1 selective inhibitor SC-560 and the non-specific COX-2 inhibitor indomethacin were used as comparison preparations. The agent of Example 1 was investigated in a concentration range of 0.1-200 μg / ml.

The test system directly evaluates the amount of prostaglandin F2a (PGF2α) formed during the reduction of tin chloride (SnCl2) PGH2, a product of the interaction of cyclooxygenase (enzyme) and arachidonic acid (substrate). The test system includes calf COX-1 and human recombinant COX-2, thereby allowing to determine the activity of pharmacological substances in relation to both enzymes.

It was found that the tool according to example 1 has a dose-dependent inhibitory effect against the enzyme COX-2. Inhibition of the enzyme at concentrations of 100 and 200 μg / ml was 43 and 55%, respectively. The IC50 of the agent of Example 1 was 67 μg / ml. With respect to COX-1 activity was not detected. Thus, the biologically active complex from the entrails of the sea urchin is a selective inhibitor of the COX-2 enzyme.

Example 7. Evaluation of specific pharmacological activity in a model of experimental acute bronchitis induced by cigarette smoke

An experimental model of acute bronchitis caused by induction of cigarette smoke for 28 days. The agent of Example 1 was administered by inhalation at a dose of 100 μg / kg of rat body weight; Ambrobene at a dose of 125 mg / kg was used as a reference preparation.

After the course use of the drug according to example 1 in the clinical blood test, a change was noted on the part of red blood - the development of hypochromic microcytic anemia, but on the part of white blood the changes were minimal, and the indices did not differ from the intact group of animals. The indices of the antioxidant system also returned to normal — the concentration of MDA malondialdehyde was minimal and slightly exceeded intact indices. A histological examination in all animals revealed the development of acute catarrhal bronchitis with an increase in 1.9 times the thickness of the bronchial mucosa, while the inflammatory changes were minimal both in the lung tissue and in the body as a whole and were comparable to the action of the comparison drug ambrobene. In the leukocyte formula, compared with the intact group, a slight increase in the total number of leukocytes with a predominance of granulocytes was revealed.

Information sources

1. Handbook on the chemical composition and technological properties of algae, invertebrates and marine mammals / ed. V.P. Bykova. - M.: VNIRO Publishing House, 1999 .-- 262 p.

2. Vorobev V.V. Creation of bioactive pharmacological substances and medicines from marine hydrobionts // Bulletin of Biotechnol. - 2009. - T.4, No. 1. - S.33-38.

3. Bhakuni D.S., Rawat D.S. Bioactive marine natural products: Anamaya Publishers, New Delhi, India. 2005.382 p.

4. Pat. RU 2432956 C1, A61K 35/56, B01D 11/02, 08/08/2010.

5. Pat. RU 2262859, C2, A23J 1/04, A23L 3/30, A23K 1/10, 08.22.2003.

6. Pat. RU 2236155, C2, A23L 1/30, A23L 1/305, A23L 1/333, 08/05/2002.

7. Pat. RU 2231272, C2, A23L 1/33, 04/19/2002.

8. Pat. RU 2215532, C2, A61K 35/56, A61P 15/00, 10/10/2001.

9. Haug T., Kjuul A.K., Styrvold O.B. et al. Antibacterial activity in Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa (Holothuroidea), and Asterias rubens (Asteroidea) // J Invertebrate Pathology. - 2002 .-- 81 (2). - P.94-102.

Claims (3)

1. An agent with anti-inflammatory and immunotropic activity, characterized in that it is an alcohol extract obtained from the insides of sea urchins after separation of the gonads, containing 10-15% peptides, 35-45% amino acids, 4-8% phospholipids, microelements, with this has the following characteristics: the molecular weight of the peptide fractions determined by polyacrylamide gel electrophoresis is 19-15 kDa, 6-3.5 kDa and less than 3.5 kDa. 2. The tool according to claim 1, where the raw materials used are the insides of sea urchins in fresh, frozen or dry form. 3. The tool according to claim 1, obtained after separation of the gonads by extraction of the insides, their extraction with ethyl alcohol in a ratio of 1: 1 ÷ 3 at a temperature of 0 to 10 ° C for 2 hours to 10 days, separation of the liquid part to obtain water alcohol extract, distillation of alcohol, drying.