RU2545703C1 - Method for producing glycolised polypeptide agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for producing an anti-inflammatory glycolised polypeptide agent with the use of commercial sea urchin (Strongylocentrotus droebachiensis) waste. A method involves extracting sea urchin inners in ethanol, separating the extract in a separator or a centrifuge, evaporating it, filtering the concentrate, performing gel chromatography, separating a fraction having molecular weight 5.5-7.0 kDa and drying it in the certain environment. The produced agent possesses the pronounced anti-inflammatory action.

EFFECT: method enables improving purity of glycolised polypeptides.

3 cl, 4 tbl, 1 dwg, 4 ex

Description

The invention relates to the processing technology of natural objects and relates to a method for producing funds based on glycosylated polypeptides from waste processing of commercial sea urchins Strongylocentrotus droebachiensis.

The biological diversity of the oceans is an almost unlimited source for the search for new biologically active compounds. Substances isolated from marine animals, algae and bacteria have a wide range of biological activity, including antibacterial, anticoagulant, anti-inflammatory, antiallergic, antifungal, antiplatelet, immunomodulating, antitumor, proapoptotic and other activities.

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 p.].

During the extraction of mollusk tissues, unique components are distinguished with antitumor activity. The bivalve mollusk extract of Mercenaria is able to inhibit the growth of malignant neoplasms. The dialyzed extract from the tissues of the mercenary in an in vitro experiment suppressed the growth of sarcoma 180 and Krebs sarcoma [Pat. US 3655875, 04/11/1972].

A known method of producing a carbohydrate-protein complex with antitumor and immunostimulating activity obtained from the mantle of the scallop using extraction with saline followed by dialysis and drying [Pat. RU 2121844, publ. November 20, 1998]. Known biologically active food supplement, which is a hydrolyzate from the waste of cutting scallop.

A method for producing a biologically active additive by acid hydrolysis of scallop butchering was also proposed. After hydrolysis, neutralization and evaporation of the mass are carried out. Then carry out cooling and re-neutralization of the mass. Ready hydrolyzate is separated from the precipitate [US Pat. RU 2192149 C1, publ. November 10, 2002].

Sea urchins are typical representatives of invertebrates and a source of a number of interesting biologically active substances [Vorobev V.V. Creation of bioactive pharmacological substances and medicines from marine hydrobionts // Bulletin of Biotechnol. - 2009. - T.4, No. 1. - S.33-38].

According to Y. Nishida et al. (2007) in the digestive tract of the sea urchin contains a large number of substances of protein nature with a wide range of molecular weights, including low molecular weights in the region of 10 kD [Nishida Y., Suzuki K., Kumagai Y. et al. Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus // Biochimie. - 2007 .-- Vol.89, N8. - P.1002-1011].

A known method of producing a glycopeptide having anti-inflammatory action from tissue of the gastrointestinal tract of animals, including mechanical cleaning of raw materials, freezing, grinding, extraction with a solution of acetic acid, filtration, processing of the filtrate with acetone, lyophilization of the obtained precipitate containing the target product, dissolving it in distilled water followed by further cleaning [SU 1697315 A1, publ. 04/10/1999].

Known peptide conjugate containing a glycosylated peptide that is covalently linked to a fragment of polyethylene glycol (PEG) through an intact glycosyl linking group. The invention provides a new chemical compound containing a branched PEG molecule, a sialyl linking group and a peptide with reduced antigenicity [RU 2460543 C2, publ. 09/10/2012].

A known method of obtaining biologically active substances of glycopeptide nature by treating the culture fluid of lactic acid bacteria with acidic proteolytic enzymes, the subsequent separation by centrifugation of the culture fluid into biomass and whey, which are processed separately. Milk whey is separated by ultrafiltration into high and low molecular weight fractions. The high molecular weight whey fraction is lyophilized. The low molecular weight whey fraction is evaporated to give lactic acid and its salts. The biomass is suspended in ammonia water and separated by centrifugation into a liquid phase and a solid. The liquid phase is concentrated by ultrafiltration. A solution of acetic acid zinc is added to the resulting concentrate to obtain a zinc-containing complex. The solid phase (biomass) for destruction of the cell walls is subjected to ultrasonic treatment or processing "freezing-thawing". Then boil, cool, add lysozyme and hydrolyze. The hydrolyzate is acidified with acetic acid and separated into solid and liquid phases by centrifugation. The solid phase obtained from the hydrolyzate is dried and receive a preparation containing the remains of cell walls and undissolved glycopeptide. The liquid phase obtained from the hydrolyzate is fractionated by ultrafiltration into a high molecular weight fraction and a low molecular weight fraction. The obtained high molecular weight fraction is lyophilized to obtain a preparation containing lysozyme and a high molecular weight glycopeptide. The low molecular weight fraction is chromatographed, filtered, evaporated and lyophilized to obtain a preparation [RU 2304167 C2, publ. 08/10/2007].

The known method describes the preparation of a product containing glycopeptides from cells of lactic acid bacteria. However, during technological processing, the biomass of lactic acid bacteria to destroy the cell walls is subjected to ultrasound or freeze-thaw treatment, boiling and lysozyme treatment, after which water-insoluble glycopeptides are separated from the suspension of lysozyme hydrolyzate by micro-, ultra-, nanofiltration and chromatography. The use of water-insoluble glycopeptides limits their use and bioavailability. In addition, a significant amount of excipients is required to dissolve such compounds and increase their bioavailability.

Known biologically active food supplement, representing an alcoholic extract from the guts of Japanese cucumaria, containing triterpene glycosides, hexosamines and minerals and possessing antioxidant, immunomodulating, cytostatic activity [TU 9265-075-00472021-96].

A known method of laboratory analytical production of peptides from sea urchins Echinus esculentus L. by extraction with acetone, evaporation, separation on a set of five cartridges C18 Sep-Paks (Waters, Milford, MA, USA) by successive elution of 20%, 40%, 60% and 80% acetonitrile (ACN) in 0.1% TFA. Next, 50 μl of samples obtained by eluting with 20%, 40%, 60% and 80% ACN in TFA were analyzed for the presence of SALMFamide peptides using radioimmunoassay. Sep-Pak eluates containing SALMFamide peptides were loaded onto a C8 column (Brownlee RP300, Perkin-Elmer, Boston, MA, USA; 2.1 mm × 220 mm) and eluted by HPLC with 0-80% ACN / TFA at a rate of 0.5 ml / min for 30 minutes Fractions for analysis of SALMFamide-like immunoreactivity were collected every 0.5 min [Elphick M.R., Thorndyke M.C. Molecular characterization of SALM Famide neuropeptides in sea urchins // J Exp Biol, 2005, Vol.208, P.4273-4282].

However, the known method is laboratory analytical. SALMFamide peptides with immunoreactivity were isolated as fractions by multistage HPLC in order to establish their genome. It is not possible to obtain peptides in an amount sufficient to confirm its activity by this method.

A known method of isolating fasciclin I-like protein with a molecular weight of 33 kDa from the gonads of the sea urchin Strongylocentrotus intermedius using hydrophobic chromatography and gel filtration [Sato K., Nishi N., Nomizu M. Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin Strongylocentrotus intermedius) ovaries // Arch Biochem Biophys, 2004, Vol.424 (1), P.1-10].

However, the known method allows you to select non-glycosylated protein with a molecular weight of 33 kDa from the gonads of sea urchins Strongylocentrotus intermedius, and not a low molecular weight glycosylated polypeptide.

Closest to the claimed method according to the technical essence is a method of obtaining funds containing 10-15% of peptides, 35-45% of amino acids, 4-8% of phospholipids and trace elements and obtained after separation of gonads from sea urchins, which consists in extracting the insides, their extraction with ethyl alcohol in a ratio of 1: 1-3 at a temperature from 0 to plus 10 ° C for 2 hours to 10 days, separating the liquid part to obtain a water-alcohol extract, distilling off the alcohol, drying [Pat. RU 2481119 C1, publ. 05/10/2013].

The disadvantage of this method is to obtain a complex preparation, which includes both low molecular weight oligopeptides, amino acids and minerals, and polypeptides. In addition, a significant amount of lipid compounds passes into the alcohol extract, which co-precipitate on the surface during the drying process, the process of “sticking” of the obtained product and its rapid oxidation take place.

The objective of the invention is to obtain funds based on a glycosylated polypeptide while maintaining its native properties, increasing the purity and stability of the final product.

This problem is solved in that in the known method, including the extraction of the insides of sea urchins, their extraction 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 separation of the liquid part to obtain water-alcohol extract, distillation of alcohol, drying of raw materials, according to the invention, fresh, frozen or dried insides of sea urchins, pour ethyl 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 in for 2 hours to 10 days, sediment they are removed by a separator or centrifuge, the extract is evaporated to 1 / 50-1 / 70 volume, the concentrate is passed through a filter with a pore size of 1 kDa, subjected to gel chromatography, the fraction with a molecular weight of 5.5-7.0 kDa is separated, the separated fraction, containing a glycosylated polypeptide, dried.

The technical result provided by the invention is to increase the degree of purification of the glycosylated polypeptide.

Distinctive features in the prototype method and the claimed method are:

- concentration by molecular weight and cutting off compounds with a molecular weight of less than 5.5 kDa and more than 7.0 kDa;

- fractionation and isolation of glycosylated polypeptide using gel chromatography.

Using the combination of these distinctive features allows you to increase the purity of the target product, because when concentrated using membranes with a limit of 1 kDa, you can get rid of trace elements, amino acids and oligopeptides. Further fractionation by gel filtration allows to obtain a glycosylated polypeptide of high purity.

The invention is illustrated by the following examples.

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 part is concentrated to 1/50 of the original volume on a vacuum evaporator, and then the concentrate for removing trace elements, amino acids, oligopeptides is filtered on an Amicon Ultra-15 1K - 1000 NMWL filter with a pore size of 1 kDa to a final concentration of 1 mg / ml. The concentration of peptides is determined by the method of Warburg and Christian, based on the measurement of the optical density of the solution at wavelengths of 280 nm and 260 nm and further calculation of the concentration of peptides. After the concentration step, the extract obtained is purified from other impurities by gel chromatography using Sephadex molecular sieves. The resulting liquid portion is dried. The result is about 0.55 g of dry purified funds based on glycosylated polypeptides with a molecular weight of 5.5-7.0 kDa.

The content of galactose in it is 30-35%, fucose is 1.4-1.6%, the degree of sulfation is 0.90-0.10%.

Example 2. Determination of glycosylated polypeptides using HPLC analysis on a column with a reversed phase.

The glycosylated polypeptide-based agent was analyzed by HPLC on a Jupiter C ₁₈ 300A column, particle size 5 μm, 4.6 × 250 mm in isocratic elution mode using 3% acetonitrile: 97% 0.03% trifluoroacetic acid (TFA) solution. Detection at a wavelength of 215 nm (Fig. 1).

According to the results of quantitative assessment by the method of internal normalization, the component with a retention time of about 6.27 minutes is dominant. Its content is about 80.0%.

Example 3. Anti-inflammatory effect on an experimental model of acute bronchitis in rats.

Acute bronchitis was modeled by the introduction of formalin into the nasal passages of rats [Guidelines for preclinical studies of drugs, ed. Mironova A.N. - Part one. - M .: Grif and K, 2012. - 944 p.]. Acute inflammation was induced by administering endotracheally 1% formalin solution to rats on the 1st day of the study. An agent based on glycosylated polypeptides and an ambroxol comparison drug (Ambrobene, oral and inhalation solution, Ratiopharm GmbH, Germany) were administered by inhalation within 7 days after the pathology was induced. The solvent was water for injection. The effectiveness of the drugs was evaluated by the severity of histological changes in lung tissue (table 1).

A histological evaluation of lung tissue showed that the agent according to Example 1 at doses of 19 and 38 μg / kg had a pronounced anti-inflammatory effect, which was expressed by a decrease in infiltration of the mucous and submucous membranes of the bronchi, as well as a significant decrease in the number of goblet cells by 34 and 45% compared with a control group. Most animals treated with the glycosylated polypeptide based on Example 1 at doses of 19 and 38 μg / kg revealed the easiest pathology option - post-inflammatory goblet cell hyperplasia. The effectiveness of the use of the agent according to example 1 in both doses was comparable with the effectiveness of the comparison drug ambroxol.

Example 4. Anti-inflammatory effect on a model of chronic obstructive pulmonary disease in rats.

Chronic obstructive pulmonary disease (COPD) was modeled by daily smoking of rats with tobacco smoke in the tanks of a smoking machine for 28 days [Groneberg D.A., Fan Chung K. Models of chronic obstructive pulmonary disease // Respir Res. 2004; 5 (1): 18]. The induction of chronic inflammation was induced by daily placing animals for 6 hours in the tank of a smoking machine, where concentrations of nicotine 28 mg / kg and resin 357.6 mg / kg were generated. The following groups were investigated: intact - without pathology, without treatment; control - with pathology, without treatment; a group receiving a comparison drug ambroxol against a pathology background; 3 groups receiving an agent based on the glycosylated polypeptides of Example 1, in 3 doses against a pathology background. The drugs were administered daily by inhalation from the 14th to the 28th day of the study. Evaluation of the effectiveness of the drugs was carried out according to the degree of change in antioxidant status (reduced glutathione, malondialdehyde (MDA) in blood plasma) (Table 2), the change in the number of leukocytes in bronchoalveolar lavage (table 3) and the severity of histological changes in lung tissue (table 4).

The use of glycosylated polypeptides based on Example 1 against a background of COPD protected the bronchi from the negative effects of tobacco smoke, reduced the severity of inflammation and had an antioxidant effect. Against the background of the use of the agent according to example 1 on day 28 of the development of pathology in animals, an increase in the concentration of reduced glutathione by 43-44% and a decrease in the level of MDA by 34-45% relative to the control group were noted.

Against the background of the developed pathology, a statistically significant increase in the number of leukocytes in bronchoalveolar lavage was 2 times compared with the intact group. The use of a drug based on a glycosylated polypeptide in 3 doses, as well as a comparison drug for 14 days, led to a decrease in the number of leukocytes.

When assessing the severity of inflammatory changes in lung tissue, it was found (Table 4) that the use of the agent according to example 1 led to a marked decrease in the processes of infiltration of the mucosa and submucous membranes, a complete absence of exudate in the lumen of the bronchi or a decrease in the amount of exudate containing single macrophages. Table 4 - Thickness of the bronchial mucosa, (M ± m)

Histological examination revealed a decrease in the thickness of the bronchial mucosa in the group treated with the agent according to example 1, by 34-47% relative to the control group of animals, which indicates a marked decrease in inflammatory hyperplasia of the bronchial epithelium. All the effects of the glycosylated polypeptide-based agent of Example 1 were comparable to the ambroxol comparison drug.

Claims (3)

1. The method of obtaining funds based on glycosylated polypeptides with anti-inflammatory action, including extraction of the insides of sea urchins Strongylocentrotus droebachiensis with ethyl alcohol at a ratio of raw materials: extractant 1: 1-1: 3 at a temperature from 0 to plus 10 ° C for 2 hours to 10 days, separating the extract on a separator or centrifuge, evaporating it to 1 / 50-1 / 70 volume, filtering the concentrate through a filter with a pore size of 1 kDa to remove trace elements, amino acids, oligopeptides, gel chromatography of the concentrate, separation fraction with a molecular weight of 5.5-7.0 kDa and its drying. 2. The method according to claim 1, characterized in that use fresh, frozen or dried insides of sea urchins. 3. An agent based on glycosylated polypeptides obtained by the method according to claim 1, having an anti-inflammatory effect.

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