RU2714128C1 - Composition of antimicrobial peptides prepared from muscars domestica larvae, and a method for production thereof - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: group of inventions refers to veterinary medicine, namely to pharmaceutics, and can be used for treating infections of bacterial aetiology. Method of producing antimicrobial peptides from Musca domestica larvae comprises grinding a weighed portion of the larvas in a mortar with sand to a homogeneous mass with the addition of a solution in parts consisting of 0.25 % sodium azide and a phosphate buffer, stirring for 4 hours, centrifugation, extraction of aqueous layer containing antimicrobial peptides. Then salting-out is carried out by adding to isolated solution (NH₄)₂SO₄, placing solution into freezer at t=5–10 °C for 24 hours, centrifuging for 40 minutes at t=5 °C at rate of 4,200 rpm, and chromatographic separation of peptides with separation of a fraction of a mixture of peptides with weight of 3.4–6 kDa. Obtained mixture is subjected to dialysis for 24 hours against 0.9 % sodium chloride, passed through a filter and packaged. Group of inventions also relates to a composition of antimicrobial peptides containing a mixture of antimicrobial peptides from Musca domestica larvae obtained by said method.

EFFECT: group of inventions provides preparing peptides from Musca domestica larvae having antibacterial activity.

2 cl, 1 dwg, 2 tbl

Description

Composition of antimicrobial peptides derived from Musca domestica larvae and method for its preparation

Application area

The invention relates to the field of veterinary medicine, namely to pharmaceuticals, and can be used to treat infections of bacterial etiology.

One of the important tasks of modern agriculture is the intensification of production. An increase in livestock production is achieved, inter alia, by the use of antibacterial drugs. However, this leads to the selection of antibiotic-resistant strains to known antibacterial drugs, so the search for new alternative compositions is an urgent task.

There are many different injectable compositions for treating infections of bacterial etiology in animals, for example, based on: azithromycin (RU 2512683 C2, 04/10/2014 and CN 102846649), flunixin and chloramphenicol (MY 135791 A), flunixin and enrofloxacin (EP 1875913 and CN 102697784 ), flunixin, enrofloxacin, sulfadiazine, trimethoprim and dexamethasone (CN 101829124), flunixin, enrofloxacin, trimethoprim, tylosin and sulfamethoxazole (CN 102697784), flunixin and oxytetracycline (PL 335878). All of the above compositions have a wide spectrum of action, are stable during storage, have a prolonged effect and high therapeutic effect, however, a large number of microorganisms at the given time have developed resistance to these active substances.

II. State of the art

One of the promising areas in the claimed field of application is the use of insect peptides with high antibacterial activity as the active substance. At this time, some antimicrobial peptides with high antimicrobial activity have been obtained, which are isolated from various organisms, for example: derivatives of the melanocyte stimulating hormone (US 6887846), retrocyclins (WO 044998), magainin (US 6872705), mucin derivatives (US 6790833), biocidal peptide (RU 103887, 08/06/2018 bull. No. 22), recombinant polypeptide (RU 123977, 01/17/2018 bull. No. 2), alloferon peptide (RU 2667128, 07/02/2018 bull. No. 19), a recombinant polypeptide having a serine protease activity (RU 142580, 05/07/2018 bull. No. 13), human defensins (WO 081486, US 034820, WO 98 07833, JP 288105, WO 9421672), crustacean antimicrobial peptides (US 6642203), cryptdin (WO 9616075), synthetic cationic peptides (US 6906035, US 6624140, US 6172185), defensin-like peptide of the dragonfly Aeschna cyanobacteriophobia 26, (26) Calliphora vicina (US 6337093), antibacterial peptides of the beetle Oryctes rhinoceros (US 6476189).

Currently, three main methods for producing antimicrobial peptides are used: chemical synthesis, extraction from the host organism, synthesis in the culture of producer cells.

Each method has its advantages and disadvantages.

So the technology of chemical synthesis does not allow to obtain high mass peptides, since the synthesis of a peptide with a sequence of more than 30 amino acids is difficult, however, this method allows to obtain peptides in large quantities and high purity, but has high cost and difficulty in the synthesis of long chains with three-dimensional organization.

Synthesis in the culture of producer cells usually comes down to the use of genetic engineering methods. Genetic engineering makes it possible to obtain peptides of any length, however, in some cases it does not allow to accurately reproduce the spatial structure of the peptide and usually has a significant cost of the final product.

The method of extraction from the host organism allows one to obtain entire complexes of antimicrobial peptides and ensures the most accurate reproduction of the structure of active components, however, this method does not allow peptides to be isolated in a pure form and most often they are a mixture characterized by a certain mass range.

Closest to the proposed invention is the application for invention No. 2017144051 "A method for producing a complex of antimicrobial peptides from the biomass of large wax moth larvae (Galleria mellonella)", where wax moth larvae were used as the initial substrate.

III. SUMMARY OF THE INVENTION

The aim of this invention is to provide a new pharmaceutical composition of antimicrobial peptides derived from Musca domestica larvae.

The claimed invention uses the latest method for the isolation of antimicrobial peptides from Musca domestica larvae.

The technical result of the invention consists in the isolation of peptides having antimicrobial activity from Musca domestica larvae by fractional salting out method with further separation on a BioSep SEC S2000 chromatographic column.

The method for producing a pharmaceutical composition based on antimicrobial peptides from Musca domestica larvae consists of three main stages:

1. Salting out

2. Chromatographic separation

3. The creation of the final pharmaceutical composition.

Salting out or extraction of water soluble peptides from a biological sample (Musca domestica larvae).

A portion of larvae (Musca domestica) weighing 3 g is ground in a mortar with sand 5 g to a homogeneous mass. Next, in the process of grinding, we add in parts a solution consisting of 0.5 ml of sodium azide 0.25% and 20 ml of phosphate-salt buffer and mix for four hours. Next, the obtained samples are centrifuged at 4200 rpm and an aqueous layer containing antimicrobial peptides is taken. For salting out, it is necessary to add 2.8 g of (NH4) 2So4 to the isolated solution per 10 ml of solution (sample). Next, complete dissolution of the salt is achieved. After which the solution is placed in a freezer at a temperature of 5-10 ° C for 24 hours. Next, the samples are centrifuged for 40 minutes at t = 5 ° C at a speed of 4200 rpm. After centrifugation, the aqueous layer is used for chromatographic separation.

The second step in the preparation of the pharmaceutical composition is the chromatographic separation of peptides and their purification.

Separation was carried out under the following conditions: BioSep S2000 column 300 × 2120 mm, wavelength 280 nm, loop volume 1575 μl, eluent 0.1 M phosphate buffered saline, flow rate 1.0 ml / min, column temperature 25 ° C .

A sample of the test solution was injected into the chromatographic column using a syringe. The analysis time for one sample takes 60 minutes. A mixture of peptides with antimicrobial activity was isolated at a retention time of 18-22.5 minutes. A total of six fractions were collected for the studies, presented in table 1 and figure 1.

The retention time was determined according to the standards analyzed individually before administration of the test sample, the retention time of porcine insulin with a retention time of 18 minutes and a mass of 6 kDa and insulin from the pancreas of the bovine 22.5 minutes, weight 3.4 kDa. Thus, a mixture of antimicrobial peptides from Musca domestica larvae with a mass of 3.4-6 kDa was isolated. Next, the resulting mixture was dialyzed for 24 hours against a 0.9% sodium chloride solution.

Third stage

To create the final pharmaceutical composition, the resulting solution was diluted with 0.9% sodium chloride in water to a peptide concentration of 1.12 mg / ml, after which a 9: 1 (by volume) 10% aqueous solution of benzyl alcohol was added and the obtained pharmaceutical composition was passed through a 0.45 μm filter, and then packaged in sterile vials.

Preparation of culture medium and physiological saline

Cooking BCH. To prepare 1 liter of MPB, 20 g of culture medium are weighed, added to 1 liter of distilled water and mixed. The solution is put on fire, heated to complete dissolution and boiling.

Preparation of a suspension of microorganisms

Antibacterial activity was determined according to the method of MUK 4.2.1890-04 Determination of the sensitivity of microorganisms to antibacterial drugs. Methodical instructions.

The following cultures were selected for the experiment: Bacillus cereus ATCC 10702, Candida albicans RKSPU-401 / ISTS-885-653, Staphylococcus aureus ATCC 6538 (209-P), Salmonella typhimurium 1626.

It was revealed that the protein fraction No. 2 obtained from the larva of Musca domestica has antimicrobial activity (Table 2). It is observed that this fraction is active in relation to all the studied strains.

Thus, it was revealed that the protein fraction No. 2 obtained from the larvae of Musca domestica has antimicrobial activity.

Claims (2)

1. A method of obtaining antimicrobial peptides from Musca domestica larvae, including grinding a sample of 3 g larvae in a mortar with sand 5 g to a homogeneous mass with the addition of a solution consisting of 0.5 ml of 0.25% sodium azide and 20 ml of phosphate buffer in parts , stirring for 4 hours, centrifugation at 4200 rpm, selection of the aqueous layer containing antimicrobial peptides, salting out by adding 2.8 g of (NH ₄ ) ₂ SO ₄ to the selected solution per 10 ml of solution, placing the solution in a freezer chamber at t = 5-10 ° C for 24 hours, centrifugation is ongoing 40 minutes at t = 5 ° C at a speed of 4200 rpm, chromatographic separation of peptides in BioSep S2000 columns 300 × 2120 mm at a wavelength of 280 nm, a loop volume of 1575 μl, using an eluent of 0.1 M phosphate buffered saline, at a flow rate of 1 ml / min and a column temperature of 25 ° C, isolation of a fraction of a mixture of peptides with a mass of 3.4-6 kDa with a retention time of 18-22.5 min, dialysis of the resulting mixture for 24 hours against a 0.9% solution sodium chloride, passing through a filter and packaging. 2. The composition of antimicrobial peptides obtained from Musca domestica larvae having antibacterial activity, containing a mixture of antimicrobial peptides from Musca domestica larvae obtained by the method according to claim 1, at a concentration of 1.12 mg / ml, 10% aqueous solution of benzyl alcohol and a buffer system comprising a 0.9% aqueous solution of sodium chloride as an osmotic pressure regulator, wherein a solution of peptides in a 0.9% aqueous solution of sodium chloride and a 10% aqueous solution of benzyl alcohol are taken in a volume ratio of 9: 1.

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