PL234591B1 - Method for isolating snail mucus, composition and application of the Arion rufus snail mucus - Google Patents

Abstract

The subject of the application is a method of isolating the Arion rufus snail mucus, the composition and also the use of the mucus for the production of preparations with bacteriostatic and bactericidal properties. The preparations can be applied to the skin and mucous membranes externally and internally. The described method uses a commonly available species of snail, is harmless to animals and, thanks to the use of aqueous solutions, does not cause waste generation.

Description

Description of the invention

The subject of the invention is a method of isolating mucus, a composition containing this mucus, and its use for the production of preparations with antibacterial properties. The preparations can be applied to the skin and mucous membranes, externally and internally.

It is known from the patent application No. BG 20100110665 to use the antibacterial properties of the mucus and hemolymph of shell snails, Helix aspersa and Rapana venosa. However, these are species that do not occur naturally in Poland, what is more, their breeding requires artificial and often costly conditions, which means that their use to obtain compounds with biological activity is significantly limited.

Unexpectedly, it turned out that the mucus of snails Arion rufus exhibits bacteriostatic activity against bacteria that are pathogenic for humans, causing inflammation of the gastrointestinal tract, which in some cases may develop into a general disease process involving internal organs and sepsis.

From the patent applications WO2007124625 and US2010048457, a method of isolating specific molecules (glycoproteins and polysaccharides) from the body of snails (glycoproteins and polysaccharides) is known, which consists in homogenizing whole snails and then extracting these molecules with ethanol. However, shaking in ethanol can degrade the mucus proteins, is harmful to snails, difficult to dispose of and harmful to the environment.

The aim of the invention was to develop a method of isolating the mucus itself, allowing the snails to be reused for the production of mucus, with the use of an iso-osmotic solution towards snails, harmless to proteins, the environment and easy to dispose of.

The snail of the species Arion rufus belongs to the salivary family (Arionidae), their body is elongated, brown-brown in color, with a darker foot and antennae. Their Polish name is the red slug. They are abundant in Poland, are not protected species and are considered pests. It is a species that lives near human settlements such as gardens, orchards, and parks. It is a species that feeds on almost every type and part of garden plants, both cultivated and ornamental, mainly their ground parts. For this reason, it is considered a pest.

The essence of the method according to the invention is that live Arion rufus snails are shaken in a physiological NaCl solution in such a way as to allow the snail to be washed away from the mucus, while surviving the individuals. Shaking is continued until the nature of the solution changes and is as uniformly gelatinous as possible. The weight-volume ratio of the weight of the snails to the amount of NaCl solution should be selected so as to maintain the stabilization of proteins in the mucus. Subsequently, after the animals are caught, the solution is centrifuged and the obtained supernatant is dialyzed to remove salt and lyophilized to remove the solvent.

Preferably, the weight / volume ratio of the weight of the snails to the amount of NaCl solution is from 1: 5 to 1:15.

The invention also relates to a composition that contains mucilage isolated according to the method according to the invention and, optionally, carriers and / or adjuvants and / or diluents.

Preferably the composition is in the form of ointments, creams, gels, pastes, solutions, liniments, suspensions and emulsions. It is particularly advantageous to formulate a gel which, due to its hydrophilic character, corresponds to the properties of mucus.

It is also of interest to use Arion rufus snail mucus for the production of a non-drug medical device that prevents the occurrence, inhibits growth or controls bacteria, preferably of the genus Escherichia and / or Salmonella.

The invention also relates to the medical use of Arion rufus snail mucus for the preparation of medicaments for the treatment of bacterial infections, especially Escherichia and / or Salmonella, preferably E. coli and S. typhimurium, of a bacterial etiology.

Snail mucus preparations can be applied externally and internally to the skin and mucous membranes.

Tests on human fibroblasts confirmed the non-toxicity of this species of snail mucus.

High availability in natural conditions in the country and the ease of breeding this species of snail are the advantages of the presented invention. The developed method of mucus isolation is non-invasive and harmless to snails. The isolation method is also less harmful to the proteins contained in the slime

This is due to the omission of ethanol, as in other known methods. Additionally, since all solutions used in the mucus production process are water solutions, no harmful waste is produced.

Examples of possible compositions are given below by the compositions of the selected preparations.

Composition of the preparation 1 Mucus Methylcellulose 4000 cp Purified water 0.2 parts by mass 4.0 parts by mass 95.8 parts by mass Composition of the preparation 2 Mucus Methylcellulose 1500 cp Purified water 0.2 parts by mass 4.0 parts by mass 95.8 parts by mass Composition of the preparation 3 Mucus Methylcellulose 4000 cp Glycerol Purified Water 0.2 parts by mass 4.0 parts by mass 5.0 parts by mass 90.8 parts by mass Composition of the preparation 4 Mucus Methylcellulose 4000 cp Propylene glycol-1,2 Purified water 0.2 parts by mass 4.0 parts by mass 10.0 parts by mass 85.8 parts by mass Composition of the preparation 5 Mucus Methylcellulose 4000 cp Polyoxyethylene 200 glycol Purified Water 0.2 parts by mass 4.0 parts by mass 15.0 parts by mass 80.8 parts by mass Composition of the preparation 6 Mucus Methylcellulose 4000 cp Glycerol Purified Water 0.2 parts by mass 4.0 parts by mass 15.0 parts by mass 80.8 parts by mass Composition of the preparation 7 Mucus Methylcellulose 1500 cp Polyoxyethylene 200 glycol Purified Water 0.2 parts by mass 4.0 parts by mass 5.0 parts by mass 95.8 parts by mass Composition of the preparation 8 Mucus Methylcellulose 1500 cp Propylene glycol-1,2 Purified water 0.2 parts by mass 4.0 parts by mass 5.0 parts by mass 90.8 parts by mass Composition of the preparation 9 Mucus Methylcellulose 1500 cp Glycerol Purified Water 0.2 parts by mass 4.0 parts by mass 5.0 parts by mass 90.8 parts by mass

PL 234 591 B1

Isolation of Arion rufus snail mucus

P r z k ł a d 1

Arion rufus snails are harvested in the wild, e.g. in home gardens. It is a nocturnal species and harvesting takes place at night. The snails are kept in earthen vessels with access to oxygen, sunlight, fresh water and food in the form of garden plants.

The mucus is obtained as follows: snails in the amount of 6 adults per replicate (approx. 40 g) are placed in a glass vessel, then they are supplemented with a 0.9% buffered NaCl solution at room temperature, in a ratio of 1: 9 by weight -volume. The snails prepared in this way are carefully shaken on a rotary laboratory shaker, made by Elpan - water bath shaker type 357. The snails are not damaged, and the mucus flows out of their bodies osmotically. Shaking is carried out for 20 minutes at 150 rpm, resulting in a relatively homogeneous gelatinous solution, while the condition of the snails indicates that they have been completely washed. The animals are then harvested.

The resulting mixture is centrifuged on a Sigma machine for 15 minutes at 15,000 rpm to remove impurities.

After this stage, the dialysis process is carried out at 10 ° C (12 water changes, every 3 hours with a night break), aimed at removing salt from the preparation in the beakers, and after its completion, the whole is lyophilized until the water is completely removed.

P r z k ł a d 2

The procedure is as in Example 1, except that the agitation of the screws in NaCl solution is carried out for 30 minutes at 120 rpm and in a weight / volume ratio of the screws to NaCl of 1:15.

The next step is as in example 1.

P r z k ł a d 3

The procedure is as in Example 1, except that the snails are agitated in NaCl solution for 10 minutes at 180 rpm and in a weight / volume ratio of the snails to NaCl 1: 5. The next step is as in example 1.

In turn, the centrifugation sludge from both replicates was examined for microbiological reasons. The results of swabs taken from the snail bodies before the mucus isolation confirm the negligible presence of live bacteria in the mucus of the studied snails and the large number of bacteria in the sludge obtained after centrifugation at the mucus isolation stage. This is in line with the assumption that centrifugation is a step to remove any contaminants and bacteria. Moreover, it should be added that snails, as mentioned above, live in an environment contaminated with human and domestic activities, which could cause the accumulation of pathogenic microorganisms in the epidermal mucus of snails. However, such a situation does not take place, as can be seen from the results of the swabs attached in Table 1. Based on the swabs, a decision was made to check the antibacterial properties in relation to selected pathogenic microorganisms. The microorganisms selected for the study are microorganisms that commonly occur in the environment and cause infections. The focus was on 4 strains that are the most common and, as in the case of S. aureus, in many cases resistant to currently available antibiotics. For this purpose, 96-well plate survival test.

PL 234 591 Β1

Table 1

type of inoculation studied species of snail score direct differential snail body- ^ 4non rufus (Example 1) barren body of the snail-zlrżon rufus (Example 2) Escherichia coli - not many Enterococcus spp, - singly Serratia marcescens - not many centrifugation sludge - Arion rufus Enterococcus spp. - in great numbers Serratia marcescens - in great numbers inoculation after multiplication snail body -. / non rufus (Example 1) ie above snail body- ^ 4rion rufus (example 2) centrifugation sludge - Arion rufus mycological examination snail body- / rion rufus (example 1) no growth of yeast and mold fungi snail body - Zrzon rufus (Example 2) centrifugation sludge - Arion rufus

Study of the antibacterial properties of Arion rufus snail mucus.

Tests confirming the bacteriostatic properties of A. rufus snail mucus were carried out using the disc diffusion method. The lyophilized mucus was dissolved in water to give a concentration of 2 mg of mucus / ml of the solution. Mueller Hinton II Agar microbial culture plates were prepared. GRASO® BIOTECH companies. A suspension of bacteria with a turbidity of 0.5 according to the McFarland scale was plated on the plates. The bacteriostaticity of the mucus was tested on 4 reference bacterial strains, i.e .:

- Salmonella typhimurium ATCC 1402

- Escherichia coli ATCC 25922

- Staphylococcus aureus MSSA ATCC 29213

- Salmonella enteritidis ATCC 13076

Sterile disks with a diameter of 6 mm were placed on the agar plates with inoculated bacteria. Each disc was loaded with 5 μΙ or 10 μΙ of the appropriate concentration of the tested mucus. The plates were placed in an incubator at 37 ° C and left under these conditions for 22 h. After this time, the plates were removed from the incubator and visually inspected for any zones of growth inhibition.

The study of the antibacterial properties of Arion rufus snail mucus with simultaneous MIC determination.

A sterile 96-well plate was prepared. 200 μΙ of GRASO® BIOTECH BHI Broth was applied to each well. 2 µL of liquid McFarland 1 turbidity was added to each well. To the thus obtained suspension, 20 μΙ of suitably diluted aqueous solutions of A. rufus snail mucus were added so that the final concentrations in the wells were respectively:

- 2 mg of lyophilized mucus / ml

- 1 mg / ml

- 0.5 mg / ml

- 0.25 mg / ml

- 0.125 mg / ml

- 0.0625 mg / ml

PL 234 591 Β1

Each concentration was tested in triplicate. A control was also performed, also in triplicate, which was the medium with the addition of a bacterial suspension with 1 McFarland turbidity.

After all solutions and liquids had been applied to the plate, it was tightly sealed with parafilm to protect against evaporation and placed in an incubator at 37 ° C and left there for 20 hours. After this time, the plates were removed and the turbidity in each well was measured using plate reader.

The results are shown in Figure 1.

Survival% - A. rufus mucus \ control «2 mg / ml = 1 mg / ml» 0.5 mg / ml? .. 0.25 mg / ml »0.125 mg / ml» 0.0625 mg / ml

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E.ccli S. aureus S. typhimurium S. enteritidis test micro-organism

Diagram 1. Result of the A. rufus mucus plate test.

In the case of Arion rufus snail mucus, the obtained mucus affects the survival rate by inhibiting the growth of Escherichia coli and Salmonella typhimurium bacteria. In the case of E. coli and S. typhimurium 230, the mucus concentration in the sample, amounting to 2 mg / ml, inhibited the growth of these bacteria by 28 and 12%, respectively. Arion rufus snail mucus is the most effective in the fight against E. coli, acting in a way that inhibits their growth, in each of the tested concentrations.

Claims (6)

Patent claims 1. Method of isolating snail mucus, characterized in that the living Arion rufus snails are shaken in a physiological NaCl solution in a way that allows washing the snail from the mucus, while surviving the individuals and allowing visual assessment of changes in the nature of the solution, the weight-volume ratio of the snails mass the amount of NaCl solution should be adjusted to maintain the stabilization of proteins in the mucus, then, after catching the animals, the solution is centrifuged, and the obtained supernatant is dialyzed to remove salt and freeze-dried to remove the solvent. 2. The method according to p. The method of claim 1, wherein the weight / volume ratio of the snails weight to the amount of NaCl solution is from 1: 5 to 1:15. 3. A composition comprising the isolated mucus according to the method of claim 1. 1 and 2, and optionally carriers and / or auxiliaries and / or diluents. 4. A composition according to claim 1 A composition according to claim 3, in the form of ointments, creams, gels, pastes, solutions, liniments, suspensions and emulsions. 5. Use of Arion rufus snail mucus for the production of a medical device for preventing, growth-inhibiting or controlling bacteria, especially of the genus Escherichia and / or Salmonella. 6. The use of Arion rufus snail mucus for the preparation of medicaments for the treatment of bacterial infections, especially Escherichia and / or Salmonella, especially E. coli and S. typhimurium.