RU2322993C1 - Method for preparing pharmaceutical preparation possessing antibacterial effect - Google Patents

Abstract

FIELD: medicine, immunology.

SUBSTANCE: invention relates to a method for preparing immunologically active substances. Method involves washing out worms, removal of their intestine content, cutting living worms for pieces of size 2-3 cm and the following drying worms. Before cutting and drying earthworms are subjected for activation, dried for about 24 h in ventilated site, then for about 5-10 h in drying chamber at 50 ± 5°C and then dries earthworms are milled to powder-like state. Invention provides preparing a preparation showing the enhanced antibacterial effect.

EFFECT: improved preparing method.

3 cl, 1 tbl, 1 dwg

Description

The present invention relates to medicine, more specifically to a method for producing immunologically active substances - pharmaceutical preparations from earthworms. These drugs are effective in the treatment of infections, gerontological, oncological, functional and many other diseases. They owe their healing effect to the unique earthworm immune system that formed in response to extreme environmental conditions. The earthworm immune system is represented by many specific immune cells, an antibiotic, enzymatic system, a system of complementary proteins, localized mainly in coelomic fluid.

A known method of obtaining an immunostimulant / RF Patent No. 2091072, A61K 35/50, 1979 /, in which the squid or cuttlefish ganglia are homogenized in water acidified with acetic acid to a pH of 3.5-4.5 and containing 0.1% zinc chloride . The mixture is incubated for 28-48 hours. After separation of the supernatant, it is subjected to sterilizing filtration. The sterile filtrate is concentrated 5.5 times by ultrafiltration through a porous material with a separation limit of 1000-5000 D and subjected to purification in three times the volume of water, alkalized to pH 9.0-10.0. The purified concentrate is again concentrated 2 times on the same porous material and subjected to sterilizing filtration. Then sterile filling of the preparation is carried out and freeze-dried.

A known method of obtaining the drug "Dilongjing" / Patent CN 1114202, A61K 35/64; A61K 35/56; 1996 /, including washing live earthworms, separation by centrifugation, freezing with a dry vacuum to obtain a dry powder, and loading into capsules, or mixing with honey to obtain a liquid for oral administration.

A known method of obtaining a therapeutic drug for thrombosis / US Patent No. 5,186,944, A61K 35/56, 1993 /. The method includes cleaning the living earthworms from soil (dirt) on the surface of the body and fecal dirt in the digestive tract, placing the worms in an acidic aqueous solution having a hydrogen index of from 3 to 6.5 for from 0.1 to 5 hours; placement in a vessel that is frozen at a temperature of from -10 ° C to -60 ° C. Subsequent drying by sublimation at this temperature from 5 to 12 hours under vacuum, vacuum drying is carried out stepwise from 5 to 15 hours at 20 - 30 ° C, at 0.01, 0.2 mm Hg, from 10 to 20 hours at 35 - 50 ° C, at 0.1-0.5 mm Hg and from 0.1 to 19 hours at 70 - 80 ° C, at 0.001-5 mm Hg.

A known method of obtaining a dry powder from earthworms and antihyperlipemic, anti-diabetic, antihypertensive and antihypotonic preparations containing dry powder of earthworms as an active ingredient / US Patent No. 5024844, A61K 35/56, 1991 /. A species of living earthworms is placed in fresh water with an aqueous solution of not more than 0.3% by weight, preferably not more than 0.1% by weight, of at least one composition selected from organic acids such as acetic acid, citric acid, succinic acid, malic acid, tartaric acid and lactic acid; mineral acids such as phosphoric acid, sulfuric acid and hydrochloric acid; and sodium or potassium salts of these acids for a period of from 0.5 to 72 hours, preferably from 1 to 40 hours. The above solution can also be characterized as a slightly acidic aqueous solution having a hydrogen index of from 3 to 6.5. Thus, the digestive tract of living earthworms is mostly freed from the soil. Then live earthworms are washed with water to remove any soil (dirt) from the surfaces of the body. After that, live wet earthworms are placed at a low temperature of -5 ° C or lower, preferably -10 ° C-60 ° C. Then the fixed suspension is dried by freezing and dried by vacuum, from 10 to 60 hours. Thus, a sterile (sterile) dried earthworm powder was obtained. The dried earthworm powder contains approximately 18 amino acids, including aspartic acid, threonine, serine (alpha-amino-beta-hydroxypropionic acid), glutamic acid, pyrrolidine-alpha-carboxylic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine , tyrosine, tryptophan, lysine, histidine.

The closest analogue is a method for producing pharmaceutical preparations from earthworms / Second International Scientific and Practical Conference "Earthworms and Soil Fertility", March 17-19, 2004, Vladimir, Russia. Kovrov, publishing house "X-PRESS", 2004, 293 pp. (hereinafter [1]), SUN ZHENJUN Pharmaceutical value and use of earthworms in China, p. 49 /. The collected earthworms are cut into 1-2 cm pieces, moistened with water for half a minute and washed in water for 1-2 minutes, and the resulting mass is dried in strong sun. Dried worms are placed in a wooden box and stored in a ventilated place.

A disadvantage of the known methods is the complexity of the preparation of preparations and their insufficiently effective antimicrobial properties.

The objective of the invention is to provide a method for producing a pharmaceutical preparation from earthworms, allowing to obtain a preparation with more effective antimicrobial properties.

The problem is achieved in that in a method for producing activated pharmaceutical preparations, including washing the worms, freeing them from intestinal contents, cutting live worms into 2-3 cm pieces and then drying, the earthworm is preliminarily activated before being cut before drying, it is dried for about a day in a well-ventilated place, then about 5-10 hours in an oven at 50 ± 5 ° C, then dried worms are crushed to a powder state.

Activation is carried out by preliminary placing the worms in an aqueous suspension with a high titer of the non-pathogenic strain Yarrowia lipolytica, for 30-60 minutes, followed by washing them in several acidified waters.

Activation is done by pre-placing the worms in the freezer in a container of water for 20 minutes.

The immune, antimicrobial and enzymatic systems of the earthworm have the ability to increase their effectiveness under the influence of both adverse environmental factors, including favorable ones. To obtain activated pharmaceuticals, it is necessary to subject some earthworms to manipulations - the raw materials for obtaining the preparations.

The method is as follows:

Worms taken from the soil are thoroughly washed in several sterile waters. It is kept during the day by hunger in 0.5 l cans with wet filter paper to free the intestines from soil lumps. Then the earthworm is pre-activated. After this, live worms are cut into pieces 2-3 cm long, dried for about a day in a well-ventilated place, then about 5-10 hours in an oven at 50 ± 5 ° C. The dried worms are crushed in a sterile dry mortar to obtain a powder. The resulting preparation is stored in a refrigerator in tightly sealed vials.

Activation is done in two ways.

The first method is that sexually mature individuals of the earthworm Eisenia fetida Andrei Bouche are placed in an aqueous suspension with a high titer of the non-pathogenic strain of Yarrowia lipolytica, for 30-60 minutes, then washed in several acidified waters.

The second method is that earthworms are placed in a freezer in a container of water for 20-30 minutes with a water volume of 50 ml, avoiding icing. Then the worms are warmed to room temperature.

Evaluation of changes in the effectiveness of the drug in comparison with the usual is carried out using the disk method:

In Petri dishes, thick suspensions of this pathogenic microorganism, related to non-pathogenic or conditionally pathogenic, or a similar microorganism in a volume of 0.5 ml, are applied to a specific nutrient medium in order to subsequently obtain continuous lawns of these cultures (for example, RPA (fish peptone agar) for Staphylococcus sp.)).

After 30 minutes, when the substrates dried up, they put on the substrate sterile paper disks with a diameter of 0.5 cm, impregnated with a suspension of the preparations of control and activated on physical. solution. To prepare the suspension take 30 mg of the drug and 0.3 ml of physical. solution.

After 30 minutes, the plates are turned over, placed in a thermostat and cultured at a temperature of 31 ° C. The next day, the results are looked at: in areas where bacteriostatic reactions occurred, an uneven, weak growth of the bacterial lawn was observed. The experiment is carried out in at least 3 replicates with the calculation of the confidence interval.

Measure the radius of the inhibition of growth of the bacterial lawn from the center of the disk maximum and minimum, find the average value. Compare the radii obtained from control and activated drugs. The difference is judged on the presence of the activation effect / Revue on the antibacterial immunity of earthworms. Wang Chong, Zheng Dong Mei, Song Zhenjun. Materials of the 2nd International Scientific and Practical Conference "Earthworms and Soil Fertility" in Vladimir, March 17-19, 2004 /.

The table shows the changes in the effectiveness of the drug compared with the usual (control) prepared according to the traditional (without our modifications) technique.

In our case, the control was the preparation of dry powder from earthworms, prepared according to the traditional method, but without prior activation. Analyzing the data in the table, it is seen that activated preparations based on earthworm (preparation 2, 3) more actively inhibit the growth of lawn crops than control preparation 1. Moreover, test cultures such as Y. lipolytica and Staphylococcus sp have the highest sensitivity ., according to the clarity of the zone of suppression of growth of the lawn around the paper disk: the zone is clear, well visible. This feature of the test cultures is very important with various assessment methods.

Table Lawn-forming microbiological culture Drug No. 1, control Preparation No. 2, activation by cooling Drug No. 3, activation of Staphylococcus sp. The radius of the zone of absence of growth of the bacterial lawn (from the center of the disk), mm Yarrowia lipolytica 5 ± 0.75 (3rd day), 18 ± 2.5 (3rd day), 14 ± 2.38 (3rd day), 15 ± 2.2 (6th day) 38 ± 5.7 (6th day) 40 ± 6.4 (6th day) Pseudomonas aeruginosa 11 ± 1,5 30 ± 4.8 - Staphylococcus sp. 10 ± 1.7 42 ± 6.3 37 ± 5.8 Rhodococcus elitropolis 6 ± 0.8 35 ± 5.2 35 ± 5,6 Bacillus thuringiensis 18 ± 2,3 15 ± 2 20 ± 2.8

It can also be said that the most effective method of activating the antimicrobial properties of earthworms is cooling: the radii of the zones of suppression of the growth of lawns of all test cultures under the influence of drug 3 (cooling) are the largest in comparison with the effect of other drugs on the same lawns.

The drug obtained after activation by heating is more effective than the control drug, but in the series of activated drugs the weakest. This is most likely due to the fact that temperature is not an activating factor, but a depressant for earthworms. In the works of other authors / N.V. Kuzmina, N.E. Koschenkova, E.N. Kubarev, G.A. Osipov. Materials of the 2nd International Scientific and Practical Conference "Earthworms and Soil Fertility" in Vladimir, March 17-19, 2004 / data on the long-term effect of elevated temperatures on earthworms are given, leading to a decrease in their antimicrobial properties and an increase in the proportion of pathogenic microorganisms in the structure microbial community of the intestinal tract of earthworms.

The most sensitive to the action of all drugs is Staphylococcus sp., The least - R. elitropolis and B. thuringiensis.

During the experiment, a well-defined bacteriostatic effect of the drug was noted - activation of staphylococcus on lawns with Yarrowia lipolytica. To prove that this drug inhibits the growth of the lawn due to its own antimicrobial properties, and not due to the antagonistic interactions between Yarrowia lipolytica and St. sp, an antagonism test was performed. It turned out to be negative (see drawing). The suspension of drug 1 (activation by staphylococcus) was also plated on RPA in order to detect the presence of St. sp in the preparation: St. sp was not sown.

Preparations obtained from earthworms subjected to slow cooling: for 20-30 minutes in a container of water in the freezer, they showed the strongest antimicrobial properties against B. thuringiensis, R. erythropolis, Staphylococcus sp. P. aeruginosa, Yarrowia lipolytica in comparison with the control drug and at the level of the drug activated by Staphylococcus sp.

Since staphylococcus is a conditionally pathogenic strain, we suggest replacing it with non-pathogenic Y. lipolytica - non-toxic yeast, the producer of the oil-destructive drug Devoroyl.

The proposed method allows to obtain a drug with more effective antimicrobial properties and simplify the method of its production.

Claims (3)

1. A method of obtaining a pharmaceutical preparation having an antimicrobial effect, including washing the worms, freeing them from intestinal contents, cutting live worms into 2-3 cm pieces and subsequent drying of the worms, characterized in that the earthworm is preliminarily activated before being cut before drying, dried about a day in a well-ventilated place, then about 5-10 hours in an oven at 50 ± 5 ° C, then dried worms are crushed to a powder state. 2. The method according to claim 1, characterized in that the activation is carried out by preliminary placing the worms in an aqueous suspension with a high titer of the non-pathogenic strain of Yarrovia lipolitica for 30-60 minutes, followed by washing them in several acidified waters. 3. The method according to claim 1, characterized in that the activation is carried out by pre-placing the worms in the freezer in a container of water for 20-30 minutes, avoiding icing, followed by warming them to room temperature.

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