RU2762154C1 - Composition and method for producing medicinal films exhibiting antimicrobial activity - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to the chemical and pharmaceutical industry, namely, to a composition for producing medicinal films exhibiting antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aegidinosa, Bacillus cereus, and to a method for production thereof. A composition for producing medicinal films exhibiting antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aegidiposa, Bacillus cereus, including a solution of chlorhexidine, agar-agar, glycerin, sea buckthorn oil, a canadian goldenrod grass infusion, and mexidol, at a certain ratio of components. A method for producing medicinal films from the above composition, exhibiting antimicrobial activity, characterised by the fact that agar-agar is added to a 0.05% solution of chlorhexidine and allowed to swell for 15 to 20 minutes, mexidol is dissolved in 5 to 10 ml of water, glycerin is dissolved in an alcohol infusion of the canadian goldenrod grass, after the agar-agar is swollen, sea buckthorn oil, the solution with mexidol and the infusion with glycerin are added thereto and heated in a water bath at a temperature of 50 to 60°C while constantly stirring for the time required to form a homogeneous mixture, a layer with a thickness of 3 to 4 mm is formed from the resulting mixture and dried, forming a medicinal film.

EFFECT: above composition exhibits effective antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aegidiposa, Bacillus cereus.

2 cl, 1 dwg, 3 tbl

Description

Technology area

The group of inventions relates to the chemical-pharmaceutical industry and concerns methods of obtaining medicinal phyto films from natural raw materials (tincture of canadian goldenrod and sea buckthorn oil), enriched with mexidol and chlorhexidine additives, which can be used as an external antimicrobial agent for the prevention and treatment of inflammatory diseases of the oral mucosa ...

State of the art

For the treatment of inflammatory diseases of the periodontal and oral mucosa, a variety of antimicrobial and antiseptic drugs are widely used (Ivanenko I.G. Pharmacotherapy for periodontal diseases // Periodontology. 2001. - No. 1-2 (19-20). - P.8) ... Many researchers note the advantage of using antimicrobial agents of plant origin, which have a milder effect compared to synthetic analogs, fewer unwanted side effects, and the absence of allergic reactions (Gorbatova E.A., Lometskaya T.N., Manuilov B.M. from plant materials in the complex treatment of periodontal diseases // Institute of Dentistry. 2000. - 196. - S. 32-33).

Collagen plates FARMADONT I, II, III (Produced by JSC "Green Dubrava") are presented on the market. FARMADONT I contains cosmetic collagen, extracts of macklea, sage, rose hips, chamomile, ichthyokol (dry collagen crushed from the swimming bladders of sturgeon fish and large catfish), crab collagenase. FARMADONT II contains collagen, extracts of chamomile, valerian, arnica, mint, ichthyokol, crab collagenase. FARMADONT III contains collagen, extracts of aloe vera, St. John's wort, plantain, ichthyokol, crab collagenase. The product is non-sterile plates of a spongy structure, rather thick, the content of various plant components in them complicates the standardization process, in addition, the inclusion of such components as extracts of sage and St. reduces pharmacological anti-inflammatory activity. Also, the joint appointment of components containing alkaloids (macklea) and extracts rich in tannins (sage) is not recommended, since alkaloids, which are nitrogen-containing compounds of a basic nature, enter into a precipitation reaction with tannins (Soboleva V.A. Difficult and incompatible recipes in prescriptions. Ministry of Health of Ukraine. 2015, 41 pages), which leads to the loss of the pharmacological action of each of the reacting components.

Known composition and method of producing phytofilms with antimicrobial and anti-inflammatory activity (RF Patent No. 2155071. "Method for producing medicinal phytofilms"). According to this method, aqueous-alcoholic extracts from the herb of Echinacea purpurea, or the fruits of milk thistle, or buds of various types of poplar, or lavender flowers, or sage leaves, or silver birch buds, or calendula flowers, or lemon balm herb are used as medicinal substances. , or licorice roots, or common lilac bark, or rhizomes of Rhodiola rosea, or tarragon herb, or monarda herb, and methylcellulose (MC) (film former), dimethyl sulfoxide (DMSO) (penetrator) are used as auxiliary substances along with gelatin and glycerin and polyvinylpyrrolidone (PVP) (osmotically active agent). Separately prepared solutions of MC and DMSO, glycerin and PVP are mixed, homogenized, water-alcohol extracts from plant raw materials are added, a heated gelatin solution is introduced and the aged mixture is poured into substrates.

The disadvantage of the known solutions is the use of dimethyl sulfoxide as a penetrator, since it is capable of causing itchy dermatitis, skin rashes, rarely bronchospasm, which makes it impossible to use this agent for periodontal diseases complicated by allergic reactions, as well as the multicomponent nature of the medicinal plant materials used, which often have the same pharmacological action, which makes it difficult to carry out the standardization of these phytofilms.

Closest to the proposed group of inventions are the composition and method of producing films for the treatment of inflammatory diseases of the periodontal and oral mucosa (RF Patent No. 2618392. "Dental phytofilms for the treatment of inflammatory diseases of the periodontal and oral mucosa"). The composition of the films as active substances includes oil extracts of calendula, yarrow herb, echinacea dry extract, sanguirithrin, as a film former and plasticizer - hydroxyethyl cellulose (HEC) and glycerin, as auxiliary substances - cremophor and sodium saccharinate, mint essential oil. Films are prepared as follows. Sanguirithrin, sodium saccharinate and echinacea dry extract are dissolved in the calculated amount of water purified at a temperature of 70-75 ° C. HEC is poured onto the surface of the resulting solution and dissolved with stirring. After dispersion, the thick polymer solution is cooled with stirring to room temperature until a thick gel is formed. Glycerin is added to the finished polymer solution with stirring. In a separate evaporating cup, the oil extract of calendula flowers, yarrow herb and mint oil are mixed with cremophor. A solution of polymer and oils is combined to form a gel (after deaeration), applied in an even layer on a glass substrate and dried at room temperature to a residual moisture content of 5%.

The disadvantage of this remedy is the possibility of developing allergic reactions to sanguirithrin, as well as a feeling of bitterness in the mouth and a possible burning sensation.

It should be noted that the number of products obtained from plant materials with antimicrobial and anti-inflammatory effects is limited. With many of them, with infected skin lesions, combined use with antibiotics is practiced, the use of which can be accompanied by dermatitis, allergies and candidiasis.

The technical problem solved by the invention is the development of a new antimicrobial, anti-inflammatory agent - phytofilm containing a complex of biologically active substances obtained by introducing a mixture of different polarity containing sea buckthorn fruit oil and canadian goldenrod tincture, which has antimicrobial activity, as well as the introduction of mexidol and chlorhexidine, which will expand the range of products for external use based on plant materials.

Disclosure of invention

The technical result of the group of inventions is the development of films for external use based on plant raw materials (phytofilms) with antimicrobial activity. Films made in accordance with the proposed method are easy to use, do not require self-dosage of the drug. EFFECT: simplification of the composition of the composition while maintaining the therapeutic effect. As additional advantages of the proposed films, it should be noted the presence of a mild refreshing taste due to the introduction of the extract from the raw material of goldenrod.

The technical result is achieved due to the development of a composition for obtaining medicinal films with antimicrobial activity, including a solution of chlorhexidine, agar-agar, glycerin, sea buckthorn oil, alcohol tincture of canadian goldenrod herb and Mexidol, taken at the following ratio of components, wt%:

Chlorhexidine solution 0.05% 60-80 Agar agar 7.5-15.5 Glycerol 4.5-8 Sea buckthorn oil 4.5-8 Alcoholic herb tincture canadian goldenrod 4.5-8 Mexidol 0.15-0.8

The technical result is also achieved through the development of a method for producing medicinal films from the above-described composition, according to which agar-agar is added to a 0.05% chlorhexidine solution and left to swell for 15-20 minutes, Mexidol is dissolved in 5-10 ml of water, glycerin is dissolved in alcoholic tincture of canadian goldenrod herb, after the agar agar swells, sea buckthorn oil, a solution with mexidol and a tincture with glycerin are added to it and heated in a water bath at temperatures of 50-60 ° C with constant stirring for the time necessary to form a homogeneous mixture. A layer with a thickness of 3-4 mm is formed from the resulting mixture and dried to form a medicinal film with a thickness of 0.1-0.3 mm.

The composition of the proposed phytofilm includes sea buckthorn oil, obtained from the fruits of sea buckthorn, the components of which are carotenoids, tocopherols, phytosterols, polyunsaturated fatty acids, which cause pharmacological action, which is manifested in the stimulation of reparative processes in cases of skin and mucous membrane lesions of various etiologies (wound, radiation, ulcerative) (OV Trineeva, AI Slivkin, IA Samylina. Research on the development of draft pharmacopoeial monographs on fruits and oil of sea buckthorn buckthorn // Bulletin of Voronezh State University. Ser. Chemistry. Biology. Pharmacy 2016, No. 3. Pp. 126-133).

An alcoholic extract from the herb of canadian goldenrod contains tannins, flavonoids, phenol carboxylic acids, which cause its anti-inflammatory, wound healing and antimicrobial action (Kolodziej B., Kowalsky K., Kedzia B. Antibacterial and antimutagenic activity of extracts aboveground parts of three virago species , S.canadensis, SSgigantea // Journal of Medicinal Plants Research / -2011 / -V / 5, No. 31 / -P / 6770-6779 /, Suleimanova F.Sh., Nesterova OV, Matyushin A.A. Historical experience and perspectives of using the herb of Canadian goldenrod (Solidago canadensis L.) in medicine // Health and education in the XXI century. - 2017. - No. 4, T. 19. - P.142-149.).

Chlorhexidine is a well-known antiseptic that exhibits bactericidal action against gram-positive and gram-negative bacteria, virucidal action against lipophilic viruses (Vidal.ru Directory of medicines., Zemlyanichenko M.K., Lebedeva S.N. Use of chlorhexidine-containing agents for the prevention of dental diseases. Saratov Journal of Medical Scientific Research. 2011. Vol. 7, No. 1).

Mexidol is a drug that has an antihypoxic, membrane-protective, nootropic, anticonvulsant and anxiolytic effect, increases the body's resistance to stress (Vidal.ru Directory of medicines). It is known that Mexidol restores microcirculation of blood in the gums, relieves swelling, reduces bleeding gums and accelerates the healing of wounds, including purulent ones (T.I. Lemetskaya, E.M. Kuzmina, T.V. Sukhova, Yu.A. Petrovich The use of the drug Mexidol in the prevention and complex treatment of inflammatory diseases of the oral cavity. A guide for dentists. Moscow, 2008. P.20).

Brief Description of Drawings

The invention is illustrated by illustration, where in FIG. 1 shows samples of films made in accordance with the invention.

Implementation of the invention

The proposed composition for obtaining medicinal films with antimicrobial activity includes a solution of chlorhexidine, agar-agar, glycerin, sea buckthorn oil, alcoholic tincture of canadian goldenrod herb and mexidol, taken at the following ratio of components, wt%:

Chlorhexidine solution 0.05% 60-80 Agar agar 7.5-15.5 Glycerol 4.5-8 Sea buckthorn oil 4.5-8 Alcoholic herb tincture canadian goldenrod 4.5-8 Mexidol 0.15-0.8

Table 1 lists examples of formulations that were used to prepare medicinal films.

Table 1 Components of the composition Compound one 2 3 Chlorhexidine solution 0.05%, ml 50 40 45 Agar-agar, g 5 10 5 Glycerin, g 3 5 4.5 Mexidol, g 0.25 0.5 0.1 Sea buckthorn oil, g 3 5 4 Tincture of canadian goldenrod herb, ml 3 4 5

The proposed method for producing medicinal films is as follows.

Agar-agar (7.5-15.5 wt.%) Is placed in a chlorhexidine solution 0.05% (60-80 wt.%) For swelling for 15-20 minutes. Mexidol (0.15-0.8 wt%) dissolved in 5-10 ml of water. Glycerin (4.5-8 wt.%) dissolved in an alcoholic tincture from the herb of canadian goldenrod (4.5-8 wt.%). After the agar-agar swells, sea buckthorn oil is added to it (4.5-8 wt.%), A solution with Mexidol and a tincture with glycerin, and heated in a water bath at a temperature of 50-60 ° C, stirring constantly. Heating in a water bath is continued until a homogeneous mixture is formed (5-7 minutes). Next, the hot mixture is poured into molds (or onto a flat glass surface) in a thin layer with a layer thickness of 3-4 mm and left to dry for a day.

Tincture from herb goldenrod canadensis can be obtained by any method known from the prior art, for example, by maceration, using alcohol or alcohol-water extraction, while alcohol in various concentrations, preferably 70% ethyl alcohol, can be used.

An example of a specific implementation of the method

Agar-agar weighing 5 g was added to a 0.05% chlorhexidine solution with a volume of 50 ml and left to swell for 15 minutes. Mexidol in the amount of 0.25 g was dissolved in 10 ml of water. Glycerin weighing 3 g was dissolved in 3 ml of alcoholic tincture from the herb of canadian goldenrod. After swelling of the agar-agar, 3 g of sea buckthorn oil, a solution with Mexidol and a tincture with glycerin were added to it and heated in a water bath at a temperature of 50-60 ° C, stirring constantly. Heating in a water bath was continued for 5 min until a homogeneous mixture was formed. Next, the hot mixture was poured onto a flat glass surface with a thin layer 3-4 mm thick and left to dry for a day.

For the preparation of tincture of the herb of canadian goldenrod used two extractants - ethyl alcohol 70% and ethyl alcohol 96% (2 parts - 70% alcohol, 1 part - 96% alcohol), taken in the total ratio of raw materials: extractant 1: 5. In this case, the grass of canadian goldenrod was crushed to a particle size passing through a sieve with a diameter of 2 mm. The resulting plant powder was poured in the first portion of 70% ethyl alcohol and infused for 24 hours, the resulting extract was poured, after which the raw material was re-poured with a second portion of 70% ethyl alcohol and also infused for 24 hours, the resulting extract was poured and mixed with the first extract. Then 96% ethyl alcohol was added to the raw material, infused for 24 hours, purified by precipitation at a temperature of 8 ° C for 24 hours, followed by filtration through a paper filter. The third extract thus obtained was added to the first two extracts and mixed.

Table 2 shows the characteristics of the films obtained in accordance with the claimed method.

table 2 Level of quality Determination method The resulting result Description The evaluation of the appearance of the films is determined by visual inspection. Films are homogeneous, thin, elastic plates, rectangular, golden-yellow in color, due to the joint presence of sea buckthorn oil and goldenrod tincture Film dimensions Determine the geometric dimensions of the film in mm: length, width, thickness, which must be fixed. Determination is carried out by measuring with a micrometer. 30 ± 1 mm
10 ± 1 mm
0.1-0.3 mm (0.2 ± 0.1) Mass uniformity The determination is carried out in accordance with the requirements of the General Pharmacopoeia Monograph "Uniformity of the mass of dosage forms". 0.058 ± 0.0029 Dosing uniformity Films must comply with the requirements of the General Pharmacopoeia Monograph "Dispensing Uniformity" The content of mexidol in the films was determined by HPLC according to the method developed by A.A. Matyushin.
The content of rutin in the films was determined by HPLC according to the GOST method.
Mexidol not less than 49.49 mg / g Rutin not less than 1.49 mg / g
(techniques are described below) Dissolution The test is carried out to confirm the appropriate release of the active substance or substances by one of the methods described in the General Pharmacopoeia Monograph "Dissolution for solid dosage forms" Films swell within 3-5 minutes, do not dissolve in water, release active ingredients within 15 minutes Loss on drying or Water. The determination is carried out in accordance with the General Pharmacopoeia Monograph "Loss in mass on drying" or the General Pharmacopoeia Monograph "Determination of water". 50 % pH of the solution. The test is carried out for films made of biodegradable materials in accordance with the requirements of the General Pharmacopoeia Monograph "Ionometry". 6.8-7.1 (6.95 ± 0.15)

To confirm the anti-inflammatory, antioxidant properties of the obtained films, they were studied for the presence of such marker compounds as mexidol and rutin, indicating the presence of these properties. Below are the protocols for the study of samples by high performance liquid chromatography (HPLC).

Determination of Mexidol by HPLC with photometric detection .

Preparation for analysis

Preparation of working standard solutions. To prepare a standard solution, 200 μl of a 5% solution of Mexidol was taken and placed in a volumetric flask V = 100 ml, then 50 ml of a 0.1% solution of formic acid was added, mixed thoroughly and the volume was brought to the mark with the same solvent.

A portion of 500 mg of the drug is placed in a volumetric flask V = 50 ml, the volume is brought to the mark with a 0.1% solution of formic acid. The flask is placed in an ultrasonic bath for 5 minutes. An aliquot fraction (1 ml) of the obtained solutions of samples and model mixtures was centrifuged at a speed of 14000 rpm for 10 min.

The sample for HPLC analysis is filtered through a 0.45 μm membrane filter. Chromatographic analysis conditions: chromatographic column - octadecyl silica gel 5 µm, 250 × 4.6 (Phenomenex Luna 5 µm C18 (2)); the volume of the injected sample is 10 ml, the flow rate of the eluent: 1.0 cm3 / min. PP: (A): 90% trifluoroacetic acid solution, pH 2.5; (B): 10% acetonitrile. Detection is carried out with a UV detector at a wavelength of λ = 297 nm.

Determination of rutin by reverse phase (RP) HPLC with photometric detection.

Additional reagents: Rutin CAS 207671-50-9 - R5143 Sigma-Aldrich Chemie GmbH (≥95.0%).

Preparation for analysis: to prepare solutions of standard samples with a concentration of 0.1 mg / cm3, accurate weighed portions of 10.0 ± 0.1 mg of rutin are transferred into volumetric flasks V = 100 ml, half filled with methanol, stirred until the substances are completely dissolved, and brought to labels and mix thoroughly.

A portion of 500 mg of the drug (film) is placed in a volumetric flask V = 50 ml, the volume is brought to the mark with 50% ethanol solution. The flask is placed in an ultrasonic bath for 5 minutes.

An aliquot of the solution obtained after the dissolution test is filtered through a membrane filter with a pore size of 0.45 μm.

Chromatographic analysis conditions: chromatographic column - octadecyl silica gel 5 µm, 250 × 4.6 (Phenomenex Luna 5 µm C18 (2)); eluent feed rate: 1.0 cm ³ / min; the volume of the injected sample: 10 mm ³ ; mobile phase: (A): 80% trichloroacetic acid, pH 2.5; (B): 20% acetonitrile. Detection was carried out with a UV detector at a wavelength λ = 365 nm.

The study of the antimicrobial properties of the finished films was carried out using a method based on the ability of medicinal substances to diffuse into agar contaminated with test cultures of microorganisms and suppress the growth of the latter (Handbook on microbiological and virological research methods, edited by Birger M.O. - M.: Medicine 1982 , Pp. 172-177). The experiment used bacterial strains of the American Type Culture Collection.

Inoculum was prepared from an 18 hour agar culture, adjusting for turbidity to 0.5 McFarland standard. The broth culture obtained in this way was subjected to a 10-fold dilution with isotonic sterile sodium chloride solution, which corresponded to a final concentration of 1-2 * 10 7 CFU / ml. The inoculum was applied to Petri dishes with a dense nutrient medium using sterile cotton swabs, which were immersed in the studied suspension of microorganisms. Inoculation on the surface of the agar medium was carried out by strokes, with periodic displacement of the Petri dish by 60 °. Disks obtained from the studied phyto-films with a diameter of 6 ± 0.1 mm were carefully placed on the surface with the applied test cultures of microorganisms with sterile tweezers. As objects of comparison, we used disks of the same size, obtained on the basis of film-formers (carriers) without the addition of active components. At the end of the placement of the discs, the phyto-films Petri dishes were transferred to a thermostat and incubated for 18 hours at a temperature of 37 ° C. At the end of the incubation time, the growth zone retardation was calculated, taking into account the generally accepted methods (Labinskaya A.S., Blinkova L.P., Eshchina A.S. General and sanitary microbiology with the technique of microbiological research - M .: Medicine 2004. .; Guidelines for the study of the antimicrobial activity of pharmacological substances / T.A. Guskova et al. // Guidelines for experimental (preclinical) study of new pharmacological substances. - M .: Remedium. 2000. - P. 264-272.). The results of studying the antimicrobial action of the films are presented in Table 3.

Table 3. Average values of the delay in the growth zones of microorganisms by discs obtained on the basis of medicinal films containing the proposed composition and placebo films. Films under investigation The values of the diameter of the zone of inhibition of the growth of microorganisms (mm) Staphylococcus aureus
(АТСС 25923) Escherichia coli
(АТСС 25922) Pseudomonas aeruginosa
(АТСС 27853) Bacillus cereus
(АТСС 10702) Phytofilm with the proposed composition 16.9 11.6 8.6 12.8 Chlorhexidine solution film 8.3 6.1 6.8 7.4 Media based film 0.0 0.0 0.0 0.0

Thus, the films obtained in accordance with the invention have a pronounced antimicrobial effect against gram-positive cocci of the genus Staphylococcus, spore-forming microorganisms of the genus Bacillus, as well as a moderate antimicrobial effect against Pseudomonas aeruginos and Escherichia coli.

The bioadhesion of experimental film samples was determined by the method based on determining the force of separation of a steel plate with a film fixed on it (7 * 2 cm in size) from the mucous surface with an area of 14 cmI. The inner surface of the cheek of a pig, which was removed immediately after the slaughter of the animal, was used as a model of the mucosa. The experiment was carried out in two ways: without wetting the buccal mucosa and with wetting with a phosphate buffer solution pH 7 (prepared according to the method of the State Pharmacopoeia of the Russian Federation XIV) to approximate the conditions of the buccal mucosa.

In the experiment, a lever mechanism was used, to the upper (movable) part of which the test sample was attached, and to the lower (fixed) part, a sample of the mucous membrane of the cheek of a pig was attached.

The film sample was fixedly fixed on the upper metal plate. A fragment of the buccal mucosa was fixed motionlessly on the bed, then a metal plate with the studied film and the mucous membrane were aligned so that there were no gaps between them. In the course of the experiment, the weight applied to the counterweight of the lever mechanism was recorded, with the help of which the experimental sample of the dental film was detached from the mucous membrane.

Detachment stress (σ) is the amount of force per unit area of the film required to detach from the mucous membrane surface:

where F is the force applied to detach the film from the mucous membrane (equal to the gravity of the loads at which the separation occurred), S is the area of the film in contact with the mucous membrane.

When evaluating The following results were obtained for the studied films: the tear-off stress of the films is 5831 ± 187 Pa and 6794 ± 211 Pa when the mucous membrane is wetted with a buffer solution. The results show that the wetting of the mucosa increases the degree of pull-off stress. The tear-off stress of the investigated film is average in comparison with films prepared from other polymers. It can be concluded that the films have average mucoadhesive properties.

Thus, the proposed method for producing films is simple to implement, while the resulting films have a pronounced antimicrobial effect and moderate adhesive properties. In addition, due to the sea buckthorn oil and Mexidol included in the films, the films allow combining anti-inflammatory and antioxidant properties.

Claims (3)

1. Composition for obtaining medicinal films with antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosа, Bacillus cereus, including a solution of chlorhexidine, agar-agar, glycerin, sea buckthorn oil, tincture of canadian goldenrod herb and mexidol wt%: Chlorhexidine solution 0.05% 60-78.85 Agar agar 7.5-15.5 Glycerol 4.5-8 Sea buckthorn oil 4.5-8 Alcoholic herb tincture canadian goldenrod 4.5-8 Mexidol 0.15-0.8 2. A method of obtaining medicinal films from the composition according to claim 1, having antimicrobial activity, characterized in that agar-agar is added to a 0.05% chlorhexidine solution and left to swell for 15-20 minutes, mexidol is dissolved in 5-10 ml of water , glycerin is dissolved in an alcoholic tincture of canadian goldenrod herb, after the agar-agar swells, sea buckthorn oil, a solution with mexidol and a tincture with glycerin are added to it and heated in a water bath at a temperature of 50-60 ° C with constant stirring for the time required for the formation homogeneous mixture, a layer 3-4 mm thick is formed from the resulting mixture and dried to form a medicinal film.

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