RU2595880C1 - Polymer film with naphthoquinone complex of biologically active substances of red-rooted gromwell - Google Patents

Abstract

FIELD: packaging industry.

SUBSTANCE: invention relates to polymer film with naphthoquinone complex of biologically active substances recovered from cell culture of red-rooted gromwell. Along with above complex of biologically active substances film contains glycerine, methyl cellulose, sodium-carboxymethyl cellulose, polyvinylpyrrolidone and purified water. Besides, said film is characterised by that at stage of making combined polymer base of methylcellulose and sodium-carboxymethyl cellulose together with glycerine is added 0.5 g of polyvinyl pyrrolidone, and then into ready polymer base added 0.68 ml of 5 % alcohol solution of naphthoquinone complex of red-rooted gromwell, that enables achieving its concentration in film of 400 mcg/cm², then mixture is homogenised, poured on standard glass substrate with area of 85 cm² and dried in drying chamber for 6-8 hours at temperature of not more than +50 °C.

EFFECT: invention provides wider spectrum of action and high antimicrobial activity of polymer films with shikonin and its ethers.

1 cl, 3 dwg, 1 tbl, 6 ex

Description

The invention relates to medicine, to the section of pharmacy, and can be further used for the prevention and treatment of infectious and inflammatory periodontal diseases, as well as in therapeutic practice in order to increase the efficiency and reduce the time of treatment of wound infections, ulcerative and erysipelas soft tissue lesions.

An increase in the range of medicines for wound healing, anti-inflammatory, antimicrobial action is an urgent task for medicine and pharmacy. This is due, first of all, to the increased antibiotic resistance of microorganisms - the causative agents of infectious diseases, which stimulates the scientific community to search for new antimicrobial substances [Kozlov RS Selection of resistant microorganisms using antimicrobial agents: the concept of "parallel damage" // Clinical Microbiology and Antimicrobial Chemotherapy. - 2010. - T. 12, No. 4. - S. 284-294]. In addition, there is growing interest in biologically active substances (BAS) of plant origin, which have a pronounced pharmacological effect with low toxicity and a low likelihood of developing adverse side effects inherent in some modern chemotherapeutic drugs [Minina S.A., Kaukhova I.E. Chemistry and technology of herbal remedies: a training manual. - 2nd ed., Revised. and add. - M.: GEOTAR-Media, 2009. - 560 p.: Ill.].

One of the promising objects of research, in our opinion, is the naphthoquinone complex of biologically active substances, which is contained mainly in the roots of the red-root passeriformes - Lithospermum erythrorhizon Sieb. et Zucc., representative of the Boraginaceae family - Boraginaceae. This complex includes naphthoquinone shikonin, its chemical formula: 5,8-dioxi-2 (1-hydroxy-4-methylpentyl-3) -1,4-naphthoquinone. The complex also includes esters of shikonin, such as acetyl chiconin, isovaleryl chiconin, isobutyryl chiconin, propionyl chiconin, β, β-dimethyl acryl chiconin and other shiconin derivatives.

Shikonin and its esters have been known since ancient times as red natural pigments and as promising drugs. So, in a review article by Papageorgiou et al. [Papageorgiou V.P., Assimopoulou A.N., Couladouros E.A., Hepworth D., Nicolaou K.C. The chemistry and biology of alkannin, shikonin, and related naphthazarin natural products // Angewandte Chemie International Edition in English. - 1999. - Vol. 38. - P. 270-301] describes the pharmacological properties of these substances and discusses the possibilities of their medical use. The authors of the article also refer to other independent studies in which it was proved that shikonin and its esters have anti-inflammatory, wound healing and antimicrobial effects.

However, to date, the use of the naphthoquinone BAS complex in medicine is limited due to the lack of a convenient dosage form. It is known in drug manufacturing technology that many organic polymers, for example cellulose derivatives such as methyl cellulose (MC), sodium carboxymethyl cellulose (Na-CMC), are capable of releasing active substances over a period of time, providing a local effect of the substance on the area of the pathological focus and providing a prolonged action of the dosage form [Pharmaceutical technology. Technology of dosage forms: textbook / II. Krasnyuk, G.V. Mikhailova, T.V. Denisova, V.I. Sklyarenko; under the editorship of I.I. Krasnyuk, G.V. Mikhailova. - M .: GEOTAR-Media, 2013. - 656 p.].

At present, it has become possible to isolate the undeclared naphthoquinone complex of the biologically active substances of the red-root beehive from cell culture and its further application as a part of polymer systems for local treatment of mucous membranes and skin, which is a promising area in medicine.

A well-known analogue is a patch for the oral mucosa containing shikonin extract from the roots of the red-root passeriformes (Application No. 1-226823, Japan, А61К 35/78, 9/70 from 09/11/89). An analogue is also known - a hygienic composition for caring for the oral cavity containing shikonin (Application No. 59-24993, Japan, А61К 7/16, 7/26, 31/35, publ. From 02.15.84, prototype). The composition contains as an active ingredient shikonin with a purity of 80%, to obtain which the raw material containing shikonin is extracted with an organic solvent, hydrolyzed using an aqueous alkali solution and precipitated with acid. The disadvantages of these inventions is their one-component nature, because it is known that shikonin in combination with the sum of its esters has a more pronounced pharmacological effect, as well as a lack of natural plant materials, as the red-rooted passerine is a rare, endemic plant with a limited habitat in Japan, China and The Far East of the Russian Federation, listed in the Red Book of the Amur Region.

An analogue is also known - a means for treating wounds containing collagen and shikonin for use in surgical practice: dressing "Geschispon" (RF patent No. 2071788). The disadvantage of this invention is the inability to use on the mucous membranes of the oral cavity in inflammatory processes.

Shikonin oil is known as an analogue (RF patent No. 21411840), however, the disadvantages of this invention are the inconvenience of applying to the mucous membranes of the oral cavity due to the lack of adhesive properties of the oil and, as a consequence, the inability to provide a prolonged effect of the active component on the area of the pathological focus due to regular washing with saliva.

The closest analogue to the prototype of the invention is the well-known method of manufacturing polymer films with shikonin and its derivatives [Basharov A.Ya., Bulgakov V.P. Dental polymer films based on shikonin esters // Forests and forestry in modern conditions: Vseros materials. conf. from the international participation / resp. ed. A.P. Kovalev. - Khabarovsk: Publishing house of FGU "DalNIIILKh, 2011. - S. 152-153], which consisted of the following: polymer solutions - methyl cellulose, sodium carboxymethyl cellulose and their mixture were prepared in a concentration of 3-4%, and then subjected to sterilization at 100 ° C for 30 minutes to reduce the natural contamination of polymers. As a plasticizer, glycerin was used in an amount of 0.7-0.9 g per 1.0 g of polymers. The sum of shikonin esters was introduced into the solution of the finished polymer in the form of a 0.25% solution on 95% ethanol. The finished composition was poured onto a glass substrate so that after drying of the polymer a film with a thickness of 110-120 microns was formed. The content of the sum of shikonin esters in the finished films was 85 μg per 1 cm ^{2 of} film. Thus, using the described technique, it is possible to obtain polymer films on three different polymer bases with a content of naphthoquinone complex of 85 μg / cm ² . However, the disadvantages of the described methods are:

1. A low concentration of the introduced alcoholic solution of the sum of shikonin esters 0.25%, which requires the introduction of a relatively larger volume of the alcoholic solution into the polymer solution, resulting in excessive dilution of the polymer and, as a result, the occurrence of deformations during the drying process, which reduces the quality of the finished product.

To avoid unwanted dilution of the polymer and deformation of the films during drying, it is proposed to reduce the volume of the introduced alcoholic solution of shikonin esters by increasing its concentration.

2. The disadvantage is also the low dose of the naphthoquinone complex of biologically active substances in the finished films. A microbiological study of determining the sensitivity of microorganisms to shikonin and its esters in the composition of films of this composition with a dose of 85 μg / cm ² did not reveal antimicrobial activity in these film samples. At the same time, according to published data, shikonin and its esters have antimicrobial activity [Isabel Andújar, José Luis Ríos, Rosa María Giner, María Carmen Recio. Pharmacological properties of shikonin - a review of literature since 2002 // Planta Medica. - 2013 .-- Vol. 79. - P. 1685-1697]. Therefore, the concentration of the naphthoquinone complex of biologically active substances in the composition of the above films of 85 μg / cm ² is insufficient to provide an antimicrobial effect in clinical practice.

To expand the spectrum of action and increase the antimicrobial activity of polymer films with shikonin and its esters, we suggest increasing the concentration of the naphthoquinone complex in the films by introducing a more concentrated shikonin-containing alcohol solution into the polymer. The introduction of a more concentrated alcoholic solution will allow you to enter a smaller, in comparison with the prototype, the volume of the alcoholic solution of the sum of shikonin esters in the polymer.

3. The next drawback is the lack of substances in the composition of the films described above that contribute to a better release of the naphthoquinone complex.

In order to improve the process of releasing the active component from the polymer film, it was decided to introduce polyvinylpyrrolidone into its composition due to its known technological and physical properties. Polyvinylpyrrolidone - a water-soluble polymer from the monomer N-vinylpyrrolidone, also known as polyvidone or povidone, which is widely used in the technology of drugs as a binder, and also due to the formation of water-soluble complexes improves the solubility and bioavailability of drugs [Pharmaceutical technology. Technology of dosage forms: textbook / II. Krasnyuk, G.V. Mikhailova, T.V. Denisova, V.I. Sklyarenko; under the editorship of I.I. Krasnyuk, G.V. Mikhailova. - M .: GEOTAR-Media, 2013. - 656 p.].

The objective of the invention: to develop and produce a polymer film of the optimal composition containing a naphthoquinone complex of biologically active substances - shikonin and its esters, isolated from a cell culture of the red-root passeriformes - Lithospermum erythrorhizon Sieb. et Zucc., with expected antimicrobial activity.

The technical result consists in the manufacture of a polymer film of the optimal composition containing a naphthoquinone complex of biologically active substances - shikonin and its esters, isolated from the cell culture of the red-roe virus - Lithospermum erythrorhizon Sieb. et zucc.

The technical result is achieved as follows. At the first stage, an undeclared naphthoquinone complex of biologically active substances is isolated from the callus culture of red-root passeriformes cells - Lithospermum erythrorhizon Sieb. et Zucc., grown on an artificial nutrient medium according to the established method [Bulgakov V.P., Kozyrenko M.M., Fedoreyev S.A. et al. Shikonin production by p-fluorophenylalanine resistant cells of Lithospremum erythrorhizon // Fitoterapia. 2001. 72. 394-401]. Isolation is carried out according to a known method [Zhuravlev Yu.N., Fedoreev S.A., Bulgakov V.P., Muzarok T.I., Beresneva N.V., Golovko E.I. Anti-inflammatory and antimicrobial agent "Shikonin oil" // Patent of the Russian Federation No. 2141840, 1999. Bull. No. 33]. To do this, a weighed sample of the red-roe roe web cells is extracted in the Soxhlet with hexane until the raw material is completely discolored, then the hexane is distilled off, and the weighed portion is dried to constant weight in a vacuum oven. In this case, the extraction is a bright red viscous mass.

At the second stage of the proposed methodology, the obtained extraction of the naphthoquinone complex of biologically active substances is dissolved in 95% ethanol to a concentration of 5%, and thus, the concentration of the shikonin-containing alcohol solution for introduction into the polymer film increases by 20 times compared to the described prototype [Basharov A.Ya. , Bulgakov V.P. 2011] The use of such a solution concentration of the sum of shikonin esters can significantly reduce the amount of alcoholic shikonin-containing solution introduced into the polymer, which will not lead to excessive polymer dilution and subsequent deformation of the films during drying.

At the third stage, a combined solution of polymers of methyl cellulose and sodium carboxymethyl cellulose is prepared according to the procedure described earlier in the application “The optimal basis for a polymer film with a naphthoquinone complex of biologically active substances of the red-rooted foxhawk” by authors Taran L.M., Basharova A.Ya., Slobodenyuk E.V., Bulgakova V.P., Strelnikova N.V., sent April 21, 2015. To do this, 0.45 g of methyl cellulose and 7 drops are added to 25 ml of purified water brought to a temperature of + 70-90 ° C. glycerin, additionally according to the proposed method ke add 0.5 g of polyvinylpyrrolidone. Then, according to the previously described methodology, the mixture is placed in a refrigerator at a temperature of + 8-10 ° C for 30 minutes to swell the polymer. Then the mixture is thoroughly mixed, a second polymer is uniformly poured onto the surface with a thin layer - 0.45 g of sodium carboxymethyl cellulose and left to swell at room temperature + 22-24 ° C for 30-40 minutes. Then the mixture is homogenized and placed in the refrigerator at + 8-10 ° C for 24 hours. After a day, re-homogenize the mixture.

At the fourth stage, according to the proposed method, 0.68 ml of a 5% alcohol solution of the sum of shikonin esters are introduced into the prepared polymer solution, homogenized and poured onto a standard glass substrate with an area of 85 cm ² . Dried in an oven at a temperature of not more than + 50 ° C for 6-8 hours. After complete drying, the finished product is a bright, transparent, flexible polymer film of bright red color with a concentration of naphthoquinone complex of 400 μg / cm ² , which is shown in FIG. 1. Pack films in individual sterile plastic bags.

To determine the antimicrobial activity of the produced polymer films, a microbiological study was conducted on the basis of the bacteriological laboratory of KGBUZ Regional Clinical Hospital No. 1 named after professors S.I. Sergeeva ”, Khabarovsk (chief physician S. S. Pudovikov). 48 strains of microorganisms of twelve different species were studied. The determination was carried out by the standard disk-diffusion method, placing a film sample of 1 cm ² on the surface of the Muller-Hilton nutrient medium with a seeded daily culture of bacteria and fungi (McFarland turbidity 0.5), and kept in a thermostat for 24 hours at + 37 ° C . After daily exposure, the result of the presence or absence of a zone of growth inhibition of microorganisms in mm was taken into account. The experiment was carried out four times and the average value was taken as the final result. The results are shown in Table 1. Thus, a microbiological experiment confirmed the presence of antimicrobial activity in films containing a naphthoquinone complex at a concentration of 400 μg / cm ² for some clinically significant types of microorganisms: Staphylococcus aureus, Enterococcus faecalis, Kocuria varians. A slight antimicrobial effect was found in relation to Enterococcus faecium, Hafnia alvei, Pseudomonas aeruginosa, Bacteroides fragilis, Candida albicans, Candida krusei, Candida tropicalis. He was found antimicrobial effect against species of Escherichia coli, Proteus mirabilis.

The advantages of the invention are:

- the possibility of using films with an optimal concentration of the naphthoquinone complex of the biologically active substances of the red-root beetle, providing an antimicrobial effect;

- the possibility of using films for the treatment of inflammatory processes on the mucous membranes of the oral cavity and skin based on the properties of shikonin and its esters;

- harmlessness;

- the possibility of using films both in combination with other drugs, and without them;

- the ability to provide local prolonged action on the mucous membranes due to the properties of polymers;

- strong fixation of films in the area of the pathological focus;

- the absence of mechanisms of addiction to shikonin and its esters in microorganisms, which makes it possible to reuse films and extend the course of treatment if necessary;

- ease of use of films: the possibility of patient use independently in any conditions.

Example 1

Polymer films were made according to the proposed method, the amount of introduced shikonin-containing alcohol solution of 5% was 0.17 ml, which ensured the concentration of the naphthoquinone complex in the films of 100 μg / cm ² . To determine the antimicrobial activity of the manufactured polymer films with a concentration of 100 μg / cm ² a microbiological study was conducted. We studied 48 strains of microorganisms of twelve different species, including museum cultures and ATCC strains. The determination was carried out by the standard disk-diffusion method, placing a film sample with an area of 1 cm ² on the surface of a Muller-Hilton nutrient medium with a 24-hour crop seeded with a lawn (turbidity 0.5 according to McFarland) and kept in a thermostat for 24 hours at a temperature of 37 ° C. After daily exposure, the result of the presence or absence of a zone of growth inhibition of microorganisms around the film sample was taken into account. The experiment was carried out four times and the average value was taken as the final result. The results are shown in Table 1. Thus, the activity of these film samples was confirmed only against two types of bacteria: Staphylococcus aureus, Enterococcus faecalis.

Example 2

Polymer films were made according to the proposed method, the amount of introduced shikonin-containing alcohol solution of 5% was 0.34 ml, which ensured the concentration of the naphthoquinone complex in the films of 200 μg / cm ² . A microbiological study was conducted to determine the antimicrobial activity of the manufactured polymer films of a given concentration. We studied 48 strains of microorganisms of twelve different species, including museum cultures and ATCC strains. The determination was carried out by the standard disk-diffusion method, placing a film sample with an area of 1 cm ² on the surface of a Muller-Hilton nutrient medium with a 24-hour crop seeded with a lawn (turbidity 0.5 according to McFarland) and kept in a thermostat for 24 hours at a temperature of 37 ° C. After daily exposure, the result of the presence or absence of a zone of growth inhibition of microorganisms around the film sample was taken into account. The experiment was carried out four times and the average value was taken as the final result. The results are shown in Table 1. Thus, the activity of these film samples was revealed only against three types of bacteria: Staphylococcus aureus, Enterococcus faecalis, Kocuria varians.

Example 3

Polymer films were made according to the proposed method, the amount of introduced shikonin-containing alcohol solution of 5% was 0.51 ml, which ensured the concentration of the naphthoquinone complex in the films of 300 μg / cm ² . To determine the antimicrobial activity of the manufactured polymer films with a concentration of 300 μg / cm ² a microbiological study was conducted. We studied 48 strains of microorganisms of twelve different species, including museum cultures and ATCC strains. The determination was carried out by the standard disk-diffusion method, placing a film sample with an area of 1 cm ² on the surface of a Muller-Hilton nutrient medium with a 24-hour crop seeded with a lawn (turbidity 0.5 according to McFarland) and kept in a thermostat for 24 hours at a temperature of 37 ° C. After daily exposure, the result of the presence or absence of a zone of growth inhibition of microorganisms around the film sample was taken into account. The experiment was carried out four times and the average value was taken as the final result. The results are shown in Table 1. Thus, the activity of these film samples was revealed against three types of bacteria: Staphylococcus aureus, Enterococcus faecalis, Kocuria varians. Moreover, the observed zones of growth inhibition exceeded those of film samples with a concentration of 200 μg / cm ² described in example 2.

Example 4

Polymer films were made according to the proposed method, the amount of introduced shikonin-containing alcohol solution of 5% was 0.68 ml, which ensured the concentration of the naphthoquinone complex in the films of 400 μg / cm ² . To determine the antimicrobial activity of the manufactured polymer films with a concentration of 400 μg / cm ² a microbiological study was conducted. We studied 48 strains of microorganisms of twelve different species, including museum cultures and ATCC strains. The determination was carried out by the standard disk-diffusion method, placing a film sample with an area of 1 cm ² on the surface of a Muller-Hilton nutrient medium with a 24-hour crop seeded with a lawn (turbidity 0.5 according to McFarland) and kept in a thermostat for 24 hours at a temperature of 37 ° C. After daily exposure, the result of the presence or absence of a zone of growth inhibition of microorganisms around the film sample was taken into account. The experiment was carried out four times and the average value was taken as the final result. The results are shown in Table 1. Thus, the activity of these film samples was revealed against ten types of microorganisms: Staphylococcus aureus, Enterococcus faecalis, Kocuria varians, Enterococcus faecium, Hafnia alvei, Pseudomonas aeruginosa, Bacteroides fragilis, Candida albicans, Candida krusei. Moreover, the observed growth retardation zones of Staphylococcus aureus, Enterococcus faecalis, Kocuria varians exceeded those in the samples of films with a concentration of 300 μg / cm ² described in example 3.

Example 5

Polymer films were made according to the proposed method, the amount of introduced shikonin-containing alcohol solution of 5% was 0.85 ml, which ensured the concentration of the naphthoquinone complex in the films of 500 μg / cm ² . To determine the antimicrobial activity of the manufactured polymer films with a concentration of 500 μg / cm ² a microbiological study was conducted. We studied 48 strains of microorganisms of twelve different species, including museum cultures and ATCC strains. The determination was carried out by the standard disk-diffusion method, placing a film sample with an area of 1 cm ² on the surface of a Muller-Hilton nutrient medium with a 24-hour crop seeded with a lawn (turbidity 0.5 according to McFarland) and kept in a thermostat for 24 hours at a temperature of 37 ° C. After daily exposure, the result of the presence or absence of a zone of growth inhibition of microorganisms around the film sample was taken into account. The experiment was carried out four times and the average value was taken as the final result. The results obtained are shown in Table 1. The results of the experiment did not significantly differ from the results of the previous experiment described in Example 4. Thus, according to the results of the experiments, it was proved that a polymer film with a concentration of naphthoquinone complex of 400 μg / cm was optimal ² .

Example 6

Polymer films were made according to the proposed method, with a concentration of naphthoquinone complex of 400 μg / cm ² , of two types: with polyvinylpyrrolidone and without polyvinylpyrrolidone. Then, a study was conducted to determine the rate of release of the naphthoquinone complex from the films by the standard disco-diffuse method. A film sample with an area of about 1 cm ^{2 was} placed on the agar surface and the diameter of the diffusion zone of the active substance, determined by the bright red color of the agar, was measured for two hours with an interval of 30 minutes. As a result of the experiment, it was revealed that the sample containing polyvinylpyrrolidone contained the naphthoquinone complex faster, since after two hours the diameter of the diffusion zone of this sample was 20 mm, which is shown in FIG. 2, and the diameter of the diffusion zone of the sample not containing polyvinylpyrrolidone was 12 mm, as shown in FIG. 3.

Claims (1)

A polymer film with a naphthoquinone complex of biologically active substances of the red-root passeriformes - Lithospermum erythrorhizon Sieb. et Zucc., including a naphthoquinone complex of biologically active substances of the red-root beehive isolated from cell culture, glycerin, methyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, purified water, characterized in that at the stage of manufacturing the combined polymer base from methyl cellulose and sodium carbide glycerol 0.5 g of polyvinylpyrrolidone is added, and then 0.68 ml of a 5% alcohol solution of naphthoquinone complex of the red-root of the rabbit is added to the finished polymer base th, which allows to achieve a concentration of naphthoquinone complex film composed of 400 g / cm ² and ensure its antimicrobial activity, then the mixture was homogenized, cast onto a glass substrate a standard area of 85 cm ² and dried in an oven for 6-8 hours at a temperature of not more than + 50 ° C, stored under aseptic conditions.

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