RU2286800C1 - Antibacterial filler for female absorbent articles, method for production and uses thereof - Google Patents

Abstract

FIELD: medicine, in particular gynecology.

SUBSTANCE: claimed filler contains 1 % hydrogel of chitosan with molecular mass of 300-700 kDa and deacetylation ratio of at least 89 %, as well as metronidazole and dioxydine; and 1 l of composition contains (g): dry chitosan chlorohydrate 10; metronidazole 1.250-1.665; dioxydine 2.5; and balance: distilled water. Method for filler production includes stirring of 10 g dry chitosan chlorohydrate in 300 ml of distilled water heated up to 50°C for 20-30 min until full dissolution. Then gradually under intense agitation 250-333 ml of metronidazole for intravenous injection is added, mixture is thoroughly stirred for 5 min, further gradually under intense agitation 250 ml of 1% dioxydine solution is added, mixture is thoroughly stirred for 10 min and packed in container made of dark glass or plastics for storage. Methods for treatment of female genitals diseases by using absorbent articles containing filler of present invention also are disclosed.

EFFECT: non-toxic filler with prolonged antiinflammation and antibacterial effects.

3 cl, 3 tbl

Description

The invention relates to medicine, namely to obstetrics and gynecology, in particular to hygiene products used to treat inflammatory diseases of the female genital organs, and can be used as an antibacterial filler for a female sanitary pad or tampon.

Known antibacterial composition for local treatment of bacterial vaginosis based on a 3.5% gel form of chitosan ascorbate containing metronidazole at a dose of 2 mg in 1 ml [1]. The gel base of chitosan in the form of a 3.5% concentration is convenient for insertion into the vagina on a tampon in the treatment of inflammatory diseases of the internal genital organs of a woman. However, the high viscosity of the solution makes it technically difficult to quickly inject its small volume into the dressing (gasket) in order to uniformly impregnate the product. The composition of the known gel does not provide funds that reduce the seeding of microbial flora belonging to different classes of microbes (both anaerobes and aerobes, both gram-positive and gram-negative microflora).

Known methods of using 2-8% chitosan gel as an anti-inflammatory agent in the field of dentistry in the treatment of deep caries as a medical pad [2] and in the treatment of destructive forms of periodontitis as chitosan-containing paste when it is introduced into the transapical region of the tooth [3].

A known method of membrane dialysis of purulent-inflammatory diseases of soft tissues, where a low molecular weight 15% solution of chitosan ascorbate was used as a filler in the capsule membrane [4]. Both in dental diseases and in the treatment of purulent-inflammatory diseases of soft tissues, chitosan or its derivative of organic acid in high concentrations was introduced by the authors either into the wound cavity or into a cellulose acetate membrane with a pore diameter of 2.5-2.8 nm, transmitting substances with a molecular mass of less than 10,000 daltons [5]. The task of obtaining an antibacterial filler for feminine sanitary napkins containing funds in subtherapeutic dosages was not posed by the authors.

Known composition on a candle or gel basis for normalizing the internal microflora of the body with antimicrobial and anti-inflammatory effects, including a dry lyophilized mass of live lactobacilli, miramistin and water [6]. However, chitosan, which has nontoxicity, high biocompatibility, biodegradability, the properties of a system for transferring drugs through hostile biological environments, sorption ability, was not used by the authors as a gel base for feminine sanitary napkins or tampons. In addition, the composition does not include funds that provide an antimicrobial effect on those pathogenic types of microbes that disseminate in the area of the female external genital organs.

As the polymers used to treat diseases of the female genital organs, the collagen sponge Sangvikol is used after tissue treatment (cervical pseudo-erosion) with a plasma-chemical nitric oxide gas stream [7]. In the composition of the filler for feminine sanitary napkins, collagen polymer was not used. In addition, the use of protein preparations as a part of the filler is problematic, since the technology for manufacturing sanitary napkins involves the use of drying ovens with a temperature regime of over 100 ° C.

The closest to the claimed technical solution for the totality of signs are:

1) a biological composition for the treatment of wounds "Collachite", containing, as well as the claimed invention, chitosan and antiseptic preparations of synthetic origin, which can be made in the form of a film or sponge, which are biodegradable agents [8];

2) a dressing for the treatment of wounds in the form of a film containing, in the same way as the claimed invention, chitosan, an organic acid, an antimicrobial or antiseptic drug [9];

3) a hydrocolloid application coating containing, as well as the claimed invention, chitosan, organic acid and water [10].

All these technical solutions are associated with the closure and treatment of wound defects, made in the form of a sponge or film and were not offered as a filler for feminine sanitary napkins or tampons. In addition, the technical (mechanical) introduction of these compositions in the form of a film or sponge into a woven or non-woven material with its uniform distribution is difficult. The content of crosslinking agents in the proposed compositions in the form of epichlorohydrin or glutaraldehyde is undesirable in view of their high toxicity (tissue, cell poison). For the treatment of wounds, the authors of the patents [9, 10] achieve a decrease in the adhesion force to the wound surface and the adsorption force of less than 400%, while using filler for a sanitary pad or tampon requires high adhesion and sorption ability to the mucous membrane and skin of the external genital organs .

The filler for a sanitary pad or tampon should be of easily flowing consistency, highly adhesive to the mucous membrane or skin, have a high sorption capacity, be transparent and not impair the aesthetic characteristics of the product, have antibacterial properties with respect to the pathogenic residual microflora of the vagina and external genital organs, contain subtherapeutic concentrations antibacterial agents, preserve the lactobacillary microflora of the vagina, cause fast pain medication and a counterweight inflammatory effects, to be a non-toxic composition, easy to dose in small doses when included in the composition of a pad or tampon.

The invention is aimed at solving the problem of creating an effective filler for a female sanitary napkin or tampon for the treatment of inflammatory diseases of the female genital organs and should have the following advantages:

- absolute non-toxicity;

- prolonged anti-inflammatory and antibacterial effects;

- selective effect on the microflora of the female genital organs while maintaining the number of viable beneficial lactobacilli;

- fast analgesic effect;

- uniform therapeutic effect on the mucous membranes and skin of the female genital organs;

- shape stability of the gel composition;

- transparency and lack of composition odor;

- high availability of antibacterial agents through mucous and skin barriers.

The objective is achieved due to the fact that the antibacterial filler for a feminine sanitary pad or swab contains 1% hydrogel of chitosan hydrochloride with a molecular weight of 300-700 kDa and a degree of deacetylation of at least 89%, which includes metronidazole and dioxidine, and include dry chitosan hydrochloride - 10 g, metronidazole - 1,250-1,665 g, dioxidine - 2.5 g, distilled water - the rest, moreover, the method of filler preparation involves mixing 10 g of dry chitosan hydrochloride in 300 ml of distilled water heated to 50 ° C with using a high-speed mixer for 20-30 minutes until completely dissolved, then gradually add 250-333 ml of metronidazole for intravenous injection with vigorous stirring, mix thoroughly for 5 minutes, then gradually add 250 ml of a 1% dioxidine solution with vigorous stirring, mix 5 min, the volume of the gel obtained is adjusted to 1 liter with distilled water, mixed thoroughly for 10 minutes, placed for long-term storage in a container made of dark glass or plastic, and when using a filler for treatment Ia pelvic inflammatory disease comprise administering to a tampon in the vagina and / or the sanitary napkin to 5 ml of a 1% gel of chitosan hydrochloride, metronidazole and dioxidine, allowed to stand for 12 hours in the procedure for 7 days.

A method of obtaining a filler is as follows.

Upon receipt of 1 liter of gel filler, 10 g of dry chitosan hydrochloride of molecular weight 300-700 kDa and a degree of deacetylation of 89-98% are completely dissolved with vigorous stirring using a high-speed stirrer for 20-30 minutes in 300 ml of distilled water heated to 50 ° C. Then, 250-333 ml of metronidazole for intravenous injection (1 ml - 5 mg of the drug) (1250-1665 mg) is gradually added into the gel of chitosan hydrochloride thus obtained, and the mixture is mixed thoroughly for 5 minutes, gradually in the resulting solution stirring make 250 ml of a 1% solution of dioxidine. Stirring is continued for 5 minutes. The volume of the obtained chitosan gel was adjusted with distilled water to 1 liter with vigorous stirring for 10 minutes. The gel is placed for long-term storage in a container of dark glass or plastic.

We used nutrient media for the cultivation of microorganisms: meat-peptone agar (MPA) and 0.7% nutrient agar, 2% and 0.7% bifidum agar, samples of chitosan gels, clinical strains of microorganisms: Escherichia coli, Enterococcus faecalis, Candida albicans and reference strain Staphylococcus aureus 209 P, lactobacilli, which are part of the official drug "Lactobacterin dry", dyes, ethyl alcohol 96%.

Antimicrobial properties were tested on the following samples of chitosan gels:

- Sample No. 1 - 0.5% aqueous solution of chitosan hydrochloride (molecular weight 70 kDa, degree of deacetylation of 87%) + 0.25% solution of dioxidine

- Sample No. 2 - 0.625% solution of ascorbic acid

- Sample No. 3 - 0.25% dioxidine solution

- Sample No. 4 - 0.5% aqueous solution of chitosan hydrochloride (molecular weight 300 kDa, degree of deacetylation 89%) + 0.25% solution of dioxidine

- Sample No. 5 - 1% aqueous solution of chitosan hydrochloride (molecular weight 300 kDa, deacetylation degree 89%) + 0.25% dioxidine solution

- Sample No. 6 - 0.5% aqueous solution of chitosan ascorbate (molecular weight 700 kDa, degree of deacetylation 98%) + 0.25% dioxidine solution

- Sample No. 7 - 1% aqueous solution of chitosan ascorbate (molecular weight 700 kDa, degree of deacetylation 98%) + 0.25% dioxidine solution

- Sample No. 8 - 0.5% aqueous solution of chitosan hydrochloride (molecular weight 700 kDa, degree of deacetylation 98%) + 0.25% dioxidine

- Sample No. 9 - 1% aqueous solution of chitosan hydrochloride (molecular weight 700 kDa, degree of deacetylation 98%) + 0.25% dioxidine

- Sample No. 10 - 0.5% aqueous solution of chitosan hydrochloride (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole

- Sample No. 11 - 1% aqueous solution of chitosan hydrochloride (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole

- Sample No. 12 - 0.5% aqueous solution of chitosan hydrochloride (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole + 0.25% dioxidine

- Sample No. 13 - 1% aqueous solution of chitosan hydrochloride (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole + 0.25% dioxidine

- Sample No. 14 - 0.5% aqueous solution of chitosan ascorbate (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole

- Sample No. 15 - 1% aqueous solution of chitosan ascorbate (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole

- Sample No. 16 - 0.5% aqueous solution of chitosan ascorbate (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole + 0.25% dioxidine

- Sample No. 17 - 1% aqueous solution of chitosan ascorbate (molecular weight 700 kDa, degree of deacetylation 98%) + 1,665 mg / ml metronidazole + 0.25% dioxidine

- Sample No. 18 - a solution of metronidazole 1,665 mg / ml

- Sample No. 19 - solution of metronidazole 1,250 mg / ml

Antimicrobial analysis is as follows.

In sterile Petri dishes under aseptic conditions, MPA is poured in a volume of 15 ml. After the medium has solidified, a 0.7% nutrient agar is poured in a volume of 4 ml with 0.1 ml of a suspension of one of the microorganism cultures taken in the experiment at a concentration corresponding to the turbidity standard 10 ⁹ . For the cultivation of lactobacilli, bifidum agar was selected, respectively 2% 15 ml and 0.7% 4 ml. In the first series of experiments, wells with a diameter of 1 cm are punched with a sterile puncher in two-layer agar, into which gel samples in a volume of 0.1 ml per well are introduced using an automatic micropipette. In the second series of experiments, gel samples in the form of droplets with a volume of 0.01 ml are applied onto the surface of a two-layer agar using an automatic micropipette. To control the sterility of the gel samples, two-layer agar is used without suspension of microorganisms. The results are evaluated after 24 and 48 hours of cultivation in a thermostat at a temperature of 37 ° C. The results of experiments with lactobacilli are evaluated after 72 hours of cultivation in desiccators at a temperature of 37 ° C.

The research results showed:

Sample No. 1.

When a sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth (32, 32 and 30 mm in diameter, including the diameter of the hole) with clearly defined boundaries (bactericidal effect in the center of the zone and bacteriostatic in the periphery).

After 48 hours, a statistically indistinguishable increase in the diameter of the zones was noted (up to 33, 33 and 32 mm, respectively), their boundaries became clearer (i.e., prolongation of bactericidal action was observed). When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed with unclear boundaries (23, 23 and 22 mm in diameter with a droplet diameter of 7-8 mm). After 48 hours, a statistically indistinguishable increase in the diameter of the zones was noted (up to 24, 25 and 23 mm, respectively), their boundaries became clearer (the result is the same as in the experiment with the introduction of the gel into the wells).

When a sample was added to the wells after 24 hours, against the background of continuous growth of S. aureus 209 P (intensity ++++), zones of lack of growth (25, 25 and 26 mm in diameter, including the diameter of the hole) with clearly defined boundaries (bactericidal effect in the center of the zone and bacteriostatic in the periphery). After 48 hours, a marked increase in the diameter of the zones was not observed (26, 25 and 26 mm, respectively), their boundaries became clearer, secondary growth was noted: 1-2 small colonies in each zone (maintaining the bacteriostatic effect of the gel). When the sample was applied to the surface of the agar against a background of continuous uniform growth (intensity ++++) immediately under the applied gel and by 2-3 mm around the periphery, a meager growth (intensity ++) was observed. After 48 hours, the picture is unchanged (i.e., the bacteriostatic effect persists).

In the experiment with E.faecalis, uniform growth was observed over the entire surface of the agar (intensity +++), regardless of the method of application of the gel. In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Thus, this gel sample, due to dioxidine, has a pronounced bactericidal effect on E. coli, and after 48 hours, the action of dioxidine is prolonged. On S.aureus 209 P, the sample has a less pronounced effect, but the bacteriostatic effect persists for 48 hours. The sample has no inhibitory effect on the growth of E.faecalis and C. albicans, which is associated with the insensitivity of these cultures to dioxidine and the use of chitosan with a low molecular weight. The sample does not have a visible antimicrobial effect on lactobacilli.

Sample No. 2.

There was continuous uniform growth of S. aureus 209 P, E. faecalis and E. coli (intensity ++++, +++ and ++++, respectively) over the entire surface of the agar, regardless of the method of application of the sample. After 48 hours, the growth pattern is maintained. In the experiment with C. albicans, when the sample was introduced into the wells after 24 hours against the background of uniform continuous growth (+++ intensity), growth intensification zones (++++) with blurred borders (approximately 33-34 mm in diameter, including the diameter of the hole) were noted ) After 48 hours, an increase in the diameter of the zones was recorded (up to 37, 38 and 39 mm), their boundaries became clearer (prolongation of the stimulating effect of ascorbic acid). When the sample was applied to the surface of the agar, fuzzy growth intensification (++++) was observed in the center of the cup and more meager growth (+++) along the periphery. In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Thus, ascorbic acid stimulates the growth of yeast fungi of the genus Candida and does not have a visible effect on other studied cultures.

Sample No. 3.

When a sample was added to the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed with clearly defined boundaries (34, 34 and 33 mm in diameter, including the diameter of the hole) - a pronounced bactericidal effect. After 48 hours, no increase in the diameter of the zones was noted, the boundaries became less clear, secondary growth was observed: 1-2 small colonies in the zone (weakening of the antimicrobial effect). When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth with clearly defined boundaries (24, 23 and 22 mm in diameter with a droplet size of 7-8 mm) were noted. After 48 hours, no increase in the diameter of the zones was noted, their boundaries became blurred, secondary growth was observed to 10-20 small colonies in the zone (a marked weakening of the antimicrobial effect is observed).

When a sample was introduced into the wells after 24 hours, against the background of continuous growth of S. aureus 209 P (intensity ++++), zones of lack of growth were observed with clearly defined boundaries (28, 29 and 28 mm in diameter, including the diameter of the hole). After 48 hours, no increase in the diameter of the zones was observed, their boundaries became less clear, there was a secondary growth of 10-20 small colonies in each zone (marked weakening of the antimicrobial effect). When applying the sample to the surface of the agar against a background of continuous uniform growth (intensity ++++) immediately below the applied gel and by 2-3 mm at the periphery, a moderate decrease in growth intensity (++) was observed. After 48 hours, a slight decrease in growth rate (++), i.e. a decrease in antimicrobial activity is observed.

In the experiment with E.faecalis, uniform growth (intensity +++) was observed over the entire surface of the agar, regardless of how the gel was applied. In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Thus, dioxidine has a more pronounced antimicrobial effect on E. coli and less pronounced on S.aureus 209 P, while the effect of dioxidine decreases markedly over time. Dioxidine does not have a visible effect on E. faecalis, C. albicans and lactobacilli.

Sample No. 4.

When a sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth (34, 35 and 38 mm in diameter, including the diameter of the hole) with unclear boundaries were observed (bactericidal effect in the center of the zone and bacteriostatic in the periphery). After 48 hours, a statistically indistinguishable increase in the diameter of the zones was noted (up to 35, 37 and 40 mm, respectively), their boundaries became clearer (i.e. prolongation of bactericidal action was observed). When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed directly under the applied gel and a decrease in growth intensity to (+) by 3 mm along the periphery of the drop; after 48 hours, the growth inhibition zone to (+) along the periphery of the drop increased to 4-5 mm (prolongation of the antimicrobial effect of the gel is noted).

When a sample was added to the wells after 24 hours, against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of lack of growth (21.23 and 22 mm in diameter, including the diameter of the hole) with unclear boundaries were observed: along the periphery of the zone bactericidal action, decrease in growth intensity to + over 5-6 mm (bactericidal effect in the center of the zone and pronounced bacteriostatic in the periphery). After 48 hours, a marked increase in the diameter of the zones was not observed, their boundaries became clearer (preservation of the antimicrobial effect of the gel). When applying the sample to the surface of the agar against the background of continuous uniform growth (intensity ++++) immediately under the applied gel and by 2-3 mm around the periphery of the drop, there was a lack of growth, the boundaries of the zone are not clearly marked. After 48 hours, the boundaries of the zone of lack of growth became clearer (i.e., the antimicrobial effect persists).

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours, the picture is unchanged (i.e., the bactericidal effect is preserved).

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, the growth rate decreases immediately below the applied gel to (++). After 48 hours, the picture is unchanged (bacteriostatic effect is maintained).

In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Thus, this gel sample, due to dioxidine, has a pronounced bactericidal effect on E. coli, and after 48 hours, the action of dioxidine is prolonged. On S.aureus 209 P, the sample has a less pronounced effect, however, the prolongation of the antimicrobial effect is noted. By increasing the molecular weight of chitosan, the sample has a bactericidal effect on the growth of E.faecalis and a bacteriostatic effect on yeast-like fungi of the genus Candida, similarly to sample No. 8. The sample does not have a visible antimicrobial effect on lactobacilli.

Sample No. 5.

When a sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth (35, 35 and 37 mm in diameter, including the diameter of the hole) with clearly defined boundaries (bactericidal effect in the center of the zone and bacteriostatic in the periphery). After 48 hours, a statistically indistinguishable increase in the diameter of the zones was noted (up to 36, 37 and 38 mm, respectively), their boundaries became clearer (i.e., prolongation of bactericidal action was observed). When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed directly under the applied gel and a decrease in the growth intensity to (+) by 4 mm around the periphery of the drop; after 48 hours - the zone of growth inhibition to (+) along the periphery of the drop 4-5 mm and then - the decrease in growth intensity to (++) by 2-3 mm from the border of the previous zone (prolongation of the antimicrobial action of the gel is noted).

When the sample was added to the wells after 24 hours against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of incomplete lack of growth were noted (2-4 colonies were preserved within the zone of 21, 22 and 22 mm in diameter, including diameter wells) with fuzzy defined boundaries (reduced bactericidal effect). After 48 hours, a decrease in growth intensity to (+++) over 4-5 mm was observed along the periphery of the zone (prolongation of the antimicrobial effect of the gel). When applying the sample to the surface of the agar against the background of continuous uniform growth (intensity ++++) immediately under the applied gel and by 2-3 mm around the periphery of the drop, there was a lack of growth, the boundaries of the zone are not clearly marked. After 48 hours, the boundaries of the zone of lack of growth became clearer (the antimicrobial effect persists).

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours, the picture is unchanged (bactericidal effect is maintained).

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. When applying the gel to the surface of the agar against the background of continuous uniform growth (intensity +++) after 24 hours, the growth rate decreases immediately below the applied gel to (+). After 48 hours, the picture is unchanged (bacteriostatic effect is maintained).

In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with an intensity of ++++.

Thus, this gel sample, compared with the previous one, has a less pronounced antimicrobial effect on S. aureus 209 P at the periphery, which is associated with a decrease in the release of dioxidine in agar. However, this prolongs the antimicrobial action, which is especially clearly seen in the example of E. coli. The gel acts on the culture of E.faecalis and on yeast-like fungi of the genus Candida similarly to sample No. 9, because dioxidine does not affect their growth. The sample does not have a visible antimicrobial effect on lactobacilli.

Sample No. 6.

When a sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth (33, 33 and 34 mm in diameter, including the diameter of the hole) with unclear boundaries were observed (bactericidal effect in the center of the zone and bacteriostatic in the periphery). After 48 hours, there was no increase in the diameter of the zones (33, 33 and 34 mm, respectively), secondary growth along the periphery: 2-3 colonies in the zone (i.e., a decrease in antimicrobial activity compared with samples No. 10-11) . When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were recorded directly under the applied gel and a decrease in growth intensity to (++) by 4-5 mm around the periphery of the drop; after 48 hours, there was no growth immediately under the applied gel, a decrease in growth intensity to (++) by 6–7 mm along the periphery of the drop was noted, and then there was an increase in the growth inhibition zone (intensity +++) by another 2–3 mm (prolonged, but less pronounced antimicrobial effect than samples No. 10-11).

When the sample was introduced into the wells after 24 hours against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of decrease in growth intensity to (+) by 3-4 mm along the periphery of the hole and further by 4-5 mm to (++). After 48 hours, the picture is unchanged (bacteriostatic effect along the periphery of the well). When applying the sample to the surface of the agar against a background of continuous uniform growth (intensity ++++) immediately under the applied gel and 2 mm around the periphery of the drop, there was a lack of growth, the boundaries of the zone are not clearly marked. After 48 hours, immediately under the applied gel, there was no growth, by 2 mm on the periphery - a decrease in the growth rate to (++) (a bactericidal effect is observed directly under the applied gel and a weak bacteriostatic effect on the periphery).

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. After 48 hours, growth is entirely. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours, the picture is unchanged (i.e., the bactericidal effect is preserved).

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, uniform continuous growth (intensity +++) was observed over the entire surface of the agar, with the exception of 1-2 mm along the periphery of the hole; outside the growth retardation zone, there was a growth intensification zone to (+ +++) with a length of 3-4 mm. After 48 hours, the growth inhibition zone is not visible, the zone of growth intensification is less clearly defined. When applying the gel to the surface of the agar against the background of continuous uniform growth (intensity +++) after 24 hours, the growth rate decreases immediately below the applied gel to (+), along the periphery - the zone of growth intensification to (++++) 2-3 mm . After 48 hours, the picture is unchanged, the zone of growth intensification is less clearly defined (i.e., the bacteriostatic effect remains). In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Thus, this gel sample, in comparison with samples No. 10-11, has a prolonged, but less pronounced antimicrobial effect on S. aureus 209 P and E. coli on the periphery, which is associated with a decrease in the release of dioxidine in agar with an increase in the molecular weight of chitosan . The gel acts on the yeast-like fungi of the genus Candida bacteriostatically. The sample does not have a visible antimicrobial effect on lactobacilli.

Sample No. 7.

When a sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed (32, 33 and 33 mm in diameter, including the diameter of the hole) with unclear boundaries (bactericidal effect in the center of the zone and bacteriostatic in the periphery). After 48 hours, there was practically no increase in the diameter of the zones (32, 33 and 34 mm, respectively), secondary growth along the periphery: 2-4 colonies in the zone (i.e., a decrease in antimicrobial activity compared with samples No. 10-12 ) When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed directly under the applied gel and a decrease in growth intensity to (++) by 4-5 mm around the periphery of the drop; after 48 hours, there was no growth directly under the applied gel, a decrease in growth intensity to (+++) was observed by 6-7 mm along the periphery of the drop (a clear decrease in antimicrobial activity was observed).

When a sample was added to the wells after 24 hours, against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of decrease in growth intensity to (++) by 2-3 mm along the periphery of the hole and further by 3-4 mm to (+++). After 48 hours, the picture is unchanged (bacteriostatic effect along the periphery of the well). When applying the sample to the surface of the agar against a background of continuous uniform growth (intensity ++++) immediately under the applied gel, there was a lack of growth. After 48 hours, the picture is unchanged (bactericidal effect directly under the applied gel and a weak bacteriostatic effect on the periphery).

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours, the bactericidal effect persists.

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, uniform continuous growth (intensity +++) was observed over the entire surface of the agar, with the exception of 1-2 mm along the periphery of the hole; outside the growth retardation zone, there was a growth intensification zone to (+ +++) with a length of 3-4 mm. After 48 hours, the growth inhibition zone is not visible, the growth intensification zone is less clearly defined (maintaining the stimulating effect of this sample on yeast-like fungi). When applying the gel to the surface of the agar against the background of continuous uniform growth (intensity +++) after 24 hours, single colonies (2-3 in the zone) immediately below the applied gel, on the periphery the zone of growth intensification to (++++) 2- 3 mm. After 48 hours, the picture is unchanged, the zone of growth intensification is less clearly defined (a pronounced bacteriostatic effect remains). In experiments with lactobacilli, when a sample was introduced into the wells, continuous growth was observed over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (+++) was noted.

Thus, this gel sample, compared to the previous one, has a less pronounced antimicrobial effect on S. aureus 209 P and E. coli at the periphery, which is associated with a decrease in the release of dioxidine in agar with an increase in chitosan concentration. On the culture of E. faecalis, this sample, when in direct contact, is bactericidal. The gel has a more pronounced bacteriostatic effect on yeast-like fungi of the genus Candida than the previous sample; the stimulating effect along the periphery of the bacteriostatic zone of the gel is preserved. The sample has a weak bacteriostatic effect on lactobacilli.

Sample No. 8.

When a sample was added to the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth (37, 37 and 40 mm in diameter, including the diameter of the hole) with clearly defined boundaries (bactericidal effect in the center of the zone and bacteriostatic in the periphery). After 48 hours, a statistically indistinguishable increase in the diameter of the zones was noted (38, 38 and 40 mm, respectively). When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed directly under the applied gel and a decrease in growth intensity to (+) by 4-5 at the periphery of the drop. After 48 hours, there was no growth directly under the applied gel, a decrease in growth intensity to (+) by 5-6 mm along the periphery of the drop was noted, and then there was an increase in the growth inhibition zone (intensity ++) by another 2-3 mm (prolonged antimicrobial effect).

When the sample was introduced into the wells after 24 hours against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of decrease in the growth intensity to (+) by 5-6 mm were observed around the periphery of the hole with blurred borders. After 48 hours, the growth rate decreases to (+++) by 4-5 mm outside the previously designated zones (prolongation of the bacteriostatic effect along the periphery of the well). When applying the sample to the surface of the agar against a background of continuous uniform growth (intensity ++++) immediately below the applied gel and 2-3 mm around the periphery of the drop, there is a lack of growth, the boundaries of the zone are not clearly marked. After 48 hours, outside the previously designated zones, the growth rate decreases to (+++) by 2-3 mm (bactericidal effect directly under the gel and a weak prolonged bacteriostatic effect on the periphery). In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours, the picture is unchanged (bactericidal effect).

In the experiment with C. albicans, when the gel was introduced into the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the well, there was no growth intensification. After 48 hours, the picture is unchanged (there is no stimulating effect on the periphery). When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, the growth rate decreases directly under the applied gel to 3-5 small colonies. After 48 hours, the picture is unchanged (bacteriostatic effect).

In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Thus, this gel sample, in comparison with samples based on chitosan ascorbate with the same molecular weight and concentration, has a more pronounced antimicrobial effect on dioxidine sensitive cultures. The gel acts on yeast-like fungi of the genus Candida bacteriostatically, but does not have a stimulating effect on the periphery of the bacteriostatic zone. The sample does not have a visible antimicrobial effect on lactobacilli.

Sample No. 9.

When a sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed (32, 33 and 34 mm in diameter, including the diameter of the hole) with unclear boundaries (bactericidal effect in the center of the zone and bacteriostatic in the periphery). After 48 hours, there was no increase in the diameter of the zones, but the boundaries became clearer. When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were recorded directly under the applied gel and a decrease in growth intensity to (++) by 3-4 mm around the periphery of the drop. After 48 hours, directly under the applied gel, there was no growth, a 5–6 mm increase in the periphery of the drop showed a decrease in growth intensity to (++) and then an increase in the growth inhibition zone (intensity +++) by another 2–3 mm (prolonged antimicrobial effect )

When the sample was introduced into the wells after 24 hours against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of decrease in the growth intensity to (+) by 5-6 mm were observed around the periphery of the hole with blurred borders. After 48 hours, the growth rate decreases to (+++) by 3-4 mm outside the previously designated zones (prolongation of the bacteriostatic effect along the periphery of the well). When the sample was applied to the surface of the agar against the background of continuous uniform growth (intensity ++++) immediately below the applied gel, there was a lack of growth, the boundaries of the zone are not clearly defined, and the growth intensity decreases to (++) by 2-3 mm on the periphery. After 48 hours, outside the previously designated zones, the growth rate decreases to (+++) by 2-3 mm (bactericidal effect immediately under the applied gel and weaker than the previous sample, prolonged bacteriostatic effect on the periphery).

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours, the picture is unchanged (bactericidal effect).

In the experiment with C. albicans, when the gel was introduced into the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the well, there was no growth intensification. After 48 hours, the picture is unchanged (there is no stimulating effect on the periphery). When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours, the picture is unchanged (bactericidal effect is maintained). In experiments with lactobacilli, when a sample was introduced into the wells, continuous growth was observed over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (+++) was noted.

Thus, this gel sample, in comparison with the previous one, has a less pronounced antimicrobial effect on dioxidine sensitive cultures, which is associated with a slower release of dioxidine in agar due to an increase in chitosan concentration. The gel acts bactericidal on yeast-like fungi of the genus Candida and does not have a stimulating effect on the periphery. The sample has a weak bacteriostatic effect on lactobacilli.

Sample No. 10.

When a sample was introduced into the wells, uniform continuous growth of S.aureus 209 P (intensity ++++), E.faecalis (intensity +++), E.coli (intensity ++++) and C. albicans (intensity ++ +) over the entire surface of the agar with the exception of the zone of 1-2 mm along the periphery of the hole. After 48 hours of cultivation, the results are unchanged.

When applying the sample to the agar surface after 24 hours against a background of continuous growth of E. coli (intensity ++++), S.aureus 209 P (intensity ++++) and E.faecalis (intensity +++) directly under the applied gel there was a lack of growth, after 48 hours - the picture is unchanged (bactericidal effect remains). Against the background of continuous growth of C. albicans (intensity +++), a decrease in growth intensity (4-5 small colonies) was noted immediately after the applied gel after 24 hours, the picture was unchanged after 48 hours (i.e., the bacteriostatic effect remains). In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Sample No. 11.

When a sample was introduced into the wells, uniform continuous growth of S.aureus 209 P (intensity ++++), E.faecalis (intensity +++), E.coli (intensity ++++) and C. albicans (intensity ++ +) over the entire surface of the agar with the exception of the zone of 1-2 mm along the periphery of the hole. After 48 hours, the cultivation results were unchanged.

When the sample is applied to the agar surface after 24 hours against a background of continuous growth of E. coli (intensity ++++), S.aureus 209 P (intensity ++++), E.faecalis (intensity +++) and C. albicans (intensity +++) immediately under the applied gel, there was a lack of growth, after 48 hours - the picture was unchanged (bactericidal effect remains).

In experiments with lactobacilli, when a sample was introduced into the wells, continuous growth was observed over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (+) was noted.

Sample No. 12.

When a sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth (36, 37 and 39 mm in diameter, including the diameter of the hole) with clearly defined boundaries were noted. After 48 hours, a statistically indistinguishable increase in the diameter of the zones was noted (37, 38 and 40 mm, respectively). When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed directly below the applied gel and a decrease in growth intensity to (+) by 4-5 mm around the periphery of the drop. After 48 hours, there was no growth directly under the applied gel, a decrease in growth intensity to (+) by 4-5 mm along the periphery of the drop was noted, and then an increase in the growth inhibition zone (intensity ++) by another 2-3 mm was noted.

When the sample was introduced into the wells after 24 hours against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of decrease in the growth intensity to (+) by 5-6 mm were observed around the periphery of the hole with blurred borders. After 48 hours, there is a decrease in growth intensity to (+++) by 4-5 mm beyond the previously indicated. When applying the sample to the surface of the agar against a background of continuous uniform growth (intensity ++++) immediately below the applied gel and 2-3 mm around the periphery of the drop, there is a lack of growth, the boundaries of the zone are not clearly marked. After 48 hours, outside the previously designated zones, the growth rate decreases to (+++) by 2-3 mm.

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours - the picture is unchanged.

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, uniform continuous growth (intensity +++) over the entire surface of the agar except 1-2 mm along the periphery of the hole, there was no growth intensification. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, the growth rate decreases directly under the applied gel to 3-5 small colonies. After 48 hours - the picture is unchanged.

In experiments with lactobacilli, when a sample was introduced into the wells, continuous growth was observed over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (++) was noted.

Sample No. 13.

When a sample was added to the wells after 24 hours, against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed (32.32 and 33 mm in diameter, including the diameter of the hole) with unclear boundaries. After 48 hours, there was no increase in the diameter of the zones (32, 33 and 34 mm, respectively), the boundaries were clear. When applying the sample to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++) - the zone of lack of growth immediately below the applied gel and a decrease in growth intensity to (++) by 3-4 mm along the periphery of the drop. After 48 hours, there was no growth directly under the applied gel, a decrease in growth intensity to (++) by 4-5 mm along the periphery of the drop was recorded, and then an increase in growth inhibition zone (intensity +++) by another 2-3 mm was recorded. When the sample was introduced into the wells after 24 hours against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of decrease in growth intensity to (+) by 4-5 mm were observed around the periphery of the hole with blurred borders. After 48 hours, there is a decrease in growth intensity to (+++) by 3-4 mm beyond the previously indicated. When applying the sample to the surface of the agar against a background of continuous uniform growth (intensity ++++) directly below the applied gel, there is a lack of growth, the boundaries of the zone are not clearly defined, and the growth intensity decreases to (++) by 2-3 mm on the periphery. After 48 hours, outside the previously designated zones, the growth rate decreases to (+++) by 2-3 mm.

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours - the picture is unchanged.

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, uniform continuous growth (intensity +++) over the entire surface of the agar except 1-2 mm along the periphery of the hole, there was no growth intensification. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours - the picture is unchanged.

In experiments with lactobacilli, when a sample was introduced into the wells, the growth was continuous over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (+) was noted.

Sample No. 14.

When the sample was introduced into the wells, uniform continuous growth of S.aureus 209 P (intensity ++++), E.faecalis (intensity +++) and E.coli (intensity ++++) was observed over the entire surface of the agar except for zone 1 -2 mm on the periphery of the hole. After 48 hours, the results are unchanged.

When applying the sample to the agar surface after 24 hours against a background of continuous growth of E. coli (intensity ++++), S.aureus 209 P (intensity ++++), and E.faecalis (intensity +++) immediately below the applied gel showed a lack of growth, after 48 hours - the picture is unchanged (bactericidal effect is preserved).

In the experiment with C. albicans, when the sample was introduced into the wells against the background of uniform continuous growth on the cup surface (intensity +++), more intensive growth (++++) of microorganisms was observed along the periphery of the hole (zones 24, 24 and 25 mm in diameter, including hole diameter). When a sample was applied to the agar surface against a background of uniform continuous growth (+++ intensity), moderate inhibition of microorganism growth (+) was observed directly under the applied gel; a growth intensification zone (++++) of 2-3 mm width was observed around the periphery. After 48 hours, the results are unchanged.

In experiments with lactobacilli, when a sample was introduced into the wells, continuous growth was observed over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (+) was noted.

Sample No. 15.

When the sample was introduced into the wells, uniform continuous growth of S.aureus 209 P (intensity ++++), E.faecalis (intensity +++) and E.coli (intensity ++++) was observed over the entire surface of the agar except for zone 1 -2 mm on the periphery of the hole. After 48 hours, the results are unchanged.

When applying the sample to the agar surface after 24 hours against a background of continuous growth of E. coli (intensity ++++), S.aureus 209 P (intensity ++++), and E.faecalis (intensity +++) immediately below the applied gel showed a lack of growth, after 48 hours - the picture is unchanged (bactericidal effect is preserved).

In the experiment with C. albicans, when the sample was introduced into the wells against the background of uniform continuous growth on the cup surface (intensity +++), more intensive growth (++++) of microorganisms was observed along the periphery of the hole (zones 23, 23 and 24 mm in diameter, including hole diameter). When the sample was applied to the surface of the agar against the background of uniform continuous growth (intensity +++) immediately below the applied gel, there was a lack of growth of microorganisms, along the periphery there was a growth intensification zone (++++) 2-3 mm wide. After 48 hours, the results are unchanged.

In experiments with lactobacilli, when a sample was introduced into the wells, continuous growth was observed over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (+) was noted.

Sample No. 16.

When a sample was added to the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth (37, 37 and 38 mm in diameter, including the diameter of the hole) with clearly defined boundaries were noted. After 48 hours, a statistically indistinguishable increase in the diameter of the zones was noted (37, 38 and 39 mm, respectively). When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed directly below the applied gel and a decrease in growth intensity to (+) by 4-5 mm around the periphery of the drop. After 48 hours, there was no growth directly under the applied gel, a decrease in growth intensity to (+) by 4-5 mm along the periphery of the drop was noted, and then an increase in the growth inhibition zone (intensity ++) by another 2-3 mm was noted

When the sample was introduced into the wells after 24 hours against the background of continuous growth of S.aureus 209 P (intensity ++++), zones of decrease in the growth intensity to (+) by 5-6 mm were observed around the periphery of the hole with blurred borders. After 48 hours, there is a decrease in growth intensity to (+++) by 3-4 mm beyond the previously indicated. When applying the sample to the surface of the agar against the background of continuous uniform growth (intensity ++++) immediately under the applied gel and by 2-3 mm around the periphery of the drop, there was a lack of growth, the boundaries of the zone are not clearly marked. After 48 hours, outside the previously designated zones, the growth rate decreases to (+++) by 2-3 mm.

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, uniform continuous growth (intensity +++) over the entire surface of the agar except 1-2 mm along the periphery of the hole. After 48 hours - no change. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours - the picture is unchanged.

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, uniform continuous growth (intensity +++) was observed over the entire surface of the agar, with the exception of 1-2 mm along the periphery of the hole; outside the growth retardation zone, there was a growth intensification zone to ( +++) 3 mm long. After 48 hours, the growth inhibition zone is not visible, the zone of growth intensification is less clearly defined. When applying the gel to the surface of the agar against the background of continuous uniform growth (intensity +++) after 24 hours, immediately under the applied gel - single colonies (3-4 in the zone), along the periphery - the zone of growth intensification (up to ++++) 2 -3 mm. After 48 hours, the picture is unchanged, the zone of growth intensification is less clearly defined (a pronounced bacteriostatic effect remains).

In experiments with lactobacilli, when a sample was introduced into the wells, continuous growth was observed over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (++) was noted.

Sample No. 17.

When the sample was introduced into the wells after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed (32.33 and 33 mm in diameter, including the diameter of the hole) with clearly defined. After 48 hours, there was no increase in the diameter of the zones, the boundaries became clear. When the sample was applied to the agar surface after 24 hours against the background of continuous growth of E. coli (intensity ++++), zones of lack of growth were observed immediately below the applied gel and a decrease in growth intensity to (++) by 3-4 mm along the periphery of the drop. After 48 hours, there was no growth directly under the applied gel, a decrease in growth intensity to (++) by 4-5 mm along the periphery of the drop was noted, and then an increase in growth inhibition zone (intensity +++) by another 2-3 mm was noted.

When a sample was introduced into the wells after 24 hours, against the background of continuous growth of S. aureus 209 P (intensity ++++), zones of decrease in growth intensity to (+) by 3-4 mm were observed along the periphery of the hole with blurred borders. After 48 hours - a decrease in growth rate to (+++) by 4 mm beyond the previously indicated. When applying the sample to the surface of the agar against the background of continuous uniform growth (intensity ++++) immediately below the applied gel, there was a lack of growth, the boundaries of the zone are not clearly marked, along the periphery by 2-3 mm - a decrease in the growth intensity to (++). After 48 hours, outside the previously designated zones, the growth rate decreases to (+++) by 2-3 mm.

In the experiment with E. faecalis, when the gel was added to the wells after 24 hours, a uniform continuous growth (intensity +++) was observed over the entire surface of the agar except 1-2 mm along the periphery of the hole. After 48 hours - the picture is unchanged. When applying the gel to the surface of the agar against a background of continuous uniform growth (intensity +++) after 24 hours, there is no growth directly under the applied gel. After 48 hours - the picture is unchanged.

In the experiment with C. albicans, when the gel was added to the wells after 24 hours, uniform continuous growth (intensity +++) over the entire surface of the agar except 1-2 mm along the periphery of the hole, outside the growth retardation zone, the growth intensification zone to (+ +++) 3 mm long. After 48 hours, the growth inhibition zone is not visible, the growth intensification zone is not clearly defined. When applying the gel to the surface of the agar against the background of continuous uniform growth (intensity +++) after 24 hours, directly under the applied gel - single colonies (2-3 in the zone), along the periphery - the zone of growth intensification to (++++) 2 mm After 48 hours, the picture is unchanged, the zone of growth intensification is less clearly defined (a pronounced bacteriostatic effect is maintained).

In experiments with lactobacilli, when a sample was introduced into the wells, the growth was continuous over the entire surface with an intensity of (++++). When applying the gel to the surface of bifidum agar in the form of drops directly below the applied gel, a decrease in the growth rate to (+) was noted.

Samples No. 18 and No. 19.

In experiments with lactobacilli in both cases, continuous growth was observed over the entire surface with intensity (++++).

Thus, the used subtherapeutic doses of metronidazole do not inhibit the growth of lactobacilli. Experience with other test cultures was not conducted due to their notorious insensitivity to metronidazole.

Based on the research, the following is stated:

1. Gels containing officinal antibacterial agents in subtherapeutic doses do not have an antimicrobial effect on lactobacilli - the main representatives of the normal microflora of the vagina of women of childbearing age.

2. Ascorbic acid stimulates the growth of yeast-like fungi of the genus Candida outside the zone of bacteriostatic action of chitosan in connection with its dissociation from the polymer. The addition of dioxidine reduces the stimulating effect of the acid.

3. The gel of chitosan hydrochloride containing dioxidine and metronidazole in subtherapeutic doses has a prolonged antimicrobial effect on microorganisms sensitive to it, and there is no stimulation of the growth of Candida yeast-like fungi.

4. Gels containing chitosan with a higher molecular weight and / or higher concentration have a greater degree of prolongation of antimicrobial activity. Adding metronidazole to the gel does not change the effect of the drug on the aerobic flora. Thus, the sample No. 13 in vitro meets the stated requirements of the task.

Clinical studies of the antibacterial filler in the feminine sanitary pad and tampon were carried out on 30 patients with nonspecific vulvovaginitis, the average age of the patients was 32.2 years, of which 40% were women in the perimenopausal period, and the remaining patients in the early reproductive period. The second group (comparisons) consisted of 20 women, whose average age was 31.6 years, of which 70% were women in the early reproductive period, 30% were aged 45 and over. Before treatment, patients complained of itching, burning, a feeling of heaviness in the vagina, excessive discharge from the genital tract, dyspareunia, dysuric disorders. A gynecological examination evaluated the presence of hyperemia, swelling of the skin and mucous membrane of the external genital organs and vagina, the nature and amount of vaginal discharge. To establish the etiology of the causative agent of the disease, all patients underwent a bacteriological study of vaginal microflora, which included a bacterioscopy of vaginal smears stained by Gram, and a culture study. To indicate the presence of lactic acid microflora, the pH of the vaginal secretion was determined. If bacterial vaginosis was suspected, patients underwent an amine test with 10% KOH.

In the last decade, opportunistic microorganisms - gram-negative anaerobes (Escherichia coli, Proteus, enterobacteria, Klebsiella, Pseudomonas aeruginosa, etc.), non-clostridial anaerobes (peptococci, peptostreptococci, bacteroids) have played a leading role in the etiology of inflammatory diseases. Increasingly, microbial associations began to occur, causing a more severe process and difficulties in its treatment [11]. The pathological process is directly related to the factors of aggression of microorganisms (hemolysin, leukotoxic factors, succinic acid, muramidase, collagenase, fibrinolysin, etc.), most of the pathogens have weak immunogenicity, are not opposed by proteins due to the constant presence of a woman in the body. [12, 13]. Since hemolysin, which causes damage to the erythrocyte membrane, is one of the factors of aggression, in all patients, the rate of erythrocyte deformability (PDE) was determined in the dynamics of treatment according to the Dormandi method in the modification of Yu.I. Greenstein [fourteen].

The treatment of the study group was carried out as follows: in the treatment room after treatment of the external genital organs with a solution of an antiseptic in the vagina on a swab and / or sanitary pad, 5 ml of 1% gel of chitosan hydrochloride containing metronidazole in a dose of 1.25-1.665 g / l and dioxidine 2.5 g / l (HMD-BOL). A tampon and a panty liner are left for 12 hours.

When applied to the pad and the swab of the preparation, the gel did not spread, was evenly distributed over the surface, absorbed well and quickly, which indicates the optimal fluidity of the gel.

The second group of women (control) used gynecological capsules "Polygynax" vaginally, 1 capsule at night for 12 days. Polygynax is a complex preparation containing neomycin, polymyxin B, nystatin and dimethylpolysiloxane gel.

Table 1
Analysis of the clinical manifestations of nonspecific vulvovaginitis before treatment (%) Symptoms of Vaginitis The first group (main) The second group (control) 1. leucorrhoea one hundred one hundred 2. burning fifty 60 3. itching thirty 40 4. dysuric disorders fifty 60 5. dyspareunia 80 70

Monitoring the effectiveness of treatment (anti-inflammatory effect, analgesic, antibacterial effect, uniformity of effect on the inflammatory surface, prolonging effect) was carried out 1, 3, 5, 7, 10, 12 days and 1 month after the treatment (Table 1).

When analyzing the results of treatment in the first group of patients, complaints of itching and burning in the vagina disappeared on the 2nd day of treatment in 70% of women, on the 5th day - in 10%. Pathological discharge from the genital tract on the 5th day of treatment was not stopped in 30% of women, on the 7th day leucorrhoea persisted in 10% of women, on the 10th day the discharge corresponded to the physiological norm. Hyperemia and edema of the external genitalia persisted on the 5th day of treatment in 30% of women, on the 7th day in three women the symptom was slightly expressed, in the remaining patients there were no inflammatory manifestations on the external genitalia. Hyperemia and edema of the vaginal mucosa were absent in 70% of women on the 5th day of treatment, on the 7th day - in 90% of patients. Dysuric manifestations on day 3 were stopped in 40% of women, on day 5 - in 70%, and on day 7 of treatment were absent in all patients. The erythrocyte deformability index (PDE) (normal value does not exceed 6%) before treatment was 10%, on day 5 it was 8%, on day 10 of treatment all women were within normal limits. Analysis of the clinical manifestations of the disease in the dynamics of treatment in the first group indicates sufficient treatment efficiency and the achievement of a stable therapeutic effect already on the 7-8th day of treatment. Due to the ability of chitosan to adsorb biological active substances released during the inflammatory process, hyperemia, edema, pain syndrome quickly stop, itching and burning in the vagina disappear, which favorably affects the woman’s well-being.

In the second group of treatment, leucorrhoea was observed on the 10th day of treatment in 10% of women, itching and burning in the vagina on the 7th day of treatment persisted in 4 women, on the 10th day were absent. Hyperemia and edema of the external genital organs on the 7th day persisted in 30%, on the 10th day of treatment in 10% of women. PDE reached normal values only on the 12th day of treatment (Table 2). The dynamics of clinical manifestations in the second group indicates a longer course of the inflammatory process and the elimination of pathogens. When studying the composition of the vaginal microflora, it was found that before treatment in patients of both study groups, there was an increase in leukocytes in the vaginal secretion of more than 30 in the field of view, increased desquamation of the surface epithelium of the vaginal mucosa, a small number of Dederlein sticks, the appearance of Gram-positive cocci and Gram-negative rods , 20% of women (in the first group) found yeast cells. In tab. quantitative and qualitative composition of the contents of the vagina in the dynamics of treatment is presented.

table 2
The characteristic of clinical signs of nonspecific vulvovaginitis in the dynamics of treatment Sign Treatment of 1% HMD-BOL Polygynax treatment Days of treatment 5 7 10 12 5 7 10 12 1.white + + - - - ++ + + - - 2. itching and burning + - - - - + + - - - 3. hyperemia and edema of the external genital organs + + - ++ + + - - 4. hyperemia and swelling of the vaginal mucosa ++ + - - - ++ + + - - 5. dysuria + - - - + + - - - 6. PDE (%) 8 7 6 5 10 8 7 6 Note: (++) the sign is expressed, (+) the sign is moderate, (+ -) the sign is weak, (-) the sign is absent.

A culture study of vaginal secretion prior to treatment revealed the growth of S.aureus, E.faecalis, E.coli, C. albicans. After treatment, in the first group of the study, growth of normal microflora was observed in all women, in the second group of the study, growth of C. albicans was noted in 4 women, and growth of Bacteroides in 1 patient.

Therefore, in the first group after treatment, restoration of normal vaginal microflora was noted, characterized by the disappearance of pathogenic microflora, an increase in the number of Dederlein sticks that support colonization resistance in the vagina, while the second stage of treatment is not required - the appointment of eubiotics. In the second group of the study, treatment was complicated in 20% of women with candidiasis vulvovaginitis, while a decrease in the number of lactic acid bacteria in 70% of women was noted, which requires replacement therapy with eubiotics.

Table 3
Bacteriological study of vaginal secretion in the dynamics of treatment (%) Vaginal contents Group 1 before treatment Group 2 before treatment Group 1 after treatment Group 2 after treatment Epithelial cells moderate amount twenty 40 80 60 all the time 80 60 twenty 40 White blood cells are absent twenty twenty 40 thirty 0-10 in s / sp 40 40 60 fifty 10-30 p / sp twenty twenty 0 twenty > 30 in s / sp twenty twenty 0 0 Dederlein sticks neg. or single thirty 40 0 10 moderate amount 60 40 twenty 60 a large number 10 twenty 80 thirty Cocci flora 80 70 10 thirty Gr (-) Flora 60 60 0 10 Key cells - 10 0 0 Gonococci - - - - Yeast twenty 40 0 twenty PH meter 3.5-4.5 10 twenty one hundred 70 > 4,5 90 80 0 thirty Amine test positive 0 10 0 0 negative one hundred 90 0 0

After 1 month, all patients received a control study. Patients of the first group of the study lacked subjective signs of the disease; gynecological examination of the external genitalia without inflammation, the vaginal mucosa is pale pink, the discharge is moderate, milky. In the second group of the study, relapse of the disease was observed in 20% of women.

The gel is convenient to use as a filler for sanitary napkins and tampons, since when applied to the surface of the napkins and tampons, the gel does not spread, is evenly distributed over the surface, is well and quickly absorbed, which indicates the optimal fluidity of the gel. The gel can be stored in a dark place at room temperature, while the physical and organoleptic properties of the gel are not changed for two years.

Clinical example. Patient D., aged 19, turned to the antenatal clinic, complained of: itching, burning, discomfort in the vagina, copious discharge from the genital tract, yellowish color, dyspareunia, frequent urination. From the anamnesis: considers himself ill during the last 5 days when the above complaints appeared. From concomitant pathology - chronic salpingo-oophoritis, last exacerbation 2 months ago. In the last 2 years, there has been a violation of menstrual function as oligomenorrhea. During a gynecological examination: the external genitalia, the vaginal mucosa are hyperemic, swollen, light yellow, with an unpleasant odor, plentiful, bimanual examination is difficult: the patient complains about the pain of the study. Microscopy of vaginal secretions with preliminary Gram stain revealed a large number of epithelial cells, white blood cells more than 30 in sight, Dederlein sticks up to 10 in sight, and cocco-bacillary flora more than 50 in sight. In a cultural study, the growth of E.faecalis, E. coli was established. Diagnosed with nonspecific vulvovaginitis. Treatment was prescribed: a tampon was injected vaginally, soaked in 5 ml of a 1% gel of chitosan hydrochloride containing metronidazole at a dose of 1.25 mg / ml, dioxidine at a dose of 2.5 mg / ml, as well as a sanitary pad on the surface to which the same gel in an amount of 5 ml. The duration of application of the pad and tampon was recommended for 12 hours. Monitoring the effectiveness of treatment was evaluated at 3, 5, 7 days and a month after treatment. On the 3rd day of treatment, itching and burning in the vagina were absent, the number of vaginal discharge decreased significantly, on the 5th day of treatment hyperemia, edema of the external genital organs and vaginal mucosa were insignificant, on the 7th day of treatment, there were no subjective and objective signs of the disease, the discharge corresponded to the physiological norm. Microscopy of a vaginal secretion stained by Gram showed that epithelial cells are up to 5 in the field of view, single leukocytes, Dederlein sticks are more than 150 in the field of view, there is no pathological microflora, there are no key, yeast cells. In a culture study of vaginal secretion, an increase in normal microflora was noted. After 1 month after treatment, a control study was carried out, the patient does not complain, with a gynecological examination, the external genitalia without inflammation, the mucous membrane of the vagina is pale pink, the cervix is clean, the discharge is moderate, milky. Recommended: a repeated course of preventive treatment after 3 months.

Thus, a promising sample of gel filler for feminine sanitary napkins or tampons has been identified, which has a significant antibacterial effect with prolonging properties, high fluidity and dosage, rapid anti-inflammatory effect for vulvovaginitis, retention of lactobacillary microflora of the vagina, quick analgesic therapeutic effect shape stability, gel transparency and lack of smell.

Used Books

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Claims (3)

1. Antibacterial filler for feminine sanitary napkins or tampons, including antibacterial preparations, characterized in that it contains 1% hydrogel of chitosan hydrochloride with a molecular weight of 300-700 kDa and a degree of deacetylation of at least 89%, metronidazole and dioxidine in the following ratio per 1 liter of composition: Dry Chitosan Hydrochloride 10 g metronidazole 1,250-1,665 g dioxidine 2.5 g distilled water rest 2. The method of producing a filler according to claim 1, comprising mixing 10 g of dry chitosan hydrochloride in 300 ml of distilled water heated to 50 ° C using a high-speed stirrer for 20-30 minutes until completely dissolved, then 250-333 are gradually added with vigorous stirring ml of metronidazole for intravenous injection, mix thoroughly for 5 minutes, after which 250 ml of a 1% solution of dioxidine are gradually added with vigorous stirring, mix for 5 minutes, the volume of the gel obtained is adjusted to 1 liter with distilled water, carefully flax stirred for 10 min, placed for long term storage in containers of dark glass or plastic. 3. The use of the antibacterial excipient according to claim 1 or 2 for the treatment of inflammatory diseases of the female genital organs by introducing into the vagina on a tampon and / or sanitary pad in an amount of 5 ml of a 1% gel of chitosan hydrochloride containing metronidazole and dioxidine for 12 hours within 7 days.