RU2706218C1 - Antibacterial agent based on sphagnum moss extract - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention refers to pharmaceutical industry, namely an antibacterial agent based on a sphagnum moss extract. Agent possessing antibacterial action represents an extract of sphagnum moss, obtained by extraction of sphagnum moss with benzyl alcohol by Bosin in three percolators with infusion of raw materials in each percolator for 12 hours.

EFFECT: agent described above possesses pronounced antibacterial action.

1 cl, 4 tbl

Description

The product (extract) belongs to the field of medicine, namely to medicines with antibacterial effect.

Treatment and rational antibacterial therapy of wound lesions is determined by the appointment of various drugs with antimicrobial effects. To date, the pharmaceutical industry produces a large number of antibacterial and antifungal agents in the form of ointments, gels, powders (methyluracil ointment, nystatin, sanguirytrin, etc.), many of which have side effects in the form of gastrointestinal and allergic reactions [1] . Therefore, the development of non-toxic drugs of plant origin, allowing you to gently provide an antibacterial effect, without exerting toxic effects, is a priority.

It is known that sphagnum moss is used in folk medicine mainly as a dressing; in addition, scientific studies are currently being conducted on the use of sphagnum moss extracts as a wound healing agent.

Closest to the proposed is a tool obtained in the laboratory of GOU VPO SibGMU ROSZDRAVA, representing a 20% water-ethanol extract of Sphagnum fuscum. The tool was obtained by repercolation according to N.A. Chulkova. Two periods are distinguished: during the start-up period, one percolator is loaded daily and the finished product is not drained. An equal amount of raw material is loaded into each percolator, which is preliminarily poured with an equal amount of pure extractant (for the 1st percolator) or extraction obtained from the previous percolator (for 2nd and all subsequent percolators). The swollen raw materials are loaded into the first percolator, poured with extractant to a “mirror” and left for a day. The next day, the extracts are poured from the first percolator in two steps: the first extract is in an amount equal to the mass of raw materials loaded into the percolator used to soak the raw materials for the second percolator, and the second extract is doubled in relation to the mass of raw materials used for infusion raw materials in the second percolator. At this time, fresh extractant is supplied to the first percolator in an amount equal to the sum of the extracts. On the third day, two extracts are also collected from the second percolator: for working with raw materials intended for loading into the third percolator. Extracts from the first percolator are fed into the second percolator, and fresh extractant is again fed into it. Further, the process is carried out similarly. A day after loading the last percolator, the working period begins. At this time, the first portion of the finished product is drained from the last percolator in an amount equal to the mass of raw materials in this percolator. At the same time, all hoods are drained from the first percolator and fed to the second percolator. The raw materials in the first percolator are completely depleted. Fresh extractant is fed to a second percolator, which now becomes tail. The first percolator becomes the head one. Collection of the finished product is carried out daily from the head percolator, which is each newly loaded raw material [2].

The prototype has the following disadvantages:

• the method used for extraction is longer than the Bosin repercolation method;

• Ethyl alcohol is a more expensive extractant of plant materials in comparison with benzyl alcohol.

A new technical task is to expand the range of products based on plant materials with antibacterial effect.

The problem is solved as follows - an antibacterial agent based on sphagnum moss, containing a large amount of benzoic acid and its esters, which have an antiseptic effect.

The tool was obtained by Bosin repercolation. Extraction was performed by repercolation in 3 percolators. To do this, the crushed raw materials are placed in percolator 1, filled with benzyl alcohol and left for 12 hours. After 12 hours of infusion, the extract is drained from percolator 1 and the finished raw material is poured into it in percolator 2, while fresh extractant is fed into percolator 1. After 12 hours of infusion, extracts are drained from percolators 1 and 2. In this case, percolator 1 is replenished with fresh extractant, and percolator 2 is extracted from percolator 1, percolator 3 is extracted from percolator 2. After 12 hours, the finished extract is drained from the last percolator and combined with the extracts obtained during the working period. The finished liquid extract is filtered through a paper filter.

The qualitative composition of the product was studied on a gas chromatograph with mass spectrometric and flame ionization detectors MSClarus 500. Gas chromatographic analysis was performed on a 60 m long capillary column with an inner diameter of 0.32 mm SGE-Wax in the temperature programming mode, helium carrier gas. The device was controlled using the TurboMass program. The spectra were identified using the NIST'14 mass spectra library.

Information characterizing the chemical composition of the sphagnum extract of moss obtained as a result of the study is presented in the table.

The growth of microorganisms was estimated using two methods: disco-diffusion and photometric. Microorganism strains were used in the experiments: S. aureus ATCC 25923, P. Aeruginosa 27853 ATCC, S. Enteritidis 495, C. Albicans 24433 ATCC.

The disk diffusion method was as follows: 20 ml of molten nutrient agar was poured into sterile Petri dishes with a diameter of 10 cm. To obtain a uniform bacterial lawn, 0.2 ml of the suspension of the test culture was added to the cup on the agar surface. The liquid was evenly distributed over the surface of the cup. The excess liquid was aspirated with a Pasteur pipette, then the agar was dried in an incubator with the cup closed.

On the surface of the seeded agar at a distance of 2 cm from the edge of the cup and at an even distance from each other, paper disks soaked in extract were laid out with tweezers one at a time. At the bottom of the cup, the name of the extract with which the disk is impregnated was signed. Cups were placed in a thermostat for 24 hours. To account for the results, the diameter of the zone of growth inhibition of the microorganism around the disks was determined using a millimeter ruler, compasses, graph paper, or a special meter [3].

The absence of growth retardation indicates the resistance of the studied microorganism to this extract. Zones whose diameter does not exceed 15 mm indicate a weak sensitivity to the extract. Zones from 15 to 25 mm are found in sensitive microorganisms.

As a result of the experiment, it was found that the benzyl extract of sphagnum moss is most sensitive to S. aureus, the diameter of the growth inhibition was 21 mm and C. albicans 23 mm.

OVV photometric method was used to detect antilysocyme activity (ALA) of microorganisms. Bukharin. The biomass of the studied cultures was seeded with a standard bacteriological loop in 3 ml of a liquid nutrient medium. Crops of facultative anaerobes were cultured in an incubator at 37 ° C for 24 hours. Then, the optical density (Do) of the broth culture versus the broth (Y) was measured on a Multiskan FC photometer, wavelength 450 nm. The supernatant was separated from microbial cells by centrifugation at 3000 rpm for 15 minutes. As a test strain for the determination of ALA, a Micrococcus luteus ATCC 158307 acetonated agar culture was used. The culture was dissolved in physiological saline and adjusted to the optical density of the test strain to 0.300 (0.28-0.32). On 1/5 M phosphate buffer (pH = 6.2), a lysozyme solution with a concentration of 20 μg / ml was prepared.

The supernatant of the studied microorganisms with a volume of 0.9 ml was mixed with 0.1 ml of the prepared lysozyme solution and incubated for 60-120 min at 37 ° C. Then, 50 μl of the mixture of supernatant with lysozyme was placed in a polystyrene plate in vertical rows. Upper and lower wells - controls: a mixture of nutrient broth and lysozyme. 200 μl of a micrococcus suspension with a multichannel pipette was added to the wells of each row of the tablet. Measure the optical density 30 and 150 seconds after the introduction of micrococcus into the wells [4, 5].

From the presented data, the extract of sphagnum moss promotes an increase in ALA in S. aureus, it follows that, despite the suppression of the growth of the microorganism, this extract cannot be applied to it, since it increases the resistance of the microorganism to one of the factors of local protection of the human and animal organisms . In relation to P. aeruginosa and C. albicans, the extract helps to reduce ALA by 1.08 μg / ml * OD and 1.39 μg / ml * OD.

Experimental studies of the hemolytic activity of microorganisms were carried out using a standard strain: S. aureus ATCC 25923, P. Aeruginosa 27853 ATCC, S. Enteritidis 495, C. Albicans 24433 ATCC. The initial level of hemolytic activity (GA) was determined photoelectrocolorimetrically by the ability of supernatants of the studied microorganisms to lyse human erythrocytes of group 0 (I) Rh (+) [6]. All solutions and culture media were prepared on deionized water. A study of the effect of moss extract on HA bacteria was carried out on a Shedler broth.

Bacteria for the experiment were grown for 24 h on beveled meat and peptone agar at 37 ° C, washed off with physiological saline, the resulting microbial suspension was titrated to an optical density of 0.500-0.510 opt. units

In 2 ml of sterile Shedler broth 0.2 ml of extract, 0.2 ml of 5% suspension of human erythrocytes of group 0 (I) Rh (+) in physiological saline and 0.1 ml of microbial suspension were added. Instead of microbial suspension, 0.1 ml of sterile saline was added to control tubes. Crops were cultured at 37 ° C for 2 hours and centrifuged for 15 min at 3000 g. The optical density above the sediment was measured relative to water (λ = 543 nm). GA was expressed in%.

A decrease in hemolytic activity is observed in S. aureus by 6.4%; P. aeruginosa 3.9%. The hemolytic activity in relation to S. enteritidis, the extract is not affected.

Information sources

1. Galimzyanov F.V. Local treatment and rational antibacterial therapy of infected wounds. Tutorial. - Yekaterinburg, 2013 .-- 75 p.

2. Ponomarev V.D. Extraction of medicinal raw materials. - M .: Medicine, 1976 .-- 202 p.

3. MUK 4.2.1890-04 Determination of the sensitivity of microorganisms to antibacterial drugs.

4. Bukharin O. V., Valyshev A. V., Elagina N. N. et al. Photometric determination of antilysocyme activity of microorganisms // ZhMEI. - 1997. - No. 4. - S. 117-120.

5. Bukharin OV, Vasiliev N.V. Lysozyme and its role in biology and medicine. - Tomsk: Publishing house of Tomsk University, 1974. - S. 207.

6. Bukharin O. V., Stadnikov A. A., Usvyatsov B. Ya., Khanina E. A. Features of the interaction of bacteria with red blood cells and their role in the development of infectious anemia // Zh. microbiol. 2006. No4. - S. 25-28.

Claims (1)

An agent with an antibacterial effect, characterized in that it is an extract of sphagnum moss obtained by extraction of sphagnum moss with benzyl alcohol according to Bosin in three percolators while infusing the raw materials in each percolator for 12 hours.