RU2345794C2 - Disinfectant - Google Patents

Abstract

FIELD: medicine; chemistry.

SUBSTANCE: disinfectant contains polyhexamethylene guanidine hydrochloride, ferrous iron salts and strong acids salts, water-containing solvent. Water containing solvent is water or aqueous solution with additives, such as neonol, hexamethylene tetramine, propylene glycol ester, urea.

EFFECT: higher bactericidal efficiency and wide potential applications.

6 cl, 5 tbl, 1 dwg, 6 ex

Description

The invention relates to the field of biotechnology, and in particular to preparations with biocidal properties and, therefore, promising as a disinfectant to combat bacterial and fungal infections of wood, woven and other materials, as well as residential and industrial premises, as well as methods for their use and can be used in medicine, veterinary medicine, textile and construction industries, as well as related industries.

Currently, to combat infection of the surfaces of objects with microorganisms, drugs such as metaflor, formaldehyde, alkamon, chloride hydrocarbons, etc. are used. (GB 1476730, 1977, DE 2820409, 1981). Disinfection is carried out by washing surfaces with a solution of biocides or by spraying.

However, these drugs do not have a prolonged effect, are dangerous for humans and the environment, ineffective in low concentrations.

One of the most promising groups of biocides is various derivatives of guanidine (GB 821113, 1959; SU 1184296, 1983; P.A. Gembitsky Synthesis of metacide. Chem. Prom. 1984, No. 2, pp. 18-19; SU 1687261, 1991), combining good biocidal properties with relative low toxicity. Among these derivatives, polyhexamethylene guanidine (PHMG) and its salts with acids, in particular hydrochloride (PHMG-GC) or phosphate (PHMG-F) (SU 247463, 1968; SU 1698061, 1991), proposed to combat bacterial contamination, are most known.

The most famous polyhexamethylene guanidine hydrochloride (PHMG-GC), which received the name chemical name metocide, has low toxicity and high antimicrobial properties for use in the food industry, medicine, veterinary medicine, and everyday life. Compositions containing methocide are produced in Russia under various trademarks - Polisept, Fogucid, BIOR-1, Teflex and a number of others (Eng. Pat 821213, 1959, CL 15 (2) G, AS USSR 1184296, 1983, C. D06M 14/04, P. A. Gembitsky Synthesis of Metacid, Chemical Prom. 1984, No. 2, pp. 18-19. Pat. USSR 1687261, 1991, C. A61L 2/16; A.S. USSR 247463 , 1968, class A61L 2/16, AS USSR 1698061, 1991, class B27K 3/34).

As a rule, the preparation of PHMG-GC is carried out by polycondensation of dicyandiamide (DCDA) with ammonium chloride (XA), and then with hexamethylene diamine (HMDA). In this case, the intermediate formed guanidine hydrochloride (HCH) contains, as impurities, derivatives of cyanuric acid (or melamine) - amelide and ameline. GHC is not purified from these impurities and they are present in the final product. (Gembitsky P.A., Limanov V.E. et al. Journal of Applied Chemistry, 1975, 48, p. 1833; Gembitsky P.A., Boksha L.F., Zhuk D.S. // Chemical Industry, 1984, N2, p. 82; Safonov G.A., Gembitsky P.A., Rodionov A.V. // Chem. Prom., 1989, N 12, p. 1448).

However, the use of existing drugs based on PHMG requires relatively high concentrations of active principle. In addition, there is a high corrosive activity of PGMG-GC and a relatively short duration, as well as the presence of low molecular weight toxic substances in its composition.

A disinfectant is known, which is an aqueous solution of a mixture of PHMG-GC and alkyldimethylbenzylammonium chloride (RU 2182889, 2001). The resulting product is a mixture of polymerhomologists of different molecular weights, including low molecular weight, characterized by relatively high toxicity and reduced antimicrobial activity, and also contains residual components of the synthesis, including hexamethylenediamine (HMDA), characterized by high toxicity (residual content in the product 1%, 2nd hazard class according to GOST 12. 1.007-76, D ₅₀ 250 mg), the presence of allergic properties.

The disadvantage of this tool is its high corrosivity, toxicity and relatively low efficiency.

The closest in technical essence to the claimed preparation is the Teflex disinfectant developed by the authors, which represents a 5-20% solution of PHMG-GC - 5-20 (% by weight) in an aqueous solvent. The aqueous solvent contains auxiliary additives that improve the properties of PHMG-GC - neonol, hexamethylenetetramine, propylene glycol ether, urea, as well as impurity synthesis components, in particular ameline and amelide.

The problem solved by the authors was to create a less toxic and more effective and stable during operation of the drug.

The technical task was the introduction of substances capable of increasing the biocidal activity of PHMG and binding toxic impurities. In this case, the products of their interaction are either removed from the solution or form practically non-toxic compounds.

It was found that the technical result was achieved by introducing from the composition of the product from 0.001 to 2% of ferrous ions calculated on the basis of PHMG. Divalent iron ions are introduced, as a rule, in the form of water-soluble iron salts with strong acids - sulfuric, hydrochloric, nitric, acetic, etc. For economic reasons, iron sulfate is most commonly used.

The resulting product is usually sold in the form of a concentrate containing about 50% of the mass. PHMG-GC and 0.001-5% of the mass. iron salts in an aqueous solvent. As the latter, water (“Teflex-I” agent) or an aqueous solution of auxiliary additives used in the “Teflex” agent — neonol, hexamethylenetetramine, propylene glycol ether and urea (“Teflex-Extra” agent) is used.

In this case, its composition includes (% mass.):

PGMG-X 3-20 Salts Fe ⁺⁺ 0.0001-0.5 Neonol 5-15 Hexamethylenetetramine 0.5-5 Propylene glycol ether 5-15 Urea 0.5-5 Water rest

The drug is prepared as follows. PHMG-GC at a temperature of 100 ° C is poured into an aqueous solution containing a given amount of Fe ⁺⁺ ions, after which the mixture is kept under stirring for 1-4 hours.

The complexes of low molecular weight polymerization products that have surfaced on the surface are removed mechanically. The resulting product at a concentration of PHMG of about 50% is sold under the name "Teflex ^® -industrial (concentrate)", with a content of PGMG 4% - under the trademark "Anti-mold ^® -Teflex".

A feature of the claimed product along with increased purity (about 99% by dry matter) is the formation of a complex between PHMG and iron salts, which has increased biocidal activity.

To confirm the formation of the complex, a quantitative measurement of the complexing ability of PHMG macromolecules with respect to the sulfate molecules of two valence iron was carried out by spin-exchange titration. The essence of the spin-exchange titration method is shown in the drawing, which shows the dependence of the EPR (electron paramagnetic resonance) line width of the probe — the free radical of 2,2,6,6-tetramethyl-4-hydroxypiperidin-1-oxyl (R) on the concentration of ferrous sulfate in the absence and in the presence of the complexing polymer PGMG-GF.

From the analysis of the drawing it can be seen that titration of an aqueous solution of R with a solution of iron sulfate in the absence of a polymer leads to the usual linear concentration-related broadening of the ESR lines of the probe R due to spin exchange with iron aqua complexes. At the same time, when PHMG-GC is added to the solution, titration with a solution of FeSO ₄ does not broaden the ESR lines of the R probe up to a certain concentration of FeSO ₄ , which depends on the concentration of the added polymer.

Upon reaching this critical concentration, linear broadening of the concentration begins again with the same angular coefficient. This dependence of AN on C is explained by the fact that the first portions of Fe ²⁺ form strong complexes with the polymer in which the iron atoms are shielded by the polymer core from collisions with the R probe. The amount of iron sulfite is determined from the corresponding inflection point of the curve ΔН = f (C), which the polymer can bind into a complex. It turned out to be 0.06 mg / ml of a 50% aqueous solution of PHMG-GC.

The formation of such a complex leads to the structuring of the system and the almost complete disappearance of fluidity at a temperature of 14 ° C.

The resulting product retains transparency and practical colorlessness, but is characterized by low fluidity, increased viscosity and oiliness.

The resulting composition is practically non-toxic (111 class of toxicity), does not accumulate in the body. The tests performed showed its higher efficiency compared to commercially available preparations based on PHMG. In particular, the composition has proven effective in disinfecting viruses.

Industrial applicability and properties of the resulting composition were illustrated by the following examples.

Example 1. Obtaining and properties of the compositions. To 1 kg of PHMG-GC at a temperature of 100 ° C was added 1 l of an aqueous solution containing predetermined amounts of FeSO ₄ and other auxiliary substances, after which the mixture was kept under stirring for 1-4 hours. The complexes of low molecular weight polymerization products that floated to the surface were removed mechanically. The resulting product was diluted to the required working concentration of PHMG-GC.

The composition of the obtained samples are shown in table 1.

Table 1 Composition No. Time h The composition,% wt. PGMG-GC FeSO ₄ neonol Gmt PGE urea Water one 3 0.0001 96,9999 2 10 0.03 89.97 3 twenty 0.5 79.5 four 5 0.01 5 5 fifteen 0.5 69.49 5 10 0.01 10 3 10 3 63,99 6 5 0.01 fifteen 0.5 5 5 69.49

Example 2. Comparison of the bactericidal action of the compositions. On the basis of the Department of Microbiology, Immunology and Virology, St. Petersburg Medical Academy I.I. Mechnikov conducted comparative tests of the biocidal action of the claimed compositions according to example 1 by research using the method of holes in agar. The results were recorded by the diameter of the zone of lack of growth of test microorganisms around the wells to a dilution of 1/200 and by the radius of the absence of growth around the dilutions of 1/500, 1/1000.

Used dilutions of preparations: without dilution, 1/10, 1/20, 1/50, 1/100, 1/500, 1/1000.

Test microbes were bacteria - Staphylococcus aureus (St.aur), Escherichia coli (E. coli); mold fungi - Aspergillus fumigatus (Asp.fm), Penicillium chrysogenum (Pen.ch).

The results are shown in table 2 and show the zone of absence of growth in mm.

table 2 The effect of composition on biocidal characteristics A drug b / rv 1/10 1/20 1/100 1/200 1/500 1/1000 Comp.1 26 eighteen fifteen 12 10 1,5 2 Comp.2 29th twenty eighteen fourteen 13 2.5 four Comp.3 28 17 fifteen 12 10 one 1,0 Comp.4 32 16 fourteen eleven 9 2 0.5 Comp.5 29th 22 17 12 9 3 1,5 Comp.6 27 eighteen 16 12 10 2 1.5

The results obtained indicate that compositions 2 and 5 are the most effective, on which basic studies were subsequently conducted.

Example 3. Comparison of the bactericidal action of the claimed and industrially produced drugs.

On the basis of the Department of Microbiology, Immunology and Virology, St. Petersburg Medical Academy I.I. Mechnikov conducted comparative tests of the biocidal action of the PGMG-GC preparations: Polisept, composition 2 of Example 1 and Teflex at the same concentrations of PHMG-GC.

We used the method of ten-fold dilutions of substances in broth (from 10 ^-1 to 10 ^-6 degrees) with the addition of a thick suspension of cultures and subsequent incubation at 37 ° C for 18 h for bacteria and yeast-like fungi. After 18 hours of incubation, molds were kept at 22 ° С for five days. The research results showed that the minimum inhibitory concentration of whole substances was:

Table 3. The effect of the nature of preparations based on PHMG-HX on biocidal activity A drug S.aureus E.coli You.antracis Candida spp. Scopulariosis spp. Polysept 2 × 10 ^-6 2 × 10 ^-3 2 × 10 ^-1 2 × 10 ^-3 2 × 10 ^-4 Comp.2 2 × 10 ^-6 2 × 10 ^-4 2 × 10 ^-2 2 × 10 ^-5 2 × 10 ^-6 Teflex 2 × 10 ^-6 2 × 10 ^-3 2 × 10 ^-1 2 × 10 ^-3 2 × 10 ^-4

The results showed that the claimed composition has higher biocidal characteristics compared with the known drugs based on PHMG-GC.

Example 4. Compared the protective effect of chloride (MC), phosphate (FC) and acetate (AC) PHMG obtained by the method of example 1 for composition No. 2.

For testing, bleached cellulose was cut into squares (S = 1 cm ² ) and sterilized in an air sterilizer at 180 ° C for 60 minutes. In preliminary experiments, the toxicity of the used cellulose for test strains of microorganisms was checked.

In the experiment, reference strains of E. coli, S. aureus and B. subtilis var. anthracoides used to assess the quality of disinfectants, as well as strain Aspergillus niger No. 23.

Working disinfectant solutions were prepared immediately before use. The drug was dissolved in hot sterile tap water. To prepare a working solution of the preparation FC and AC, a concentrated (20%) solution of the drug was diluted with sterile tap water 20 times.

Test disinfectants 24 hours before the start of the experiment were applied to sterile test objects at the rate of 0.1 ml of 1% solution per test object, which corresponds to a dose of 1 mg / cm ² (10 g / m ² ). The objects were dried in a thermostat at 37 ° C. Evaluation of the activity of the drugs was carried out 1 day, 7 days and 1 month after processing the test objects.

The strains of E. coli and S.aureus were grown on meat and peptone agar at 37 ° C for 18-24 hours. B. subtilis var. anthracoides were grown on the same medium at 37 ° C for 48 hours. Aspergillus niger No. 23 was grown on Saburo agar at 37 ° C for 48 hours, and then at 22 ° C for another 48 hours. A working suspension of microorganisms was prepared by successive 10-fold dilutions of the initial suspensions with a concentration of 10 ⁹ CFU / ml (10 units according to the standard turbidity). During the experiments, the microbial load was 10 ⁶ CFU / test object. As a control, test objects not treated with disinfectants were used. Two exposures of test objects were used, 1 hour and 24 hours. After exposure, test objects were transferred with sterile forceps to test tubes and meat and peptone broth and incubated in an incubator. All experiments were performed in triplicate. A positive result was the lack of growth in test tubes after incubation. The results are shown in table 4.

Table 4. Antimicrobial activity of compositions based on salts of GMPG. Microorganism test Exposure, h Time after treatment, days A drug MP FC AC E.coli 1 hour 1 day +++ +++ +++ 24 h +++ +++ +++ 1 hour 7 days +++ +++ +++ 24 h +++ +++ +++ 1 hour 30 days +++ +++ +++ 24 h +++ +++ +++ S.aureus 1 hour 1 day +++ +++ +++ 24 h +++ +++ +++ 1 hour 7 days +++ +++ +++ 24 h +++ +++ +++

1 hour 30 days +++ +++ +++ 24 h +++ +++ +++ B.subtilis var. anthracoides 1 hour 1 day +++ +++ ++ - 24 h +++ +++ +++ 1 hour 7 days +++ +++ - ++ 24 h +++ +++ +++ 1 hour 30 days +++ +++ +++ 24 h +++ +++ ++ - Aspergillus niger No. 23 1 hour 1 day +++ +++ +++ 24 h +++ +++ +++ 1 hour 7 days +++ +++ +++ 24 h +++ +++ +++ 1 hour 30 days +++ +++ + - + 24 h +++ +++ +++

The data obtained showed that the preparations MC, FC and AC have pronounced germicidal activity, including against spore-forming bacteria and molds.

Example 5. In FGU "S. Petersburg NIIEM them. Pasteur ”of the Ministry of Health of the Russian Federation, the possibility of using the composition as an antiviral agent was studied in accordance with the methods described in the collection of“ Methods for testing disinfectants to assess their safety and effectiveness ”, M., 1998, part 2. Vaccine polio virus type 1, per. N ~ 584/02, obtained from the Collection of the National Institute of Biological Standards and Control, UK, code No. 95/602, the virus titer is 7.5 lg TCD ₅₀ .

Used cell culture Ner-2, obtained from the Institute of poliomyelitis and viral encephalitis them. M.P. Chumakova.

Investigated sample 5 obtained in example 1 in the form of a 3% solution. The exposure time with a disinfectant is 30 minutes, the neutralizer is 80% cattle serum (cattle).

In the course of the study, suspension experiments were performed without protein load and with a protein load of 40% cattle serum, and the virus was treated with a disinfectant and treated after exposure to a mixture of the virus with a disinfectant and a neutralizer.

Infection of sensitive cell cultures treated with the virus was carried out during incubation at 37 ° C in an atmosphere of 5% CO ₂ and 98% humidity for 5-7 days. In the absence of a CPP, an additional passage was performed on cell culture.

The following controls were used: cell culture during the entire observation period, virus in the development from 10 ^-1 to 10 ^-7 ; disinfectant; neutralizer. The research results are shown in table 5.

Table 5 The results of the study of the virucidal effect of composition 5 Substrates Disinfection time, min Virus titer in lg TCD ₅₀ in experiments Number 1 Number 2 Number 3 Virus (control) - 7.0 7.0 6.7 Protein-Free Virus thirty <1.0 <1.0 <1.0 + disinfectant + neutralizer Protein Load Virus thirty <1.0 <1.0 <1.0 + disinfectant + neutralizer

It is shown that the composition is not toxic to cells. In this case, complete inhibition of poliomyelitis virus reproduction was found at a 3% concentration of the studied composition at an exposure of 30 min.

Example 6. In St. Petersburg GUZ "City Diagnostic Center (Virological)", tests were carried out using the composition No. 2 as a disinfectant for adenovirus.

Type 6 adenovirus and the HEP-2 transplantable cell culture obtained from the collection of cell cultures and viruses from the RAMI Research Institute of Influenza were used. Previously, adenovirus underwent 2 passages on tissue culture in order to restore infectious activity.

To perform studies, a cell culture of HEP-2 was grown in penicillin vials with coverslips. As a growth medium, we used DMEM Needle medium supplemented with 10% cattle serum, and as a supporting medium, we used DMEM Needle medium with 2% cattle. Preparing a working solution of composition 2 based on distilled water. For the study, equal volumes of the viral suspension were taken with a dilution of 10 ^-1 and dysenfiktant, mixed and left for exposure for 30 minutes.

Coverslips were used for fluorescence antibody (MFA) testing with an adenovirus fluorescent diagnosticum produced by the Research Institute of Influenza RAMS, the liquid phase was used for PCR testing for the presence of adenovirus DNA using the diagnostic kit produced by TsNIIE MZRF.

1 ml of the mixture was mixed with 5 ml of a neutralizer - 0.5% sulfonol solution and left for 30 min, after which 0.2 ml of the mixture was introduced into penicillin vials with cell culture.

The following controls were simultaneously performed:

1. Cell culture is not infected.

2. A cell culture infected with a virus with a dilution of 10 ^-1 .

3. Cell culture with the addition of a disinfectant (instead of a suspension of the virus and a neutralizer, an appropriate volume of medium is added).

4. Cell culture with the addition of a neutralizer (instead of a suspension of the virus and disinfectant, an appropriate volume of medium is added).

The bottles were kept at 37 ° C, viewing was performed under a light microscope daily for 5 days, sampling for studies was carried out in the first (24 hours after infection) and fifth day. It was shown by light microscopy that in the experiment for 5 days no destruction of the cell monolayer was observed, using the MFA method, it was shown that specific granular fluorescence localized in the cytoplasm of HEP-2 cells was observed only in the drug - virus control.

Similar results were obtained by PCR. The test results indicate that a 3% solution of the composition under conditions of normal protein loading has virucidal activity against adenovirus.

Analysis of the data given in the examples showed that the proposed biocidal composition is characterized by higher efficiency and a wide range of potential applications, in particular for use against viruses, bacteria and various fungi.

Claims (6)

1. A disinfectant based on salts of polyhexamethylene guanidine and an aqueous solvent, characterized in that it additionally contains from 0.001 to 2% of ferrous ions per polyhexamethylene guanidine. 2. The disinfectant according to claim 1, characterized in that it contains the following ingredients, wt.%:
Polyhexamethylene guanidine hydrochloride 3-50 Ferrous salts 0.001-5 Water solvent rest 3. The disinfectant according to claim 1, characterized in that it contains water as an aqueous solvent. 4. The disinfectant according to claim 1, characterized in that as an aqueous solvent it contains an aqueous solution of neonol, hexamethylene tetramine, propylene glycol ether and urea in the following ratio of ingredients, wt.%:
Polyhexamethylene guanidine hydrochloride 5-20 Ferrous salts 0.0001-0.5 Neonol 5-15 Hexamethylenetetramine 0.5-5 Propylene glycol ether 5-15 Urea 0.5-5 Water rest 5. The disinfectant according to claim 1, characterized in that it contains water-soluble iron salts with strong acids as a source of ferrous ions. 6. The disinfectant according to claim 5, characterized in that it contains ferrous sulfate as salts of ferrous iron.

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