Antimicrobial agent

FIELD: medicine.

SUBSTANCE: compound 2-(1'-hydroxy-4'-isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanoate having structural formula I: is applicable as an antimicrobial agent.

EFFECT: using the compound of formula I possessing antimicrobial activity provides treating infectious processes caused by susceptible organisms.

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The invention relates to medicine and can be used in medical practice as a means of having antimicrobial activity for the treatment of infectious processes caused by susceptible microorganisms. Summary of the invention - the use of Terminalia of formula (I) 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methyladenine, possessing anti-microbial activity.

Modern pharmaceutical market offers a wide range of antimicrobial, antiseptic, disinfectants and drugs of both synthetic and natural origin.

Herbal drugs, as a rule, are a combination of biologically active compounds and may have a complex multi-directional action.

To date, it has accumulated a significant amount of information about the healing properties of natural terpenoids synthesized by various plants (Pavlov I. N. The reduction of stability of coniferous forests of Siberia to the root pathogens as a result of contemporary increases in the surface layer air temperature and soil / I. N. Pavlov. O. A. Barabanov, S. S. Kulakov, V. V. Eremin et al. // Boreal coniferous zone. - 2011. - Vol. 28. - Vol. 1-2. - Pp. 47-54). The number of individual representatives with an established chemical structure�blunt terpenoids exceed all other classes of natural compounds. This class of natural substances revealed a wide spectrum of biological activity (Startsev V. A. Synthesis and biological activity of monoterpenoids montenevoso series / V. A. Startsev, L., E. Nikitina, E. V. > Ministry of economy et al. // Chemistry for sustainable development. - 2009. - Volume 17. - Vol. 5. - P. 539-545; Khasanov, V. V. Study of the composition and antioxidative activity of products of water-steam distillation of Siberian fir / V. V. Khasanov, G. L. Ryzhova, T. T. Kuryaeva K. A. Dychko // Chemistry of plant raw material. - 2009. - No. 4. - P. 83-88; Mosul V. I. Investigation of the essential oil MyrtuscommunisL. / V. I. Mosul, V. S. Share, L. I. Sloboda Ukraine // Actual problems of pharmaceutical and medical science in practice. - 2011. - Vol. XXIV. - No. 2. P. 30 - 32; Stepanenko I. S. Study of antimicrobial activity of some natural terpenoids / I. S. Stepanenko, S. V. Shatkin, I. V. Akulin, V. N. Kargaev, L. E. Nikitina // Materials of all-Russian scientific-practical conference "Actual problems of biomedical disciplines." - Saransk, 2012. - P. 147-150; Akulin V. I. Pharmacological properties of Terminalia montenevoso series / I. V. Akulin, Garaev R. S., L. E. Nikitina, N. P. Artemova, I. S. Stepanenko // international journal of applied and fundamental research. - 2012. - No. 2. - S. 83). Essential oil and oleoresin of conifers, due to the high content of monoterpenoids, are an important source procedeveloping active substances (V. Khasanov The study of the composition and antioxidative activity of products of water-steam distillation of Siberian fir / V. V. Khasanov, G. L. Ryzhova, T. T. Kuryaeva K. A. Dychko // Chemistry of plant raw material. - 2009. - No. 4. - P. 83-88; Pavlov. The reduction of stability of coniferous forests of Siberia to the root pathogens as a result of contemporary increases in the surface layer air temperature and soil / I. N. Pavlov, A. A. Barabanov, S. S. Kulakov, V. V. Eremin et al. // Boreal coniferous zone. - 2011. - Vol. 28. - Vol. 1-2. - S. 47-54; Mosul V. I. Investigation of the essential oil MyrtuscommunisL. / V. I. Mosul, V. S. Share, L. I. Sloboda Ukraine // Actual problems of pharmaceutical and medical science in practice. - 2011. - Vol. XXIV. - No. 2. P. 30 - 32).

In the last two decades as a result of increased attention to natural terpenoids a list of medicinal substances was enlarged due to the appearance of medicines on their basis (Pat. 2197994 of the Russian Federation, IPC7A61L 2/16. The disinfectant "Receptek" / Slimina K. N. [and others]; applicant and patentee of Scientific.-issled. vet. Institute of non-Chernozem zone of the Russian Federation, LTD. Nauch.-prod. firm Lewis-NN, sue Center. Nauch.-issled. and design Institute of forest-chem. prom. - No. 2000113057/13; Appl. 25.05.2000; publ. 10.02.2003; Pat. 2054945 of the Russian Federation, IPC6A61K 35/78. The tool Amisil-1", possessing anti-inflammatory, antibacterial and wound healing activity / N. Pinigin.M. [and others]; applicant and patentee �enigine N. M. - No. 95109894/14; Appl. 28.06.1995; publ. 27.02.1996; Pharmacological activity of the essential oil of Bupleurum gibraltaricum: Anti-inflammatory activity and effects on isolated rat uteri / M. A. Ocete [et al.] // J. of Ethnopharmacology. - 1989. - Vol. 25, Iss. 3. - P. 305-313; Balsamic remedy for local application, has antimicrobial, anti-inflammatory and analgesic action (business information office P. A. [and others]; applicant and patentee, too NPF "Lewes", Institute of labour hygiene and occupational diseases center, Siberian division of the Russian medical Academy. - No. 94025798/14; Appl. 12.07.1994; publ. 27.08.1997; Pat. 2054945 of the Russian Federation, IPC6A61K 35/78. The tool Amisil-1", possessing anti-inflammatory, antibacterial and wound healing activity / N. Pinigin.M. [and others]; applicant and patentee N. Pinigin.M. - No. 95109894/14; Appl. 28.06.1995; publ. 27.02.1996).

It is known that terminalhead - 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol - has fungicidal and anti-inflammatory action (Pat. 2431479 OF THE RUSSIAN FEDERATION, IPC SS 321/24, SS 319/08, A61L 2/16, AK 35/78. 2-(1'-Hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol, possessing fungicidal and anti-inflammatory action / Nikitina L. E. and others; applicant and patentee Kazan state medical University, No. 2010130104/15, Appl. 19.07.2010, publ. 20.10.11).

Antimicrobial properties of the inventive compound 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methyladenine in the literature describing�s.

The purpose of the invention is an effective compound having antimicrobial activity.

This goal is achieved by the invention, namely, 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol (formula I).

Study of antimicrobial activity of 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylalanine

The study of Terminalia (I) 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methyladenine, original (+)-1,2-limonene oxide and its structural analogue (+)-limonene held in respect of the 14 museums of the test strains of microorganisms and 610 experimental strains of microorganisms isolated from material collected from patients with nonspecific and specific diseases of the respiratory and urinary tract, gastrointestinal tract.

Characteristics of the studied microorganisms. As the test microorganisms used collection strains: Salmonella enteritidis 5765 ADS, Shigella sonnei S-shape 20, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa 453, Escherichia coli M17 strain, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Staphylococcus aureus 906, Enterococcus faecalis 2919 ATCC, Citrobacter freundii 101/57, Proteus vulgaris 222, Klebsiella pneumoniae 9172, Bacillus cereus, 96, Streptococcus pyogenes 1238 ATCC. Collection strains used in the present study, obtained from the collection of the Museum of living cultures of the state research Institute of them. L. A. Tarasevich.

Staphylococcus aureus ATCC 29213, Staphylococcus aureus 906 n�and the growth of a dense nutrient medium: conditions for cultivation - environment No. 1, 2; t=36±1°C for 18-20 hours. The colony is smooth, with smooth edges, with an evenly Golden pigmentation. With the growth in liquid nutrient medium: conditions for the cultivation of environment No. 3, pH 7,2-7,4, 18-20 hours. Produces a uniform clouding of the broth without film and slimy sludge on the bottom. Microscopy: uniform size arranged in the form of bunches cocci, with well-pronounced gram-positive coloring.

Escherichia coli ATS, Escherichia coli M17 strain during growth on nutrient dense environment: the conditions for growing environment No. 1, 2; t=36±1°C for 18-20 hours. The colony PLANO-convex opal-cloudy 0,3-0,5 cm in diameter, with smooth or slightly wavy edges, may be mucoid colonies. During growth on lactose-containing differential nutrient media environment No. 4, 5; t=36±1°C, 18-20 hours - colonies with greenish metallic sheen (Magenta gloss). With the growth in liquid nutrient medium: conditions for the cultivation of environment No. 3, pH 7,2-7,4, 18-20 hours. There is diffuse clouding and the formation of sludge or film. Microscopy: gram-negative small Bacillus with slightly rounded ends and peritracheal located flagella.

Bacillus cereus 96 the growth of a dense nutrient medium: conditions for growing environment No. 1, 2; t=36±1°C for 18-20 hours. Colonies with a rough edge, formed of interwoven fibers. With the growth in liquid Pete�individual environment: conditions for cultivation - Wednesday No. 3, pH 7.8 to 8.0, 18-20 hours. Growth in the form of a wrinkled film, the whole environment is transparent without sediment. Microscopy: thin with rounded ends wand, located singly or in short chains, with well-defined gram-positive coloring.

Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa 453, with an increase of nutrient dense environment: the conditions for growing environment No. 1, 2; pH 7,2-7,-4, t=36±1°C for 18-20 hours. The colony is broad, vague, transparent, with rough edges. With the growth in liquid nutrient medium: conditions for the cultivation of environment No. 3, pH 7,2-7,4, 18-20 hours. Growth in the form of a uniform haze, thick film; Wednesday may acquire a yellowish-green color. Microscopy: single sticks or short chains with well-defined gram-negative overtones.

Streptococcus pyogenes 1238 ADS, cocci irregular circular shape, are in the form of chains or in pairs, the dimensions of 0.5-2.0 μm. On biochemical properties are separated by S. bovis, S. agalactia, S. mutans, etc. During growth on nutrient dense environment: the conditions for growing environment 6, 7; No., t=36±1°C for 18-20 hours. Colonies small shiny.

Klebsiella pneumoniae 9172, opportunistic bacteria, straight gram-negative rods (0.3 to 1.0 and 0.6-6 µm), located singly, in pairs or short chains. With the growth on nutrient media: growth conditions for the cultivation of the environment No. 1, 2; t=36±1°C for 18-20 hours. Colony �rupen, convex, moist, mucous, often merging with each other.

Proteus vulgaris 222, opportunistic bacteria, straight gram-negative rods with rounded ends (0,4-0,8 and 1,0-3,0 µm). Growth conditions for the cultivation environment No. 1, 2, etc.; t=36±1°C for 18-20 hours. Forms a creeping colony with spikes. During growth on lactose-containing differential nutrient media No. 4, 5; t=36±1°C, 18-20 hours - clear, slightly pinkish colonies show the ability to apply and give confluent growth.

As an experienced studied strains of microorganisms (No. 610), isolated from material collected from patients of the State budget institution of healthcare of the Republic of Mordovia "Republican infectious clinical hospital" of Saransk with diseases of the respiratory, urinary and gastrointestinal tract. Source of release of pathogens served as urine, sputum, mucus from the throat, nose and nasopharynx, feces, sectional material.

Table 1
Characteristics of the studied clinical strains of microorganisms
№ p/pThe microorganism strainThe number of studied strainsIsol�were protected from material research
1234
1Staphylococcus aureus92Mucus from throat, nose, nasopharynx, urine, feces, sectional material
2Staphylococcus epidermidis36Mucus from throat, nose, nasopharynx, urine, feces, sectional material
3Streptococcus pyogenes20Mucus from throat, nose, nasopharynx, sectional material
4Streptococcus pneumoniae18Mucus from throat, nose, nasopharynx, sectional material
5Streptococcus salivarius20Mucus from throat, nose, nasopharynx, sectional material
6Streptococcus uberis21Mucus from throat, nose, nasopharynx, sectional material
7Streptococcus mitis 20Mucus from throat, nose, nasopharynx, sectional material
8Streptococcus agalactia23Mucus from throat, nose, nasopharynx, sectional material
9Streptococcus sanguinis24Mucus from throat, nose, nasopharynx, sectional material
10Streptococcus mutans23Mucus from throat, nose, nasopharynx, sectional material
11Escherichia coli64Mucus from throat, nose, nasopharynx, urine, feces, sectional material
12Klebsiella pneumoniae32Mucus from throat, nose, nasopharynx, urine, feces, sectional material
13Klebsiella oxytoca19Mucus from throat, nose, nasopharynx, urine, feces, sectional material
14Enterococcus faecalis18 Urine, feces, sectional material
15Enterococcus faecium18Urine, feces, sectional material
16Salmonella enteritidis24Faeces
17Shigella sonnei12Faeces
18Enterobacter cloaceae19Faeces, sectional material
19Enterobacter aerogenes18Faeces, sectional material
20Proteusvulgaris24Urine, feces, sectional material
21Proteus mirabilis18Urine, feces, sectional material
22Citrobacter freundii18Urine, feces, sectional material
23 Pseudomonas aeruginosa49Mucus from throat, nose, nasopharynx, urine, feces, sectional material

Characterization of nutrient medium. Nutrient medium for cultivation and determination of the antimicrobial activity of the compounds under study must meet the following requirements:

- contain optimal concentrations of all the elements required by the microbial cell to ensure the life processes and reproduction: macro - and microelements, organic sources of carbon, nitrogen, inorganic salts, vitamins and other growth factors;

- possess a certain moisture content (20%), since water is necessary to carry out all metabolic processes, in addition, it serves as a source of hydrogen and oxygen for microbial cells;

- must be isotonic, that is, the concentration of salts in the environment must match the one in the microbial cell (for most microorganisms to 0.5%, for halophilic - 3%);

- the concentration of hydrogen ions (pH) in the environment must be optimal and consistent with this type of microbe (range 4,5-8,5);

- redox potential of the medium (Eh) must meet the needs of a microorganism (for anaerobes - 0,120-0,060 In, for aerobes - more In 0,080);

- a nutrient medium must be STERI�encourages creativity.

Wednesday's No. 1. Meat-peptone agar (MPA) ZAO "NITF"

Compositiong/l
Peptone10,0
Sodium chloride5,0
Water meat1000,0 ml
Agar-agar2,0-20,0

The final pH (at 25°C) 7,3±0,2

Used for the cultivation unpretentious microorganisms for the preparation of special media.

Wednesday No. 2. Nutrient agar (M) HiMedia Laboratories Pvt. Limited

Compositiong/l
Peptone5,0
Sodium chloride5,0
Extract of beef1,5
Extract yeast1,5
Agar-agar15,0
Distilled water1000,0 ml

The final pH (at 25°C) 7,4±0,2

Used for the cultivation of nariko�Levi microorganisms.

Wednesday No. 3. Meat-peptone broth (BCH) ZAO "NITF"

Compositiong/l
Peptone10,0
Sodium chloride5,0
Water meat1000,0 ml

The final pH (at 25°C) 7,3±0,2

Used for the cultivation unpretentious microorganisms for the preparation of special media.

Wednesday No. 4. Wednesday Endo (commercial) FS 42-350498 ZAO NICF"

Compositiong/l
Pancreatic hydrolysate sprat11,5
The extract of fodder yeast0,86
Lactose12,9
Magenta main0,22
Sodium sulphate disodium0,48
The sodium sulfite0,83
Sodium chloride3,6
Carbonate on�Riya 0,01
Agar-agar9,6
Distilled water1000,0 ml

The final pH (at 25°C) 7,3±0,2

Microorganisms able to degrade lactose (ectoparasiticide) form colonies are bright pink or red-crimson color, often with a metallic sheen. Lactosonegative microbes give rise in the form of colorless or slightly pink colonies.

Wednesday No. 5. Wednesday Endo (M029R) Hi Media Laboratories Pvt. Limited

Compositiong/l
Peptic parivar animal tissue10,0
Lactose10,0
Potassium hydrophosphate3,5
Sodium sulfite2,5
Magenta main0,5
Agar-agar0,5
Distilled water1000,0 ml

The final pH (at 25°C) 7,5±0,2

The sodium sulfite and basic fuchsin have an overwhelming effect on �analogically microorganisms. Lactose is decomposed by microorganisms to aldehyde and acid. The aldehyde, in turn, liberates fuchsin from the fuchsin-sulfite complex, increasing red staining of the colonies. In Escherichia coli, this reaction is very pronounced and is accompanied by the crystallization of Magenta that appears greenish metallic sheen (Magenta gloss) colonies.

Wednesday No. 6. The base Columbia blood agar (M144) Hi Media Laboratories Pvt. Limited

Compositiong/l
Peptone (special)23,0
Corn starch1,0
Sodium chloride5,0
Agar-agar10,0
Distilled water1000,0 ml

The final pH (at 25°) of 7.3±0,2

To prepare blood agar aseptico make sterile defibrinating the blood of the RAM (up to 5 vol.%). Mix thoroughly.

The presence of special peptone provides rapid and abundant growth of fastidious microorganisms. Corn starch serves as an energy source and simultaneously neutralizes toxic metabolites. �Aranya blood allows to evaluate the ability of microbes to hemolysis and provides them with Gemina (X-factor), which is essential for the growth of many bacteria.

Wednesday No. 7. Whey agar

As the base used is 1.2-1.5% agar (pH of 7.4) prepared with broth, fish hydrolysate, broth, parivara of Law (amine nitrogen in the broth 150-180 mg/ml) or on dry nutrient agar for special purposes. To 80 ml of molten and cooled to a temperature of 50°C agar add 20 ml inactivated at 56°C for 30 minutes in serum.

Wednesday No. 8. Agar Mueller-Hinton (MHA) (M) Hi Media Laboratories Pvt. Limited

Compositiong/l
Hood beef300,0
Acid hydrolysate of casein17,0
Starch1,5
Agar-agar17,0
Distilled water1000,0 ml

The final pH (at 25°) of 7.3±0,2

To prepare blood agar aseptico make sterile defibrinating the blood of the RAM (up to 5 vol.%). Mix thoroughly.

Supports the growth of many microorganisms. Starch serves as a "protective colloid" against toxic substances, viranative�required in the growth process.

Wednesday No. 9. The broth Mueller-Hinton (MHB) (M) Hi Media Laboratories Pvt. Limited

Compositiong/l
Hood beef300,0
Acid hydrolysate of casein17,0
Starch1,5
Distilled water1000,0 ml

The final pH (at 25°) of 7.4±0,2

For the preparation of a blood broth aseptico make sterile defibrinating the blood of the RAM (up to 5 vol.%). Mix thoroughly.

Supports the growth of many microorganisms. Starch serves as a "protective colloid" against toxic substances produced in the growth process.

Characteristics of research methods. The study of the sensitivity of microorganisms to limonene, 1,2-limonene oxide and terminalid (I), regardless of the particular method, conducted in a consistent performance of several stages (Definition of sensitivity of microorganisms to antibacterial preparations (HOWTO MOOK 4.2.1890-04) // Clinical Microbiology and antimicrobial chemotherapy. - 2004. - Volume 6. - No. 4. - S. 306-359):

- preparation of culture media;

- PR�the manufacture of suspension of microorganisms (inoculum);

- inoculation;

- incubation;

- accounting and interpretation of results.

Disk diffusion method also includes the step of overlapping disks on a dense nutrient medium.

To assess the sensitivity used is specially designed for the purpose of environment (environment No. 8, 9), permitted for use in the Russian Federation in the established order and its characteristics meet the requirements. Intralaboratory quality control environment was performed in all environments, permitted for use in the Russian Federation in the prescribed manner.

View nutrient medium for the evaluation of sensitivity was determined by the chosen method of research (agar or broth) and views of the tested microorganism.

Selected nutrient medium for determining the sensitivity prepared from the dry environment of industrial production in accordance with the manufacturer's instructions. After autoclaving, the culture medium was immediately poured into sterile tubes or Petri dishes. The agar was poured into cups with a layer thickness of 4 mm (a Cup with a diameter of 100 mm is required 25 ml of agar, a Cup with a diameter of 90 mm 20 ml). Cups were left at room temperature for gelation. Cooked this way the Petri dishes used immediately.

The concentration of suspension of microorgani�mA was 1.5×10 8CFU/ml Optical density of bacterial suspension with a concentration of 1.5×108CFU/ml by visual inspection according to the standard turbidity of 0.5 on Mac-Farland. Monitoring the optical density of the suspension was carried out by densitometry. We used commercial standard turbidity. Bacterial suspension was prepared from agar cultures.

To prepare the inoculum used pure daily culture of microorganisms grown on dense nutrient media. 've selected a few of the same type, clearly isolated colonies grown on non-selective nutrient dense environments. Loop transferred a small amount of material from the tops of the colonies into a tube containing sterile saline, adjusted to a density of inoculum precisely to 0.5 standard Mac-Farland. The inoculum was used within 15 min after preparation.

The method of serial dilutions in broth - macromethod (test-tube)

Testing was performed in a volume of 1 ml of each dilution of the investigated compounds, with a final concentration of the studied microorganism of about 5×105CFU/ml.

MHB for determination of sensitivity was poured 0.5 ml in each tube. The number of tubes amounted to nine pieces plus one to set a "negative" control, i.e., ten.

The working solution SPS�CSOs compounds were prepared from the basic solution with the use of liquid culture media - MHB. Then the working solution in an amount of 0.5 ml using a micropipette with a sterile tip was introduced into the first test tube containing 0.5 ml of broth. Was mixed thoroughly and a new sterile tip was transferred to 0.5 ml of a solution of tested compound in the broth a second tube that originally contained 0.5 ml of broth. This procedure was repeated until it was cooked all the required number of dilutions. From the last test tube and 0.5 ml of broth was removed.

Thus, received a number of test tubes with solutions of the tested compound, the concentration of which differed in the adjacent tubes 2 times.

For inoculation used a standard microbial suspension equivalent to a 0.5 standard Mac-Farland diluted 100 times on MHB, then the concentration of the microorganism in it was about 106CFU/ml with 0.5 ml of inoculum was introduced into each tube containing 0.5 ml of the appropriate dilutions of a compound in a single tube with 0.5 ml MHB without antibiotic (negative control). The final concentration of the microorganism in each tube was approximately 5×105CFU/ml Inoculum was added to the test tubes for dilutions of compound no later than 15-30 min after preparation.

The tube was closed with a sterile cotton-gauze plugs and everything except the tubes "negative�" control, incubated in a normal atmosphere at 37°C for 16-20 or 20-24 h (depending on the type of test microorganism). Vial negative control was placed in a refrigerator at 4°C when kept before accounting for the results.

To determine whether growth of the microorganism tubes of crops were viewed in transmitted light. The growth of culture in the presence of the tested compound compared to the reference sample tube (negative control) containing the original inoculum and kept in the refrigerator. The minimum inhibiting concentration (BMD) was determined by the lowest concentration of tested compound that inhibits visible growth of the microorganism. Minimum bactericidal concentration (MBC) was determined by seeding from test tubes on a dense nutrient medium, followed by incubation of seeds in a normal atmosphere at 37°C for 16-20 or 20-24 h (depending on the type of test microorganism).

For convenience in the analysis of the data evaluation activity of the test compounds was determined in the crosses according to the following scheme (Pershin, G. N. Methods of experimental chemotherapy / G. N. Pershin // M.: Medicine, 1971. - 541 p.): "+ + + " - abundant growth; "++" - deep or surface growth strains less abundant; "+" - weak growth - inactive less 50-30%; "+/-,0" - more than 70% of the delay or the lack of growth� culture, compliance with the "negative control".

Disk diffusion method (DDM)

To determine the sensitivity of the DDM used MOSS (for streptococci with the addition of sheep blood) prepared according to the manufacturer's instructions. The thickness of the layer of agar in a Cup total of 4.0±0.5 mm, which was achieved by entering into a Petri dish with a diameter of 90 mm is strictly 20 ml of agar, 100 mm diameter and 25 ml of agar. Before filling, the molten medium of the Petri dish was mounted on a horizontal surface (adjusted by level, without hollows and bumps). After filling the cups were left at room temperature for gelation. Used a freshly made Cup, which prior to inoculation were dried by incubation at 37°C with lid ajar for 10-20 min Prior to inoculation was controlled by the absence of the liquid condensate on the inner surface of the lids.

To determine the sensitivity of the DDM used standardized drives ND-PMP-1 from cardboard filter technical GOST 6722-75 (fgun "Saint-Petersburg scientific research Institute of epidemiology and Microbiology. L. Pasteur" of the Federal service for supervision of consumer rights protection and human welfare).

Storage drives ND-PMP-1 from cardboard technical filtration was carried out in sealed packaging�e of the manufacturer in a dry dark place at a temperature of 2-8°C during the entire shelf life of the kit. A small batch of discs used in daily work, kept at a temperature of 25°C not more than 15 days tightly sealed so as to ensure the impossibility of getting into the vial of moisture, in addition, for extra protection from moisture in bottles (cartridges) with the discs contains a special desiccant (silica gel).

Before using the bottles with the discs were maintained at room temperature 18-25°C for 1 h to prevent the formation of condensate on the inner wall of the vial. Opened a bottle with the discs stored at 2-8°C during the entire shelf life of the set, while maintaining the color indicating silica gel from light blue to dark blue. Immediately before use, the disks were impregnated with the studied compound. As a control, the discs impregnated with: 1) distilled water; 2) dioxydinum (production of "Biosynthesis", solution for topical use, endotracheal and intravenous injection, 10 mg/ml), antibacterial drug, a derivative of quinoxaline.

To determine the sensitivity of the DDM used a standard inoculum corresponding to a density of 0.5 on the standard Mac-Farland and containing approximately 1.5×108CFU/ml Inoculum was used within 15 min after preparation. To inoculate the prepared cups with'agaro� using sterile cotton swabs industrial production (wand-swab (plastic-cotton) CITOSWAB sterile in individual packaging). The swab was immersed in a standard suspension of the organism, then the excess inoculum was removed by pressing the swab against the sides of the tube. Inoculation was performed dashed movements in three directions, rotating the Petri dish at 60°.

No later than 15 minutes after inoculation on the surface of the nutrient medium was applied to the discs impregnated with the studied compounds and distilled water. Applique discs was performed using sterile tweezers. The distance from the disk to the edge of the Cup and between the discs was 15-20 mm For uniform contact with the surface of the agar discs were gently pressed with tweezers.

Directly after application of discs to the surface of the nutrient medium in Petri dishes were placed in a thermostat upside down and incubated at 37°C for 18-24 hours (depending on the type of test microorganism).

After the incubation cups were placed upside down on a dark matte surface so that the light fell on them at an angle of 45° (given in reflected light). The diameter of zones of growth retardation was measured with an accuracy of 1 mm using a ruler pattern (Hi Antibiotic Zone Scale (PW 2097) (Hi Media Laboratories Pvt. Limited, India)).

The degree of activity to the tested compounds was determined in the crosses according to the following scheme (Determination of sensitivity of microorganisms to antibacterial preparations (HOWTO MOOK 4.2.189004) // Clinical Microbiology and antimicrobial chemotherapy. - 2004. - Volume 6. - No. 4. - S. 306-359): "+++" high activity is the diameter of the zone of stunted growth of more than 25 mm; "++" active - zone diameter of stunting 16-25 mm; "+" inactive - the diameter of the zone of stunting 10-15 mm; "+/-, 0" - disabled: the diameter of the zone of stunted growth of less than 10 mm and the total absence.

Statistical processing of obtained results was performed with analysis of variance, the reliability of the results was evaluated using the method for the determination of t-criterion of student. Reliable considered results with P≤0.05 (Fisenko V. Guidance on experimental (preclinical) study of new pharmacological substances / Under the editorship of V. P. Fisenko, B. In Arzamastseva, And E. Babayan et al. - Moscow: "Remedium", 2000. - 398 S.; lang, T. A. How to describe statistics in medicine / T. A. lang, M. Secic; per. s angl. under the editorship of V. P. Leonov. - M.: Practical medicine, 2011. - 480 C.). We used a personal computer and standard software package Statistica 6.0.

The results of the study

Study of antimicrobial activity of the test compounds by the method of serial dilutions in broth

A study of the antimicrobial activity of (+)-limonene, (+)-1,2-limonene oxide, terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methyladenine against reference strains of Staphylococcus aureus 29213, Escherichia coli 25922, Pseudomona aeruginosa 27853, Streptococcus pyogenes 1238.

As the comparison drug was used antimicrobial drug dioxidine (derived di-N-oxigenacion), widely used in medical practice. This drug has a high chemotherapeutic activity in vitvo to model infections with similar pathogenesis to pathological processes in humans (purulent meningitis, pyelonephritis, septicopyemia) and caused by strains of anaerobic bacteria that are resistant (including multidrug-resistant) to drugs of other classes, including strains of Pseudomonas aeruginosa and methicillinsensitive staphylococci. Dioxidine is characterized by a broad spectrum antibacterial with bactericidal action, active against gram-positive and gram-negative aerobic pathogenic bacteria. Shown activity dioksidina against Mycobacterium tuberculosis (Padejskij E. N. Antibacterial drug dioxidine: peculiarities of biological action, and significance in the treatment of various forms of purulent infection / E. N. Padejskij // Infection and antimicrobial therapy. - 2011. - Vol. 3 - No. 5. - S. 105-155).

The results of antimicrobial activity of the test substances are shown in table 2.

+++
Table 2
The definition of the minimum�'s inhibitory concentrations (MPC) of the test compounds by the method of serial dilutions in liquid nutrient medium
Test 1 cultureStaphylococcus aureus 29213Escherichia coli 25922Pseudomonas aeruginosa 27853Streptococcus pyogenes 1238
analytelemon1,2-limonene oxideterpene-sulfidelemon1,2-limonene oxideterpene-sulfidelemon1,2-limonene oxideterpene-sulfidelemon1,2-limonene oxideterpene-sulfide
The concentration of substances in the nutrient medium (mg/ml32,0000000000000
16,0000000+/-1+/-10000
8,00000+/-1+/-++++0+/-100
4,0000+++/-++++++0++00
2,0+/-100+++++ +/-1+++++++/-1++++/-1+/-1
1,0+++/-1+/-+++++++++++++++++++++++
0,5++++++/-1+++++++++++++++++++++++++++
0,25++++++++++++++++++++++++++++++++
0,125++++++++++++++++++++++++++++++++++++
"Negative" control+/-+/-+/-+/-
Note: -1the titer of activity, "+++" - abundant growth; "++" - deep or surface growth strains less abundant, growth is weak - less inactive 50-30% (+); more than 70% of the delay or the lack of growth in culture compared with control(+/-, 0).

For product comparison dioksidina IPC relative to strains of Staphylococcus spp. is 125,0-1000,0 µg/ml, Escherichia coli 8,0-250,0 mg/ml, Pseudomonas spp. 125,0-1000,0 mg/ml, Streptococcus spp. 64,0-1000,0 mg/ml (Padejskij E. N. Antibacterial drug dioxidine: peculiarities of biological action, and significance in the treatment of various forms of purulent infection / E. N. Padejskij // Infection and antimicrobial �Arabia. - 2011. - Vol. 3 - No. 5. - S. 105-155).

From table 2 it is seen that (+)-limonene has a bacteriostatic effect on S. aureus 29213 at a concentration of 2.0 mg/ml and at a concentration of 4.0 mg/ml completely inhibits the growth of culture. A bacteriostatic effect on E. coli 25922 (+)-limonene is shown at the concentration of 4.0 mg/ml and at a concentration of 8.0 mg/ml, it completely inhibits the growth of culture. Bacteriostatic effect on P. aeruginosa 27853 investigated the presence of monoterpene has a concentration of 16.0 mg/ml and at a concentration of 32.0 mg/ml completely inhibits the growth of culture. (+)-limonene has a bacteriostatic effect on S. pyogenes 1238 at a concentration of 8.0 mg/ml and at a concentration of 16.0 mg/ml completely inhibits the growth of culture.

(+)-1,2-limonene oxide has a bacteriostatic effect on S. aureus 29213 at a concentration of 1.0 mg/ml and at a concentration of 2.0 mg/ml completely inhibits the growth of culture. A bacteriostatic effect on E. coli 25922 (+)-1,2 limonene oxide exhibits at a concentration of 8.0 mg/ml and at a concentration of 16.0 mg/ml completely inhibits the growth of culture.

Bacteriostatic effect on P. aeruginosa 27853 investigational compound produces a concentration of 16.0 mg/ml and at a concentration of 32.0 mg/ml completely inhibits the growth of culture. (+)-1,2 limonene oxide has a bacteriostatic effect on S. pyogenes 1238 at a concentration of 2.0 mg/ml and at a concentration of 4.0 mg/ml and fully�will gebirol the growth of culture.

Terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methyl-ethanoate bacteriostatic effect on S. aureus 29213 at a concentration of 1.0 mg/ml and at a concentration of 2.0 mg/ml completely inhibits the growth of culture. A bacteriostatic effect on E. coli 25922 of Terminalia (I) occurs at a concentration of 2.0 mg/ml and at a concentration of 16.0 mg/ml completely inhibits the growth of culture. Bacteriostatic effect on P. aeruginosa 27853 investigational compound produces a concentration of 2.0 mg/ml and at a concentration of 4.0 mg/ml completely inhibits the growth of culture. 2-(1'-Hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol bacteriostatic effect on S. pyogenes 1238 at a concentration of 2.0 mg/ml and at a concentration of 4.0 mg/ml completely inhibits the growth of culture.

All tubes with "negative control" there was a delay of growth of the test cultures. Conducted 5 sequences of experience.

Thus, for IPC (+)-limonene amounted to about S. aureus 29213 - 2.0 mg/ml, E. coli 25922 - 4,0 mg/ml, P. aeruginosa 27853 - 16,0 mg/ml S. pyogenes 1238 - 8.0 mg/ml.

IPC for (+)-1,2-limonene oxide - S. aureus 29213 - 1.0 mg/ml, E. coli 25922 - 8.0 mg/ml, P. aeruginosa 27853 - 16,0 mg/ml S. pyogenes 1238 - 2.0 mg/ml.

IPC for terminalhead - S. aureus 29213 - 0.5 mg/ml, E. coli 25922 - 2.0 mg/ml, P. aeruginosa 27853 - 2.0 mg/ml S. pyogenes 1238 - 2.0 mg/ml.

Table 3
Antimicrobial activity of the drug comparison dioksidina and researched substances
The investigated test microorganism straindioxidine (mg/ml)(+)-limonene (mg/ml)(+)-1,2 limonene oxide (mg/ml)terminalid (I) (mg/ml)
Staphylococcus aureus 292130,125-1,02,01,00,5
Streptococcus pyogenes 12380,064-1,08,02,02,0
Escherichia coli 259220.008 to 0.250 kg4,08,02,0
Pseudomonas aeruginosa 278530,125-1,016,016,02,0

The investigated compounds have antimicrobial activity. Comparative analysis of the results showed that the investigated various compounds for antimicrobial activity, the highest activity against the test strains of micro�of Organismo showed terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol. Its IPC is 4 times lower than that of (+)-limonene, and 2 times lower than that of (+)-1,2 limonene oxide, against Staphylococcus aureus; 2 times lower than that of (+)-limonene, and 4 times lower than the (+)-1,2 limonene oxide, against Escherichia coli; 8 times lower than that of (+)-limonene, (+)-1,2 limonene oxide, relative to P. aeruginosa and 4 times lower than the (+)-limonene, against pyogenic streptococci.

Study of antimicrobial activity of the investigated compounds disco-diffusion method

The study of Museum strains of microorganisms obtained the following results (table.4). In a control experiment in 100% of cases there was a continuous growth of the studied microorganisms.

The reference drug dioxidine showed high activity against the test strains of Citrobacter freundii 101/57, Klebsiella pneumoniae 9172, 222 and Proteus vulgaris Bacillus cereus 96 (P<0,05). Also sensitive to the comparison drug were Staphylococcus aureus 906, Streptococcus pyogenes 1238, Salmonella enteritidis 5765 ATCC, Shigella sonnei S-shape 20, Pseudomonas aeruginosa 453 (P<0,05). On the background of application dioksidina significant delays the growth of Escherichia coliM17, Enterococcus faecalis 2919 ATCC were observed.

(+)-Limonene was active against Streptococcus pyogenes 1238, Escherichia coli M17, Salmonella enteritidis 5765 ATCC, Citrobacter freundii 101/57, Proteus vulgaris 222 (P<0,05). High sensitivity to this compound showed Klebsiella pneumoniae 9172 and Bacillus cereus 96 (P<0,05). Against Staphylococcus aureus 906, Enterococcus faecalis 299 ATCC, Shigella sonnei S-shape 20 and Pseudomonas aeruginosa 453 (+)-limonene was not active.

(+)-1,2 Oxide limonene showed activity against the test strains Staphylococcus aureus 906, Enterococcus faecalis 2919 ATCC, Escherichia coli M17 strain, Salmonella enteritidis 5765 ATCC, Shigella sonnei S-shape 20, Citrobacter freundii 101/57, Pseudomonas aeruginosa 453 (P<0,05). The high activity of the (+)1.2-limonene oxide was observed against Klebsiella pneumoniae 9172, Proteus vulgaris 222, Bacillus cereus 96 (P<0,05).

Sensitive to terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol was the test strains Staphylococcus aureus 906, Streptococcus pyogenes 1238, Enterococcus faecalis 2919 ATCC, Salmonella enteritidis 5765 ATCC, Shigella sonnei S-shape 20, Citrobacter freundii 101/57, Klebsiella pneumoniae 9172, Proteus vulgaris 222, Pseudomonas aeruginosa 453 (P<0,05). High sensitivity for semisynthetic derivative of limonene was observed in Escherichia coli M17 strain and Bacillus cereus 96 (P<0,05).

The sensitivity of experimental strains of microorganisms to the tested compounds was as follows (table.5). Clinical strains of S. aureus were statistically significantly sensitive K (+)-limonene (P<0,05). Zone of stunting in 43% of the strains ranged from 16 to 25 mm and 27% above 25 mm.

(+)-1,2 Oxide limonene showed relatively low activity of the studied strains of S. aureus, 75% of them are areas of stunting did not exceed 15 mm.

Table 4
Study of antimicrobial activity of the test compounds relative to the test strains of microorganisms disk diffusion method
The test strain / activitycontrol(+)-limonene(+)-1,2-limonene oxideterminaliddioxidine
Staphylococcus aureus 9060+++*++*++*
Streptococcus pyogenes 12380++*+++*++*
Enterococcus faecalis 2919 ATCC0+++*++*+
Escherichia coli M17 strain0++*++*+++*+
Salmonella enteritidis 5765 ATCC0++*++* ++*++*
Shigella sonnei S-shape 200+++*++*++*
Citrobacter freundii 101/570++*++*++*+++*
Klebsiella pneumoniae 91720+++*+++*++*+++*
Proteus vulgaris 2220++*+++*++*+++*
Pseudomonas aeruginosa 4530+++*++*++*
Bacillus cereus 960+++*+++*+++*+++*
Note: * - difference from control is statistically significant at P<0,05; "+++" high activity diameter of zone of stunted growth of more than 25 mm; "++" active - zone diameter of stunting 16-25 mm; "+" inactive - the diameter of the zone of stunting 10-15 mm; "+/-, 0" - disabled: the diameter of the zone of stunted growth of less than 10 mm and the total absence.

Terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methyl-ethanoate was active about 57% of clinical S. aureus strains (P<0,05). But 43% of the studied strains on the use of Terminalia gave a growth delay not exceeding 15 mm.

The investigated strains of S. epidermidis in 64% were sensitive K (+)-limonene (P<0,05). 55% of the area of stunting ranged from 16 to 25 mm, 9% above 25 mm. 23% of the studied strains were not sensitive to the studied presence of monoterpene.

(+)-1,2 limonene Oxide had a statistically significant antimicrobial effect on clinical strains of S. epidermidis (P<0,05). Moreover, about 40% of the strains showed a high activity, the remaining 60% gave a zone of stunted growth from 16 to 25 mm.

Statistically significantly sensitive to 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol was studied clinical strains of S. epidermidis (P<0,05). About 37% of the microorganisms of this type of terminalid was inactive.

Members of the family Streptococcaceae were not sensitive to the tested compounds. Been studied clinical strains of childbirth and Stretococcus Enterococcus.

(+)-Limonene had no statistically significant antimicrobial effect on clinical strains of S. pyogenes (70% - zone growth delay not exceeding 15 mm); S. pneumoniae (70% - zone growth delay not exceeding 15 mm, 30% - insensitive); S. uberis (65% - zone growth delay not exceeding 15 mm); S. salivarius (55% - zone growth delay not exceeding 15 mm); S. mitis (75% - zone growth delay not exceeding 15 mm); S. agalactia (64% zones of growth retardation is not more than 15 mm); S. sanguinis (78% - zone growth delay not exceeding 15 mm); S. mutans (73% - zone growth delay not exceeding 15 mm); E. faecalis (73% - zone growth delay not exceeding 15 mm); E. faecium (64% - zone growth delay not exceeding 15 mm).

(+)-1,2 limonene Oxide had no significant antimicrobial effect on clinical strains of S. pneumoniae (70% - zone growth delay not exceeding 15 mm, 30% - insensitive); S. uberis (75% - zone growth delay not exceeding 15 mm); S. salivarius (75% - zone growth delay not exceeding 15 mm); S. mitis (80% zone of stunting is not more than 15 mm); S. sanguinis (60% zone of stunting is not more than 15 mm); S. mutans (100% zones of growth retardation is not more than 15 mm); E. faecalis (60% zone of stunting is not more than 15 mm); E. faecium (96% - zone growth delay not exceeding 15 mm). But with respect to S. pyogenes (+)-1,2 oxide limonene showed a statistically significant antimicrobial effect (P<0,05). 58% of the investigated strains were sensitive K (+)-1,2 limonene oxide, 42% were highly sensitive. 50% of clinical strains of S. agalactia �and the background of the application (+)-1,2 limonene oxide gave a zone of stunted growth from 16 to 25 mm.

The investigated strains of S. epidermidis in 64% were sensitive K (+)-limonene (P<0,05). 55% of the area of stunting ranged from 16 to 25 mm, 9% above 25 mm. 23% of the studied strains were not sensitive to the studied presence of monoterpene.

(+)-1,2 limonene Oxide had a statistically significant antimicrobial effect on clinical strains of S. epidermidis (P<0,05). Moreover, about 40% of the strains showed a high activity, the remaining 60% gave a zone of stunted growth from 16 to 25 mm.

Statistically significantly sensitive to terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol was studied clinical strains of S. epidermidis (P<0,05). About 37% of the microorganisms of this type of terminalid was inactive.

Members of the family Streptococcaceae were not sensitive to the tested compounds. Been studied clinical strains of the genera Streptococcus and Enterococcus.

(+)-Limonene had no statistically significant antimicrobial effect on clinical strains of S. pyogenes (70% - zone growth delay not exceeding 15 mm); S. pneumoniae (70% zone delay growth of over 15 mm, 30% - insensitive); S. uberis (65% - zone growth delay not exceeding 15 mm); S. salivarius (55% - zone growth delay not exceeding 15 mm); S. mitis (75% - zone growth delay not exceeding 15 mm); S. agalactia (64% zone of stunting not �ore than 15 mm); S. sanguinis (78% - zone growth delay not exceeding 15 mm); S. mutans (73% - zone growth delay not exceeding 15 mm); E. faecalis (73% - zone growth delay not exceeding 15 mm); E. faecium (64% - zone growth delay not exceeding 15 mm).

(+)-1,2 limonene Oxide had no significant antimicrobial effect on clinical strains of S. pneumoniae (70% - zone growth delay not exceeding 15 mm, 30% - insensitive); S. uberis (75% - zone growth delay not exceeding 15 mm); S. salivarius (75% - zone growth delay not exceeding 15 mm); S. mitis (80% zone of stunting is not more than 15 mm); S. sanguinis (60% zone of stunting is not more than 15 mm); S. mutans (100% zones of growth retardation is not more than 15 mm); E. faecalis (60% zone of stunting is not more than 15 mm); E. faecium (96%) - zone growth delay not exceeding 15 mm). But with respect to S. pyogenes (+)-1,2 oxide limonene showed a statistically significant antimicrobial effect (P<0,05). 58% of the investigated strains were sensitive K (+)-1,2 limonene oxide, 42% were highly sensitive. 50% of clinical strains of S. agalactia on the background of application (+)-1,2 limonene oxide gave a zone of stunted growth from 16 to 25 mm.

Terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol showed statistically significant antimicrobial effect on clinical strains of S. pyogenes, S. pneumoniae, E. faecalis (P<0,05). 65% of strains of S. pyogenes, 60% of strains of S. pneumoniae, 80% of E. faecalis strains were sensitive to the tested drug. Amid p�change of Terminalia (I) of the investigated strains of S. uberis in 63% gave a zone of stunted growth is not more than 15 mm, S. salivarius - 75%, S. mitis - 71%, S. agalactia - 100%, S. sanguinis - 65% of the S. mutans - 75%, E. faecium - 100%.

The most pronounced activity of the investigated compounds showed relatively the representatives of the family Enterobacteriaceae.

(+)-Limonene showed a statistically significant antimicrobial effect on clinical strains of E. coli, S. enteritidis, S. sonnei, E. aerogenes, K. pneumoniae, K. oxytoca (P<0,05). Studied strains of E. coli in 49% were sensitive to the tested presence of monoterpene, 31% were highly sensitive. 89% of S. enteritidis strains were sensitive to the (+)-limonene. Regarding clinical strains of S. sonnei (+)-limonene 30% active, 70% were highly active. 100% of the investigated strains of E. aerogenes with application of (+)-limonene gave a stunted growth from 16 to 25 mm. of the Studied strains of K. pneumoniae in 57% were sensitive to the tested presence of monoterpene, 29% were highly sensitive. 33% of the investigated strains of K. oxytoca with application of (+)-limonene gave a stunted growth from 16 to 25 mm, and 17% above 25 mm. To 100% (+)-limonene was inactive or low relative to the studied strains of P. vulgaris, P. mirabilis, E. cloaceae. Clinical strains of C. freundii in 74% with application of (+)-limonene was given a growth delay not exceeding 15 mm.

(+)-1,2 Oxide limonene showed a statistically significant antimicrobial effect on clinical strains of E. coli, S. enteritidis, S. sonnei, C. freundii, E aerogenes, K. pneumoniae, K. oxytoca, P. vulgaris, P. mirabilis (P<0,05). Studied strains of E. coli in 57% were sensitive to the tested compound in 43% - high sensitivity (P<0,05). 44% of S. enteritidis strains were sensitive to the (+)-1,2 limonene oxide and 56% sensitive. Regarding clinical strains of S. sonnei (+)-1,2 limonene oxide 25% were active, 75% were highly active. Clinical strains of C. freundii in 85% on the background of the application (+)-1,2 limonene oxide gave a stunted growth from 16 to 25 mm, 15% - more than 25 mm. 75% of tested strains of E. aerogenes on the background of application (+)-1,2 limonene oxide gave a stunted growth from 16 to 25 mm. of the Studied strains of K. pneumoniae in 34% were sensitive to the tested compound in 55% is highly sensitive. 26% of the investigated strains of K. oxytoca on the background of application (+)-1,2 limonene oxide gave a stunted growth from 16 to 25 mm, 74% - higher than 25 mm. P. vulgaris and P. mirabilis were sensitive to the tested drug, and its use of the zone of stunted growth of the studied strains was in 64 and 76%, respectively, from 16 to 25 mm, 22 and 14% respectively - higher than 25 mm. To 100% (+)-1,2 limonene oxide was inactive about the examined strains of E. cloaceae.

Terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol showed a statistically significant antimicrobial effect on clinical strains of E. coli, S. enteritidis, S. sonnei, C. freundii, K pneumoniae, K. oxytoca (P<0,05). Studied strains of E. coli in 67% were sensitive to the investigated 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylalanine. 90% of the S. enteritidis strains were sensitive to the tested terminalid. Regarding clinical strains of S. sonnei terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol in 87% were active. 100% of the investigated strains of C. freundii with the use of Terminalia gave a stunted growth from 16 to 25 mm. of the Studied strains of K. pneumoniae in 63% were sensitive to the tested compound. 53% of the investigated strains of K. oxytoca on the use of Terminalia gave a stunted growth from 16 to 25 mm. In 100% terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol was inactive regarding the studied strains of P. vulgaris, P. mirabilis, E. cloaceae, E. aerogenes.

The investigated compounds were less active against clinical strains of P. aeruginosa.

The data indicate the presence of antimicrobial activity of (+)-limonene, (+)-1,2 limonene oxide and, in particular, the sulfide of limonene against the test strains of gram-negative and gram-positive bacteria.

Terminalid 2-(1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylethanol showed broad spectrum antimicrobial activity.

Use 2-1'-hydroxy-4'-Isopropenyl-1'-methylcyclohexyl-2'-thio)-methylalanine
formula I:

as an antimicrobial.



 

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9 cl, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: for the purpose of treating allergy in an individual characterised by cytokine Th1 and Th2 imbalance, β-hydroxy-β-methylbutyrate (HMB) is introduced into the individual in an amount effective to reduce cytokine Th1 and Th2 production imbalance. What is also presented is a method of treating allergy in the individual belonging to a risk group of developing allergy.

EFFECT: elimination, delay and reduction of the rate of the onset or severity of allergy-related symptoms due to increase of the cytokine Th1 content with no increase of the cytokine Th2 content.

11 cl, 5 dwg, 8 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a pharmaceutical combination and to its use for treating an infection caused by influenza virus. The declared composition contains a pyrazine derivative of formula wherein R1 and R2 are identical or different, and each represents a hydrogen atom or a halogen atom; and R3 represent a hydrogen atom or a protective group for amino group or its salt, and, and a neuraminidase inhibitor. The neuraminidase inhibitor is specified in oseltamivir, zanamivir, peramivir or CS-8958.

EFFECT: invention provides preparing the combination which shows strong antiviral activity, smaller side effects and applicable for treating influenza.

12 cl, 8 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a compound of general formula (I), in the form of optical isomers or their mixtures, and its pharmaceutically acceptable salt, which show activity as α2δ ligand and affinity to a subunit of α2δ potential-dependent calcium channels. In formula each R1, R2, R2', R4, R5, R8 and R8' independently represents hydrogen atom, halogen atom or C1-C6 alkyl group; each R6 and R7 independently represents hydrogen atom or C1-C6 alkyl group; or R2 and R2' together with carbon atom whereto attached form C3-C7 cycloalkyl group; R3 represents hydrogen atom, C1-C6 alkyl group, C1-C6 alkylhalogenide group, C1-C6 alkoxy-C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C1-C6 alkylsulphanyl-C1-C6 alkyl group, C2-C7 acyloxy-C1-C6 alkyl group or C3-C7 cycloalkyl group.

EFFECT: preparing the pharmaceutical composition for treating and/or preventing pain.

25 cl, 19 tbl, 43 ex

FIELD: medicine.

SUBSTANCE: to improve functional-aesthetic results of the plastic surgery of post-traumatic eyelid deformations, starting from 7-8 day from the moment of carrying out blepharoplasty of the post-traumatic cicatrical eyelid deformation, immediately after the removal of operational sutures, on the operated zone of the eyelid performed is a procedure of magnetic-photophoresis of a medical solution, made from 3000 ME of longidaze, 3 ml 25% solution of dimethylsulphoxide and 1 ml 0.25% solution of derinate, with 2 ml of the prepared solution being applied on an autodermal transplant and healthy skin surrounding it, with up to 1 cm indent from the wound edge, after 3-5 minutes a gauze pad, soaked with 2 ml of the remaining prepared solution, is applied on the processed surface of skin with the further 10 minute impact by a contact method by a travelling pulsed magnetic field of the apparatus "AMO-ATOS" with the frequency of pulse consecution of 5-10 Hz. Then, after the pad is removed, without the time interval, borders of the autodermal transplant and healthy skin closing are processed by infrared laser irradiation of a range of 0.89 mcm of the apparatus UZOR-2K with the frequency of the pulse repetition rate of 1500 Hz, in accordance with a contact labile method, 1 minute on each field, with carrying out 1 procedure daily for 10 days.

EFFECT: improvement of the quality of skin transplant engraftment, absence of secondary eyelid deformations, and reduction of the post-operational treatment duration.

1 tbl, 2 ex

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