Thymol-containing composition useful in treatment of patients suffering from drug-resistant bacterial infections

FIELD: pharmaceutics.

SUBSTANCE: composition is constituted by effective amount of thymol obtained from plant Trychyspermum ammi, mint oil combination of mint oil containing required amounts of monoterpenes and isolated from Mentha spicata and Mentha arvensis, and typical additives. Invention also relates to preparation of the composition by mixing above ingredients and to method of treating patients by administering therapeutically effective amount of the composition.

EFFECT: created therapeutic agent overcoming drug resistance of bacteria and minimized or even eliminated secondary adverse effect of drug.

19 cl, 12 tbl, 11 ex

 

The technical field

The present invention relates to a new synergistic composition useful for the treatment of drug resistant bacterial infections, which contains an effective amount of thymol, a blend of essential oils of Mentha arvensis and Mentha spicata or monoterpenoid components in an appropriate ratio and conventional additives. This composition is useful for treatment of drug resistant enteric and systemic infections. The composition is characterized by increased activity of thymol, includes thymol in combination with the specified oil, which contains a rare mixture of carvone, limonene and menthol. This invention relates also to methods for obtaining this composition and to the method of application of thymol isolated from the seeds of plants Trychyspermum amini (Ajwain), as the antibiotic composition of the fourth generation, to combat drug resistant bacteria. In particular, this invention relates to the use of compounds "thymol", isolated from the oils obtained by distillation from the seeds of plants Trychyspermum arnmi (Ajwain), for the destruction of bacteria that are resistant even to potent antibiotics third generation, and pathogenic microorganisms that are resistant to many drugs (mdr), and thus it is useful as a herbal antibiotic composition is otwartego generation plant-based.

Background of the invention

Microbial infections are the main danger for human health and are a major cause of diseases that cause high mortality among the population worldwide. In addition, infection significantly degrade human performance, affecting different metabolic functions and systems such as digestive, respiratory, urinary, circulatory, nervous system and skin. This causes constant discomfort, while it doesn't completely get rid of causing disease microorganisms. Bacterial infections pose a serious threat to the health and well-being of people of all ages. Since the 1940's, when Alexander Fleming discovered penicillin to combat pathogens and treatment of infectious diseases doctors usually use antibiotics and antimicrobial drugs. However, infectious microorganisms constantly exert a counteracting new medicines, developing resistance to used drug/antibiotic. The emergence of strains resistant to multiple drugs is a serious problem in the field of medicine. The root cause of sustainability are random mutations. Mutations can occur in the ü in the genes, responsible for discussing sensitivity to the drug. If there are not enough new antibiotics, features a new mechanism of action, humanity may be on the verge of a medical catastrophe. It is time to go to the hidden possibilities of molecules of plants, using modern means of purposeful search for drugs and antibiotics new, smarter generation, characterized by new principles of action.

New antimicrobial agents create by structural modification of existing tools, thus obtaining drugs next generation with a broader spectrum of activity and higher efficiency. Well-known examples of a family of antimicrobial agents in the next generation are the penicillins and cephalosporins. These medicines resulting from chemical modifications of the basic β-lactam ring. The cephalosporin is the drug of the third generation on the basis of tsefalotina. Similarly, since 1962, when Loescher together with his colleagues used nalidixic acid to create a quinolone, antimicrobial drugs of the first generation, the family quinolones have added drug which means third generation. In fact, nalidixic acid has found limited application in the treatment of systemic infections, and in 1970-ies were obtained minimally improved quinolones, such as oxolinic acid, pipemidinova acid and cinoxacin. In the early 1980s, there has been a breakthrough that was marked by the creation of fluorinated quinolones. First, Wolfson and Hooper created norfloxacin, hinolan second generation of 6-fluoro and 7-piperazine-substituents. Specified substance possessed more effective action against gram-negative and gram-positive bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureus. Following this were developed related molecules such drugs like ciprofloxacin, enoxacin, ofloxacin, pefloxacin, and so forth. Almost simultaneously, were established medicinal product of the third generation, such as lomefloxacin, fleroxacin, temafloxacin, tosufloxacin, substituted by one or more fluorine atoms compared to the second generation quinolones having one fluorine atom in position 6 of the main quinolones.

Permanent and unrestricted use of the quinolone/fluoroquinolone drugs gradually makes the bacteria targeted by these medicines, immune to their effects, which requires increasing doses specified the output connections for the destruction of pathogenic microorganisms. Because these drugs are synthetic compounds, they have a strong side effect. In addition, pathogens become resistant to the effects of increasing doses of these compounds, which further exacerbates the situation. To solve this problem, the applicants have developed a new method of application of thymol known plant compounds, which are used separately for the destruction of drug resistant bacteria and in combination with other antibiotics to combat the occurrence of bacterial resistance to drugs.

Among deadly bacterial infections first place is tuberculosis, which annually kills more than three million lives worldwide. Even more terrifying factor is the increase in morbidity and mortality from TB among HIV-infected subjects. The spread of tuberculosis and the frequency of lesions of this disease in persons infected with human immunodeficiency virus, in both developing and industrialized countries were forced to pay attention to the urgent need to create new medicines, better generation.

Worldwide well known that the commonly used antibiotics are nedostate is yet effective for the treatment of such diseases, as tuberculosis. Even developing countries are forced to admit that these diseases are becoming epidemic. You need to constantly search for new combinations of drugs, using active molecules from natural sources such as plants, because otherwise humanity in the new Millennium will be faced with serious problems. The most promising achievement in the field of drugs is the discovery of new molecules or new uses for known compounds that are safe and do not have any side effects. Such active biomolecules in combination with other antibiotics can kill drug resistant bacteria and simultaneously to control the further development of drug resistance in infectious microorganisms.

Thymol is widely used in various herbal preparations, starting with liquids for rinsing the mouth and medicines for intestinal disorders to infectious skin lesions. Original oil and thymol are indispensable components “Granny” home recipes to treat various ailments, as they are equally useful for both children and adults. Such tools are widely used in India and others the other continents through formal and informal dissemination of knowledge of traditional and herbal medicine.

Some relevant sources of prior art that should be put in this description of the invention to ensure uniqueness of the present invention, the following describes the different applications.

1. Composition for treating viral infections, including HIV (Pruthi et al. 1999; U.S. patent No. 5980903, November 9, 1999), and drugs of similar action (Badaway 1998; U.S. patent No. 5801153, September 1998; Rohatgi 1996, U.S. patent No. 5529778, June 1996; Hozumi et al. 1995, U.S. patent No. 5411733, may 1995).

2. Analgesic composition, temporarily overwhelming pain arthritis symptoms (Beck 1991, U.S. patent No. 5073366, December 17, 1991), and drugs of similar action (Arbir et al. 1981, U.S. patent No. 4307109, December 1981; Dubash et al. 1983, U.S. patent No. 4383986, may 1983; Seth 1985, U.S. patent No. 4540572, September 1985; Geria 1987, U.S. patent No. 4702916, October 1987; Grohe 1989, U.S. patent No. 4844902, July 1989; Bisset et al. 1989, U.S. patent No. 4847071, July 1989; Deckner et al., U.S. patent No. 4863725, July 1989).

3. Powder composition to obtain a liquid for rinsing of the mouth (Smigel et al. 1999, U.S. patent No. 4925655, March 15, 1990) and drugs of similar action (Fleischman 1936, U.S. patent No. 2035267, March 1936; Poerschke 1937, U.S. patent No. 2094671, October 1937; Welsh et al. 1970, U.S. patent No. 3518343, June 1970; Weese et al. 1974, U.S. patent No. 3821117, June 1974; Cheng 1976, U.S. patent No. 3936385, February 1976).

As can be seen from the Pref is established above links and references none of the known inventions is not described or even mentioned the possibility of using thymol-containing compositions for treating diseases caused by drug resistant bacteria/microorganisms. And at the same time well aware of the use/consumption of the substance by the person that makes it unnecessary to conduct Toxicological studies.

Thus, there is an urgent need for efficient, cost-effective tool for the treatment of serious and life-threatening bacterial infections, including tuberculosis, which has minimal side effects or no side effects. The present invention can satisfy the above requirement through the use of known natural compounds of plant origin and containing his compositions together with other antibiotics/compounds to kill bacteria, including pathogens of tuberculosis. Special analyses of biological activity helped to identify new, the use of plant compounds "thymol", isolated from oil, which is obtained by distillation from the seeds of plants Trychyspermum ammi (Ajwain), for the destruction of bacteria that are resistant even to commonly used antibiotics of the third generation, and pathogenic microorganisms resistant to multiple Carstensen means. The origin of thymol, prior experience its traditional use and available information on the consumption of the compounds indicate that this substance can be safely used as a herbal antibiotic of the fourth-generation plant-based. Therefore, applicants investigated the possibility of enhancing activity of thymol and developed a new composition, which includes thymol in combination with oils/monoterpene obtained from various species of mint (Mentha arvensis and M. spicata), containing rare combination of carvone, limonene and menthol.

Applicants have discovered new and promising opportunities for the protection of human health and the fight against microbial infections caused by drug resistant pathogens. Unlimited and excessive use of known antibiotics causes of infectious microorganisms that are resistant to one or more drugs. Applicants used the categories of resistance of bacteria to drugs as the basis for their classification depending on the risk to human health, which is much broader than traditional taxonomic classes. The invention, in particular, deals with drug resistant bacteria, fuss the surrounding due to mutations, with subsequent selection due to the constant presence of the used antibiotics. In their experiments, the applicants used the microorganisms Escherichia coli and Mycobacterium as model systems to study the emergence of resistance to quinolone and fluoroquinolone medicines and the emergence of strains resistant to multiple drugs, and found the possibility of a new application of molecules of plants (thymol) for the destruction of strains resistant to multiple drugs, and bacteria resistant to drugs of new generation, which arose under the action of the medicines. This discovery is unique and has great application prospects in the field of medicine.

The purpose of the invention

The main purpose of this invention is a novel composition useful for the treatment of drug resistant bacterial infections.

Another purpose of this invention is a new composition comprising an effective amount of thymol isolated from plants Trychyspermum ammi (Ajwain), a combination of essential oils and monoterpenes isolated from Mentha spicata and Mentha arvensis, which is useful for the treatment of bacterial infections.

Another objective of the present invention are methods of obtaining a new composition according to the present invention.

Even the ne the purpose of this invention is antibacterial, contains thymol, which is intended for the treatment of bacterial infections.

Another purpose of this invention is the use of thymol for the destruction of drug resistant bacteria.

Summary of invention

The present invention relates to a new composition containing an effective amount of thymol obtained from plants Trychyspermum ammi (Ajwain), an appropriate combination of peppermint oils obtained from Mentha spicata and Mentha arvensis, and conventional additives. This invention relates also to methods of obtaining this new composition intended for the treatment of drug resistant bacterial infections. In addition, this invention relates to antibacterial agent containing an effective amount of thymol, which is intended for the destruction of drug resistant bacteria. This invention relates also to method of application of thymol for the destruction of drug resistant bacteria.

Detailed description of the invention

This invention relates to new synergistic composition useful for the treatment of drug resistant bacterial infections, which contains an effective amount of thymol obtained from plants Trychyspermum ammi, the combination of mint oils, including with the appropriate quantity of certain monoterpenes, isolated from Mentha spicata and Mentha arvensis, and conventional additives.

In one embodiment of the invention thymol is present in an amount of from about 100 to 500 mg or 20 to 50% (wt./wt.).

In another embodiment of the invention the combination of mint oils is present in an amount of from about 0.1 to 0.5 mg.

In another embodiment of the invention the additive is chosen from the group comprising citric acid in an amount of about 2-10 mg, calcium carbonate in an amount of about 100-200 mg, gel magnesium hydroxide in an amount of about 50-100 mg, lactose in an amount of about 200-600 mg, honey in the amount of from about 0.1 to 1%, sodium glutamate in an amount of about 200 mg and sodium buffer.

In another embodiment of the invention mint oil, if necessary, diluted to 10 times.

In another embodiment of the invention peppermint oil contains lemon in the range of from about 6 to 25%, menthol in the range of from about 0.50 to 2.50 per cent., carvon in the range of from about 64,0%to 76%.

In another embodiment of the invention, the honey was diluted to 10 times.

In another embodiment of the invention the composition is effective against a group of bacteria selected from the genus Mycobacterium or Escherichia.

In another embodiment of the invention the composition is effective against bacteria resistant to drug the output means, selected from the group including ethidium bromide, isoniazid, chloramphenicol, tetracycline, rifampicin, nalidixic acid, oxolinic acid, sparfloxacin, ciprofloxacin and lomefloxacin.

New synergistic composition useful for the treatment of drug resistant bacterial infections, contains an effective amount of thymol obtained from plants Trychyspermum ammi, peppermint oil, obtained from the hybrid of Mentha spicata and Mentha arvensis, and conventional additives.

It should be noted that the synergistic composition of this invention has the surprising and unexpected antibacterial properties. Some of the ingredients of this composition does not have the properties of a composition, useful for the treatment of drug resistant bacterial infections. The composition of the present invention containing an effective amount of thymol obtained from plants Trychyspermum ammi, peppermint oil, obtained from the hybrid of Mentha spicata and Mentha arvensis, and conventional additives, has an amazing and unexpected antibacterial properties.

The composition according to the present invention can be obtained in different physical forms, such as tablets, syrup, powders, injections, etc. known in this field. To obtain such compositions in different physical forms, the basic ingredients of the composition, in h the particular connection "thymol" and a combination of essential oils, including the desired mixture of monoterpenes, mixed with the above conventional additives such as honey, monosodium glutamate, citric acid and the like, to improve therapeutic efficiency. It should also be noted that the above amounts of the respective ingredients in the composition are indicated as examples, you can use a different number of required ingredients, which can be easily determined by the person skilled in the art. The ratio between the amounts of the components in the compositions of the present invention is not critical and can vary within wide limits. The best results, of course, will be obtained from the use of thymol and a certain combination of peppermint oils in the above proportions. The optimal number of ingredients in a given composition will vary depending on the method of administration of the composition.

This invention relates also to a method for producing a composition useful for the treatment of drug resistant bacterial infections, which includes a step of mixing an effective amount of oil thymol obtained from plants Trychyspermum ammi, and the combination of peppermint oils obtained from Mentha spicata and Mentha arvensis, with conventional additives.

In one embodiment of the invention thymol is present in an amount of from prima is but 100 to 500 mg or 20 to 50% (wt./wt.).

In another embodiment of the invention mint oil is present in an amount of from about 0.1 to 0.5 mg.

In another embodiment of the invention the additive is chosen from the group comprising citric acid in an amount of about 2-10 mg, calcium carbonate in an amount of about 100-200 mg, gel magnesium hydroxide in an amount of about 50-100 mg, lactose in an amount of about 200-600 mg, honey in the amount of from about 0.1 to 1%, sodium glutamate in an amount of about 200 mg and sodium buffer.

In another embodiment of the invention mint oil, if necessary, diluted to 10 times.

In another embodiment of the invention peppermint oil contains lemon in the range of from about 6.8% to 23.2%, menthol in the range of from about 0.66 to 2,50%, carvon in the range of from about 64,0 to 76,1%.

In another embodiment of the invention honey optional diluted up to 10 times.

In another embodiment of the invention the oil thymol and mint oil is dispersed in 0.1-1% of the honey with the formation of the syrup.

In another embodiment of the invention the oil thymol and mint oil mixed with citric acid in the amount of 2-10 mg and dissolved in a buffer containing MSG, with the formation of the injection solution.

In another embodiment of the invention the composition is effective against the bacteria is' choose from the genus Mycobacterium or Escherichia.

In another embodiment of the invention the composition is effective against bacteria resistant to drugs selected from the group including ethidium bromide, isoniazid, chloramphenicol, tetracycline, rifampicin, nalidixic acid, oxolinic acid, sparfloxacin, ciprofloxacin and lomefloxacin.

This invention relates also to a method of treatment of drug resistant bacterial infections in humans, including the stage of introducing a therapeutically effective amount of a new composition to a subject in need of it.

In one embodiment of the invention the composition is administered orally or subcutaneously.

In another embodiment of the invention the composition is dissolved in 5 ml of 0.05 M solution of sodium buffer (pH 7.0)containing 200 mg of sodium glutamate, and is administered by subcutaneous injection.

In another embodiment of the invention the composition is effective against bacterial infections such as intestinal and systemic infection.

In another embodiment of the invention, the treatment consists of the administration of the composition against bacterial infections caused by bacteria resistant to drugs selected from the group including ethidium bromide isoniazid, chloramphenicol, tetracycline, rifampicin, nalidixic acid, oxolinic acid, sparfloxacin, ciprofloxacin and lomefloxacin.

In one embodiment of the invention the composition is used to treat infections caused by resistant to multiple drugs bacteria selected from the genus Mycobacterium or Escherichia.

In another embodiment of the invention the composition is used for the destruction of bacteria resistant to the group of medicines, including ethidium bromide, isoniazid, chloramphenicol, tetracycline, rifampicin, nalidixic acid, oxolinic acid, sparfloxacin, ciprofloxacin and lomefloxacin.

The present invention is the result of planned experimental work by analysis method for biological assessment of the specific activity. These studies are aimed at identifying and assessing therapeutic potential of plant compounds "thymol", isolated from oil plants "Ajwain", as the antibiotic of a new generation and the creation of herbal antibiotic composition stronger actions, in particular, is able to kill drug resistant bacteria. As a result of the experiments were obtained evidence that strains of Escherichia coli that are resistant to nalidixic acid (hinolan is the first drug of broad-spectrum) due to mutations in the gene arc, more sensitive to the effects of oils extracted from plants Trychyspermum ammi, and its main component "thymol".

The invention is described in detail in the following examples, which should not be construed as limiting in any way the volume or scope of the invention.

Example 1

Antibacterial activity of the oil extracted from plants Trychyspermum ammi, in particular, timelines and nativley fraction of the specified oil is determined on the basis of zones of inhibition of growth produced on a bacterial lawn of multi drug-resistant strains, with analyses by diffusion disks (table 1).

Table 1
The zone of growth inhibition (in mm)as determined by analyses of the diffusion disks
Strains E. ColiNaeli Trychyspermum ammiNetmania fraction oilThymol
 50 mg/ml50 mg/ml10 mg/ml20 mg/ml50 mg/ml
SA4,5-1,02,05,0
DH5α5,0-1,02,05,5
ET5,5-1,03,06,0

In the above table SA means a wild strain of E. coli, while DH5α and ET represent mutations gyr, i.e. the change of the gene encoding subunit A Gyr enzyme called DNA gyrase, which is responsible for relaxation of DNA.

These mutants resistant to quinolone due to altered DNA gyrase. Specific mutations were confirmed by genetic complementation by transferring cloned in plasmid gene gyr A. As shown in the table, the area of destruction of bacteria by thymol much more in the case of mutant strains of arcs that are resistant to the effects of the above drugs. Such activity is not detected in nativley faction considered oil.

Example 2

Sensitivity is resistant to nedivx.avi acid strains of Escherichia coli to various antibiotics, determined by the method poisonous agar

Independent mutants resistant to nalidixic acid produced in E. coli by induced and spontaneous mutagenesis in wild strain SA. The resulting mutants are tested for sensitivity not only to nalidixic acid, but also to other quinolone and fluoroquinolone medicines last generations, as resistance to drugs is authorized means of the second and third generations also occurs due to one or more mutations only in the gene arc. In the following table 2 shows cross-resistance of these mutants.

Table 2
Cross-resistance mutants NalRE. coli
Mutant

(NalR)

strains

E. coli
Growth in the poisonous environment of agar containing
 Nalidixic acidOxolinic acidSparfloxacinCiprofloxacinLomefloxacin
 50 mg/ml5 mcg/ml5 mcg/ml5 mcg/ml5 mcg/ml
CAN 101÷-÷--
CAN 102÷-÷--
CAN 103÷-÷--
CAN 104÷-÷--
CAN 105÷-÷--
CAN 106÷÷÷ --
CAN 107÷÷÷--
CAN 108÷÷÷--
CAN 109÷÷÷--
CAN 110÷÷÷--
CAN 111÷÷÷--
CAN 112÷-÷--
CAN 113÷÷÷--
CAN 114÷÷÷--
CAN 115÷÷÷--
CAN 116÷÷÷--
CAN 117÷÷÷--
CAN 118÷÷ --
CAN 119÷-÷--
CAN 120÷-÷--
CAN 121÷÷÷--
CAN 122÷÷÷--
CAN 123÷÷÷--
CAN 124÷÷÷- 
CAN 125÷÷÷--
CAN 126÷÷÷--
CA 8000 (WT)-----
÷ = the Presence of growth; - = No growth; WT = wild strain

Resistant to nalidixic acid strains grow in the presence of different antibiotics, are presented in the table above. These mutants are characterized by positive growth in the presence of the tvii high concentrations of nalidixic acid. Some of these mutants resistant to oksolinovoj acid, all strains were resistant to sparfloxacin, but none of the strains are not resistant to ciprofloxacin, drugs of the second generation, and lomefloxacin, drugs of the third generation. As expected, the control wild strain SO sensitive to all antibiotics.

Example 3

Biological evaluation of thymol on the activity against mutants NlRE. coli

The above specimens are tested for sensitivity to thymol to further confirm the hypothesis of a more effective destruction of thymol mutants NalRE. coli, which indicates the possibility of its use as a herbal antibiotic for the destruction of drug resistant bacteria. When performing these analyses thymol shows high activity against all mutants NalRresistant to one or more drugs (table 3).

CAN 118
Table 3
Analysis of the diffusion disks for determining the activity of thymol against resistant to nalidixic acid mutants of Escherichia coli
Mutant (NalRstrains E. coliThymol (zone deceleration is foster growth in mm)
 50 µg/disc100 µg/disk250 µg/disc
CAN 101Tg1,54,0
CAN 102Tg2,07,0
CAN 103Tg2,04,0
CAN 104Tg2,06,0
CAN 105Tg2,05,0
CAN 10612,04,0
CAN 10713,05,0
CAN 108Tg2,05,0
CAN 10913,06,0
CAN 11013,06,0
CAN 11113,05,0
CAN 11213,05,0
CAN 113Tg2,05,0
CAN 11413,06,0
CAN 11513,05,0
CAN 11613,05,0
CAN 11713,05,0
13,05,0
CAN 11913,06,0
CAN 12013,06,0
CAN 12113,05,0
CAN 122Tg2,05,0
CAN 12313,05,0
CAN 12413,05,0
CAN 12513,05,0
CAN 126125,0
CA 8000 (WT)124
Tg - Tracking activity (<0.5 mm); WT = wild strain

Connection "thymol" really capable of destroying all resistant to nalidixic acid mutants of E. coli with higher efficiency compared with the wild strain SA.

Example 4

Selection and study of mutants of E. coli that are resistant to lomefloxacin (fluoroquinolone drug third generation)

Induced mutants obtained after selection in the presence of lomefloxacin in a nutrient medium. The growth of resistant lomefloxacin strains of Escherichia coli are examined in the presence of various antibiotics by the method of the poison is twisted agar to determine the cross-resistance. As expected, resistant to lomefloxacin (medicine third generation) strains resistant to antibiotics of the first and second generations (table 4).

Table 4
The nature of the sensitivity of resistant lomefloxacin cells E. coli
Mutant (LomR) strains

E. coli
Growth in the poisonous environment of agar containing
 LomefloxacinNalidixic acidOxolinic acidOhlocationCiprofloxacinSparfloxacin
 1.5 mcg/m50 mg/ml5 mcg/ml20 mg/ml2 mcg/ml20 mg/ml
CAL 101÷÷÷÷-÷
CAL 102÷÷÷÷-÷
CAL 103÷÷÷÷-÷
CAL 104÷÷ ÷÷-÷
CAL 105÷÷÷÷-÷
CAL 106÷÷÷÷-÷
CAL 107÷÷÷÷-÷
CAL 108÷÷÷÷-÷
SA 8000(WT)------
÷ = the Presence of growth; - = No growth; WT = wild strain

Example 5

Biological evaluation of thymol on the activity against mutants LomRE. coli

Mutants resistant to lomefloxacin, tested the sensitivity thymol by analyzing the diffusion disks. As can be seen from table 5, thymol effectively destroy all mutant cells LomREscherichia coli, which indicates the possibility of its direct use as a drug of a new generation against drug resistant bacteria.

Table 5
Analysis of the diffusion disks for determining the activity of thymol against resistant lomefloxacin mutants of Escherichia coli
Mutant (LomrR) strains of E. coliThymol (zones of inhibition of growth in mm)
 50 µg/disc100 µg/disk
CAL 1012,04,0
CAL 1023,05,0
CAL 1032,03,0
CAL 1041,01,5
CAL 1052,04,0
CAL 1062,04,0
CAL 1072,04,0
CAL 1083,05,0
SA3,05,0
Tg = Tracking activity (<0.5 mm); WT = wild strain

Example 6

Evaluation of thymol on the activity against resistant to nalidixic acid mutants of Mycobacterium smegmatis

After receiving encouraging results of the effectiveness of thymol as an antibacterial agent against different concentrations and types of drug resistant mutants of E. coli, the authors of the present invention execute the experiments using Mycobacterium. Were used bacteria M. smegmatis, representing the fastest-growing model system designed for the study of drugs against tuberculosis. In a wild strain MS2was selected series of multi drug-resistant mutants. Resistant to nalidixic acid, the first strains were tested for sensitivity to thymol. Resistant to nalidixic acid mutants obtained by growing the wild strain of Mycobacterium smegmatis in a medium containing 50 μg/ml nalidixic acid. As can be seen from table 6, thymol can effectively destroy the strains NalRMycobacterium smegmatis, which indicates its usefulness as an effective antibacterial drugs against mycobacteria.

Example 7

Biological evaluation of thymol on the activity against mutants LomRMycobacterium smegmatis

Resistant lomefloxacin mutants get growing wild strain of Mycobacterium smegmatis in a medium containing 20 μg/ml lomefloxacin (third generation antibiotic belonging to the class of fluoroquinolones). These mutants resistant to lomefloxacin to a concentration of 60 µg/ml it is Important to note that the degree of stability achieved in mycobacterial cells as a result of mutations is very high compared to E. coli, therefore, medicinal cf is DSTV, capable of destroying such mutants LomRcritical and valuable. Very interesting is the fact that these mutants are resistant to the effects of second generation antibiotics, such as ciprofloxacin. These mutants are tested for sensitivity to thymol, the connection according to this invention, which, as can be seen from table 1, it effectively destroys resistant to several drugs on cells of Mycobacterium smegmatis, which confirms the use of thymol as a herbal antibiotic forth a new generation.

Table 7
Analysis of the diffusion disks for determining the activity of thymol against resistant lomefloxacin mutants of Mycobacterium smegmatis
Mutant (LomR) strains of Mycobacterium smegmatisLomefloxacinThymol (zones of inhibition of growth in mm)
 200 μg/disk100 µg/disk150 µg/disk
MSL 101-13
MSL 102-13
MSL 103-13
MSL 104-1 3
MSL 105-13
MSL 106-Tr3
MSL 107-13
MSL 108-13
MSL 109-12,5
MSL 110-13
MSL 111-13
MSL 112-13
MC (WT)-13
Tr = Trace activity (<0.5 mm); - = No activity; WT = wild strain

Example 8

Activity of thymol against bacterial mutants resistant to isoniazid, another new anti-TB drug

Table 8
Sensitivity to thymol isoniazid-resistant mutant of Escherichia coli cells
Strains E. coliThymol (zones of inhibition of growth in mm) 100 µg/disk
SA6
SO2
SO (revertant)8

Isoniazid is another drug widely used to treat tuberculosis because the bacteria are sensitive to the effects of this drug due to the absence of oxyR system. A wild strain of E. coli resistant to concentrations of this antibiotic (1000 µg/ml). The specified strain is subjected to mutagenesis using N’,N-methylnitrosourea order to obtain sensitive strain, which is amenable to destruction at a concentration of 250 µg/ml in broth (SAO). In addition, sensitive mutant culture detected spontaneous mutation that is resistant to isoniazid at a concentration of 1000 μg/ml of These cultures are tested for sensitivity to thymol at a concentration of 100 µg/disc, performing analysis of the diffusion disks, the results of which show that the most sensitive thymol is resistant to isoniazid reverently strain (SAO revertant).

Example 9

Activity of thymol against mutants resistant to ethidium bromide (resistant to multiple drugs)

Table 9
Strains of Mycobacterium amegmetisThymol (100 µg/disc)
Wild strain8 mm
A strain that is resistant to ethidium bromide11 mm

Mutant Mycobacterum smegmetis obtained by sequential enrichment normally sensitive wild strains to achieve a concentration of ethidium bromide in broth, equal to 12 µg/ml of This mutant grows well at the indicated concentrations of ethidium bromide, while the wild strain is destroyed when the concentration of ethidium bromide in the broth, equal to 3 mg/ml in Addition, it was found that this resistant to ethidium bromide strain is also resistant to several other drugs (mdr), in particular to antibiotics such as chloramphenicol (20 μg/ml), tetracycline (10 µg/ml) and rifampicin (40 µg/ml).

Example 10

Activity of thymol, the compounds of the present invention, at different pH values in respect of Escherichia coli SA

Table 10
ConcentrationpH (zone of growth inhibition in mm)
 4,007,009,001M NaOH
12.5 µg/disk----
25 µg/disctraces1,00--
50 µg/disc2,002,001,001,00
100 µg/disk4,006,003,002,00

From the above table it follows that the activity of the compounds according to the present invented the Yu is maximum at the neutral pH (7,00).

Example 11

Getting remasterizada synergistic antibacterial composition against resistant to multiple drugs bacteria

On the basis of the performed investigations showed that thymol is a potent bactericidal agent against resistant to several drugs of bacteria. In addition, it was found that the effect of this tool is enhanced when combining oils isolated from different species of mint Mentha arvensis and Mentha spicata. This combination of oils contains lemon (6,8-23,2%), menthol (0,66-2,45%), carvon (64,0 is 76.1%) and unidentified fraction in essential oils complement to 100% at different stages of growth. When using this combination of oils thymol in a concentration of 0.1% antibacterial activity of thymol increased by 45%. In addition, the specified oil composition according pleasant aroma of carvone with a hint of menthol. Taking into account the above obtained different compositions, are described below.

Table 11
Analysis of the diffusion disks to determine the synergistic effects of thymol with nalidixic acid against cells of E. coli
The concentration of nalidixic acid (µg/disc)The zone of inhibition of growth in mm) of the disk, containingThe concentration of thymol (µg/disc)
 Nalidixic acidCombination (nalidixic acid + thymol)Thymol 
0,2534Tr12,5
0,50612225,0
1,00910350,0
1,501110575,0
2,0012106100,0
Tr = Trace activity (<0.5 mm); WT = wild strain

Table 12
Analysis of the diffusion disks to determine the synergistic effects of thymol oil hybrids of Mentha against cells of E. Coli
Oil hybrids of Mentha (µg/disc)The zone of growth inhibition (in mm) of the disk containing theThe concentration of thymol (µg/disc)
 Oil hybrids of MenthaThe combination of oil + thymol)Thymol 
5001,0 3,050
505,03,050
0,502,53,050
Tr = Trace activity (<0.5 mm); WT = wild strain

Composition 1

1. Thymol 100-500 mg (20-50%, masmas.)

2. Essential oil of the hybrid 0.1-0.5 mg

3. Citric acid 2-10 mg

4. Calcium carbonate 100-200 mg

5. Gel magnesium hydroxide 50-100 mg

6. Lactose 200-600 mg

7. Total weight of 500-1000 mg

The ingredients are thoroughly mixed, ground into powder and Packed into gelatin capsules, which can be purchased commercially.

Composition 2

Essential oil diluted in 0-10 times.

1. Thymol 50-300 mg (10-60%) in the combination of essential oils.

This drug is impregnated with 250 mg bulbs lactose.

Composition 3

Honey diluted in 0-10 times with sterile distilled water, and add the following ingredients:

1. Thymol 50-300 mg (10-60%)

2. The combination of essential oils from different species of mint (0,1-1,0%)

Composition 4

1. Thymol 100-500 mg

2. Citric acid 2-10 mg

These two component dissolved in 5 ml of 0.05 M solution of sodium buffer (pH 7.0)containing 200 mg of sodium glutamate.

1. Composition intended for the treatment of drug resistant bacterial infections, the content is based on thymol, obtained from the plant Trachyspermum ammi, in the amount of 100-500 mg and peppermint oil in an amount of 0.1-0.5 mg, essentially containing lemon in the range of about 6 to 25%, menthol in the range of about 0.50 to 2.50 per cent., carvon in the range of about 64 to 76%, which is obtained from a hybrid of Mentha spicata and Mentha arvensis, and conventional additives.

2. The composition according to claim 1, in which the additive is chosen from the group comprising citric acid in an amount of about 2-10 mg, calcium carbonate in an amount of about 100-200 mg, gel magnesium hydroxide in an amount of about 50-100 mg, lactose in an amount of about 200-600 mg, honey in the amount of from about 0.1 to 1%, sodium glutamate in an amount of about 200 mg and sodium buffer.

3. The composition according to claim 1, in which peppermint oil, if necessary, diluted to 10 times.

4. The composition according to claim 2, in which the honey is optional diluted up to 10 times.

5. A method of obtaining a composition according to claim 1, intended for the treatment of drug resistant bacterial infections, involving the mixing thymol obtained from the plant Trachyspermum ammi, in the amount of 100-500 mg peppermint oil in an amount of 0.1-0.5 mg, essentially containing lemon in the range of about 6 to 25%, menthol in the range of about 0.50 to 2.50 per cent. and carvon in the range of about 64 to 76%, which is obtained from a hybrid of Mentha spicata and Mentha arvensis, and conventional additives.

6. The method according to claim 5, in which the additive is chosen from the group VK is causa citric acid in an amount of about 2-10 mg, calcium carbonate in an amount of about 100-200 mg, gel magnesium hydroxide in an amount of about 50-100 mg, lactose in an amount of about 200-600 mg, honey in the amount of about 0.1 - 1%, sodium glutamate in an amount of about 200 mg and sodium buffer.

7. The method according to claim 5, in which peppermint oil diluted up to 10 times.

8. The method according to claim 6, in which the honey is optional diluted up to 10 times.

9. The method according to claim 5, in which thymol and mint oil is dispersed in 0.1-1% of honey with getting the syrup.

10. The method according to claim 5, in which thymol and mint oil mixed with citric acid in the amount of 2-10 mg and dissolved in a buffer containing MSG for a solution for injection.

11. The method according to claim 5, in which the specified composition is effective against a group of bacteria selected from the genus Mycobacterium or Escherichia.

12. The method according to claim 5, in which the specified composition is effective against bacteria resistant to drugs selected from the group including ethidium bromide, isoniazid, chloramphenicol, tetracycline, rifampicin, nalidixic acid, oxolinic acid, sparfloxacin, ciprofloxacin and lomefloxacin.

13. A method for the treatment of drug resistant bacterial infections, involving the administration to a subject in need, a therapeutically effective amount of a composition according to claim 1.

14. The way p is item 13, in which the composition according to claim 1 is administered orally or by subcutaneous method.

15. The method according to item 13, in which the above composition is dissolved in 5 ml of 0.05 M solution of sodium buffer (pH 7.0)containing 200 mg of sodium glutamate, for insertion into subcutaneous injection.

16. The method according to item 13, in which the specified composition is effective against bacterial infections such as intestinal and systemic infection.

17. The method according to item 13, in which the treatment consists of the administration of the composition against bacterial infections caused by bacteria resistant to drugs selected from the group including ethidium bromide, isoniazid, chloramphenicol, tetracycline, rifampicin, nalidixic acid, oxolinic acid, sparfloxacin, ciprofloxacin and lomefloxacin.

18. The method according to item 13, which is resistant to many drugs bacteria belong to the genus Mycobacterium or Escherichia.

19. The composition according to claim 1, designed to destroy resistant to several drugs of bacteria selected from bacteria, resistant to ethidium bromide, isoniazid, chloramphenicol, tetracycline, rifampicin, nalidixic acid, oksolinovoj acid, sparfloxacin, ciprofloxacin or lomefloxacin, and thus for the effective treatment of bacterial infections in the introduction of an appropriate dose.



 

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