WO2001072317A1

WO2001072317A1 - Formulation comprising thymol useful in the treatment of drug resistant bacterial infections

Info

Publication number: WO2001072317A1
Application number: PCT/IN2000/000033
Authority: WO
Inventors: Suman Preet Singh Khanuja; Suchi Srivastava; Ajit Kumar Shasney; Mahendra Pandurang Darokar; Tiruppadiripuliyur Santha Kumar; Krishna Kumar Agarwal; Atique Ahmad; Nirmal Kumar Patra; Prachi Sinha; Sunita Dhawan; Dharmendra Saikia; Sushil Kumar
Original assignee: Council Of Scientific And Industrial Research
Priority date: 2000-03-28
Filing date: 2000-03-28
Publication date: 2001-10-04
Also published as: US6514541B2; AU2000255628A1; US20020090380A1

Abstract

Novel formulation useful in the treatment of drug resistant bacterial infections comprising an effective amount of 'Thymol' obtained from the plant Trychyspermum ammi, combination of mint oil containing appropriate amounts of monoterpene obtained from Mentha spicata and Mentha arvensis and obtaining conventional additives, a method for producing the formulation by mixing the above ingredients and a method for the treatment of drug resistant in a patient by administration of a therapeutically effective amount of the said formulation.

Description

FORMULATION COMPRISING THYMOL USEFUL IN THE TREATMENT OF DRUG RESISTANT BACTERIAL INFECTIONS

FIELD OF THE INVENTION

The present invention relates to a novel synergistic composition useful in the treatment of drug resistant bacterial infections, said composition comprises an effective amount of thymol, a mixture of the essential oils of Mentha arvensis and Mentha spicata or their monoterpene components m appropnate ratio and conventional additives The said composition is useful in the treatment of drug resistant enteric and systemic infections The formulation with enhanced activity of thymol action compπses thymol in combmation with the said oil which is a combination containing the rare mixture of carvone, hmonene and menthol The invention also provides methods of producing the said composition and a method of using thymol obtained from the seeds of the plant Trychyspermum ammi (Ajwam) as a fourth generation antibiotic formulation for control of drug resistant bacteπa More particularly, the invention relates to the use of a compound 'Thymol' isolated from the oil distilled from the seeds of the plant Trychyspermum ammi (Aj warn) to kill the bacteπa resistant to even prevalent third generation antibiotics and multi-drug resistant (mdr) microbial pathogens and thus useful as a plant based fourth generation herbal antibiotic formulation

BACKGROUND OF THE INVENTION

Microbial infections are a major cause of human health hazards and misery leading to sizeable number of human deaths globally In addition, the infections drastically affect human efficiency by incapacitating various metabolic functions and systems like digestive, respiratory, uπnary, circulatory, nervous systems and skm This then leads to continuous human - suffering till the patient is completel cured of the causative microbes Bacterial infections present a seπous threat to the health and w ell being of people of all ages Antibiotics and antimicrobial dmgs ever since the discovery of Penicillin by Alexander Fleming m the 1940s have been used by the medical practitioners to eliminate infective agents and curing the diseases However, the infective microbes have always been able to fight back ev ery new drug through development of resistance against the dnig/antibiotic m use Emergence of multiple drug resistant strains has appeared as a real problem in the field of medical science The primary cause of the development of resistance is occurrence of random mutations Mutations may occur in genes responsible for conferring sensitivity against a drug With a relative dearth of new antibiotics with novel mode of action we may find ourselves on the verge of a medical disaster It is high time to revive the hidden wonders of plant molecules with the modern tools of target based screening to develop newer advanced generation dmgs and antibiotics with novel modes of actions

Newer antimicrobials have been arising through structural modifications of existing agents leading to the development of higher generation drugs with wider spectrum of activity and enhanced potency Well-exemplified cases for evolution of higher generation families of antimicrobial agents are penicillins and cephalospoπns These are evolved by the chemical modification of the basic β-lactam ring Cephalosporm represents great-grand daughter-drug of cephalothm Similarly, since the development of nalidixic acid as the first generation antimicrobial quinolone drug m 1962 by Lesher and colleagues. members of quinolone family have also evolved upto third generation In fact, nalidixic acid found limited utility in treating systemic infections and subsequently marginally improved qumolones like oxolimc acid, pipemidic acid and cinoxacin were released m 1970^"s Then came a breakthrough in early 1980^'s with the beginning of evolution of fluoπnated qumolones First to come up was norfloxacin. a second-generation quinolone having 6-fluoπne and ^"-piperazme substituents developed by Wolfson and Hooper It had enhanced activity against both gram-negative and gram-positive bacteria like Pseudomonas aeruginosa and Staphylococcus aiireus. respectively This followed the development of sister drug molecules like ciprofloxacm. 10 enoxacm, ofloxacm and pefloxacm etc Almost concurrent has been the development of third generation agents such as lomefioxacin, fleroxacm, temafloxacm, tosufloxacin possessing one or more additional fluorine substituents as compared to second generation qumolones having a fluorine on posιtιon-6 on the basic qumolones

I i

Continuous and indiscriminate use of these quinolone floroqumolone drugs is resulted into gradual msensitivity of the bacteπa against whom these are used and thereby the pathogens requires more and more amount of these compounds as doses Being synthetic, the use of these dmgs results into 0 forced side effects Further, the pathogens are becoming resistant to even higher doses of these compounds to make the matter worse To counter this problem, the applicants have developed a novel method of using the known plant based compound thymol in isolation to kill the drug resistant bacteria and also in combination with other available antibiotics to check the 5 resistance development m the bacteria Considering the deadliest bacteπal infections, tuberculosis is world^'s leading killer claiming more than three million lives world-wide every year. More appalling is the increased incidence of this disease and moπahty among HIV positive individuals The resurgence of tuberculosis and its incidence in human immuno-defϊciency vims-positive populations in both developing and industrialized countries has focussed attention on the urgent need for development of new advanced generation drugs.

Current scenario of global awareness repeatedly flashes the ineffectiveness of the normally used antibiotics used for the cure of diseases like Tuberculosis.

These problems are now assuming epidemic proportion even in the developing countries. New drug combinations using active molecules from natural sources like plants need to be systematically explored failing which the consequences are destined to be devastating and out of control for the human race in the new millennium. Among the most promising advances in the field of drug development is discovering new molecules or novel uses of the already available compounds with known safety and without any side effects Such active biomolecules combined with other antibiotics can kill the dπig resistant bacteria and simultaneously check further development of resistance in the infectious microbes.

Use of thymol in various herbal preparations ranging from mouthwashes and enteric disorders to skin infections is common. The parent oil and thymol itself as component of grandma^'s household recipes to treat a range of common ailments has found resident place because of being equally effective for children and adults These uses have been exploited m the Indian subcontinent and also across the continents through formal and informal dissemination of traditional and herbal medicine knowledge

Some of the related or prior art that need to be specifically cited here for ^ establishing the uniqueness of our invention include the following different uses

1 Composition for the treatment of viral infections including HIV (Pruthi et al. 1999. US Patent = 5,980.903. November 9. 1999) and its related arts lo (Badaway 1998. US Patent = 5,801 ,153, September, 1998. Rohatgi 1996.

US Patent « 5.529,778. June. 1996. Hozumi et al. 1995, US Patent *? 5,41 1.733. May. 1995)

2 Analgesic composition useful m providing a temporary relief from symptoms of arthritis (Beck 1991. US Patent 5,073.366, December 17, ι 1991 ) and its related arts (Arbir et al. 1981, US Patent 4.30M09.

December. 1981. Dubash et al. 1983, US Patent ? 4,383.986. May. 1983.

Seth 1985, US Patent 4,540.5^"2, September. 1985. Geria 1987. US

Patent = 4,702.916. October, ^δ^'7. Grohe 1989, US Patent 4,844.902.

July, 1989, Bisset et al. 1989. US Patent = 4,847.071 , July, 1989, o Deckner et al. 1989. US Patent - 4.863,725. July, 1989)

3 Powder composition for forming a mouthwash (Smigel et al. 1999. US Patent - 4,925.655, Mat 15, 1990) and its related arts (Fleischman 1936. US Patent ~ 2,035.267, March. 1936. Poetschke 1937, US Patent = 2,094,6~ 1. October, 1937. Welsh et al. 1970. US Patent - 3.518.343. 5 June, 1970, Breece et al. 1974. US Patent = 3,821.1 17, June, 1974,

Cheng 1976. US Patent ≠ 3,936,385. February, 1976) As evident from the cited references and literature search, none of the known inventions have mentioned or descπbed the use of thymol and its compositions for treatment against drag resistant bacteria/microbes But at the same time, its use, consumption by human sy stem is well established and hence requiring no toxicological testing

Accordingly , there is an urgent need for an effective, less expensiv e means of treating serious and life endangering bacteπal infections, including TB. with minimal or nil side effects The present invention addresses this compelling need and provides a known herbal ingredient natural compound and its composition with other antibiotics/compounds for controlling bacterial agents, including Tuberculosis Specific target based biological activity assays could define the novel use of a plant compound "Thymol^" isolated from the oil distilled from the seeds of the plant Tn chyspermiim ammi ( Ajwain) to kill the bacteria resistant to even prevalent third generation antibiotics and multi-drug resistant (mdr) microbial pathogens Thymol^'s nature and previous traditional use as well as the available information on human consumption indicates that it can be safely used as a plant based fourth generation herbal antibiotic preparation Accordingly, the applicants have worked on the enhancement of activity of thymol action and developed a novel formulation comprising thymol m combination with the appropriate combination of oils/inonoterpenes from mints (Mentha arvensis and M spicata) containing the rare combination of carvone, hmonene and menthol The applicants have found entirely novel and far-reaching applications for overall aid to the human health care and fighting the microbial infections by drug resistant pathogens The indiscriminate and excessive use of the common av ailable antibiotics has been continuously leading to emergence of single and multiple drag resistance! s) in infectious agents The applicants have taken the classes of drag resιstance(s) in bacteria as the approach to classify them into categories of endangerment to human health, which is over and above the traditional taxonomic classes The invention deals specifically with the drag resistant bacteria arising due to mutational events followed by selection due to continued presence of the said antibiotics In the experiments, the applicants have used Escherichia coli and Mycobacterium as the model systems to monitor the evolution of resistance to quinolone and flouroquinolone drags, mdr strain emergence and found out the novel use of the plant molecule (thymol) to kill the mdr strains and advanced generation drug resistant bacteria developed in continued presence of these drags This way this is a unique finding with great utility m the field of medicine

OBJECTS

The mam object of the invention is to provide a novel formulation useful in the treatment of drag resistant bacterial infections

Another object of the invention is to provide a novel formulation comprising an effective amount of Thymol derived from the plant Tn chx spermum ammi (Ajwam) and essential oil or monoterpene combination derived from Mentha spicata and Mentha arvensis useful m the treatment of bacterial infections

Yet another is to provide methods for the preparation of the novel formulation of the invention

Another object of the invention is to provide an anti-bacterial agent comprising Thymol, useful in the treatment of bacterial infections

Still another object is to provide method of using Thymol for control of drag resistant bacteria

SUMMARY OF THE INVENTION

The present invention provides a novel formulation comprising an effective amount of Thymol obtained from the plant Trychyspermum ammi (Ajwam), appropriate mint oil combination obtained from Mentha spicata and Mentha arvensis. and conventional additives The mvention also provides methods for the preparation of the said novel formulation useful in the treatment of drag resistant bacterial infections Further, the mvention provides an anti-bacteπal agent comprising an effective amount of Thymol, useful in controlling drag resistant bacteria The invention also provides a method of using Thymol for control of drag resistant bacteria

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel synergistic formulation useful m the treatment of drag resistant bacterial infections, said formulation comprising an effective amount ^'Thymol^' obtained from the plant Tiychy spermum ammi, mint oil combination containing appropπate amounts of specific monoterpene obtained from Mentha spicata and Mentha arvensis and conventional additives In an embodiment Thymol is present m the range of about 100 to 500 mg or

20 to 50% w, w

In yet another embodiment, mint oil combination is presented an amount of about 0 1 to 0 5 mg.

In still another embodiment, the additives are selected from the group comprising Citric acid present m the range of about 2-10 mg. Calcium carbonate present in the range of about 100 -200 mg, Magnesium hydroxide gel present in the range of about 50-100 mg, Lactose present in the range of about 200-600 mg, honey in the range of about 0.1 to 1%. sodium glutamate present in the range of about 200mg and sodium buffer. In another embodiment, mint oil is diluted upto 10 trmes if desired.

In still another embodiment, the mint oil comprises Limonene tn the range of about 6 to 25%, Menthol in the range of about 0 50 to 2.50% Carvone m the range of about 64 0 to 76%

Yet another embodiment, comprises dilution of honey upto 10 times. In still another embodiment, the formulation is effective against the group of bacteria selected from the genus Mycobacterinm or Escherchia.

In yet another embodiment, the formulation is effective against bacteπa resistant to arugs selected from the group comprising Ethidium bromide. Isoniazid. Chloremphemcol.

Tetracvc ne. Rifampicin. Xalidixic acid. Oxolimc acid, Spartloxicin. Ciprofloxicin and Loamtloxicin 1 ⁾

A novel sv nergistic formulation useful m the treatment of drag resistant bacteπal directions, said foπnulaπon compπsing an effectiv e amount ^'Thymol^' obtained from the plant

ammi. mint oil obtained from a hv bπd of Mentha spicata and Mentha arvensis and conventional additives

It would be pertinent to note that the synergistic formulation ox the invention exhibited surprising and unexpected anti-bacterial propeπies The individual ingredients of the formulation do not have the propeπy of the composition useful in the treatment of dmg resistant bacteπal infections The present formulation comprising an effective amount -"Thymol^" obtained from the plant Trychyspermum ammi. mint oil obtained from a hybπd oϊ Mentha spicata and Mentha arvensis and conventional additives exhibited surprising and unexpected anti-bacterial properties

The formulation of the present invention may be derived in various physical forms such as tablets, syrup, powders, injections, etc as are known in the an In order to prepare such formulations in vaπous physical forms, the essential ingredients of the formulation namely the compound Thymol and the essential oil combination having the desired monoterpene combination are mixed with conventional additives such as honey, sodium glutamate. citric acid and the like as mentioned above to increase clinical efficacy It is also pertinent to note that the amounts of the respective ingredients of the formulation herein mentioned are only exemplary and appropriate amount of the respectiv e ingredients will vary and may be readily determined by a person skilled in the art The ratio o the amounts in the formulation of the present mv ention are not critical and vary widely The best results would of course be obtained employing Thymol and specific mint oil combination in the proportion aforementioned Optimal amounts of the ingredients of the formulation will vary with the method of administration of the formulation The invention also provides a method for the preparation of a formulation useful in the treatment of dπig resistant bacterial infections, said method comprising the step of mixing an effective amount of Thymol oil obtained from the plant Tnchyspermiim ammi, combination of mint oils obtained from vtentha spicata and Mentha arvensis with conventional additives In an embodiment. Thymol is present in the range of about 100 to 500 mg or 20 to 50% w/w

In another embodiment, mint oil is presented an amount of about 0 1 to 0 5 g

In still another embodiment, the additives are selected from the group comprising Citric acid present in the range of about 2-10 mg, Calcium carbonate present m the range of about 100 -200 mg. Magnesium hydroxide gel present in the range of about 50-100 mg, Lactose present in the range of about 200-600 mg, honey m the range of about 0 1 to 1%. sodium glutamate present in the range of about 200mg and sodium buffer In a further embodiment, mint oil is diluted upto 10 times if desired In yet another embodiment, the mint oil comprises Limonene m the range of about 6 8 to 23 2%. Menthol in the range of about 0 66 to 2 50% Carvone m the range of about 64 0 to 76 1%

In still another embodiment, honey is optionally diluted upto 10 times In a further embodiment, the Thymol oil and the mint oil are dispersed m 0 1 to 1% honey to obtain a syrup In another embodiment. Thymol oil and the mint oil are mixed with Citric acid 2 to lOmg and dissolved m a buffer containing sodium glutamate to obtain an injection

In another embodiment, the formulation is effectiv e against the group of bacteria selected from the genus Mycobactenum or scherchia

In yet another embodiment, the formulation is effective against bacteria resistant to drags selected from the group comprising Ethidium bromide.

Isomazid. Chloremphemcol. Tetracyclme, Rifampicm, Nahdixic acid.

Oxolmic acid, Sparfloxicm, Ciprofloxicm and Loamfloxicin Further, the invention also provides a method for the treatment of drag resistant bacterial infections in humans comprising the steps of administration of a therapeutically effective amount of the novel formulation to a subject in need thereof In an embodiment. the formulation is administered through oral or subcutaneous routes

In still another embodiment, the formulation is dissolved 5 ml 0 05 M Sodium buffer (pH 7 0) containing 200mg sodium glutamate to be applied as subcutaneous injection In a further embodiment, the formulation is effective against bacterial infections such as enteric and systemic infections

In yet another embodiment, the treatment comprises administration of the formulation for bacterial infections caused by bacteria resistant to drags selected from the group comprising Ethidium bromide. Isomazid. Chloremphemcol. Tetracyclme. Rifampicm. Nahdixic acid. Oxolmic acid. Sparfloxicm. Ciprofloxicm and Loamfloxicin In an embodiment, the formulation is used for treatment of infections caused by multi-drug resistant bacteπa selected from the genus Mxtobacienum or

Escherchia

In still another embodiment, the formulation is used to kill bacteπa resistant to the group of drugs comprising Ethidium bromide, Isomazid.

Chloremphemcol, Tetracyclme. Rifampicm. Nahdixic acid. Oxolmic acid.

Sparfloxicm, Ciprofloxicm and Loamfloxicin

The present invention is the consequence of planned experimentation through specific activity bioevaluation assays The mtent of the investigation has been to ascertain and evaluate the potential of plant compound thymol from the oil of ^'Ajwam^" as the advanced generation antibiotic and development of a herbal antibiotic formulation with enhanced activity particularly the activity of killing drag resistant bacteπa The expeπments progressed in finding this novel use following the first observation by us that the strains of Escherichia coli that had become resistant to nahdixic acid fa broad spectram quinolone drag) due to mutations m the gyrΛ gene rather more sensitive to the Trychyspermum ammi oil and its major component 'thymol'

The mvention is described in detail by the following examples which should not be constraed as limitations on the scope or sphere of the invention in any manner

Example 1 Antibacterial activity of the Tnchyspermiim ammi oil. thymol and non thvmol fraction of the oil was determined m terms of growth inhibition zones produced on the bacterial lawn of drug resistant strains using disc diffusion assays (Table 1 ).

Table 1 Zone of inhibition ( in mm) determined bv disc diffusion assavs

E. coli Trychysperm Non -thymol Thymol strains um ammi oil oil fraction

50 mg / ml 50 mg/ml lOmg'm 20mg/ml 50mg/ml

1

CA8000 4.5 - 1 0 2 0 5 0

DH5α 5 0 - 1 0 2 0 5 5

ET8000 5 5 - 1 0 3 0 6 0

Here, CA 8000 is the wild type strain of E. coli while DH5α and ET 8000 harbor gyr mutations i.e. the gene encoding for the Gyr A subunit of DNA gyrase enzyme responsible for DNA relaxation is modified. These mutants are resistant to the quinolone because of the altered DNA gyrase. The specific mutations were confirmed through genetic complementation by transferring the plasmid cloned gyr A. As indicated in the table the killing zone of thymol is more in case of gyr mutant strains which are resistant to the above mentioned drags. The activity was not observed in the non-thymol fraction of the oil.

Example 2

Sensitivity of Nalidixic acid resistant strains of Escherichia coli to different antibiotics by poison agar method.

Independent mutants resistant to nalidixic acid were isolated in E. coli through induced and spontaneous mutagenesis in the wild type strain CA

8000. These were then tested for the sensitivity pattern not only against nalidixic acid but also the other quinolone and fluoroqumolone drags of advanced generations since the resistance against the second and third generation drags also arises due to the mutatιon( s ) in the gyrA gene only Table 2 below shows the pattern of cross-resistance pattern of such mutants Table 2 Cross-resistance pattern of Nal^R mutants of E coli

Vlutdnt ( NaD Growth in poison agar medium containing strains of C -.« / Nalidixic CKohnic Sparfloxacm Ciprofloxacin Lomeflovicin

^0 .ΞJ ml i-vml 5μg/ml 5u2 ml 5 Lie/ml

C AN 101 - - * - -

C AN 102 - - - - -

C AN 103 - - - - -

CAN 104 - - ^j- - -

CAN 105 - - + - -

CAN 106 - ^- - -

CAN 107 - - -

CAN 108 - ~ - -

CAN 109 - -

CAN 1 10 + - -

CAN 1 11 - -

CAN 1 12 - - + - -

CAN 113 - ^ - -

CAN 1 14 + ~ - - -

C AN 1 15 - - -

C AN 1 16 - - - - -

CAN I P - - - -

C AN 1 18 - - ~ - -

CAN 1 19 - - - - -

CAN 120 - - - - -

CAN 121 ^J- - - -

CAN 122 - - - -

CAN 123 - -r - -

CAN 124 - - - - -

CAN 125 - - -^ - -

C ΛN 126 - - - - -

CA 8000 (WT) - - - - -

- = Growth. - = No growth, WT= Wild Type

The nahdixic acid resistant strains isolated were grown in presence of different antibiotics as mentioned in the above table These mutants showed positive growth in presence of high concentration of nahdixic acid Some of them were also resistant to oxolmic acid and all to sparfloxacm but none of them were resistant to second generation drag ciprofloxacm and third generation drag lomefloxacm. The control wild type strain CA8000 as expected was sensitive to these antibiotics.

Example 3: Bioevaluation of thymol for activity against Nal^R mutants of£^".

The above mutants were tested for the sensitivity against thymol to further confirm the above hypothesis that the Nal^R mutants of E. coli can be killed by- thymol rather more efficiently and hence indicating its use as a plant antibiotic to control drag resistant bacteria. Thymol m these assays clearly- showed the activity against all the Nal^R mutants whether it was single or multiple resistance (Table 3)

Table 3 : Disk diffusion assay for determining the activity of thymol against nalidixic acid resistant mutants of Escherichia coli.

Mutant ( Nal^*4) Thymol (inhibition zones in mm) strains of E. coii

50u2 dιsc lOOuε/disc 250ue/disc

CAN 101 Tr. 1.5 ! 4.0

CAN 102 Tr. , 2.0 7 0

CAN 103 Tr . 2.0 ι 4.0

CAN 104 Tr. 2.0 6 0

CAN 105 Tr. 2.0 5.0

CAN 106 2.0 4.0

CAN 107 3.0 5.0

CAN 108 Tr 2.0 5.0

CAN 109 3.0 ■ 6.0

CAN 1 10 3.0 6.0

CAN 1 1 1 3.0 5.0

CAN 1 12 3.0 5.0

CAN 1 13 Tr. 2.0 5 0

CAN 1 14 3.0 6.0

CAN 1 15 3.0 5.0

CAN 1 16 3.0 5.0

CAN 1 17 3 0 5.0 CAN 1 18 1 3 0 5 0

CAN 1 19 1 3 0 6 0

CAN 120 1 3 0 6 0

CAN 121 1 3 0 5 0

CAN 122 Tr 2 0 5 0

CAN 123 1 3 0 5 0

CAN 124 1 3 0 5 0

CAN 125 1 3 0 5 0

CAN 126 1 2 5 0

CA 800O ( WT) 1 2 4

Tr = actn irv in traces (< 0 5 mm). WT = Wild Type

In fact, the compound thymol was able to kill all the nalidixic acid resistant mutants of E. coli with greater efficiency than the wild type strain CA8000

Example 4

Isolation and characterization of mutants of£^". coli resistant to Lomefloxacm (third generation fluoroqumolone drag)

Induced mutants were isolated after selection in the presence of lomefloxacin in the growth medium. Growth of lomefloxacin resistant strains of Escherichia coli was studied in presence of different antibiotics by poison agar method to determine the cross-resistance. The lomefloxacm (third generation ) resistant strains as expected were also resistant to first generation and second generation antibiotics (Table 4).

Table 4: Sensitivity pattern of lomefloxacm resistant cells of E. coli

CA8000 (WTl

~ = Growth. - = No growth, WT= Wild Type Example 5 Bioevaluation of thymol for activity against Lom^R mutants of E. coli

The mutants resistant to lomefloxacin were tested against thymol for sesnsitivity through the disc diffusion assays. As obvious from the Table 5. thymol was effective in killing all the Lom^R mutant cells of Escherichia coli indicating its direct use as the advanced generation drag against drug resistant bacteria.

Table 5: Disk diffusion assay for determining the activity of thymol against lomefloxacin resistant mutants of Escherichia coli.

Mutant (Lom^R) Thymol (inhibition zones in mm) strains of I ^'.. coli

! 50μg disc lOOμg/disc !

CAL 101 ¹ 2.0 ! 4.0 1

CAL 102 ! 3.0 ¹ 5.0 !

CAL 103 2.0 3.0

CAL 104 i 1.0 ' 1.5

CAL 105 2.0 4.0

CAL 106 2.0 4 0

CAL 107 2 0 4 0

CAL 108 5.0

CA8000 3 0 ! 5.0

Tr = activity in traces (< 0 5 mm). WT = Wild Type

Example 6

Evaluation of thymol for activity against nalidixic acid resistant mutants of Mycobacterium smegmatis.

Upon finding the encouraging results of affectivity of Thymol as antibacterial agent against various levels and kinds of drug resistance's) developed in E. coli we planned the experiments in the Mycobacterium also. We employed M. smegmaus. the fast growing model system used for screening anti- tuberculosis drags A series of dπig resistant mutants were isolated in the wild type strain MC Initially nahdixic acid resistant strains were tested for sensitivity against thymol The nalidixic acid mutants were isolated by growing the wild type strain of Mycobacterium smegmatis m medium containing 50 μg / ml nalidixic acid As evident from Table 6, thymol could effectively kill Nal^R strains of Mycobacterium smegmatis establishing its usefulness as an effective antibacterial drug even against mycobacteπa Table 6 Disk diffusion assay for determining the activity of thymol against nahdixic acid resistant mutants of Mycobacterium smegmatis

Mutant ( NaT) Nalidixic acid Thvmol (inhibition zones in mm) strains of ι 50μg/dιsc 50μg/dιsc lOOμg/disc 250μg/dιsc cobacterium smegmatis

MSN 101 Tr

MSN 102 Tr

MSN 103 - 1 2 5

MSN 104 - 1 2 5

MSN 105 - - - 2

V1SN 106 - 1 3 5

MSN 107 - 1 3 6

MSN 108 - 1 3 5

MSN 109 - 1 2 5

MSN 110 - 1 2 5

MSN 111 - 1 2 5

MSN 112 - 1 3 5

MSN 113 Tr 2 ^

MSN 1 14 Tr 1 5 3

MSN 115 Tr 2 3

MC (WT) 1 2 4

Tr = activ in traces (< 0 5 mm) WT = Wild Tvpe

Example 7

Bioevaluation of thymol for activity against Lom^R mutants of Mycobacterium smegmatis The lomefloxacin mutants were isolated by growing the wild type strain of Mycobacterium smegmeus m medium containing 20 μg ; ml lomefloxacin ( third generation antibiotic of the fluoroqumolone category). These mutants were resistant to lomefloxacm upto 60 μg / ml. It is important to note here that the level of resistance that emerges in the mycobacteπal cells upon mutations is tremendously high compared to E. coli and the drags that can kill such Lorn¹ mutants will be of immense need and value. Interestingly these mutant were also found to be resistant against the second generation antibiotics like ciprofloxacin. These were then checked for the sensitivity against thymol, the compound of this mvention, and as indicated in the Table 7 thymol effectively killed these multiple resistant cells of Mycobacterium smegmatis and thus supporting the inference that thymol can act as the fourth/advanced generation herbal antibiotic.

Table 7: Disk diffusion assay for determining the activity of thymol against lomefloxacin resistant mutants of Mycobacterium smegmatis.

Mutant (Lorn") Lomefloxacm Thymol (inhibition zones in mm) strains of 200ug/dιsc lOOug disc 150mg/mi

Mvcobactenwn smeςmetis

MSL 101 - ₃

MSL 102 - ^

MSL 103 - ^

MSL 104 - ^

MSL 105 - 3

MSL 106 - Tr 3

MSL 107 - -;

MSL 108 -

MSL 109 - 2.5

MSL 1 10 - 1

MSL 1 1 1 - 3

MSL 1 12 - 3 MC" (WT)

Tr = activity in traces (< 0 5 mm). - = No activity. WT = Wild Type

Example 8 : Activity of thymol against bacterial mutants resistant to another frontline anti-tubercular drug isoniazid.

Table 8: Sensitivity of isoniazid resistant mutant cells of Escherichia coli against thymol

Strains of E. coli Thymol (inhibition zones in mm) lOOuε/disc

CA8000 6

CA03 2

CA03 (revert) , 8

Isoniazid is another drag widely used for controlling tuberculosis as the bacteria is sensitive to this drug due to absence of oxyR system. Wild type E. coli strain is resistant to high concentration of antibiotic (1000 μg /ml). This strain was mutagenised by N'N-methyl Nitrosoguanidine to isolate the sensitive strain which is being killed at a concentration of 250 μg /ml in broth (CA03). Further, a spontaneous mutation was detected from the sensitive mutant culture, which was resistant to 1000 μg /ml isoniazid. These cultures were tested for thymol sensitivity at a concentration of 100 μg / disc through disc difftision assay and observed that the isomazid resistant revertant strain (CACβrevert) was more sensitive.

Example 9 : Activity of thymol against Ethidium bromide resistant (mdr) mutants

Table 9

Strains of Mycobacterium smegmetis j Thymol (100 μg / disc)

A mutant of Mycobacterium smegmetis was isolated by successive enrichment of the normally sensitive wild type strains upto 12 μg ethidium bromide /ml in broth. This mutant grow well at this concentration of ethidium bromide, while the wild type is killed at 3 μg ethidium bromide /ml in broth. Further, this ethidium bromide resistant strain was found to be multiply drug resistant (mdr) against antibiotics like Chloromphenicol (20 μg / ml), Tetracycline (10 μg / ml) and Rifampicin (40 μg / ml).

Example 10 : Activity of the compound of the present invention thymol at different pH in Escherchia coli strain CA8000.

The above table clearly indicate that the activity of the present compound is maximum at neutral pH (7.00).

Example 11 : Preparation of synergistic antibacterial composition of thymol against multidrug resistant bacteria.

From our study we observed that thymol is a potent bactericide agent against multidrag resistant bacteria . Further it was observed that the potency is increased by combining the oils from mints Mentha arvensis and Mentha spicata. The oil combination had Limonene ( 6.8 - 23.2%), Menthol(0.66 - 2.45%), Carvone(64.0 - 76.1%) and unidentified fractions in the essential oils totalling to 100% at different stages of growth. When this oil combination is used at a concentration of 0.1 % of thymol the antibacterial activity of thymol is increased by 45%. Beside this the oil adds the pleasant carvone flavor to the composition with a menthol tinge. Considering this we prepared different compositions as follows.

Table 1 1 : Disk diffusion assay for determining the synergistic effect of thymol with nalidixic acid against E. coli cells

Nalidixic acid Inhibition zone (in mm) of disc containing Thvmol concentration Nalidixic Combination ι Thymol concentration (μg / disc) acid (Nal + Thymol) (μg / disc)

0.25 3 4 Tr 12.5

0.50 6 12 2 25.0

1.00 9 10 3 50.0

1.50 11 10 i 3 75.0 t 2.00 12 10 1 6 100.0

Table 12: Disk diffusion assay for determining the synergistic effect of thvmol with Mentha hvbrid oil against E. coli cells

Mentha hvbrid Inhibition zone (in mm) of disc containing Thvmol oil (μg / disc) Mentha Combination Thymol concentration hybrid oil (OiI+ Thymol) ! (μg / disc)

50 0 1.0 3.0 50

5 0 5.0 3.0 50

0.5 0 2.5 3.0 50

1

Tr = activity in traces (< 0.5 mm), WT = Wild Type

Composition 1

1. Thymol 100 -500mg (20 -50 % w/w)

2. Essential oil from the hybrid 0.1 -0.5 mg 3. Citric acid 2 - 10 mg

4. Calcium carbonate 100 -200 mg 5 Magnesium hydroxide gel 50- 100 mg

6 Lactose 200-600 mg Total weight 500 - 000 mg

The ingredients are mixed properly, powdered and packed m gelatin capsule available commercially in the market

Composition 2 The essential oil is diluted 0 to 10 times

1 Thymol 50- 300 mg ( 10-60%) m essential oil combination

These preparation are soaked m 250 mg lactose ball

Composition 3

Honey is diluted 0 to 10 times in sterile distilled water and following ingredients added

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

2 Essential oil combination from mints (0 1 to 1 0%)

Composition 4 1 Thymol 100 -500mg 2 Citric acid 2-10 mg

This two components are dissolved in 5 ml 0 05 M Sodium buffer (pH 7 0) containing 200 mg sodium glutamate

Claims

_ Nov el formulation useful m the treatment of drag resistant bacteπal infections compπsing an effective amount of Thy mol^" obtained from the plant Tnchy spermiim ammi. combination of mint oil containing appropπate amounts of monoterpene obtained from Mentha spicata and Mentha arvensis and conventional additives _ A formulation as claimed m claim 1 wherein Thymol is present m the range of about 100 to 500 mg or 20 to 50% vv/w

3 A formulation as claimed m claim 1 wherein mint oil combination is present m an amount of about 0 1 to 0 5 mg

4 A formulation as claimed in claim 1 wherein the additives are selected from the group comprising Citric acid present in the range of about 2-10 mg, Calcium carbonate present m the range of about 100 -200 mg,

Magnesium hydroxide gel present m the range of about 50-100 mg, Lactose present m the range of about 200-600 mg, honey m the range of about 0 1 to 1%. sodium glutamate present m the range of about 200mg and sodium buffer 5 A formulation as claimed m claim 1 wherein mint oil is diluted upto 10 times if desired s A foπnulation as claimed m claim 1 wherein the mint oil combination comprises Limonene m the range of about 6 to 25%. Menthol in the range of about 0 50 to 2 50% Carvone m the range of about 64 0 to 76% ^_ A foπnulation as claimed in claim 1 wherein honey is optionally diluted upto 10 times

. A formulation as claimed m claim 1 wherein the formulation is effective against the group of bacteria selected from the genus Mxconacternim or Escherchia

- A foπnulation as claimed m claim 1 wherein the formulation is effective

^ against bacteria resistant to drags selected from the group comprising

Ethidium bromide. Isoniazid. Chloremphemcol. Tetracy clme. Rifampicm.

Nahdixic acid. Oxolmic acid. Sparfloxicm. Ciprofloxicm and

Loamfloxicin

: : . A method for the preparation of a formulation as claimed in claim 1 0 and useful m the treatment of drag resistant bacterial infections, said method comprising the step of mixing an effective amount of Thymol obtained from the plant Trychyspermum ammi and mint oil combination obtained from Mentha spicata and Mentha arvensis with conventional additives. 5 ... A method as claimed m claim 10 wherein Thymol is present m the range of about 100 to 500 mg or 20 to 50% vv/w

.: . A method as claimed m claim 10 wherein mint oil is presented an amount of about 0 1 to 0 5 mg.

13 A method as claimed in claim 10 wherein the additiv es are selected from o the group comprising Citric acid present m the range of about 2-10 mg.

Calcium carbonate present m the range of about 100 -200 mg, Magnesium hydroxide gel present m the range of about 50-100 mg. Lactose present in the range of about 200-600 mg. honey in the range of about 0 1 to 1 %. sodium glutamate present the range of about 200mg and sodium buffer 5 14 A method as claimed in claim 10 wherein mint oil is diluted upto 10 times 15 A method as claimed m claim 10 wherein the mint oil combination compπses Limonene in the range of about 6 to 25%. Menthol in the range of about 0 50 to 2 50% Carvone in the range of about 64 0 to 76%

16 A method as claimed m claim 10 wherein honey is optionally diluted upto ^ 10 times

A method as claimed m claim 10 wherein the Thymol and the mint oil are dispersed m 0 1 to 1 % honey to obtain a syrap .s . A method as claimed in claim 10 wherein Thymol and the mint oil are mixed with Citric acid 2 to lOmg and dissolved m a buffer containing sodium glutamate to obtain an injection.

19 A method as claimed m claim 10 wherein the formulation is effective against the group of bacteria selected from the genus Mycobacterium or Escherchia

20 A method as claimed m claim 10 wherein the formulation is effective against bacteria resistant to drugs selected from the group comprising

Ethidium bromide, Isomazid. Chloremphemcol. Tetracyclme. Rifampicm. Nalidixic acid. Oxolmic acid, Sparfloxicm. Ciprofloxicm and Loamfloxicin

21 A method for the treatment of dπig resistant bacterial infections in a patient, compπsing the steps of administration of a therapeutically effectiv e amount of the formulation as claimed in claim 1 to a subject in need thereof

22 A method as claimed in claim 21 wherein the formulation of claim 1 is administered through oral or subcutaneous routes A method as claimed m claim 21 wherein the formulation is dissolved 5 ml 0 05 M Sodium buffer <pH 7 0) containing 200mg sodium glutamate to be applied as subcutaneous injection A method as claimed m claim 21 wherein the formulation is effective against bacterial infections such as enteric and systemic infections A method as claimed m claim 21 wherein the treatment comprises administration of the rormulation for bacteπal infections caused by bacteria resistant to drugs selected from the group comprising Ethidium bromide. Isoniazid. Chloremphemcol, Tetracyclme, Rifampicm. Na dixic acid. Oxolmic acid, Sparfloxicm, Ciprofloxicm and Loamfloxicin

A method as claimed in claim 21 , wherein the bacteria multi-drug resistant bacteria are from the genus Mycobacterium or Escherchia new use of Thymol which effectively kill the multi-drag resistant bacteria and thus effectively control bacterial infections at appropriate dose Use as claimed m claim 26, wherein the compound kills the bacteria resistant to Ethidium bromide. Isoniazid, Chloremphemcol, Tetracyclme,

Rifampicm. Nalidixic acid. Oxolmic acid, Sparfloxicm. Ciprofloxicm or Loamfloxicin AMENDED CLAIMS

[received by the International Bureau on 28 February 2001 (28 02 01 ), original claims 1-28 replaced by new claims 1 -29 (4 pages)]

Novel composition useful in the treatment of drug resistant bacterial infections comprising an effective amount of 'Thymol' and a combination of mint oil containing appropriate amounts of monoterpene obtained from Mentha spicata and Mentha arvensis along with conventional additives A composition as claimed in claim 1 wherein Thymol is obtained from the plant Trychyspermum ammi. A formulation as claimed m claim 1 wherein Thymol is present in the range of about 100 to 500 mg or 20 to 50% w/w A formulation as claimed in claim 1 wherein mint oil combination is present in an amount of about 0.1 to 0.5 mg. A formulation as claimed m claim 1 wherein the additives are selected from the group comprising Citric acid present m the range of about 2-10 mg, Calcium carbonate present m the range of about 100 -200 mg, Magnesium hydroxide gel present m the range of about 50-100 mg, Lactose present in the range of about 200-600 mg, honey in the range of about 0.1 to 1%, sodium glutamate present m the range of about 200mg and sodium buffer. A formulation as claimed m claim 1 wherein mint oil is diluted upto 10 times if desired. A formulation as claimed in claim 1 wherein the mint oil combination comprises Limonene m the range of about 6 to 25%, Menthol in the range of about 0.50 to 2.50% Carvone m the range of about 64.0 to 76% A formulation as claimed m claim 1 wherein honey is optionally diluted upto 10 times

9. A formulation as claimed in claim 1 wherein the formulation is effective against the group of bacteria selected from the genus Mycobacterium or Escherchia.

10. A formulation as claimed in claim 1 wherein the formulation is effective against bacteria resistant to drugs selected from the group comprising Ethidium bromide, Isoniazid, Chloremphemcol, Tetracyclme, Rifampicin, Nalidixic acid, Oxolinic acid, Sparfloxicm, Ciprofloxicm and Loamfloxicin.

11. A method for the preparation of a formulation as claimed in claim 1 and useful in the treatment of drug resistant bacterial infections, said method comprising the step of mixing an effective amount of Thymol obtained from the plant Trychyspermum ammi and mint oil combination obtained from Mentha spicata and Mentha arvensis with conventional additives.

12. A method as claimed in claim 11 wherein Thymol is present in the range of about 100 to 500 mg or 20 to 50% w/w.

13. A method as claimed in claim 11 wherein mint oil is presented an amount of about 0.1 to 0.5 mg.

14. A method as claimed in claim 11 wherein the additives are selected from the group comprising Citric acid present in the range of about 2-10 mg, Calcium carbonate present in the range of about 100 -200 mg, Magnesium hydroxide gel present in the range of about 50-100 mg, Lactose present in the range of about 200-600 mg, honey in the range of about 0.1 to 1%, sodium glutamate present in the range of about 200mg and sodium buffer.

15. A method as claimed in claim 11 wherein mint oil is diluted upto 10 times.

16. A method as claimed in claim 11 wherein the mint oil combination comprises Limonene in the range of about 6 to 25%, Menthol in the range of about 0.50 to 2.50% Carvone in the range of about 64.0 to 76%.

17. A method as claimed in claim 11 wherein honey is optionally diluted upto 10 times.

18. A method as claimed in claim 11 wherein the Thymol and the mint oil are dispersed in 0.1 to 1% honey to obtain a syrup.

19. A method as claimed in claim 11 wherein Thymol and the mint oil are mixed with Citric acid 2 to lOmg and dissolved in a buffer containing sodium glutamate to obtain an injection.

20. A method as claimed in claim 11 wherein the formulation is effective against the group of bacteria selected from the genus Mycobacterium or Escherchia.

21. A method as claimed in claim 11 wherein the formulation is effective against bacteria resistant to drugs selected from the group comprising Ethidium bromide. Isoniazid, Chloremphemcol, Tetracycline, Rifampicin, Nalidixic acid, Oxolinic acid, Sparfloxicm, Ciprofloxicm and Loamfloxicin.

22. A method for the treatment of drug resistant bacterial infections in a patient, comprising the steps of administration of a therapeutically effective amount of the formulation as claimed in claim 1 to a subject in need thereof.

23. A method as claimed in claim 22 wherein the formulation of claim 1 is administered through oral or subcutaneous routes.

24. A method as claimed in claim 22 wherein the formulation is dissolved 5 ml 0.05 M Sodium buffer (pH 7.0) containing 200mg sodium glutamate to be applied as subcutaneous injection.

25. A method as claimed in claim 22 wherein the formulation is effective against bacterial infections such as enteric and systemic infections.

26. A method as claimed in claim 22 wherein the treatment comprises administration of the formulation for bacterial infections caused by bacteria resistant to drugs selected from the group comprising Ethidium bromide, Isoniazid, Chloremphemcol, Tetracycline, Rifampicin, Nalidixic acid, Oxolinic acid, Sparfloxicin, Ciprofloxicm and Loamfloxicin.

27. A method as claimed in claim 22 wherein the bacteria multi-drug resistant bacteria are from the genus Mycobacterium or Escherchia.

28. A new use of Thymol which effectively kill the multi-drug resistant bacteria and thus effectively control bacterial infections at appropriate dose.

29. Use as claimed in claim 28, wherein the compound kills the bacteria resistant to Ethidium bromide, Isoniazid, Chloremphenicol, Tetracycline, Rifampicin, Nalidixic acid, Oxolinic acid, Sparfloxicin, Ciprofloxicm or Loamfloxicin.