WO2010010320A1

WO2010010320A1 - Antimicrobial compositions and methods

Info

Publication number: WO2010010320A1
Application number: PCT/GB2009/001587
Authority: WO
Inventors: Donald Campbell
Original assignee: Donald Campbell
Priority date: 2008-07-21
Filing date: 2009-06-25
Publication date: 2010-01-28
Also published as: GB0813323D0

Abstract

The present invention relates to the use of a composition comprising carvacrol, thymol and p-cymene as a therapeutic agent or disinfectant with broad spectrum antimicrobial activity. The compositions of the invention may be used as non-toxic alternatives to conventional disinfectants. The invention is an effective biocide against many pathogens including MRSA providing an improvement to current, harsher products. It may be particularly useful in personal care, health care and household products having good compatibility with human skin and a wide-ranging disinfecting effect. It is particularly applicable, but in no way limited, to antimicrobial and antifungal compositions and their use in the prevention and treatment of infections.

Description

ANTIMICROBIAL COMPOSiTlONS AND METHODS patent application GB0813323.3

Abstract patent application GB0813323.3

Field of the Invention The present invention relates to antimicrobial compositions and in particular to antimicrobial compositions containing mixtures of carvacrol, p-cymene and thymol, to methods of their formulation and to their use. It is particularly applicable, but in no way limited, to antimicrobial, antifungal compositions and their use in the prevention and treatment of infections, via hand and skin disinfection and decolonization.

Background to the Invention

The antiseptic qualities of aromatic and medicinal plants and their extracts have been recognized since antiquity, while attempts to characterize these properties in the laboratory date back to the early 1900s. Plant volatile oil are generally isolated from nonwoody plant material by distillation methods, usually steam or hydrodistillation, and are variable mixtures of principally terpenoids, specifically monoterpenes [C₁₀] and sesquiterpenes [C₁₅] although diterpenes [C₂₀] may also be present, and a variety of low molecular weight aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones and exceptionally nitrogen- and sulphur-containing compounds, coumarins and homologues and phenylpropanoids. Terpenes are generally considered to be amongst the chemicals responsible for the medicinal, culinary and fragrant uses of aromatic and medicinal plants. Most terpenes are derived from the condensation of branched five-carbon isoprene units and are categorized according to the number of these units present in the carbon skeleton. Treatment of infectious diseases is primarily done with antibiotics, sulphonamides, steroid hormones and antifungal compounds. Viral infections are more difficult to treat and limited drugs are available that are effective against these diseases without significant side effects. In the case of bacterial and fungal disease, an increasing problem is the numbers of resistant organisms, some of which can be resistant to multiple drugs. These necessitate the use of antimicrobial drugs that have more toxic side effects often leading to liver and kidney damage.

This is a particular problem in hospitals today where patients, who may or may not be immunosuppressed or have their immune system otherwise compromised, are often subject to acute and chronic fungal and bacterial infections, some of which may be life threatening. For example, multi-resistant organisms such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) are real problems in hospitals, causing high rates of infection in patients, lmmunisuppressed patients, (e.g. those undergoing cancer therapy) are especially susceptible to such microbial infections. As well as adversely affecting the clinical outcomes of infected patients, such bacterial and fungal infection impacts substantially on the health economy through increased health resource utilization for management of infection.

S. Aureus is a common pathogenic commensal bacterium found in warm, moist areas of the body, particularly the nose, axilla and perineum. Approximately 30% of the population are colonised with the bacterium - that is they carry S. Aureus but it does not cause them harm and they do not require treatment. According to the patients Association the figure for healthcare workers who are carriers could be as high as 80%. However, within the hospital environment this means that potentially both patients and staff can act as a reservoir and source for the spread of infection to susceptible individuals.

S. Aureus causes a range of infections from superficial abscesses and boils to the more serious infections of bacteraemia, osteomyelitis, septicaemia and pneumonia. MRSA causes the same types of infection and again people can be colonised with the bacterium. S. Aureus is a gram-positive bacterium with a thick cell wall able to adapt to the presence of antibiotics and develop resistance by infiltrating the antibiotic molecule and disrupting its structure. Certain strains of S. Aureus became resistant to penicillin and an alternative antibiotic was found - methicillin. However shortly after the introduction of methicillin in 1960 resistant strains were reported and incidences increased with many serious outbreaks in hospitals. Methicillin is now used as a screening process in the laboratory for sensitivity testing and hence the title MRSA. Gradually MRSA became more resistant and reports of outbreaks increased, with epidemic strains (EMRSA) being reported in London in the mid 1980's. (EMRSA 3 is often isolated from pressure sores; EMRSA 15 is often associated with pulmonary infections). MRSA is now endemic in many hospitals.

MRSA can only be treated with the glycopeptide group of antibiotics such as vancomycin, which are expensive, can only be given intravenously and are associated with renal impairment. New strains of vancomycin resistant S. Aureus (VRSA) are now emerging.

To maintain an infectious disease in a human population, the pathogen must be transmitted from one host or source to another. One mode of transmission of MRSA is by direct contact, usually with another person's hands. MRSA has also been isolated from people's hands after touching contaminated material or equipment. Because air is not a suitable medium for the growth of a pathogen, any pathogen that is airborne must have originated from a source such as humans, other animals, plants, soil, food, or water. In airborne transmission the pathogen is truly suspended in the air and travels a meter or more from the source to the host. The pathogen can be droplet nuclei, coughing, sneezing, vocalization and dust. Droplet nuclei are small particles 1 to 4 um in diameter. Droplet nuclei can remain airborne for hours or days and can travel long distances. Dust is also an important route of airborne transmission. It's estimated that 80% of dust in public buildings is dead skin cells. The presence of MRSA in peoples noses and throats can be attributed to the inhalation of contaminate dust particles.

At times, a pathogen adheres to dust particles and contributes to then number of airborne pathogens when the dust resuspended by some disturbance. A pathogen that can survive for relatively long periods in or on dust can be the source of hospital acquired infections.

MRSA may also be released in to the atmosphere on skin squames and fibres from clothing and carried as dust particles. About 300 million dead skin squames are shed daily, with as many as 10,000 per minute shed during excessive movement, (cells are microscopic and can pass through the weave of clothing) of which 10% may carry micro-organisms. Therefore it is essential that all equipment and inanimate objects are kept clean and free from dust. Its is furthermore essential that all staff, patients and anybody having business in the hospital maintain a regular skin hygiene regime. Bacteria have evolved protein adhesives called s layers and fimbria, normal bathing procedures do not effectively remove them.

The single most effective method of preventing and controlling the spread of MRSA, and other drug resistant strains, is by the effective decontamination of hands, and in particular decolonization of skin, after patient contact of surfaces in the patient's environment, including the patient's own skin.

There have been reports in the literature of studies to investigate the antimicrobial activity of certain essential oils. For example US 2003/0225003 (Ninkov) describes the use of essential oils, and their derivatives, obtained from the Labiatrae family.

The common name for members of the Labiatae, a large family of chiefly annual or perennial herbs, is the mint family, which is classified in the division Magnoliphyta, class Magnoliopsida, order Lamiales. The mint family includes about 200 genera, such as, for example, Salvia (sage), Rosmarinus (rosemary), Mentha (mint), Ocimum (basil), Thymus (thyme), Marrubium (hoarhound), Monarda (horse mint), Trichostema (bluecuris), Teucrium, Hyptis, Physostegia, Lamium (henbit), Stachys, Scutellaria (skull-cap), and Nepeta (catmint). Members of the related Verbenaceae family include Lippia (Mexican Oregano) and Lycopus. The plants in the mint family are typically shrubby or climbing, although some exist as small tress. The plants are found throughout the world.

The diversity of these plants, and the large number of chemical compounds found in each oil make it very difficult to determine a suitable starting point from which to explore the activity of individual components in these complex mixtures. Furthermore, there is considerable variability in the relative chemical composition within oil from plants of the same family, depending upon where they are grown.

One way to make allowance for this natural variation is to blend essential oils obtained from two plant species. This approach is described in WO2005/087244 (The University of Manchester). This describes compositions comprising at least two essential oils selected from a group of oils derived from a plant genus independently selected from: Palargonium; Cymbopogon; Mentha; Aniba; Lavandula; Cinnamonum; Origanum; Litsea; Citrus; melissa; Pogostemon; Santalum; Valeriana; and Rosa, for use as a medicament. Whilst this type of composition may go some way to guard against natural variation, by including a larger number of potentially active individual chemical components, it certainly does not direct towards an individual or small number of key actives that might be formulated into a useful composition. Rather it teaches that mixtures are required to produce the desired broad spectrum of activity.

Even when oils from individual species have been studied, there are widely varying results. One oil that has been the subject of such studies is the essential from Origannan vulgare in an article by Raduoiene et al in Biologija 2005 Nr 4 pp 53-58.

The authors examined plant material (inflorescences and leaves) of 21 accessions collected during the flowering period (July 2001) from the field collection of the

Medicinal and Aromatic Plants of the Institute of Botany, Vilnius, Lithuania. Voucher specimens of each field accession were deposited in the Herbarium of the Institute of

Botany (BILAS, Vilmius, Lithuania). Essential oils were obtained from the air-dried inflorescences and leaves by hydrodistillation for two hours. Analyses of the essential oils were carried out by GC and GC/MS.

The authors found that the essential oils in their study contained very low amounts of carvacrol and thymol, two compounds previously considered to show antimicrobial activity. They therefore concluded the antimicrobial activity of the oils in their study could be caused by other compounds. They did not go on to elucidate what those other compounds might be.

This result is borne out by results from The University of Manchester, and the Laboratories of Dr Peter Warn, an inventor on WO2005/087244. In their hands although Origanum oil exhibited some antimicrobial activity against some bacteria and fungi, it did not demonstrate high levels of activity against MRSA (unpublished communication).

Against this background, and contrary to expectation, the inventor has surprisingly found that certain mixtures of carvacrol p-cymene and thymol have good antimicrobial activity against a variety of organisms, including a wide number of multi- resistant bacteria and fungi. P-cymene itself posses no antimicrobial properties, the presence of the aromatic hydrocarbon has an activating effect on the hydroxyl group. Such mixtures therefore find particular application in consumer product preparations and as disinfectants. Summary of the Invention

According to a first aspect of the present invention there is provided a composition comprising a mixture of carvacrol p-cymene and thymol for use as an agent having antimicrobial/biocidal action in a consumer product preparation.

Preferably the ratio of carvacrol present in the composition is 42% to 87% v/v and the thymol is present in the composition from 2% to 43% v/v and the p-cymene is present in the composition from 1% to 18% v/v

Preferably carvacro p-cymene and thymol, in combination, comprise at least 80% by weight of the total phenolic and aromatic component of the composition, and more preferably carvacrol p-cymene and thymol, in combination, comprise at least 90% by weight of the total phenolic and aromatic component of the composition.

Preferably the consumer product preparation is selected from the group comprising soap, hand wash, body wash, shower gel, bath gel, bath oil, bubble bath, shampoos, creams including hand cream, lotions, cleaning preparations and disinfectants, including disinfectant sprays and wipes, bath towelettes, sponges, and alcohol rubs.

Preferably the carvacrol is derived from natural sources, and more preferably the carvacrol is derived from a species of origanum.

Preferably the carvacrol is derived from origanum onites.

Preferably the thymol is derived from natural sources and more preferably the thymol is derived from a species of Thymus.

Preferably the P-cymene is derived from natural sources as the precursor to carvacrol

In an alternative preferred embodiment the carvacrol, p-cymene and/or thymol are synthetically prepared.

Preferably the antimicrobial/biocidal action comprises an antibacterial action.

Preferably the antibacterial action is against a bacteria selected from the group comprising Escherichia CoIi, klebsiella pneumoniae, pseudomonas aeruginosa, vancomycin resistant enterococcus, vancomycin-intermediate and resistant S aureus (VIRA + VRSA), Helicobacter pylori and staphylococcus aereus and including MRSA, PVL MRSA USA 400 (ST1), PVL USA 300 (ST8), PVL MRSA Europe (ST80 Clone), Candida albicans, E. aerogenes and A. niger.

Alternatively the antimicrobial/biocidal action comprises an antifungal action.

Preferably the antifungal action is against a fungus selected from the group comprising Aspergillus; Candida; Cryptococcus; Coccidiodes; Encephalitozoon;

Fusarium; Pneumocystis; Histoplasma; Blastomycetes; Pythium; Trichophyton;

Mucorales; Rhizopus; Mucor; Rhizomucor; Absidia; Apophysomyces;

Cunninghamella; Saksenaea; Scytalidium; Malassezia; Madurella; Pseudallescheria;

Sporothrix; Basidiobolus; Penicillium; Acremonium; Alternaria; Bipolaris; Aureobasidium; Exophilia; Scedosporium; Xylohypha; Paecilomyces; and

Scopulariopsis.

Preferably the carvacrol p-cymene/thymol component comprises 0.02% to 10% v/v of the total preparation.

More preferably the carvacrol p-cymene/thymol component comprises 0.02% to 5% v/v of the total preparation, and more preferably the carvacrol p-cymene/thymol component comprises 0.1% to 2% v/v of the total preparation.

According to a second aspect of the invention there is provided use of a composition comprising a mixture of carvacrol, p-cymene and thymol in the manufacture of medicament for the treatment of a microbial infection.

Preferably the ratio of carvacrol to thymol by volume is less than 10, and more preferably the ratio of carvacrol to thymol is less than 5.

Preferably carvacrol, p-cymene and thymol, in combination, comprise at least 80% by weight of the total phenolic and aromatic component of the composition, and more preferably carvacrol, p-cymene and thymol, in combination, comprise at least 90% by weight of the total phenolic and aromatic component of the composition.

Preferably the consumer product preparation is selected from the group comprising soap, hand wash, body wash, shower gel, bath gel, bath oil, bubble bath, cosmetics, moisturizers, sun block, toothpastes, mouthwashes, shampoos, creams including hand cream, lotions, cleaning preparations and disinfectants, including disinfectant sprays and wipes, bath towelettes, sponges, and alcohol rubs.

Preferably the carvacrol p-cymene and thymol are derived from natural sources. Preferably the carvacrol is derived from a species of origanum, and more preferably the carvacrol is derived from origanum onites.

Alternatively the carvacrol, p-cymene and thymol are synthetically prepared.

Preferably the antimicrobial/biocidal action comprises an antibacterial action.

Alternatively the antimicrobial/biocidal action comprises an antifungal action.

Fusarium; Pneumocystis; Histoplasma; Blastomycetes; Pythium; Trichophyton;

Mucorales; Rhizopus; Mucor; Rhizomucor; Absidia; Apophysomyces;

Scopulariopsis.

Preferably the carvacrol/thymol/p-cymene component comprises 0.01% to 10% v/v of the total preparation.

Preferably the carvacrol, p-cymene and thymol component comprises 0.02% to 5% v/v of the total preparation the carvacrol p-cymene and thymol comprises 0.1% to 2% v/v of the total preparation.

According to a further aspect of the present invention there is provided a method of preventing or treating a microbial contamination on an object or a surface in need thereof said method comprising applying to said object or said surface a composition as defined herein. Preferably the object or surface is the skin of a human subject.

Detailed Description of the Invention

The following description describes the best method known to the applicant of putting the invention into practice. However, there may be other methods by which this can be achieved.

The invention provides antimicrobial, biocidal compositions containing a mixture of carvacrol (5-isopropyl-2-methylphenol or isopropyl-5-cresol) I, thymol (5-methyl-2[1- methylethyl]phenol or isopropyl-cresol) Il and p-cymene (1-isopropyl-4- methylbenzene III.

Carvacrol Thymol P-Cymene (C10H14O) (C10H14O) (C10H14)

Compound I Compound Il Compound III

Carvacrol, p-cymene and thymol are commercially available in high purity, for example from Millenium Speciality Chemicals Inc, Jacksonville, Florida 32201 , USA, Lingerer Ltd Sealand Road Chester CH1 4LP UK, Frutarom Ltd Belasis Avenue Billingham UK TS23 1LQ₁ and Destilerias Munoz-Galvez SA Avenida Ciudad de Almeria 162 E-30010 Spain.

In any event, the isolation and purification of caracrol, p-cymene and thymol from naturally occurring essential oils by steam distillation and fractional distillation are well known. Methods for the isolation and synthesis of carvacrol, p-cymene and thymol and references thereto can be found in the Merck Index.

Compositions having antimicrobial activity are formed by mixing carvacrol, p-cymene and thymol such that the ratio of carvacrol to thymol in the mixture is less than 10. In a typical experiment, 60 to 70 grams of carvacrol is mixed 5 to 10 grams of P- cymene and with 20 grams of thymol to give a pale brown clear liquid. It should be noted that the specific densities of carvacrol and thymol are similar, being 0.976 and 0.970 respectively while p-cymene is 0.857. Thus, although these oils are usually measured out by volume, measurement by weight would give a practically identical mixture. Certainly these mixtures would have equivalent microbial activity. Carvacrol, p-cymene and thymol, whilst being infinitely soluble in each other, are practically insoluble in water. Hence other ingredients are required to formulate a useable consumer product preparation.

Care is taken in choosing the starting reagents to formulate compositions according to the invention such that carvacro p-cymene and thymol comprise the greater majority of the phenolic aromatic or monoterpene components present. Typically, carvacrol, p-cymene and thymol comprise at least 80% of the phenolic aromatic or monoterpene content of the composition, and more preferably comprise at least 90% of the composition.

A mixture of carvacrol (65 parts by volume) p-cymene (15 parts by volume) and thymol (20 parts by volume) having a purity of >95% was prepared as described above. This mixture is described hereinafter as carmenol. The density of carmenol at 20oC is 0.9343 g/cm3.

Mixtures according to the present invention may contain 42% to 87% v/v of carvacrol and 2% to 43% v/v of thymol and 1% to 18% v/v of p-cymene

A similar mixture to that prepared above but having carvacrol 70 parts and thymol 4 parts and p-cymene 16 parts was used in the following experimental determinations.

RESULTS Example 1 Antimicrobial Tests with Oily Powder, Decol Soap containing carmenol mixture

Decolonising soap comprises olive oil (60%), palm oil (20%) and coconut oil (20%). Generally the carmenol oil mixture is added to the soap solution at a rate of 1 gram oil in 100 grams soap solution.

Oily soap is made from olive pomace (vegetable residue after oil extraction) + 10% sodium hydroxide NaOH a) 1g of Oily soap powder was dissolved in 10ml of distilled water (soap powder was dissolved by vigorous vortex then centrifuged at 3500G to remove foam). This concentration makes a fairly viscous green solution containing small green and brown particles slightly thicker than liquid soap. b) 1g of Oily soap powder was dissolved in 100ml of distilled water (soap powder was dissolved by vigorous vortex then centrifuged at 3500G to remove foam). This concentration makes a free flowing light green solution slightly thinner than liquid soap. c) 1g of grated Oecol soap was dissolved in 10ml of distilled water (soap was dissolved by vigorous vortex then centrifuged at 3500G to remove foam). This concentration makes a fairly viscous white solution thicker than liquid soap. d) 1g of grated Decol soap was dissolved in 100ml of distilled water (soap was dissolved by vigorous vortex then centrifuged at 3500G to remove foam). This concentration makes a white free flowing solution similar to liquid soap. e) Carmenol mixture was diluted 1:10 in distilled water and vortexed to mix. f) Carmenol mixture was diluted 1:100 in distilled water and vortexed to mix. For all tests bacteria were grown overnight in nutrient broth (Oxoid Basingstoke UK).

Overnight cultures were washed once in phosphate buffered saline and resuspended in phosphate buffered saline to approximately 1 x 10^Λ6cfu/ml.

All tests were performed in duplicate with subcultures plated onto nutrient agar

(Oxoid Basingstoke UK). At Time=0 bacteria was mixed with diluted soap or oil and vortexed to resuspend.

Subcultures were removed at the time-points indicated and immediately pipetted onto the surface of agar plates (100ml per plate). The liquid was spread over the entire surface of the 90mm plate (containing ~20ml of nutrient agar).

All plates were incubated at 37C in air for 24-48 hours. Organisms: Staphylococcus aureus ATCC43300

(Methicillin Resistant Staphylococcus aureus) E. coli ATCC8739 All counts are colony forming units per ml

All E.coli were cleared within 2 minutes

All E.coli were cleared from decol soap within 5 minutes >99.99 cleared within 2 minutes.

All MRSA were cleared within 10 minutes contact of OLLY and within 2 minutes contact of Decol soap.

All MRSA were cleared within 5 minutes of contact of OLLY and DECOL soap

All MRSA were cleared within 2 minutes of contact of OLLY and DECOL soap

NB: different limits of detection were used in testing the 1:10 [(a) (c) and (e)] and 1 :100 solutions [(b) (d) and (f)]. For the 1:10 tests 100ml was plated undiluted and dilute 1:10 whereas in the 1 :000 tests 1:10 and 1:100 dilutions were cultured

Example 2

A similar mixture to that prepared above but having 60 to 70 parts cavacrol and 5 to 10 parts p-cymene plus 5 to 10 parts thymol was used in the following experimental determinations.

Bactericidal Activity of a Range of Products Determined using the European Standard Test method BS EN 1276:1997.

21 June 2009

Author: P. Humphreys Date:

Checked by: S. Rout « D«at_we.:

Authorised by: P. Humphreys ^Date:

Tests Carried Out By:

University of Huddersfieid School of Applied Sciences Queensgate Huddersfieid HD1 3DH

Microbiological Tests

Test Method British/European Standard BS EN 1276:1997. Dilution neutralisation

Test Procedures Full details of all the test and control procedures used are given in the Test Method

Disinfectant 80/20 Carvacrol/Thymol preparation in 2% body wash (33% Dilution)

80/20 Carvacrol/Thymol preparation in 3% handrub (50% Dilution)

Origanum NI 1% body wash micelle free (50% Dilution)

Origanum NI 1% body wash olieate (50% Dilution)

80/20 Carvacrol/Thymol preparation in 2% handrub (50% Dilution)

Batch number: N/A

Date of delivery: 26^th of June 2008

Storage conditions: 2O⁰C - 25⁰C

Active substances: As above Working Concentration: The products were tested at the concentrations outlined above. These dilutions were used to allow the products to be tested.

Interfering Substance Simulated dirty conditions:

(Organic Challenge) 3.0 g I^'1 bovine albumin (final concentration)

Temperature 2O⁰C Contact Time Tested 5 (± 10 s) minute.

Test Organisms Staphylococcus aureus 9518 (NClMB)

Culture Medium Tryptone Soya Agar (Lab011 , LabM Ltd)

Incubation Plates were incubated at 37°C for 24-48hrs.

Diluent MRD (Lab103, Lab M Ltd) Neutraliser D/E Neutralising Broth (Lab187, LabM Ltd). General Method

A standard suspension of test organisms containing 1.5 - 5.0 x 10⁸ cells ml^"1 was prepared. 1 ml of interfering substance was pipetted into a Universal bottle, followed by 1 ml of test organism suspension. The mixture was mixed and left for 2 minutes. After 2 minutes 8 ml of product was added and mixed. After a contact time of 5 minutes, a 1 g sample of the reaction mixture was weighed into a tube containing 8 ml of neutraliser and 1 ml of water and then left for 5 minutes. A 1 ml sample was then pipetted into 2 Petri dishes and mixed with 15 ml of culture medium tempered to 47 ⁰C. In order to gain a better insight into the bactericidal activity of the products under test further dilutions (10^~1 and 10^"2) of the test mixture were also plated out. After setting, the Petri dishes were incubated at 37⁰C. Colony forming units were counted after 24 and 48 hours incubation and the fraction of surviving organisms calculated.

Requirements of this standard

The product, when tested as stipulated under simulated dirty conditions (3.0 g/l bovine albumin) under the required test conditions (2O⁰C, 5 minute contact time, for the selected reference strain), shall demonstrate at least a 5 log 10 reduction in viable counts.

Results

Results from the test are summarised in Table 1 a full set of results can be found in Appendix 1. In order to pass the test a 5 log₁₀ reduction is required. As specified in the standard total kills are recorded as <15 cfu/ml, where this is the case the log reduction associated with a total kill is presented in brackets. In tests where the 10^~2 test dilution returned too many colonies to count a >300 value is employed and the lo reduction is ex ressed as a 'less than' < fi ure.

Table 1. Summary of Test Results.

Interpretation of the Results

Of the products tested, three were able to >5 log₁₀ reductions in viable counts within a 5 minute contact time. These products (80/20 Carvacrol/Thymol preparation in 3% handrub, Origanum N1 1% body wash micelle free (50% Dilution), 80/20 Carvacrol/Thymol preparation in 2% handrub (50% Dilution)) met the requirements set down in BSEN 1276. The 80/20 Carvacrol/Thymol preparation in 2% body wash generated a 3.4 log₁₀ reduction in viable counts. The viscous nature of this product required a 33% (1 :3) dilution to allow it to be used in the test. Given these results it is possible that the product may have achieved a 5 log₁₀ reduction if it had been possible to perform the test on the neat product. The Origanum NI 1% body wash olieate (50% Dilution) product did not demonstrate any significant activity (<1 log₁₀ reduction) at the dilutions tested.

Example 3

REPORT

EUPR 029 090526

Assessment of the Bacteriocidal Activity of a Series of Proprietary Hand Rubs and Body Washes (Proposal: EUP_029_090505)

FOR

Imhere Ltd CONFIDENTIAL

AUTHORS: Sandra Howsley CONTRIBUTORS: Peter Warn DATE: 26th May 2009 VERSION: 1.0 EUPROTEC Ltd. CONFIDENTIAL

EUPR_029_090526 vl.O

Bactericidal activity of various hand rubs and body washes. 26ft May 2009 Page 2 of 6

Ll PHYSICAL PROPERTIES OF COMPOUNDS RECEIVED

Euprotec received seven personal hygiene products from Imhere. All compounds were stored at room temperature following delivery. For simplicity, the soaps were labelled 1 to 7 and table 1 indicates which soap corresponds to which number. The solubility of the soaps for testing was also assessed and the results (including visual assessment) are also detailed below in table 1: 2.1 METHODS 2.1.1 Bacterial Isolate

Susceptibility testing was performed on the bacterial strain, Staphylococcus aureus (NCTC 13142). The Staphylococcus aureus strain was grown up on nutrient agar (Oxoid) at 37°C for 24 hours. 2.1.2 Preparation of the inoculum The S. aureus (NCTC 13142) inoculum was prepared by picking 5-10 distinct colonies from the culture plate and suspending in 3mL sterile water. The concentration of the inoculum was then adjusted using a spectrophotometer to an OD600nm of 0.669 which is equivalent to 1x109 colonies per mL. A 1:100 dilution in sterile water was then performed to give a working concentration of 1x107 colonies per mL. 2.13 Time Kill Assay

900μl of diluted soap (see table 1) was added to a small bijou. lOOμl of 1x107 S. aureus (NCTC 13142) colonies per mL was then added to the soap to give a final concentration of 1x106 S. aureus (NCTC 13142) colonies per mL. At 0, 0.5, 1, 2 5 10 and 30 minute intervals, 2x 100μl aliquots were removed, and lxlOOμl was plated directly on to a nutrient agar culture plate. The other 1 OOμl was diluted further 1 : 10, 1 : 100 and 1 : 1000. lOOμl aliquot of each dilution was then plated on to separate nutrient agar plates. All plates were then incubated at 37°C for 24 hours. A control assay was also performed with S. aureus (NCTC 13142) in the presence of water instead of soap, n.b. Using this methodology the minimum number of bacteria detectable is 10 colonies forming units per mL. 2.1.4 Analysis AU plates were removed from 37°C incubator after 24 hours. For each time point, the number of colonies on each plate was counted. The decrease in bacterial growth in the presence of each soap was then determined over time.

3.1 RESULTS

3.1.1 Soap nomenclature and dilutions

For simplicity each soap was given an assay number, the details of which are outlined in table 1. In addition, the soaps had to be diluted prior to using in the assay as each stock was too viscous to pipette accurately. The appearance and the dilution required prior to using in the assay are also outlined in table 1.

4.1. SUMMARY

• Personal hygiene products 1 to 6 were potent bactericidal agents within 30 seconds against S. aureus (NCTC 13142). • Personal hygiene product 7 was a less potent bactericidal agent against 5". aureus (NCTC 13142). However, some bactericidal activity was still observed by 30 minutes post incubation.

• Personal hygiene products 1-6 were able to reduce the number of S. aureus (NCTC 13142) by >99.999% in less than 30 seconds. • Antibacterial activity was assessed without the addition of specific inhibitors. Inactivation was achieved through dilution (1 :200) Dr Peter Warn Director Euprotec Incubator Building, Grafton Street, Manchester Ml 3 9XX

The Carmenol mixture of the present invention may be formulated into a variety of antimicrobial/biocidal consumer produce preparation using known methodologies.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions for topical administration. Such solutions are prepared so as to be acceptable to biological systems (isotonicity, pH,

- hydrophobicity, viscosity, among other solution properties). Liquid preparations can also be formulated in aqueous polyethylene glycol solutions. Aqueous solutions can be prepared by dissolving the antimicrobial compound in water and adding suitable colorants, flavors, stabilizing, emulsifying, and thickening agents as desired. Aqueous suspensions can be made by dispersing finely divided active components in water with materials such as cocco glucoside, potassium cocoate, potassium oleate natural or synthetic gums, resins, methyl cellulose, carboxymethyl cellulose, as well as other accepted cellulose derivatives, certain common solubilizers such as polysorbates (tween) and others that are hydrogenated castor oil based are not suitable as they inhibit the biocidal action. At all times the ph balance must be as close to neutral (ph7) as is possible. Commonly used liquid preparations include but are not limited to, soap, hand wash, body wash, cosmetics, moisturizers, shampoos, hand cream, hand rub, lotions, cleaning preparations and disinfectants, alcohol rubs, all of whose formulation and manufacture are well known in the art.

Topical ointment preparations may contain formulation of the antimicrobial compound for topical uses to treat infections. Four broad types of ointments, referred to as ointment bases, are used to deliver medicinal agents. The first is hydrocarbon base and usually includes petrolatum, which is an unctuous material having emollient qualities and a melting temperature of about 38-6O⁰C. They may also include oleaginous base along with bases made from vegetable oils or animal fats. These ointments often require addition of antioxidants and other preservatives are not as commonly used today.

A second ointment type is referred to as absorption bases. These are hydrophilic and may include anhydrous materials or alternatively hydrous bases that can absorb water. These often include lanolin, which is a complex assortment of compounds that readily absorb water. Other components of absorption bases may include stearyl alcohol, white wax, and white petrolatum. Derivatives of lanolin are available and include alcohols, dewaxed, acetylated, ethoxylated, hydrogenated, among other types of modifications. In some absorption bases cholesterol has been substituted for lanolin due to allergic reactions reported to lanolin type compounds. There are several commercially available absorption bases including, but not limited to Aquaphor, (Beirsdorf), Polysorb (Fougera), and Nivea (Beirsdorf). A third ointment type, commonly referred to as creams, is water removable base or emulsion bases. These typically have three main components: an oil phase, an emulsifier, and an aqueous phase. The medicinal agent may be part of any of the three phases or alternatively added to the final emulsion. The emulsion base hydrophilic ointment USP is an example designed to minimize any undesirable drug interactions, either physical or chemical. It contains methylparaben, propylparaben, sodium laurel sulphate, propylene glycol, stearyl alcohol, white petrolatum, and water. It is typically made according to established formulas that are well known in the art. In this illustrative example the stearyl alcohol and petrolatum make up the oil phase and give the desired smoothness and characteristics for compatibility with the skin. An enormous number of additional oil phase components are known in the art and may be used. Preservatives may be added to the aqueous phase and include, but are not limited to, methylparaben, propylparaben, benzyl alcohol, sorbic acid, or quaternary ammonium compounds. The aqueous phase in most cases exceeds the oil phase and contains the preservative materials and emulsifiers. The emulsifier may be either an anionic, cationic, or non-ionic type. The aqueous component also contains a humectant, which is often glycerine, propylene glycol, or a polyethylene glycol. These add to the overall characteristic of the preparation and maintain water content. Any necessary components for maintaining pH, buffers, antioxidants, and stabilizers are also included in the aqueous phase.

The fourth type is the soluble ointment bases that are composed of soluble ingredients. These may also be gelled aqueous solutions, which are commonly called gels. The polyethylene glycols are often the major component of these water soluble bases and are known to be inert and specifically do not cause irritation. These have characteristics ranging from liquids to soft solids to hard wax types. Commonly used polyethylene glycols include polymers designated by their average molecular weights of 400, 1500, 1600 and 6000. Various gelling agents are often included and include: cellulose derivatives, carbomers, colloidal magnesium aluminium silicate (Veegum), sodium alginate, and the propylene glycol ester of alginic acid (kelcoloid). One feature of the soluble ointment bases is that they are designed to maximize delivery of the therapeutic agent.

The preparations may also contain additives such as preservatives, vitamins, minerals, and amino acids. Suitable additives may vary depending on the desired end use.

Claims

Claims:

1. A composition comprising a mixture of Carvacrol, Thymol and p-Cymene for use as an agent having antimicrobial/biocidal action in a consumer product preparation.

2. A composition as claimed in Claim 1 wherein the Carvacrol is present in the composition from 42% to 87% v/v, the Thymol is present in the composition from 2% to 43% v/v and the p-Cymene is present in the composition from 1% to 18% v/v.

3. A composition according to any preceding claim wherein carvacrol, thymol, and p-Cymene in combination, comprise at least 80% by weight of the total phenolic and aromatic component of the composition.

4. A composition according to Claim 3 wherein carvacrol, thymol and p- Cymene in combination, comprise at least 90% by weight of the total phenolic and aromatic component of the composition.

5. A composition according to any preceding claim wherein the consumer product preparation is selected from the group comprising soap, hand wash, body wash, shower gel, bath gel, bath oil, bubble bath, moisturizers, mouthwashes, shampoos, creams including hand cream, lotions, cleaning preparations and disinfectants, including disinfectant as a spray or gel or paste or wipe, bath towelettes, sponges, and alcohol rubs.

6. A composition according to any preceding claim wherein the carvacrol and/or thymol and/or p-Cymene are derived from natural sources.

7. A composition according to Claim 6 wherein the carvacrol and/or thymol and/or p-Cymene are derived from a species of origanum or thymus.

8. A composition according to Claim 7 wherein the carvacrol and/or thymol and/or p-Cymene are derived from origanum onites or thymus vulgaris.

9. A composition according to any of Claims 1 to 5 inclusive wherein the carvacrol and/or thymol and/or p-Cymene are synthetically prepared.

10. A composition according to any preceding claim wherein the antimicrobial/biocidal action comprises an antibacterial action.

11. A composition according to Claim 10 wherein the antibacterial action is against a bacteria selected from the group comprising Escherichia CoIi, klebsiella pneumoniae, pseudomonas aeruginosa, vancomycin resistant enterococcus, vancomycin-intermediate and resistant S aureus (VIRA + VRSA), Helicobacter pylori and staphylococcus aereus including MRSA, PVL MRSA USA 400 (ST1), PVL USA 300 (ST8), PVL MRSA Europe (ST80 Clone), Candia albicans, E. aerogenes and A. niger.

12. A composition according to any of Claims 1 to 9 inclusive wherein the antimicrobial/biocidal action comprises an antifungal action.

13. A composition according to Claim 12 wherein the antifungal action is against a fungus selected from the group comprising Aspergillus; Candida; Cryptococcus;

Coccidiodes; Encephalitozoon; Fusarium; Pneumocystis; Histoplasma; Blastomycetes; Pythium; Trichophyton; Mucorales; Rhizopus; Mucor; Rhizomucor; Absidia; Apophysomyces; Cunnighamella: Saksenaea; Scytalidium; Malassezia; Madurella; Pseudallescheria; Sporothrix; Basidiobolus; Penicillium; Acremonium; Alternaria; Biopolaris; Aurebasidium; Exophilia; Scedosporium; Xylohypha; Paecilomyces and Scopulariopsis.

14. A composition according to any preceding claim wherein the carvacrol/thymol/p-Cymene component comprises 0.01% to 10% v/v of the total preparation.

15. A composition according to Claim 14 wherein the carvacrol/thymol/p- Cymene component comprises 0.02% to 5% v/v of the total preparation.

16. A composition according to Claim 14 or Claim 15 wherein the carvacrol/thymol/p-Cymene comprises 0.1 % to 2% v/v of the total preparation.

17. A composition comprising a mixture of carvacrol, thymol and p-Cymene for use as an agent having antimicrobial/biocidal action for use in a consumer product preparation substantially as herein described.

18. Use of a composition comprising a mixture of carvacrol, thymol and p- Cymene in the manufacture of medicament for the treatment of a microbial infection.

19. Use according to Claim 18 wherein the Carvacrol is present in the composition from 42% to 87% v/v, the Thymol is present in the composition from 1% to 43% v/v and the p-Cymene is present in the composition from 1% to 18% v/v.

20. A composition according to any preceding claim wherein carvacrol, thymol and p-Cymene, in combination, comprise at least 80% by weight of the total phenolic and aromatic component of the composition.

21. A composition according to Claim 4 wherein carvacrol, thymol and p- Cymene, in combination, comprise at least 90% by weight of the total phenolic and aromatic component of the composition.

22. Use according to any of Claims 18 to 21 inclusive wherein the consumer product preparation is selected from the group comprising soap, hand wash, body wash, shower gel, bath gel, bath oil, bubble bath, moisturizers, mouthwashes, shampoos, creams including hand cream, lotions, cleaning preparations and disinfectants, including disinfectant as a spray or gel or paste or wipe, bath towelettes, sponges, and alcohol rubs.

23. Use according to any of Claims 18 to 22 inclusive wherein the carvacrol and/or thymol and/or p-Cymene are derived from natural sources.

24. Use according to Claim 23 wherein the carvacrol and/or thymol and/or p- Cymene are derived from a species of origanum or thymus vulgaris.

25. Use according to Claim 24 wherein the carvacrol and/or thymol and/or p- Cymene are derived from origanum onites or thymus vulgaris.

26. Use according to any of Claims 18 to 22 inclusive wherein the carvacrol and/or thymol and/or p-Cymene are synthetically prepared.

27. Use according to any of Claims 18 to 26 inclusive wherein the antimicrobial/biocidal action comprises an antibacterial action.

28. Use according to Claim 27 wherein the antibacterial action is against a bacteria selected from the group comprising Escherichia CoIi₁ klebsiella pneumoniae, pseudomonas aeruginosa, vancomycin resistant enterococcus, vancomycin-intermediate and resistant S aureus (VIRA + VRSA), Helicobacter pylori and staphylococcus aereus including MRSA, PVL MRSA USA 400 (ST1), PVL USA 300 (ST8), PVL MRSA Europe (ST80 Clone), Candia albicans, E aerogenes and A. niger.

29. Use according to any of Claims 18 to 26 inclusive wherein the antimicrobial/biocidal action comprises an antifungal action.

30. Use according to Claim 29 wherein the antifungal action is against a fungus selected from the group comprising Aspergillus; Candida; Cryptococcus; Coccidiodes; Encephalitozoon; Fusarium; Pneumocystis; Histoplasma;

Blsatomycetes; Pythium; Trichophyton; Mucorales; Rhizopus; Mucor; Rhizomucor;

Absidia; Apophysomyces; Cunninghamella; Saksenaea; Scytalidium; Malassezia;

Madurella; Pseudallescheria; Sporothrix; Basidiobolus; Penicillium; Acremonium;

Alternaria; Bipolaris; Aureobasidium; Exophilia; Scedosporium; Xylohypha; Paecilomyces and Scopulariopsis.

31. Use according to any of Claims 18 to 30 inclusive wherein the carvacrol/thymol/p-Cymene component comprises 0.01% to 10% v/v of the total preparation.

32. Use according to Claim 31 wherein the carvacrol/thymol/p-Cymene component comprises 0.02% to 5% v/v of the total preparation.

33. Use according to Claim 31 or Claim 32 wherein the carvacrolΛhymol/p- Cymene comprises 0.1% to 2% v/v of the total preparation.

34. Use of a composition comprising a mixture of carvacrol, thymol and p- Cymene in the manufacture of a medicament for the treatment of a microbial infection substantially as herein described.

35. A method of preventing or treating a microbial contamination on an object or a surface in need thereof said method comprising applying to said object or said surface a composition as defined in any of Claims 1 to 17 inclusive.

36. A method according to Claim 35 wherein the object or surface is the skin of a human or animal subject.

37. A method of treating or preventing a microbial contamination substantially as herein described.