WO2013034581A1 - Antimicrobial composition comprising thymol and a terpinyl derivative - Google Patents

Abstract

The present invention relates to an antimicrobial composition and a method for disinfection involving the antimicrobial composition. It particularly relates to an antimicrobial composition for personal cleaning, oral care or hard surface cleaning applications. It was found that compositions comprising thymol, selected terpinyl derivatives and a carrier provide synergistic antimicrobial action. In a preferred aspect the composition also comprises 1 to 80 %-wt of surfactant.

Description

ANTIMICROBIAL COMPOSITION COMPRISING THYMOL AND A TERPINYL DERIVATIVE

FIELD OF THE INVENTION

The present invention relates to an antimicrobial composition and a method for disinfection involving the antimicrobial composition. It particularly relates to an antimicrobial composition for personal cleaning, oral care or hard surface cleaning applications.

BACKGROUND TO THE INVENTION

Sanitising and disinfecting soap or cleaning compositions are of great benefit to individuals, since proper use generally may reduce the number of germs and pathogens the individual is exposed to. Thus, such compositions may for instance play an important role in reducing the occurrence and spread of infectious diseases. Sanitising and disinfecting soap compositions comprising chlorine-based antimicrobial agents such as triclosan are known. Such compositions require a rather long contact time to provide efficacious antimicrobial action. In practice, users, in particular children, do not spend a long time on cleansing and as a result cleaning with such compositions does not provide adequate prevention from surface or topical infection or adequate protection against diseases. The user, in spite of cleaning his hands, is generally likely to end up with relatively inadequate bacterial removal from his skin. Therefore, he may cause contamination of further animate and/or inanimate surfaces and contribute to the spreading of pathogens and consequent diseases. Users in general and children in particular who wash contaminated hands before meals with slow-acting antimicrobial compositions for relatively short time are at risk of contracting diseases.

Similarly in the area of hard surface cleaning, e.g. cleaning of floors, table tops or utensils, the antimicrobial actives in the compositions are in contact with the substrate for less than a few minutes after which the surface is either wiped off or rinsed with water. These short time scales of cleaning action are ineffective in providing the desired benefit since most known antimicrobials commonly used in such products take many minutes to hours to provide the desired kill of microbes. Therefore, there is a need of providing a composition that -upon application- provides relatively more efficacious antimicrobial action during a relatively short cleaning period, preferably about 30 seconds or less. A well-established class of antimicrobially active compounds are phenolic compounds [P A Goddard and K A McCue in "Disinfection, Sterilisation and Preservation", ed. S S Block, 5^th edition, Lippincott, Williams and Wilkins, Philadelphia, 2001 pp. 255-282.]. However, not every phenolic compound is suitable as an antimicrobial agent.

Moreover, many phenols - even if they are antimicrobially active - may exhibit undesirable side-effects, such as corrosiveness, malodour and irritating or sensitising effects when applied on the human or animal skin.

A particular problem of thymol and/or terpineol is that its presence in a formulation is generally well-perceptible due to its olfactory properties. Although the latter may - at least to some extent - be appreciated in certain fragrance compositions, it is considered too intense by some users when applied at concentrations efficacious in rapid disinfection. Additionally, a lower concentration of odoriferous compounds including thymol or the availability of antimicrobial compounds that are less or not odoriferous allows greater flexibility to the manufacturer in providing alternative scents to his composition at lower doses. Hence there is a need to provide alternative antimicrobial compositions and methods that preferably require lower concentrations of thymol and/or have a more acceptable sensory profile.

WO 2010/046238 A1 describes an effective antimicrobial composition which provides rapid kill of pathogenic bacteria and which comprises 0.01 to 5% by weight of thymol, 0.01 to 5% by weight of alpha-terpineol and a carrier. WO 2010/046238 A1 also discloses a method of disinfecting a surface including the step of applying the above composition to the surface. Despite the general availability of antimicrobial compounds and compositions, there remains a continuous need to find alternative antimicrobial compositions and active compounds that are suitable for use in such compositions. In particular, alternative compositions providing fast antimicrobial action remain highly desirable in view of current consumer habits. Such alternatives may reduce the dependency on current raw materials. Moreover, in the field of antimicrobials, the availability of alternatives may reduce the risk of development of microbial resistance or insensitivity to particular antimicrobial compounds.

In addition, there is a continued need to reduce the total amount of active ingredients required in such an antimicrobial composition. This need may for instance be driven by the desire for cost-efficiency, because such compositions are particularly relevant to developing countries. Moreover, reducing the amounts may also be beneficial for environmental reasons.

Essential oils containing terpinyl acetate have been reported to show antimicrobial efficacy. For example, the essential oil obtained from the seeds and pods of Elettaria cardamomum shows antibacterial action against organisms including E. coli [G Singh, S Kiran, P Marimuthu, V Isidorov and V Vinogorova, Journal of the Science of Food and Agriculture, Volume 88, Issue 2, pp. 280-289 (2008)]. This oil is complex mixture comprising more than 70 identifiable compounds. It contains 44.3 % of alpha-terpinyl acetate and 0.1 % of alpha-terpinyl propionate, as identified by GC/MS. A chloroform oleoresin of the same seeds and pods is a similarly complex mixture and comprises among many other compounds 21 .8% alpha-terpinyl acetate, 0.1 % alpha-terpinyl butyrate, and 0.08% thymol. The antimicrobial action of these mixtures is ascribed to phenolic compounds such as thymol, carvacrol and methyl eugenol, while other constituents are believed to contribute little to antimicrobial effects. No synergistic interactions between compounds are shown. In view of the above-observed problems and drawbacks of the prior art, it is an object of the present invention to provide alternative antimicrobial compositions.

It is a particular object of the invention to provide such compositions, requiring a lower dose of antimicrobial compounds.

Similarly, it is an object of the present invention to provide an antimicrobial composition in which the olfactory contribution of the antimicrobially active compounds is reduced or in which the active compound contributes to providing a consumer-acceptable or even consumer-appreciated scent. It is another particular object of the invention to provide an antimicrobial composition which upon use yields a reduced disinfection time.

It is yet another particular object of the invention to provide an antimicrobial

composition that contributes to reducing the required contact time in a method for disinfection of a surface.

In particular, it is an object of the invention to provide an antimicrobial composition which gives improved disinfection during cleansing of surfaces of the human body, such as the skin and the oral cavity.

It is yet another object of the present invention to provide an alternative method for sanitising and/or disinfecting, in particular of surfaces. It is a further object of the invention to provide a method for disinfection with a reduced disinfection time. More specifically, it is an object of the invention to provide a method, wherein the disinfection time of the method is less than 300 seconds, preferably less than 60 seconds, and more preferably less than 15 seconds. In particular, it is an object of the invention to provide a method for disinfection that gives improved disinfection during cleansing of surfaces, in particular hard surfaces, or surfaces of the human body, such as the skin and the oral cavity.

SUMMARY OF THE INVENTION

We have now found that one or more of the above objects are met by the present invention. Thus, we have found that compositions comprising thymol and selected terpinyl derivatives provide synergistic antimicrobial action. Such compositions provide similar or more efficacious anti-microbial action, at similar or lower concentrations when compared to thymol and alpha-terpineol. In particular, we found that combinations of thymol and terpinyl derivatives according to this invention are distinctively more active than combinations of thymol and alpha-terpinyl acetate. Moreover, we found that combinations of thymol and terpinyl derivatives according to this invention are capable of very fast antimicrobial action. For instance, we found that complete microbial inactivation could be effected with compositions according to the present invention after a contact time of only 15 seconds. Accordingly, in a first aspect the invention provides an antimicrobial composition comprising:

a. 0.001 to 5% by weight of thymol,

b. 0.001 to 5% by weight of a terpinyl derivative, and

c. a carrier;

wherein the terpinyl derivative is selected from

i. alpha-terpinyl esters and amides of the structure:

beta-terpinyl esters and amides of the following structun

iii. gamma-terpinyl esters and amides of the following structure

iv. delta-terpinyl esters and amides of the following structure

x ^R< v. 4-terpinyl esters and amides of the following structure

vi. p-me rs and amides of the following structure

vii. dihydrocarvyl esters and amides of the following structure

viii. isopulegyl esters and amides of the following structure

and wherein X is N H or O, and

wherein R-i is

a linear or branched C₂ to Ci₇ alkyl group, or

a linear or branched substituted C₂ to Ci₇ alkyl group, or

a linear or branched C₂ to Ci₇ alkenyl group, or

a linear or branched C₂ to Ci₇ substituted alkenyl group, or

R₂OR₃, wherein R₂ is a linear or branched C_x alkyl group and R₃ is a linear or branched C_y alkyl group, such that x + y is between and including 2 and 17, or an alkyl ethoxylate (CH₂)_x(OC₂H₄)_yOH group, wherein x is between 1 and 10, and y is between 1 and 10, or

an aliphatic C₅ to C₇ ring, or

a substituted aliphatic C₅ to C₇ ring, or an unsaturated C₅ to C₇ ring, or

a substituted unsaturated C₅ to C₇ ring, or

an aryl group, or

a substituted aryl group, or

an aryloxy group, or

a substituted aryloxy group;

and mixtures of these terpinyl derivatives.

According to a second aspect of the invention, there is provided a method of disinfecting a surface comprising the steps of

a. applying a composition according to the invention on to the surface; and b. rinsing the surface with a suitable solvent or wiping the surface with a suitable wipe. In a third aspect, the invention provide the use of a composition according to the invention for improved hand hygiene. In a fourth aspect, the invention provides the use of a composition according to the invention for improved oral hygiene.

DESCRIPTION OF FIGURES

Figure 1 provides an isobologram, showing synergistic combinations of thymol and alpha-terpinyl propionate.

Figure 2 provides an isobologram showing synergistic combinations of thymol and alpha-terpinyl butyrate.

Figure 3 provides an isobologram showing synergistic combinations of thymol and alpha-terpinyl cyclohexanoate.

DETAILED DESCRIPTION OF THE INVENTION

For the avoidance of doubt, any feature of one aspect of the present invention may be utilised in any other aspect of the invention. The word "comprising" is intended to mean "including" but not necessarily "consisting of" or "composed of." Thus, the term "comprising" is meant not to be limiting to any subsequently stated elements but rather to optionally also encompass non-specified elements of major or minor functional importance. In other words, the listed steps or options need not be exhaustive.

Whenever the words "including" or "having" are used, these terms are meant to be equivalent to "comprising" as defined above. It is noted that the examples given in the description below are intended to clarify the invention and are not intended to limit the invention to those examples per se.

Except in the examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts of material or conditions of reaction, physical properties of materials and/or use are to be understood as modified by the word "about". Unless specified otherwise, numerical ranges expressed in the format "from x to y" are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format "from x to y", it is understood that all ranges combining the different endpoints are also contemplated.

The antimicrobial composition comprises thymol, a terpinyl derivative and a carrier. Various components of the antimicrobial composition are described below. The compositions of the present invention are preferred for non-therapeutic use, and more particularly preferred for use in cleaning surfaces of human body including skin, hair or oral cavity or for hard surface cleaning applications.

Thymol

The antimicrobial composition according to the invention comprises 0.001 to 5% by weight of thymol. The composition comprises preferably 0.005 to 5 wt-%, more preferably 0.01 to 4 wt-%, even more preferably 0.01 to 3 wt-%, still more preferably 0.01 to 2 wt-%, and still more preferably 0.01 to 0.5 wt-% of thymol. Alternatively, for instance in compositions intended to be diluted before application, the minimum preferred concentrations of thymol can be higher. For example, when washing hands with water and a composition according to the invention, the lather produced, typically is a 50 wt% dilution of the original composition. Similarly, in body wash situations, soap bars or soap liquids are typically diluted until about 8 wt% soap in water, corresponding to an approximately tenfold dilution of the product. Therefore, compositions according to the invention intended for dilution upon use preferably comprise 0.02 to 5 wt-%, more preferably 0.03 to 4 wt-%, even more preferably 0.04 to 3 wt-%, still more preferably 0.05 to 2 wt-%, and still more preferably 0.1 to 1 wt-% of thymol.

These preferred concentration ranges of thymol are important since below the preferred lower concentration of thymol, the desired fast acting antimicrobial kinetics in combination with a terpinyl derivative would not be met. At concentrations higher than the higher preferred concentrations of thymol, when in combination with a terpinyl derivative, while the kinetics of action would not be compromised, the present inventors have found that unlike in therapeutic/pesticidal/herbicidal applications where sensorial aspects are not critical, in the present application, which is preferably a personal cleaning, oral care or hard surface cleaning applications, the product is in contact with hands, mouth or other body parts, the sensorial aspects like smell and skin feel would be compromised. Thymol (also known as 2-isopropyl-5-methylphenol) may be added to the antimicrobial composition in purified form.

Alternatively, thyme oil or thyme extract comprising thymol may be added to the antimicrobial composition, while ensuring that thymol is present in the desired concentration in the composition of the present invention. Thyme oil or thyme extract is obtained from the thyme plant. Thyme plant refers to a plant belonging to the genus Thymus and includes but is not limited to the following species: Thymus vulgaris, Thymus zygis, Thymus satureoides, Thymus mastichina, Thymus broussonetti, Thymus maroccanus, Thymus pallidus, Thymus algeriensis, Thymus serpyllum, Thymus pulegoide, and Thymus citriodorus .

Thymol has the following structure:

Terpinyl derivatives

The antimicrobial composition according to the invention comprises 0.001 to 5% by weight of a terpinyl derivative. The composition comprises preferably 0.005 to 4.5 wt- %, more preferably 0.01 to 4 wt-%, even more preferably 0.02 to 3 wt-%, yet more preferably 0.03 to 2 wt-%, still more preferably 0.04 to 1 wt-% and still more preferably 0.05 to 0.5 wt-% of a terpinyl derivative. Any of these concentrations ranges may preferably be combined with any of the concentration ranges for thymol specified above. Therefore, the antimicrobial composition according to the invention for example preferably comprises:

a. more than 0.01 and less than 0.3% by weight of the thymol;

b. more than 0.05 and less than 1 % by weight of the terpinyl derivative. The terpinyl derivative may be a single compound or may be a mixture of the terpinyl compounds as detailed below. Mixtures of terpinyl derivatives are preferred, since such mixtures may show increased antimicrobial activity against a wider range of microbes. In case the composition according to the invention comprises such a mixture of terpinyl derivatives, the mixture preferably comprises at least 30%, more preferably at least 50%, even more preferably at least 70% and still more preferably at least 90% by weight of one terpinyl derivative with respect to the total weight of the terpinyl derivatives. The preferred concentrations ranges of the terpinyl derivative are important for the same reasons as the preferred concentration ranges of thymol in meeting the desired fast acting antimicrobial kinetics while not being sensorially unpleasant when used in products for personal cleaning, oral care or hard surface cleaning applications. Terpinyl radical

The term terpinyl radical denotes the terpinyl headgroup, i.e. a terpinyl parent alcohol moiety in the way in which it is comprised as a substituent in a particular terpinyl derivative. The terpinyl derivative according to the invention is selected from esters and amides with a terpinyl radical selected from alpha-terpinyl, beta-terpinyl, gamma- terpinyl, delta-terpinyl, 4-terpinyl, p-menth-1 -en-9-yl, dihydrocarvyl, and isopulegyl radicals. The structures of these derivatives are schematically depicted in Table 1 below.

Table 1

Where applicable, the different stereoisomers of these terpinyl radicals are

contemplated. Thus, compositions comprising enantiomerically pure radicals, racemic mixtures and other mixtures of different stereoisomers are equally preferable.

5

The parent terpinyl alcohol groups corresponding to these radicals are all members of the menthenol class of compounds [A L Gunatilaka, Natural products in Plants:

Chemical Diversity in the Wiley Encyclopedia of Chemical Biology, pp 1 -17 and E Breitmaier, Terpenes: flavors, fragrances, pharmaca, pheromones; p. 17, Wiley-VCH,

10 2006], also termed oxytetrahydrocymenes [F Heusler, The Chemistry of the Terpenes, trans. F J Pond, P Blakistons's son & Co, Philadelphia, 1902, p. 21] which may be defined as monohydroxy alcohol derivatives of p-menthene, with the generic formula CioH₁₇OH. Combinations of these terpineol parent alcohols are often found together in nature, because it is generally believed that their biosyntheses proceed via closely

15 related synthetic pathways. Without wishing to be bound by theory, it is believed that the mode of antimicrobial action of the derivatives of these terpinyl radicals is similar.

The terpinyl radicals may also be referred to by the alternative names as detailed below in Table 2.

20 Table 2 alpha-terpinyl p-menth-1 -en-8-yl

beta-terpinyl p-menth-8-en-1 -yl

gamma-terpinyl p-menth-4(8)-en-1 -yl

delta-terpinyl p-menth-1 (7)-en-8-yl

4-terpinyl p-menth-1 -en-4-yl

p-menth-1 -en-9-yl p-menth-1 -en-9-yl dihydrocarvyl p-menth-8-en-2-yl

isopulegyl p-menth-8-en-3-yl

In nature, the isomeric compounds alpha-terpineol, beta-terpineol and gamma-terpineol are among the most abundant terpineols and often occur together in mixtures.

Similarly, there is a relatively good availability of (mutually isomeric) alpha-, beta- and gamma-terpinyl derivatives and mixtures thereof. Therefore, preferably, the terpinyl derivative is selected form alpha-terpinyl , beta-terpinyl, and gamma-terpinyl derivatives, and mixtures thereof. Among these, alpha-terpinyl derivatives are often the most abundant compounds. Hence, the terpinyl derivative of the present invention is even more preferably selected from alpha-terpinyl derivatives and mixtures thereof. Particularly preferred are mixtures of alpha-terpinyl , beta-terpinyl, and gamma-terpinyl derivatives.

The terpinyl derivative in the antimicrobial composition according to the invention is selected from terpinyl esters and amides. That is, terpinyl derivatives according to the structures in Table 1 above, wherein X is NH or O. Preferably, the terpinyl derivative is a terpinyl ester (i.e. X = O).

Terpinyl esters are preferred, since they commonly occur in nature or may be readily synthesized from the relevant parent terpinyl alcohol and acid. Additionally, such terpinyl esters may possess pleasant sensory properties.

Alternatively, the terpinyl derivative is a terpinyl amide (i.e. X = NH). Terpinyl amides advantageously are less hydrolytically sensitive than terpinyl esters. In case terpinyl esters are selected, the terpinyl derivative is preferably selected from alpha-terpinyl esters, beta-terpinyl esters, and gamma-terpinyl esters, and mixtures thereof. More preferably, the terpinyl ester is an alpha-terpinyl ester, due to the ready commercial availability of the parent alpha-terpineol and some alpha-terpinyl esters as fine chemicals. The terpinyl ester preferably is a mixture of terpinyl esters, more preferably a mixture of alpha-terpinyl esters, beta-terpinyl esters, and gamma-terpinyl esters. Ri substituents

The antimicrobial composition according to the invention comprises a terpinyl derivative selected from the general structures in Table 1 above, wherein R-i is

a linear or branched C₂ to Ci₇ alkyl group, or

a linear or branched substituted C₂ to Ci₇ alkyl group, or

a linear or branched C₂ to Ci₇ alkenyl group, or

a linear or branched C₂ to Ci₇ substituted alkenyl group, or

R₂OR₃, wherein R₂ is a linear or branched C_x alkyl group and R₃ is a linear or branched C_y alkyl group, such that x + y is between and including 2 and 17, or an alkyl ethoxylate (CH₂)_x(OC₂H₄)_yOH group, wherein x is between 1 and 10, and y is between 1 and 10, or

an aliphatic C₅ to C₇ ring, or

a substituted aliphatic C₅ to C₇ ring, or

an unsaturated C₅ to C₇ ring, or

a substituted unsaturated C₅ to C₇ ring, or

an aryl group, or

a substituted aryl group, or

an aryloxy group, or

a substituted aryloxy group;

and mixtures of these terpinyl derivatives.

In case Ri is selected to include a substituted group or substituted ring, the

substituents can be any substituent known to the skilled person, for example halogens, hydroxyl groups, ethers, carboxylic acids, carboxylate salts, esters, amines, amides, ammonium salts, nitriles, nitrates, thiols, thioethers, sulfones, sulfates, phosphones, or phosphates. Particularly preferred substituted terpinyl derivatives are bifunctional derivatives, such as -for example- 1 ,4-bis-(alpha-terpinyl)-succinate or 1 ,6-bis-(alpha- terpinyl)-adipamide. An advantage of the terpinyl derivatives in which R-i is an alkyl group is their synthetic availability. Terpinyl derivatives in which Ri is selected from alkyl groups, aliphatic rings, aryl groups and aryloxy groups according to the invention, may advantageously provide increased organosolubility, which may be preferred in certain compositions according to the invention. Conversely, the alkyl ethoxylate (CH₂)_x(OC₂H₄)_yOH substituents may be preferred as Ri if increased water-solubility is preferred. Preferably, Ri is

a linear or branched C₂ to Ci₇ alkyl group, or

a linear or branched C₂ to Ci₇ alkenyl group, or

R2OR₃, wherein R₂ is a linear or branched C_x alkyl group and R₃ is a linear or branched C_y alkyl group, such that x + y is between and including 2 and 17, or an aliphatic C₅ to C₇ ring, or

an unsaturated C₅ to C₇ ring, or

an aryl group, or

an aryloxy group;

wherein R-i is preferably unsubstituted.

More preferably, Ri is a linear or branched C₂ to Ci₇ alkyl group, or an aliphatic C₅ to

C₇ ring, or an aryl group.

In case the carrier or the dissolution medium during later application is water-based, it may be advantageous if the terpinyl derivative is sufficiently water-soluble. The terpinyl derivative is sufficiently water-soluble if it is soluble at at least the minimum

concentration required in the antimicrobial composition according to the invention.

Therefore, even more preferably, Ri is a linear or branched C₂ to C12 alkyl group, or an aliphatic C₅ to C₇ ring, or an aryl group. Still more preferably Ri is a linear or branched

C₂ to C₄ alkyl group, or an aliphatic C₆ ring, or an aryl group.

Regarding the terpinyl derivatives of the present invention, it is particularly preferred that a preferred terpinyl radical is combined with a preferred X group and a preferred Ri group. Thus -for example- preferable terpinyl derivatives may be selected from alpha- terpinyl esters, beta-terpinylesters, and gamma-terpinyl esters, and mixtures thereof, wherein R-i is a linear or branched C₂ to Ci₇ alkyl group, or a linear or branched C₂ to Ci₇ alkenyl group, or R₂OR₃, wherein R₂ is a linear or branched C_x alkyl group and R₃ is a linear or branched C_y alkyl group, such that x + y is between and including 2 and 17, or an aliphatic C₅ to C₇ ring, or an unsaturated C₅ to C₇ ring, or an aryl group, or an aryloxy group.

Thus, it is even more preferred that the terpinyl derivative is selected from alpha- terpinyl propionate, alpha-terpinyl butyrate, alpha-terpinyl iso-butyrate, alpha-terpinyl cyclohexanoate, alpha-terpinyl valerate, alpha-terpinyl caproate, alpha-terpinyl enanthate, alpha-terpinyl caprylate, alpha-terpinyl caprate, and alpha-terpinyl laurate, and mixtures thereof.

It is similarly preferred that the terpinyl derivative is selected from mixtures of alpha- terpinyl esters, beta-terpinyl esters and gamma-terpinyl esters, wherein the esters are selected from propionate esters, butyrate esters, iso-butyrate esters, cyclohexanoate esters, valerate esters, caproate esters, enanthate esters, caprylate exters, caprate esters, and laurate esters and mixtures thereof. Alternatively, it may also be preferred that the terpinyl derivative is selected from alpha- terpinyl isovalerate, alpha-terpinyl 2-methyl-butyrate, alpha-terpinyl nonanoate, alpha- terpinyl benzoate, alpha-terpinyl anthranilate, alpha-terpinyl cinnamate, and palmitate.

Alternatively, it may also be preferred that the terpinyl derivative is selected from beta- terpinyl propionate, cis-beta-terpinyl propionate, trans-beta-terpinyl propionate, beta- terpinyl butyrate, cis-beta-terpinyl butyrate, trans-beta-terpinyl butyrate, beta-terpinyl isobutyrate, cis-beta-terpinyl valerate, trans-beta-terpinyl vlalerate, beta-terpinyl isovalerate, trans-beta-terpinyl benzoate and cis-beta-terpinyl benzoate. Alternatively, it may also be preferred that the terpinyl derivative is selected from gamma-terpinyl propionate, gamma-terpinyl butyrate and gamma-terpinyl valerate.

Alternatively, it may also be preferred that the terpinyl derivative is selected from delta- terpinyl propionate, 4-terpinyl propionate, p-menth-1 -en-9-yl propionate, dihydrocarvyl propionate, isopulegyl propionate, and isopulegyl isobutyrate.

It is particularly preferred that the terpinyl derivative is selected from

alpha-terpinyl propionate,

alpha-terpinyl butyrate,

alpha-terpinyl iso-butyrate,

alpha-terpinyl cyclohexanoate,

and mixtures thereof.

It is similarly preferred that the terpinyl derivative is selected from mixtures of alpha- terpinyl esters, beta-terpinyl esters and gamma-terpinyl esters, wherein the esters are selected from propionate esters, butyrate esters, iso-butyrate esters, cyclohexanoate esters, and mixtures thereof.

Alternatively, preferred terpinyl derivatives may be selected, which are amides.

Preferred amides are (S)-alpha-terpinyl propanamide, alpha-terpinyl-butanamide, alpha-terpinyl 2-methylpropanamide, alpha-terpinyl 2-benzamide, beta-terpinyl propanamide, gamma-terpinyl propanamide, and delta-terpinyl propanamide.

Mixtures of preferred terpinyl derivatives are also preferred.

Suitable terpinyl derivatives according to the present invention may be sourced from biological resources. For example, alpha-terpinyl esters where Ri is a C₂ or C₃ substituent, commonly occur in nature, often in combination with alpha-terpineol.

Alpha-terpinyl esters are used as fragrance and flavour components [T B Adams, J B Hallaghan, J M Putnam, T L Gierke, J Doull, I C Munnro, P Newberne, P S Portoghese, R L Smith, B M Wagner, C S Weil, L A Woods and R A Ford, Food and Chemical Technology, Volume 34, 1996, pp. 763-828], [K Bauer, D Garbe, H Surburg, "Common Fragrance and Flavor Materials", Wiley VCH, Weinheim, 1997, page 69] and several are GRAS (Generally Regarded As Safe) materials. For instance, alpha-terpinyl butyrate provides balsamic notes as described in US 2004/0242452.

Alternatively, suitable terpinyl derivatives according to the present invention may be obtained via synthetic chemical methods. Such methods are generally well-known. For example, terpinyl esters may be obtained from the parent terpinyl alcohol, by esterification methods as described in literature, e.g. [C Wiles, P Watts, S J Haswell and E Pombo-Villar, Tetrahedron 59, 10173 (2003)]

Carrier

The antimicrobial composition according to the invention comprises a carrier. The carrier is preferably selected from the group consisting of water, oil, solvent, inorganic particulate material, starch, air and mixtures thereof. The carrier is preferably from 0.1 to 99% by weight of the composition. The antimicrobial composition may be in form of a solid, liquid, gel, paste or soft solid and the carrier may be selected by a person skilled in the art depending on the format of the antimicrobial composition. Examples of inorganic particulate materials include clay, talc, calcite, dolomite, silica, and aluminosilicate. Examples of oils include mineral oils, oils of biological origin (e.g. vegetable oils), and petroleum-derived oils and waxes. The oils of biological origin are preferably triglyceride-based. Preferably, the carrier oil is not a perfume oil. Thus, the carrier oil preferably does not substantially contribute to the odour of the composition, more preferably it does not contribute to that odour. Examples of solvents include alcohols, ethers and acetone. The starch may be natural starch obtained from food grains or may be a modified starch. Air can for instance be used as a carrier when the terpinyl derivatives according to the invention and/or the thymol are atomised or otherwise dispersed as a fine mist.

Particularly preferred carriers are water or oil/solvent and even more preferred is a carrier that is a mixture of water and oil. Thus, in many of the envisaged applications like personal care/washing, oral care and hard surface cleaning, the antimicrobial composition may be formulated with either an aqueous base or a oil/solvent base. Compositions with an aqueous base (water being the carrier), may also for instance be products in gel format. Compositions with a purely oil/solvent base may for instance be products in anhydrous stick form or propellant-containing products.

Thus, the antimicrobial composition may for instance, preferably be an antimicrobial anhydrous stick personal care composition on a purely oil/solvent base wherein the composition has a water content of less than 0.01 % by weight, and wherein the composition preferably is free of water. Alternatively, the antimicrobial composition may for instance, preferably be an antimicrobial propellant-drivable personal care composition, also comprising a propellant. Air can also be used as propellant, for instance in the form of compressed or liquefied air.

However, the most preferred product format has an emulsion base (water and/or oil being the carrier) or is capable of forming an emulsion upon dilution, e.g. soap products in liquid, solid, lotion or semisolid form for hand wash, face wash, body wash, or shaving applications; toothpaste/ dentifrices for oral care applications or products for hard surface cleaning in bars or liquids form. If the product comprises an emulsion base, it preferably also comprises one or more surfactants as described below. Surfactants

The antimicrobial composition according to the invention preferably comprises from 1 to 80% by weight of surfactant. Surfactants may for instance advantageously contribute to the cleaning efficacy or the formulation stability of a composition.

Thus, the antimicrobial composition according to the invention preferably comprises a. 0.001 to 5% by weight of thymol,

b. 0.001 to 5% by weight of the terpinyl derivative according to the invention, c. a carrier, and

d. from 1 to 80% by weight surfactant.

It is particularly preferred that the antimicrobial composition comprises from 1 to 80% by weight of surfactant in combination with the one or more terpinyl derivatives, and the thymol at their more preferred concentrations as specified above.

In general, the surfactants may be chosen from the surfactants described in well-known textbooks like "Surface Active Agents" Vol. 1 , by Schwartz & Perry, Interscience 1949, Vol. 2 by Schwartz, Perry & Berch, Interscience 1958, and/or the current edition of "McCutcheon's Emulsifiers and Detergents" published by Manufacturing Confectioners Company or in "Tenside-Taschenbuch", H. Stache, 2nd Edn., Carl Hauser Verlag, 1981. Any type of surfactant, i.e. anionic, cationic, nonionic, zwitterionic or amphoteric can be used. Preferably, the surfactant is anionic, nonionic, or a mixture of anionic and nonionic surfactants. More preferably, the surfactant is anionic. A particularly preferred surfactant is soap. Soap is a suitable surfactant for personal washing applications of the antimicrobial composition of the invention. The soap is preferably C₈-C₂4 soap, more preferably a Ci₀-C₂o soap and most preferably Ci₂-Ci₆ soap. The soap may or may not have one or more carbon-carbon double bonds or triple bonds. The cation of the soap can for instance be an alkali metal, alkaline earth metal or ammonium. Preferably, the cation of the soap is selected from sodium, potassium or ammonium. More preferably the cation of the soap is sodium or potassium.

The soap may be obtained by saponifying a fat and/or a fatty acid. The fats or oils may be fats or oils generally used in soap manufacture, such as tallow, tallow stearines, palm oil, palm stearines, soya bean oil, fish oil, castor oil, rice bran oil, sunflower oil, coconut oil, babassu oil, palm kernel oil, and others. In the above process the fatty acids are derived from oils/fats selected from coconut, rice bran, groundnut, tallow, palm, palm kernel, cotton seed, soyabean, castor etc. The fatty acid soaps can also be synthetically prepared (e.g. by the oxidation of petroleum or by the hydrogenation of carbon monoxide by the Fischer-Tropsch process). Resin acids, such as those present in tall oil, may be used. Naphthenic acids are also suitable.

Tallow fatty acids can be derived from various animal sources and generally comprise about 1 to 8 wt-% myristic acid, about 21 to 32 wt-% palmitic acid, about 14 to 31 wt-% stearic acid, about 0 to 4 wt-% palmitoleic acid, about 36 to 50 wt-% oleic acid and about 0 to 5 wt-% linoleic acid. A typical distribution is 2.5 wt-% myristic acid, 29 wt-% palmitic acid, 23 wt-% stearic acid, 2 wt-% palmitoleic acid, 41.5 wt-% oleic acid, and 3 wt-% linoleic acid. Other similar mixtures, such as those from palm oil and those derived from various animal tallow and lard are also included.

Coconut oil refers to fatty acid mixtures having an approximate carbon chain length distribution of 8 wt-% C₈, 7 wt-% Ci₀, 48 wt-% C_i2, 17 wt-% C_i4, 8 wt-% Ci₆, 2 wt-% Ci₈, 7 wt-% oleic and 2 wt-% linoleic acids (the first six fatty acids listed being saturated). Other sources having similar carbon chain length distributions, such as palm kernel oil and babassu kernel oil, are included within the term coconut oil.

A typical fatty acid blend consists of 5 to 30 wt-% coconut fatty acids and 70 to 95 wt-% fatty acids ex hardened rice bran oil. Fatty acids derived from other suitable oils/fats such as groundnut, soybean, tallow, palm, palm kernel, etc. may also be used in other desired proportions. The soap, when present in solid forms of the present invention, is preferably present in an amount of 30 to 80%, more preferably from 50 to 80%, and even more preferably 55 to 75% by weight of the composition. The soap, when present in liquid forms of the composition is preferably present in 0.5 to 20%, more preferably from 1 to 10% by weight of the composition.

Other preferred surfactants are fatty acid glycinates and fatty amphocarboxylates. The fatty acid glycinates are fatty acid amides of salts of glycine, including for example sodium cocoyl glycinate. The fatty amphocarboxylates are amphoteric surfactants including for example sodium lauroamphoacetate (i.e. sodium 2-[1 -(2-hydroxyethyl)-2- undecyl-4,5-dihydroimidazol-1 -ium-1 -yl]acetate). Yet another example of suitable surfactants are derivatives of isethionates, including acylisethionates.

The antimicrobial composition of the invention is also useful in hard surface cleaning applications. In such applications, preferred surfactants are nonionic surfactants, such as C₈-C₂2, preferably C₈-Ci₆ fatty alcohol ethoxylates, comprising between 1 and 8 ethylene oxide groups when the product is in the liquid form. When the product for hard surface cleaning applications is in the solid form, surfactants are preferably selected from primary alkyl sulphates, secondary alkyl sulphonates, alkyl benzene sulphonates, ethoxylated alkyl sulphates, or alcohol ethoxylate nonionic surfactants. The composition may further comprise an anionic surfactant, such as alkyl ether sulphate preferably those having between 1 and 3 ethylene oxide groups, either from natural or synthetic source and/or sulphonic acid. Especially preferred are sodium lauryl ether sulphates. Alkyl polyglucoside may also be present in the composition, preferably those having a carbon chain length between C6 and C16. Other classes of useful surfactants include cationic surfactants, such as long chain quaternary ammonium compounds and amphoteric surfactants such as betaines and alkyl dimethyl amine oxides. Suitable surfactant concentrations in liquid forms of hard surface cleaning application are generally from about from 0.5 to 10%, preferably from 1 to 5 % by weight of the composition. In solid compositions, surfactant is preferably present in 5 to 40%, preferably from 10 to 30% by weight of the composition.

The antimicrobial composition of the invention is useful in oral care compositions e.g. in a dentifrice/ toothpaste or an oral rinse product. In such applications, preferred surfactants are anionic, nonionic or amphoteric in nature, preferably anionic or amphoteric. The anionic surfactant is preferably an alkali metal alkyl sulphate, more preferably a sodium lauryl sulphate (SLS). Mixtures of anionic surfactants may also be employed. The amphoteric surfactant is preferably a betaine, more preferably an alkylamidopropyl betaine (wherein the alkyl group is a linear Ci₀-Ci₈ chain), and most preferably is cocoamidopropyl betaine (CAPB). Mixtures of amphoteric surfactants may also be employed. Suitable surfactant concentrations in oral care application are generally from about 2% to about 15%, preferably from about 2.2% to about 10%, more preferably from about 2.5 to about 5% by weight of the total composition. Thus, it is highly preferred that the antimicrobial compositions include soap, alkyl sulphate or linear alkyl benzene sulphonate as the surfactants. More preferably, the surfactant is a soap, an alkyl sulphate or a linear alkyl benzene sulphonate. Liquid and solid compositions

The antimicrobial composition may be in form of a solid, a liquid, a gel or a paste. A person skilled in the art can prepare compositions in various formats by choosing one or more carrier materials and/or surfactant. The antimicrobial compositions of the present invention are useful for cleansing and care, in particular for skin cleansing and skin care. It is envisaged that the antimicrobial composition can be used as a leave-on product or a wash-off product, preferably a wash-off product. The antimicrobial composition of the present invention can also be used for cleansing and care of hard surfaces such as glass, metal, plastic and the like. A particularly preferred carrier is water. When water is the carrier, both liquid and solid compositions are possible. Different amounts of water may be preferred depending on the product format. When water is present, it is preferably present in at least 1 %, more preferably at least 2%, further more preferably at least 5% by weight of the

composition. When water is the carrier, a preferred liquid antimicrobial composition according to the invention comprises:

a. 0.01 to 5% by weight of the thymol,

b. 0.05 to 5% by weight of the terpinyl derivative

c. 10 to 99.9% by weight of water, and;

d. 1 to 30% by weight of surfactant.

The liquid antimicrobial composition is useful for skin cleansing, in particular for hand wash or a face wash.

When water is the carrier, a preferred solid antimicrobial composition according to the invention comprises:

a. 0.05 to 5% by weight of the thymol,

b. 0.05 to 5% by weight of the terpinyl derivative,

c. 5 to 30% by weight of water, and;

d. 30 to 80% by weight of surfactant. The solid antimicrobial composition is preferably in form of a shaped solid, more preferably a bar. The solid antimicrobial composition is particularly useful for skin cleansing in particular for hand wash or a face wash. Such a bar-shaped solid antimicrobial composition may for instance be a soap bar. Soap bar compositions are well-known and may be similar to the following non-limiting example composition, comprising 75.6 wt-% of anhydrous sodium soap, 1 .0 wt-% of glycerine, 0.5 wt-% of sodium carbonate, 0.2 wt-% of EHDP (ethane-1 -hydroxy-1 ,1 - disphosphonate) acid, 0.04 wt-% of EDTA (ethylenediaminetetraacetic acid) tetrasodium salt, 8.5 wt-% of hydrated magnesium silicate (Talc), 0.7 wt-% of sodium chloride, 0.05 wt-% of dyes, 0.75 wt-% perfume, 0.05 to 10 wt-% of antimicrobial agents, and water up to 100 wt-%.

Alternatively, inorganic particulate material is also a suitable carrier. When inorganic particulate material is the carrier, the antimicrobial composition is in a solid form. Preferably the inorganic particulate material is talc. When the inorganic particulate material is talc, the solid antimicrobial composition is particularly useful as a talcum powder for application on face or body. According to another alternative, a solvent (different from water ) is a preferred carrier. Although any solvent can be used, alcohol is a preferred solvent. Short chain alcohols -in particular ethanol, propanol, and isopropanol- are particularly preferred as carrier for an antimicrobial wipe or an antimicrobial hand sanitiser composition. Solvents like ethanol and isopropanol generally show antimicrobial efficacy

themselves. However, they are also volatile and may readily evaporate during application of the composition. Thus, their levels on the surface that is treated might even reduce until below the minimum level required for antimicrobial action, before the minimum period needed for disinfection has passed. In contrast, the thymol and the terpinyl derivatives according to the present invention are much less volatile and may therefore yield prolonged antimicrobial action after applying them to the skin.

Additional ingredients

The composition may further comprise various additional ingredients known to a person skilled in the art. Such additional ingredients include but are not limited to: perfumes, pigments, preservative, emollients, sunscreens, emulsifiers, gelling agents, thickening agents, humectants (e.g. glycerine, sorbitol), sequestrants (e.g. EDTA) or polymers (e.g. cellulose derivatives for structuring such as methyl cellulose) Thymol and some of the terpinyl derivatives according to the invention are known for their olfactory properties, and have been applied for instance in perfume compositions. However, the present invention is directed towards antimicrobial compositions.

Therefore, the composition is preferably not a perfume composition. Here, a perfume composition is defined as a composition comprising a plurality of olfactory components, where these components are solely intended to provide the composition with a harmonious scent.

Effect of the invention

The inventors have surprisingly found that while thymol alone and terpineol alone do not individually provide the fast antimicrobial kinetic action, a combination of thymol and terpineol at the selective concentrations provides a synergistic antimicrobial action which is especially important in a wash off processes where the contact time of the antimicrobial actives with the surface is low, i.e. of the order of less than 5 minutes, preferably less than 2 minutes, further more preferably less than a minute and in many cases less than 15 seconds. Favourably, such wash off processes include a surfactant for the cleaning action. To the further surprise of the inventors, while the surfactant alone does not provide the fast antimicrobial kill at the concentration present in wash off processes, it provides for further improvement in extent of reduction in microbial counts on the surface in the short period of time when surfaces are washed with a composition comprising a terpinyl derivative, thymol and additionally surfactant. Thus, while on the one hand surfactant is generally known to be responsible for washing off dirt and also antimicrobial actives used in the composition, in the present invention, it provides a highly useful additional benefit in that it enhances the reduction of microbial count in a composition comprising a combination of thymol and a terpinyl derivative alone.

Synergistic combinations of thymol and terpinyl analogues

Thymol and selected terpinyl derivatives may be capable of antimicrobial action when used in isolation. However, we have now surprisingly found that compositions according to the invention, comprising a combination of thymol and terpinyl derivatives are capable of synergistic antimicrobial action.

The antimicrobial action of two or more active compounds is considered additive if the combined action merely results from the addition of the effects the individual components would have in isolation. In contrast, the antimicrobial action of two or more active compounds is considered to be synergistic if the combined effect of the two or more compounds is stronger than expected based on the assumption of additivity. Without wishing to be bound by theory, it is believed that the antimicrobial action of the one compound may be enhanced by the action of the other compound and vice versa. Such enhanced antimicrobial action may manifest itself for instance by the fact that lower concentrations of active compounds are required to obtain complete microbial kill, or alternatively, that the same extent of microbial kill is arrived at in a shorter time. Whether an antimicrobial composition comprising two or more active compounds is capable of synergistic antimicrobial action may for instance be determined following the procedures and using the criteria as outlined in Example 2 below. Typically, evidence of synergistic antimicrobial action may be provided at concentrations below the minimum biocidal concentrations of each of the components when taken individually. However, it is generally believed that synergistic action can still occur when the concentration of one or more of the active compounds is raised above its minimum biocidal concentration (when taken individually).

The antimicrobial composition according to the present invention preferably comprises thymol and a terpinyl derivative according to the invention at concentrations at which they are capable of synergistic antimicrobial action. Thus, the concentrations of the thymol and of the terpinyl derivative in the antimicrobial composition are preferably such that, when the composition is diluted or dissolved with a suitable medium during use, (e.g. when washing hands with water and a composition according to the invention) the concentration in the diluted or dissolved mixture is still sufficient to be antimicrobially efficacious. That is, to be capable of synergistic antimicrobial action, the concentrations of the thymol and the terpinyl derivative in the composition (C_COmp, thymol, and Ccom , ter , respectively) are preferably such that upon application, at a given concentration of one of the thymol and the terpinyl derivative in the application medium (Cmed, thymol, or C_med, terp), the other component is available at at least a minimum concentration. Here, the application medium denotes the medium in which the antimicrobial action desirably takes place. For example, in personal care applications like hand-washing, the composition may be a solid soap bar. In that case, C_COmp refers to the concentration of the component in the soap bar, whereas C_meci refers to the concentration in the lather. The optimum concentrations may for instance be determined by a protocol as described for the examples below. It is generally preferred that the concentrations of the thymol and the terpinyl derivative in the composition according to the invention are equal to or higher than the optimal concentrations in the application medium, because in many typical applications, the composition is either used pure or is diluted to form the application medium.

Thus, when for instance antimicrobial action against E. Coli is desired, the data of Example 2 may be used to determine preferable medium concentrations C_meci- For example, if the terpinyl derivative is alpha-terpinyl propionate, and C_meci, thymol is selected as 0.025 %(w/v), C_med, ter_P preferably is at least 0.1 %(w/v) or vice versa. Similarly, if Cmed.ter is selected as 0.15 %(w/v), C_meci, th_ym is preferably at least 0.0125 %(w/v) or vice versa.

Alternatively, the desired antimicrobial effect may be obtained by the selecting a ratio of the respective concentrations of the thymol and the terpinyl derivative. Thus, the antimicrobial composition according to the invention preferably comprises the thymol and the terpinyl derivative in a concentration ratio (thymohterpinyl) of between 1 :2 and 1 :12, wherein the concentration is expressed as weight percent.

A further additional advantage of the present invention is that it is observed that treatment of a surface with a composition comprising a terpinyl derivative and thymol, surprisingly enable continued protection of the surface against growth of microbes for a substantial period of time thereafter.

Effect of including surfactant

Favourably, compositions suitable in wash-off processes as described above include a surfactant for the cleaning action. To the further surprise of the inventors, while the surfactant alone does not provide the fast antimicrobial kill at the concentration present in wash off processes, it provides for further improvement in extent of reduction in viable microbial counts on the surface in the short period of time when surfaces are washed with a composition comprising the terpinyl derivate, thymol and additionally surfactant. Thus, while surfactant is generally known to be responsible for washing off dirt and also antimicrobial actives used in the composition, in the present invention, it provides a highly useful additional benefit in that it enhances the reduction of viable microbial count in a composition comprising a combination of the terpinyl derivative and thymol alone.

Method according to the invention

According to the second aspect, the invention relates to a method of disinfecting a surface comprising the steps of

a. applying a composition according to the invention on to the surface; and b. rinsing the surface with a suitable solvent or wiping the surface with a suitable wipe.

Preferably, the surface is skin. Thus, for example, a surface like the hands, face, body, or the oral cavity is contacted with the composition of the invention. Alternatively, the surface is any hard surface. Typically, such hard surfaces are surfaces that commonly require cleaning and preferably also require sanitisation or disinfection. Such surfaces may be found in many household or industrial environments, and may include for example kitchen and bathroom surfaces, table tops, floors, walls, windows, utensils, cutlery, and crockery. Such surfaces may be made from many different materials, including for instance plastics, wood, metal, ceramics, glass, concrete, marble, and painted surfaces.

The composition may be applied to the surface by any suitable means known to the skilled person. For instance, a suitable means may be pouring, dropping, spraying or wiping in case of liquid compositions.

Preferably, the method includes diluting or dissolving the composition with a suitable solvent, preferably water, before or whilst applying the composition to the surface. The method according to the invention also includes the step of rinsing the surface with a suitable solvent or wiping the surface with a suitable wipe. This step serves to remove the composition from the surface. Here, removing the composition also encompasses partially removing the composition, because traces of the composition may remain on the surface. In many typical situations, including washing of the skin or hard-surface cleaning, it is acceptable or sometimes even desirable if part of the composition - in particular certain active ingredients - remains on the surface.

Therefore, this step preferably involves removing at least 5%, more preferably at least 10%, even more preferably at least 25%, still more preferably at least 50% and yet more preferably at least 75% of the composition by weight.

The solvent for rinsing the surface is preferably water but could also be for example a mixture of water and alcohol. It is then rinsed preferably with sufficient amounts of water after a pre-determined period of time to remove any visible or sensory residue of the composition. Alternately an alcohol wipe or a water/alcohol impregnated wipe may be used to wipe the surface to be visibly free of the anti-microbial composition. The step of rinsing the substrate is preferably carried out less than 5 minutes, more preferably less than 2 minutes, further more preferably less than a minute and in many cases even more preferably less than 15 seconds after the step of applying the composition on the substrate. Even though partial microbial kill may be almost instantaneous upon application of the composition according to the invention, it is preferred that the step of removing the composition from the surface is started out at least 5 seconds, preferably at least 10 seconds, more preferably at least 15 seconds after commencement of the step of applying the composition on the surface, in order to effect optimal antimicrobial action. Combinations of these times into time intervals are preferred too. Therefore, it is particularly preferred that the step of removing the composition from the surface (i.e. step b) is started between 2 minutes and 5 seconds, more preferably between 1 minute and 10 seconds, even more preferably between 30 and 10 seconds and still more preferably between 20 and 15 seconds after commencement of the step of applying the composition on the surface (i.e. step a).

Disinfection time

These times between applying the composition and rinsing or wiping are preferably related to the disinfection time, in order to ensure optimal cleansing and sanitising of the surface. Therefore, the invention preferably relates to a method, wherein the disinfection time T is less than 300 seconds, preferably less than 60 seconds, and more preferably less than 15 seconds; wherein T is defined as the time that elapses from the moment of adding the composition to a microbial culture until the number of microbes per unit volume of the culture is reduced by a factor of 100 000; and wherein the initial number of microbes preferably exceeds about 100 000 000 microbes per millilitre and wherein the composition is preferably a liquid composition. The disinfecting action (which can be expressed in terms of the disinfection time T) of the method is preferably determined according to the protocol of Example 2 as described hereinafter. This test relates to a standardised test environment in which the microbial culture is kept in suspension. Alternatively, one of the test methods as described in WO 2010/046238 may for instance be applied to establish the disinfecting action.

Such test methods may preferably also be used by the skilled person to determine the optimal concentrations of the thymol and the terpinyl derivative in an antimicrobial composition according to the present invention.

Alternatively, since the method is directed towards surface disinfection, the disinfection time may also be determined by test methods involving a surface. Therefore, the invention preferably relates to a method according to the present invention, wherein the surface disinfection time T2 is less than 60 seconds, preferably less than 15 seconds, wherein T2 is defined as the time starting from the moment of applying the composition to the surface to be disinfected after which the number of microbes per unit area is reduced by a factor of 10000 (i.e. a 4 log reduction), wherein the initial number of microbes preferably exceeds 10³, more preferably 10⁵, and even more preferably 10⁷ microbes per square centimetre. Such tests may for instance be performed as described in WO 2010/046238, or as described in European Standards EN 13697:2001 and EN 1500:1997.

Use according to the invention

The invention preferably provides for non-therapeutic benefits. Thus, for instance, the invention relates to use of an antimicrobial composition according to the present invention for faster reduction in microbial count.

Thus, there is provided use of an antimicrobial composition according to the invention for improved hygiene of surfaces of the human body. Such surfaces include e.g. skin, hands and the oral cavity. According to a preferred aspect, the invention relates to use of a composition according to the invention for improved hand hygiene. According to another preferred aspect, the invention relates to use of a composition according to the invention for improved oral hygiene. EXAMPLES

The invention is illustrated by the following non-limiting examples.

Example 1 : Synthesis of alpha-terpinyl esters

Thymol, alpha-terpineol and alpha-terpinyl acetate were purchased as fine chemicals from Sigma Aldrich.

Other esters were synthesised by reaction of the alpha-terpineol alcohol with the appropriate acid chloride using a literature method [C Wiles, P Watts, S J Haswell and E Pombo-Villar, Tetrahedron 59, 10173 (2003)]. Alternatively, esters were similarly synthesised starting from a commercial mixture of terpinyl esters, comprising about 70 wt-% of alpha-terpineol, 4 wt-% of beta-terpineol and 26 wt-% of gamma-terpineol.

Alpha-terpineol (5 g, 32 mmol) was dissolved in dry THF (40 ml) and to it was added a suspension of sodium hydride (60% dispersion in oil, 1.44g, 36 mmol) in dry THF (20 ml). The mixture was then stirred at room temperature under an atmosphere of argon for 15 minutes before addition of the acid chloride (1 .4 equivalents, 45 mmol) and then stirred at room temperature under argon for 2-3 hours. After this period, the reaction was quenched by dropwise addition of water to the reaction vessel until effervescence ceased. Saturated sodium bicarbonate solution (30 ml) was then added and the aqueous phase was separated and washed with dichloromethane (3 x 50 ml). Organics were then combined, dried using a hydrophobic frit and concentrated to dryness. TLC indicated the presence of impurities so the crude materials were purified by column chromatography, eluting with a gradient of petroleum ether and ethyl acetate (varying from 2 to 10% ethylacetate). Relevant fractions were identified by TLC, combined and concentrated to dryness yielding the desired products whose structures were confirmed by NMR.

Example 2: Assessment of antimicrobial efficacy

General method for assessment of antimicrobial synergy

The efficacies of antimicrobial agents can be usefully compared by determining the Minimum Biocidal Concentration (MBC). The MBC is defined as the lowest absolute concentration of the particular active that provides complete kill (zero bacterial growth). The differing behaviours of inhibitory antimicrobials in isolation and mixtures have been widely explored using the concept of the Fractional Concentration and Fractional Inhibitory Concentration (FIC). See for instance JRW Lambert and R Lambert, J. Appl. Microbiol 95, 734 (2003); T. Jadavji, CG Prober and R Cheung, Antimicrobial Agents and Chemotherapy 26, 91 (1984), and WO 2004/006876. These parameters can be defined as follows:

Concentration of component a tested in the mixture

FC (component a) =

MIC (component a tested as a single active) MIC (component a tested in the mixture)

FIC (component a) =

MIC (component a tested as a single active) By analogy the Fractional Biocidal Concentration (FBC) is given by:

MBC (component a tested in the mixture)

FBC (component a) =

MBC (component a tested as a single active)

The interactions between antimicrobials can be additive, synergistic or possibly antagonistic depending on whether the efficacy of the combination is equivalent to, greater than or less than that obtained for the same total concentration of the individual components when tested alone.

These relationships can be expressed mathematically by summing the fractional MBC values for all the components present in the mixture to give the "fractional biocidal index":

∑FBC = FBC(component 1 ) ⁺ FBC(_COmponent 2) ⁺ FBC(_COmponent 3) ⁺■■■ etc

such that

∑FBC≥ 1 corresponds to additive or antagonistic bactericidal activity ∑FBC < 1 corresponds to synergistic bactericidal activity Experimental method

Antimicrobial efficacy is tested against a representative pathogenic bacterial organism, Gram negative Escherichia coli. Concentrations of actives are expressed in terms of the percentage weight/volume (%w/v) throughout Example 2.

5

Bacterial stock

An overnight culture of Escherichia coli (10536 strain) was prepared in 50 ml total volume of TSB broth, grown for ca. 18 hrs at 37°C and shaken at 150 rpm. 1 ml of this overnight E. coli culture was transferred to 50 ml of fresh TSB broth and incubated at

10 37°C at 150 rpm for ca. 4 hours. This culture was separated into equal volumes and centrifuged at 4000 rpm for 15 minutes, washed with sterile saline (0.85% NaCI), centrifuged once more and re-suspended in saline to give a final concentration of 0.8 OD₆2o equivalent to about 10⁸ cells per millilitre for this particular organism. Here, OD₆2o indicates the absorbance of a sample in a cuvette of 1.0 cm path length at a

15 wavelength of 620 nm. This bacterial stock was used for assaying against antimicrobial actives (in triplicate).

Protocol

The following assay describes the testing of 8 materials using 6 dilutions across half of 20 a 96-well micro titre plate (MTP). Using this approach it is possible to assay 16 actives (without replicates) with one full dilution plate, replicating this set up in two halves of the plate columns, 1 -6 and 7-12.

1 M solutions of the test actives were prepared in dimethylsulphoxide (DMSO). Stock 25 solutions of the actives at 1.1 1 times the desired final concentration were prepared by diluting the DMSO solutions in water, so that for example a 0.89% w/v solution was prepared for a desired "in test" concentration of 0.8% w/v in order to allow for the further dilution of the active when the bacterial suspension is added (dilution from 270μΙ to 300 μΙ), as described below.

30

Aliquots (270 μΙ) of the materials at 1 .1 1 times the final concentration were dispensed into the wells of the MTP along one column (A1 -H1 ). This MTP was labelled as the "Screening plate". In another MTP, labelled as the "Dilution plate", 270μΙ of D/E neutralising solution from DIFCO Composition was added to column 1 . The composition of the neutralising solution was as follows: pancreatic digest of casein, 5.0g/L; Yeast Extract, 2.5 g/L; Dextrose, 10 g/L, sodium thioglycollate, 1.0 g/L, sodium thiosulphate, 6.0 g/L; sodium bisulphite, 2.5 g/L; Polysorbate 80, 5.0 g/L; lecithin 7.0 g/L; bromocresol purple, 0.02 g/L with a pH in the range 7.6±0.2.

270μΙ of tryptone diluent solution was added to all the remaining wells of the Dilution MTP (columns 2-6).

Bacterial stock (30 μΙ) was then added to the prepared 270 μΙ of the solution of actives in the Screening Plate and mixed, using a multichannel pipette with 8 tips to aspirate and dispense the same volume of bacterial stock in parallel to 8 wells in rows A-H. After a contact time of 15 seconds, the mixtures were quenched by transferring 30μΙ volumes of the mixtures into the 270μΙ D/E neutralising solution in the prepared dilution plate, using aspiration to mix. After exactly 5 minutes in the D/E neutralising solution, 30μΙ volumes were transferred from column 1 to column 2 of the Dilution MTP and mixed, before transferring further 30μΙ volumes from column 2 into column 3. This process was repeated serially diluting the bacteria across the plate to column 6.

30μΙ volumes from each well in the Dilution MTP were transferred onto pre-labelled segment of Tryptone Soya Agar (TSA) plates starting from the lowest bacterial concentration (highest dilution, column 6) to the highest bacterial concentration (column 1 ). The TSA plates were allowed to stand for ca. 2 hours so that the 30μΙ inocula spots could dry and the plates were then inverted and incubated overnight at 37°C before enumerating the bacterial colonies at the labelled dilutions to determine the effects of the actives on bacterial growth.

Calculation of results

Mean bacterial survival numbers N_MBS (expressed in Log CFU/ml) are obtained by first determining the segment of the TSA plate where the number of bacterial colonies is countable. From the colony number in this segment, N_MBS is calculated by the formula:

N_MBs = log{ Ncoi - 10^DF - 100 / 3 }

Here, N_C0| is the colony count, and DF is the dilution factor taken from the MTP-well corresponding to the TSA plate segment (i.e. DF may range from 1 for the quench, to 6 for the highest dilution). The factor 100/3 is a conversion factor from the volume of the inocula spot to one millilitre.

Every assay test was performed in triplicate. The reported mean bacterial survival results are the average of such a triplet, the error is the corresponding standard deviation.

Thus, a value of N_MBS of about 7 corresponds to a count of about 3 colonies from the fifth dilution well, i.e. with DF = 5. Such a count of about 7 is generally observed when bacteria are exposed to non-biocidal materials. In case no surviving colonies are observed in any segment of the TSA plate, this is interpreted as complete kill and a value of N_MBS=0 is reported.

Validation

All test results were validated by running every test assay in parallel with four control experiments on the same MTP. All control experiments are executed exactly according to the above protocol, but with the following active ingredients:

A 0.025 %(w/v) thymol

B 0.15 %(w/v) alpha-terpineol

C 0.025 %(w/v) thymol + 0.15 %(w/v) alpha-terpineol

D no active component

The control experiments A, B and D validate a test assay by not showing bacterial kill, whereas control experiment C, comprising a synergistic combination of thymol and alpha-terpineol according to WO 2010/046238 A1 validates a test assay by showing complete bacterial kill. A reference experiment according to the above protocol, but without active component, showed that DMSO does not affect bacterial growth at the concentrations present in the test solutions in this protocol ( <5 % (w/v) ), as can be seen in Table 3.

Table 3

DMSO in water Mean bacterial survival Standard

(% w/v) [log CFU/ml] deviation

4.5 8.2 0.1

3.6 8.4 0.2

2.7 8.2 0.1

1 .8 8.5 0.2 0.9 8.6 0.1

0.0 8.5 0.1

Results

The above method was applied to asses the antibacterial efficacy of terpinyl esters. In each of the below examples, the terpinyl ester, as prepared from the above described mixture of terpineols, comprised about 70 wt-% of alpha-terpinyl ester, 4 wt-% of beta- terpinyl ester, and 26 wt-% of gamma-terpinyl ester, all with the same acid residue. Therefore, in the results presented below, the mixtures are identified by their main constituent, the alpha-terpinyl ester. The reported concentrations refer to the total of the terpinyl esters. Table 4 shows the antibacterial activities of the terpinyl esters, both alone and in conjunction with thymol.

Table 4: Antibacterial activities of alpha-Terpineol and alpha-Terpinyl esters alone and in combination with thymol

Example Thymol Concentration of NMBS Standard concentration terpineol derivative [log deviation C_THYMOL(% W/V) CTERP(% W/V) CFU/ml]

2:1 ^* 0.075 0 0 0

2:2^* 0.05 0 0 0

2:3^* 0.025 0 > 7 0.1

2:4^* 0 0.5% alpha-terpineol 0 0

2:5^* 0 0.4% alpha-terpineol 0 0

2:6^* 0 0.3% alpha-terpineol 7 0.2

2:7^* 0 0.15% alpha-terpineol 7 0.2

2:8^* 0.025 0.15% alpha-terpineol 0 0

2:9^* 0.025 0.6% alpha-terpinyl

formate 4.3 0.2

2:10^* 0.075 0.15% alpha-terpinyl

acetate > 7 0.1

2:1 1 ^* 0 0.25% alpha-terpinyl

0 0 propionate

2:12^* 0 0.15% alpha-terpinyl

7 0.2 propionate 2:13 0.025 0.1 % alpha-terpinyl

0 0 propionate

2:14 0.0125 0.15% alpha-terpinyl

0 0 propionate

2:15^* 0 0.15% alpha-terpinyl

0 0 butyrate

2:16^* 0 0.075% alpha-terpinyl

7 0.2 butyrate

2:17 0.025 0.05% alpha-terpinyl

0 0 butyrate

2:18 0.0125 0.1 % alpha-terpinyl

0 0 butyrate

2:19^* 0 0.25% alpha-terpinyl

0 0 cyclohexanoate

2:20^* 0 0.15% alpha-terpinyl

7 0.2 cyclohexanoate

2:21 0.025 0.1 % alpha-terpinyl

0 0 cyclohexanoate

2:22 0.0125 0.15% alpha-terpinyl

0 0 cyclohexanoate

^* Examples (2:1 ) to (2:12), (2:15), (2:16), , (2:19), and (2:20) are comparative examples

Comparative examples

Determination of the parameter∑FBC, which is used as measure of the synergistic antimicrobial action of compositions according to the present invention, requires determination of the Minimum Biocidal Concentrations (MBCs) of the relevant actives first. As described above, the MBC for an active can be defined as the lowest concentration of the active that provides zero bacterial bacterial survival. Data for examples (2:1 ) to (2:3) demonstrate that the MBC value for thymol is 0.05% (w/v). For alpha-terpineol, compositions (2:4) to (2:7), show that the MBC is 0.4% w/v. The same analysis has been carried out for other terpinyl esters and is summarised in Table 5 below. Table 5: Minimum Biocidal concentrations of antimicrobial components

It is clear from the data in Table 5 that the propionate, butyrate and cyclohexanoate esters of alpha-terpineol are more efficacious antimicrobials than alpha-terpineol itself and can be used at lower concentrations.

Synergistic interactions

The tested combinations of thymol with alpha-terpineol and alpha-terpinyl esters provide complete bacterial kill in the examples (2:8), (2:13), (2:14), (2:17), (2:18), (2:21 ), and (2:22). Using the MBC values listed in Table 5 above, the fractional MBC values for the components present in these mixtures and the experimental∑FBC of the compositions can be calculated in order to discriminate between combinations providing evidence of synergistic effects, as opposed to additive biocidal effects. The results from this analysis are given in Table 6.

Table 6: Extent of synergistic interactions between binary compound mixtures for compositions providing complete bacterial kill

Terpinyl Ex. Thymol T.D. ∑FBC Evidence derivative MBC FBC^a MBC FBC^b of

(T.D.) %(w/v) %(w/v) Synergy⁰ alpha-terpineol 2:8^* 0.05 0.5 0.4 0.38 0.88 Yes alpha-terpinyl 2:13 0.05 0.5 0.25 0.4 0.9 Yes propionate

alpha-terpinyl 2:14 0.05 0.25 0.25 0.6 0.85 Yes propionate alpha-terpinyl 2: 17 0.05 0.5 0.15 0.33 0.83 Yes

butyrate

alpha-terpinyl 2: 18 0.05 0.25 0.15 0.67 0.92 Yes

butyrate

alpha-terpinyl 2:21 0.05 0.5 0.25 0.4 0.90 Yes

cyclo- hexanoate

alpha-terpinyl 2:22 0.05 0.25 0.25 0.6 0.85 Yes

cyclo- hexanoate

* Example (2:8) is a comparative example.

^A FBC of thymol : C_Thymoi/MBC_Thymoi

cCriterion for synergy: (∑FBC <1 )

For Examples (2:8), (2:13), (2:14), (2:17), (2: 18), (2:21 ) and (2:22), the∑FBC value is below 1 , thus providing evidence for synergistic interactions, according to the set criteria. Therefore, these examples show how the antimicrobial efficacy of thymol and terpinyl derivatives according to invention are enhanced when they are applied together. Such synergies allow for reductions in the concentrations of the antimicrobials required to achieve complete kill. For example 0.05% w/v thymol is required to achieve complete bacterial kill when tested in isolation but this can be reduced four-fold to 0.0125% when used in combination with 0.1 % alpha-terpinyl butyrate or 0.15% alpha- terpinyl propionate or cyclohexanoate.

The isobolograms in Figures 1 , 2, and 3 provide a graphic representation of the synergy: The data points correspond to the lowest tested concentrations of the terpinyl derivative at a given thymol concentration (and vice versa) that yield complete microbial kill. The straight lines connecting the MBCs of the thymol and terpinyl derivative, respectively, constitute the hypothetical isobole in case the contributions of the thymol and the terpinyl derivative were purely additive. The positions of the data points corresponding to complete microbial kill below these lines clearly show the synergistic antimicrobial efficacy. Example 3: Antimicrobial efficacy of alpha-terpinyl benzoate and thymol

The antimicrobial efficacy of combinations of alpha-terpinyl benzoate and thymol against E. coli was assessed using the same methodology as in Example 2. The results are summarised in Table 7, reporting both the bacterial survival numbers (N_Bs) for the individual replicates and the resulting mean bacterial survival numbers (N_MBS)-

Table 7: Antibacterial activities of alpha-terpinyl benzoate alone, and in combination with thymol in water

Comparative examples (3:1 ) to (3:5) show that complete kill could not be reached at the tested concentrations with alpha-terpinyl benzoate as the only active compound. Without wishing to be bound by theory, it is hypothesised that the limited solubility of alpha-terpinyl benzoate in water in the absence of any solubility-enhancing compounds means that complete bacterial kill cannot be effected by alpha-terpinyl benzoate at its saturation concentration. Therefore, a minimum biocidal concentration of alpha-terpinyl benzoate cannot be specified, and it is not possible to calculate values of∑FBC for any of the compositions of Examples (3:6) to (3:10). Nevertheless, Examples (3:6) to (3:10) show that the reported combinations of thymol and alpha-terpinyl benzoate lead to a 5 log to 7 log reduction of the mean bacterial survival numbers, that is the antimicrobial efficacy is strongly enhanced when compared to thymol or alpha-terpinyl benzoate taken individually (N_MBS = 7.43 ± 0.12 for compositions with 0.03 % w/v thymol as the sole antimicrobial active). The replicates show that for many individual tests of Examples (3:6) to (3:10), complete kill was observed, even at concentrations of alpha-terpinyl benzoate as low as 0.03 % w/v, as shown in Example (3:10). Therefore, it is believed that the results of Examples (3:6) to (3:10) are consistent with synergistic antimicrobial efficacy of thymol and alpha-terpinyl benzoate.

Example 4: Automated assessment of efficacy in surfactant base

Sample preparation

In these examples, the efficacy of combinations of thymol and terpineol and terpinyl benzoate were tested in a surfactant cleansing formulation comprising 2.85% sodium cocoyi glycinate and 1.85% sodium lauroamphoacetate. This corresponds to a 50% in use dilution with water of a typical neat formulation containing 5.7% cocoyi glycinate and 3.7% % sodium lauroamphoacetate during hand washing. Solutions were prepared such that the concentrations of the surfactant components and test actives were 1.1 times the final desired concentration in order to allow for dilution with the bacterial inoculum in the test. The solutions were manually adjusted to pH 10.0 by dropwise addition of sodium hydroxide solution, as measured with a pH meter at ambient temperature. Solutions of the thymol and/or terpinyl esters were prepared at a maximum of 24 hours before testing. Test methodology

The efficacy of the combinations of the present invention was determined against the same bacterium as in Example 2, Escherichia coli (£. coli - ATCC #10536), at a concentration of approximately 1 x 10⁸ bacteria per ml_. Tests were conducted using standard microtiter plate assays using an automated liquid handling system. 270μΙ of the surfactant test solution was pipetted into each well of the microtitre plate (Nunc F Gamma Irradiated 96F untreated microtitre plates of clear polystyrene) and 30μΙ of the bacterial suspension was then added. After exactly 15 seconds of bacterial exposure, a 30μΙ volume of bacterial cells was withdrawn and transfered to 270μΙ of D/E quench solution. After 5 minutes in the D/E quench, the optical density (OD) was measured for each plate in turn at two specific wavelengths (450nm and 590nm). These provide a dual check of antimicrobial activity, as the OD₄₅₀ reading is specific for the yellow colour of D/E quench when bacterial growth is observed, whereas OD₅₉₀ is specific for the initial purple colour of the D/E quench which is retained if no bacterial growth is observed. After the time zero OD

measurements, plates were then incubated at 37°C overnight (16 hours) before repeating the OD measurements. Delta OD values were calculated by subtracting the OD values at 16 hours from the initial value at time zero from those at time = 16 hours. Bacterial growth is observed as an increase in OD₄₅₀ and a decrease in OD₅₉₀. To identify antibacterially efficacious systems (those preventing appreciable bacterial growth after incubation), the following threshold changes in OD readings have been adopted: if (1 ). OD₄₅₀ increases by less than 0.2 absorbance unit (AU) on incubation and (2). OD₅₉₀ decreases by less than 0.35 AU on incubation. Conversely, where OD₄₅₀ increases by more than 0.1 AU and OD₅₉₀ decreases by more than 0.1 AU after incubation, corresponding to a colour shift from purple to yellow, the test system allows bacterial growth and is not deemed efficacious. Four replicate measurements in the same plate have been made for each test system. The number of replicate wells showing either bacterial growth or no growth is also readily assessed by eye by following the colour change. Thymol and terpineol were tested both alone and in combination for comparison purposes.

Dose responses for individual components and binary mixtures of actives at a fixed concentration ratio were generated by sequential dilution of liquors with further surfactant solution to obtain a series of endpoints ranging from 0.2 to 0.025% of thymol and 0.5% to 0.063% of the terpinyl ester.

Table 8: Antibacterial activities of terpinyl esters alone, and in combination with thymol in model surfactant solution

Ex. r >-»thymol ^(a> r ERP m DeltaOD DeltaOD N rep ^(e)

(% w/v) (% w/v) (450 nm) ^(c) (590 nm) ^(d)

Mean S.D.^(f) Mean S.D^(f)

4:1 ^* 0 0 -0.60 0.02 0.64 0.02 4

4:2^* 0.2 0 -0.54 0.02 0.64 0.02 4

4:3^* 0.175 0 -0.54 0.02 0.56 0.02 4

4:4^* 0.15 0 -0.57 0.01 0.55 0.01 4 Ex. r >-»thymol ^(a> r ERP m DeltaOD DeltaOD N rep ^(e)

(% w/v) (% w/v) (450 nm) ^(c) (590 nm) ^(d)

Mean S.D.^(f) Mean S.D^(f)

4:5^* 0.125 0 -0.58 0.01 0.55 0.01 4

4:6^* 0.1 0 -0.58 0.00 0.54 0.02 4

4:7^* 0.075 0 -0.59 0.01 0.54 0.01 4

4:8^* 0.05 0 -0.58 0.03 0.55 0.01 4

4:9^* 0.025 0 -0.55 0.01 0.65 0.02 4 :10^* 0 0.5% terpineol -0.45 0.02 0.64 0.02 4 :1 1 ^* 0 0.4% terpineol -0.47 0.01 0.59 0.00 4 :12^* 0 0.35% terpineol -0.48 0.01 0.58 0.01 4 :13^* 0 0.3% terpineol -0.51 0.01 0.57 0.01 4 :14^* 0 0.25% terpineol -0.54 0.01 0.56 0.1 4 :15^* 0 0.2% terpineol -0.56 0.01 0.55 0.01 4 :16^* 0 0.15% terpineol -0.57 0.01 0.55 0.01 4 :17^* 0 0.1 % terpineol -0.53 0.02 0.67 0.02 4

4 18 0.2 0.5% terpineol 0.20 0.02 0.23 0.04 0

4 19 0.175 0.438% terpineol 0.16 0.02 0.17 0.02 0

4 20 0.15 0.375% terpineol 0.13 0.01 0.16 0.01 0

4 21 0.125 0.313% terpineol -0.20 0.35 0.36 0.23 2 :22^* 0 0.5% terpinyl benzoate -0.42 0.01 0.66 0.01 4 :23^* 0 0.4% terpinyl benzoate -0.44 0.02 0.58 0.01 4 :24^* 0 0.35% terpinyl benzoate -0.47 0.02 0.57 0.01 4 :25^* 0 0.3% terpinyl benzoate -0.50 0.02 0.56 0.01 4 :26^* 0 0.25% terpinyl benzoate -0.52 0.02 0.56 0.01 4 :27^* 0 0.2% terpinyl benzoate -0.54 0.02 0.56 0.01 4 :28^* 0 0.15% terpinyl benzoate -0.58 0.02 0.52 0.02 4 :29^* 0 0.1 % terpinyl benzoate -0.55 0.02 0.63 0.02 4

4:30 0.2 0.5% terpinyl benzoate 0.13 0.01 0.17 0.01 0

4:31 0.175 0.438% terpinyl benzoate 0.13 0.01 0.17 0.01 0

4:32 0.15 0.375% terpinyl benzoate -0.34 0.01 0.57 0.02 4

4:33 0.125 0.313% terpinyl benzoate -0.41 0.01 0.55 0.02 4

4:34 0.1 0.25% terpinyl benzoate -0.44 0.01 0.57 0.02 4

4:35 0.075 0.188% terpinyl benzoate -0.48 0.02 0.55 0.02 4 Ex. thymol ERP DeltaOD DeltaOD ¹¹¹ rep

(% w/v) (% W/V) (450 nm) ⁽ (590 nm) ⁽

Mean S.D. (f) Mean S.D (f)

4:36 0.05 0.125% terpinyl -0.52 0.02 0.53 0.02

benzoate

4:37 0.025 0.063% terpinyl benzoate -0.51 0.01 0.64 0.02

Examples marked with an asterisk (^*) are comparative examples

(e) Thymol concentration

(f) Concentration of terpineol or terpinyl ester

(g) DeltaOD (450 nm) = OD₄₅₀ (time = 16 hours) - OD₄₅₀ (time zero)

(h) DeltaOD (590 nm) = OD₅₉₀ (time = 16 hours) - OD₅₉₀ (time zero)

(i) N_rep = No. of replicates showing growth (out of 4)

(j) S.D. = standard deviation

Table 9: Minimum biocidal concentrations of antimicrobial components in sodium cocoyl glycinate + 1 .85 % sodium lauroamphoacetate solution at pH 10

Results

Examples (4:18), (4:19) and (4:20) show no bacterial growth resulting from the combinations of thymol with alpha terpineol at concentrations where the individual components are ineffective, as seen from the examples (4:2 to 4:17). No growth is achieved in 2 out of 4 replicates for concentrations in Example (4:21 ). Examples (4 and (4:31 ) show that the combinations of alpha-terpinyl benzoate with thymol are effective at the same concentrations as (4:18) and (4:19), respectively, although Example (4:32) is not as efficacious as Example (4:20).

Claims

Claims

1 . An antimicrobial composition comprising:

a. 0.001 to 5% by weight of thymol,

b. 0.001 to 5% by weight of a terpinyl derivative, and c. a carrier;

wherein the terpinyl derivative is selected from

i. alpha-terpinyl esters and amides of the structure:

beta-terpinyl esters and amides of the following structure

gamma-terpinyl esters and amides of the following structure

iv. delta-terpinyl esters and amides of the following structure

v. 4-terpinyl esters and amides of the following structure

vi. p-me rs and amides of the following structure

vii. dihydrocarvyl esters and amides of the following structure, and

isopulegyl esters and amides of the following structure;

and wherein X is NH or O; and

wherein R-i is

a linear or branched C₂ to Ci₇ alkyl group, or

a linear or branched substituted C₂ to Ci₇ alkyl group, or

a linear or branched C₂ to Ci₇ alkenyl group, or

a linear or branched C₂ to Ci₇ substituted alkenyl group, or

R₂OR₃, wherein R₂ is a linear or branched C_x alkyl group and R₃ is a linear or branched C_y alkyl group, such that x + y is between and including 2 and 17, or an alkyl ethoxylate (CH₂)_x(OC₂H₄)_yOH group, wherein x is between 1 and 10, and y is between 1 and 10, or

an aliphatic C₅ to C₇ ring, or

a substituted aliphatic C₅ to C₇ ring, or an unsaturated C₅ to C₇ ring, or

a substituted unsaturated C₅ to C₇ ring, or

an aryl group, or

a substituted aryl group, or

an aryloxy group, or

a substituted aryloxy group;

and mixtures of these terpinyl derivatives.

An antimicrobial composition according to claim 1 ,

wherein the terpinyl derivative is a terpinyl ester

An antimicrobial composition according to claim 2,

wherein the terpinyl derivative is selected from alpha-terpinyl esters, beta- terpinylesters, and gamma-terpinyl esters, and mixtures thereof.

An antimicrobial composition according to claim 3

wherein the terpinyl derivative is an alpha-terpinyl ester

An antimicrobial composition according to claim 3 or 4

wherein R-i is

a linear or branched C₂ to Ci₇ alkyl group, or

a linear or branched C₂ to Ci₇ alkenyl group, or

R₂OR₃, wherein R₂ is a linear or branched C_x alkyl group and R₃ is a linear or branched C_y alkyl group, such that x + y is between and including 2 and 17, or an aliphatic C₅ to C₇ ring, or

an unsaturated C₅ to C₇ ring, or

an aryl group, or

an aryloxy group.

An antimicrobial composition according to claim 5

wherein R-i is

a linear or branched C₂ to C₄ alkyl group, or

an aliphatic C₆ ring, or

an aryl group.

7. An antimicrobial composition according to claim 6, wherein the terpinyl derivative is selected from

alpha-terpinyl propionate,

alpha-terpinyl butyrate,

alpha-terpinyl iso-butyrate,

alpha-terpinyl cyclohexanoate,

and mixtures thereof.

8. An antimicrobial composition according to any one of claims 1 to 7 comprising from 1 to 80% by weight of surfactant.

9. An antimicrobial composition according to claim 8 wherein the surfactant is anionic, nonionic, or a combination of anionic and nonionic surfactants.

10. An antimicrobial composition according to claim 8 or 9 wherein the surfactant is a soap, an alkyl sulphate or a linear alkyl benzene sulphonate.

1 1 . A solid antimicrobial composition according to claim 8 comprising:

a. 0.05 to 5% by weight of the thymol,

b. 0.05 to 5% by weight of the terpinyl derivative,

c. 5 to 30% by of weight water, and;

d. 30 to 80% by weight of surfactant.

12. A method of disinfecting a surface comprising the steps of

a. applying a composition according to any one of claims 1 to 1 1 on to the surface; and

b. rinsing the surface with a suitable solvent or wiping the surface with a suitable wipe.

13. A method according to claim 12, wherein the disinfection time T is less than 300 seconds, preferably less than 60 seconds, and more preferably less than 15 seconds; wherein T is defined as the time that elapses from the moment of adding the composition to a microbial culture until the number of microbes per unit volume of the culture is reduced by a factor of 100 000; and wherein the initial number of microbes preferably exceeds about 100 000 000 microbes per millilitre and wherein the composition is preferably a liquid composition.

14. Use of a composition according to any of claims 1 to 1 1 for improved hand hygiene.

15. Use of a composition according to any of claims 1 to 1 1 for improved oral hygiene.