Field of the invention

This invention relates to an antimicrobial composition. The invention more particularly relates to a personal care composition e.g those for hair, body and face wash which provides anti-microbial efficacy. It more particularly relates to a cleansing composition for hair and scalp comprising actives that interact to provide synergistic antimicrobial efficacy for anti-dandruff benefits. Background of the invention

The invention relates to an anti-microbial composition useful for cleaning of any body part but especially suitable for hair and scalp. Hair care compositions generally provide cleansing or conditioning benefits or a combination of the two. Such compositions typically comprise one or more cleansing surfactants which generally aid in cleaning the hair and/or the scalp free of undesirable soil, particles, and fatty matter.

Additionally, anti-dandruff benefit has been provided through hair care compositions. Dandruff is an issue that affects many people globally. The condition is manifested by the shedding of clumps of dead skin cells from the scalp. These are white in colour and provide an aesthetically displeasing appearance. A factor that contributes to dandruff are certain members of the Malassezia yeasts. To combat these, anti-dandruff products have been developed in the form of hair cleansing shampoos. An example of a known anti-dandruff shampoo comprises sodium lauryl ether sulfate (an ethoxylated anionic surfactant) in combination with an anti-dandruff agent. Typical anti-dandruff agents used in hair care are metal pyrithione e.g zinc pyrithione (ZPTO), octopirox (piroctone olamine), azole antimicrobials (e.g. climbazole), selenium sulfide and combinations thereof. The present inventors have carried out experiments to see if synergy could be obtained by combining the above actives. They tried a few and find that it is not straightforward to expect known actives to behave synergistically when combined for the same (anti-fungal) activity.

While the problem of dandruff is mitigated to a large extent through use of the above actives in such shampoos, there is a need for enhancing the efficacy of these actives. The present inventors have found through a combination of exploring very many possible mechanisms by which the efficacy of one of the above actives (zinc pyrithione) can be enhanced together with extensive experiments on possible enhancer candidates that a certain class of compounds having alcoholic groups (e.g. honokiol, magnolol or paeonol) displays such enhancing properties. The synergistic antimicrobial combination of this invention can also be formulated into products for skin cleansing e.g. body and face care. So in addition to anti-fungal activity, the present invention also provides anti-bacterial efficacy against organisms like S. Aureus. W014131 19 (Johnson and Johnson) discloses compositions comprising an antimicrobial active comprising honokiol and/or magnolol and a carboxylic acid. Also provided are methods of use thereof.

US2010143515 (Mary Kay) discloses a composition comprising at least two of the following ingredients: Magnolia extract, honokiol, humulus lupulus extract, hesperidin methyl chalcone, gotu kola, dipeptide valyl-tryptophane, palmitoyl tetrapeptide-3, corylus avellana bud extract, centella asiatica extract, cucumis sativa extract, morus alba extract, hibiscus sabdariffa flower extract, vitis vinifera extract, ascorbyl glucoside, citrus medica limonum extract, avena sativa kernel extract, hydrolyzed soy protein, aniseed myrtle extract, tasmania lanceolata leaf extract, artemisia abrotanum extract, or citrus grandis fruit extract or any combination thereof. Also disclosed are methods of treating skin conditions by topically applying the composition to skin.

CN 104225603 (Tianjin Bokni Technology Dev Co Ltd) discloses paeonol and glyceryl polyether complex compound that has good bactericidal effect.

The above publications do not disclose a combination of the actives of the present invention nor is there any indication that they can be combined to provide synergistic anti-microbial activity.

It is thus an object of the present invention to provide for a personal care composition that exhibits synergistic antimicrobial activity as compared to the individual components. Summary of the invention

According to the first aspect of the present invention there is a synergistic antimicrobial composition comprising

(i) A polyvalent metal salt of pyrithione; and

(ii) 0.1 to 10% of an alcoholic compound selected from honokiol, magnolol, or paeonol.

The second aspect of the present invention relates to a method of inhibiting the growth of Malassezia furfur comprising the step of applying a composition as claimed in the first aspect, on the scalp of an individual.

Detailed description of the invention

These and other aspects, features and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims. For the avoidance of doubt, any feature of one aspect of the present invention may be utilized in any other aspect of the invention. The word "comprising" is intended to mean "including" but not necessarily "consisting of" or "composed of." In other words, the listed steps or options need not be exhaustive. 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. Similarly, all percentages are weight/weight percentages unless otherwise indicated. Except in the operating and comparative examples, or where otherwise explicitly indicated, all numbers in this description and claims 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". 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. In other words, in specifying any ranges of values, any particular upper value can be associated with any particular lower value.

The disclosure of the invention as found herein is to be considered to cover all embodiments as found in the claims as being multiply dependent upon each other irrespective of the fact that claims may be found without multiple dependency or redundancy.

Where a feature is disclosed with respect to a particular aspect of the invention (for example a composition of the invention), such disclosure is also to be considered to apply to any other aspect of the invention (for example a method of the invention) mutatis mutandis.

By 'an antimicrobial composition' as used herein, is meant to include a composition for topical application to skin, hair and/or scalp of mammals, especially humans. Such a composition is generally applied on to the desired topical surface of the body for a period of time from a few seconds to up to 24 hours. When the period of time of application is low say of the order of a few seconds to a few minutes after which the composition is rinsed off with water or wiped away, such a composition is known as a cleansing composition or a wash-off composition. When the composition is applied for longer period of time say from several minutes to up to 24 hours and washed off usually during the process of normal personal cleaning, such a composition is known as a leave-on composition. The composition as per the present invention may be of the wash-off or of the leave-on type. Of the two, it is preferably of the wash-off type. It includes any product applied to a human body for also improving appearance, cleansing, odor control or general aesthetics. The composition of the present invention can be in the form of a liquid, lotion, cream, foam, scrub, gel, or bar. Non-limiting examples of such compositions include wash-off shampoos, conditioners, shower gels, hand wash liquids or gels or bars. The composition of the present invention is most preferably a shampoo or a conditioner. It is most preferably used for preventing or alleviating the symptoms of dandruff on the scalp and/or hair.

The present invention more particularly relates to an antimicrobial cleansing composition. A preferred aspect is an anti-dandruff hair care composition. It comprises synergistic anti-fungal action of the polyvalent metal salt of pyrithione e.g. zinc pyrithione with the alcoholic compound claimed in the present invention. The preferred polyvalent metal salt of pyrithione is zinc pyrithione (ZPTO) which is shorthand for zinc 1 -hydroxy-2-pyridinethione.

The polyvalent metal salt of pyrithione is represented by the following general formula:

In the case of zinc pyrithione, M is the metal cation zinc. Zinc pyrithione is preferably present in 0.1 to 5.0%, more preferably from 0.1 to 2.0% based on weight of the composition. ZPTO is a particulate material. While the particle size is not critical to achieve the benefits of the present invention, the particle size of ZPTO is preferably from 0.25 to 8 micrometer, more preferably from 0.5 to 8.0 micrometer, and further more preferably from 1.0 to 7.5 micrometer. ZPTO is commercially available from Kolon Life Science Inc., Sino Lion (USA) Ltd, Lonza, and other suppliers.

The alcoholic compound is preferably chosen from magnalol, honokiol or paeonol which have the chemical structures given below.

Honokiol

Paeonol

Honokiol is commercially available from Sigma-Aldrich Co. LLC, with purity≥98%. The compound has been extracted from seed cones, and leaves of trees belonging to the genus Magnolia, native to many regions of the globe.

Magnalol is commercially available from Sigma-Aldrich Co. LLC, with purity≥95%. The compound is an isomer of honokiol, which exists at the level of a few percent in the bark of species of Magnolia.

Paeonol is commercial available from Sigma-Aldrich Co. LLC, with purity of 99%. The compound is found in peony such as Paeonia suffruticosa. The alcoholic compound is preferably present 0.1 to 5%, more preferably 0.2 to 3.0% based on the total weight of the composition. According to a preferred aspect of the present invention the alcoholic compound is magnalol. It is expected that the alcoholic compound is included in the composition at purities higher than 90% preferably higher than 95%. The present inventors have found that extracts of parts of trees belonging to the genus Magnolia are found to have levels of only a few percentage of the alcoholic compounds magnalol and honokiol. Thus, such extracts per se when added to the composition of the invention would not give the desired synergistic efficacy with zinc pyrithione. It is necessary to purify the extract to high concentrations of the alcoholic compounds before such efficacy can be obtained. Similar purification of extract of Paeonia is necessary for synergy of zinc pyrithione with paeonol.

It is preferred that the weight ratio of zinc pyrithione to the alcoholic compound in the composition of the invention is from 1 :10 to 10:1 , preferably 1 :5 to 5:1 . Without wishing to be bound by theory, the inventors believe that the anti-microbial efficacy of ZPTO is enhanced by the alcoholic compounds through disruption of microbial membrane integrity.

The composition of the invention preferably comprises a cosmetically acceptable carrier. According to one aspect the cosmetically acceptable carrier comprises water. According to another preferred aspect, the carrier additionally comprises a surfactant.

According to a further preferred aspect of the present invention, the composition is either a shampoo or a hair conditioner.

Shampoo

As per an especially preferred aspect of the invention, the composition is a shampoo. The composition of the invention especially shampoos are formulated with an anionic surfactant e.g. an alkyl sulphate and/or ethoxylated alkyl sulfate surfactant. These anionic surfactants are preferably present at a level of from 1 to 20%, preferably 2 to 16%, further more preferably from 3 to 16% by weight of the composition. Preferred alkyl sulfates are C8-18 alky sulfates, more preferably C12-18 alkyl sulfates, preferably in the form of a salt with a solubilising cation such as sodium, potassium, ammonium or substituted ammonium.

Preferred alkyl ether sulfates are those having the formula: RO(CH2CH20) _n S03M;

wherein R is an alkyl or alkenyl having from 8 to 18 (preferably 12 to 18) carbon atoms; n is a number having an average value of greater than at least 0.5, preferably between 1 and 3, more preferably between 2 and 3; and M is a solubilising cation such as sodium, potassium, ammonium or substituted ammonium. An example is sodium lauryl ether sulfate (SLES). SLES having an average degree of ethoxylation of from 0.5 to 3, preferably 1 to 3 is especially preferred.

Shampoo compositions according to the invention may comprise one or more further anionic cleansing surfactants which are cosmetically acceptable and suitable for topical application to the hair. Examples of further suitable anionic cleansing surfactants are the alkaryl sulphonates, alkyl succinates, alkyl sulphosuccinates, alkyl ether sulphosuccinates, N-alkyl sarcosinates, alkyl phosphates, alkyl ether phosphates, and alkyl ether carboxylic acids and salts thereof, especially their sodium, magnesium, ammonium and mono-, di- and triethanolamine salts. The alkyl and acyl groups generally contain from 8 to 18, preferably from 10 to 16 carbon atoms and may be unsaturated. The alkyl ether sulphosuccinates, alkyl ether phosphates and alkyl ether carboxylic acids and salts thereof may contain from 1 to 20 ethylene oxide or propylene oxide units per molecule.

Typical anionic cleansing surfactants for use in compositions of the invention include sodium oleyl succinate, ammonium lauryl sulphosuccinate, sodium lauryl ether sulphosuccinate, sodium dodecylbenzene sulphonate, triethanolamine dodecylbenzene sulphonate, lauryl ether carboxylic acid and sodium N-lauryl sarcosinate.

Suitable preferred additional anionic cleansing surfactants are sodium lauryl ether sulphosuccinate(n)EO, (where n is from 1 to 3), lauryl ether carboxylic acid (n) EO (where n is from 10 to 20).

Mixtures of any of the foregoing anionic cleansing surfactants may also be suitable. A composition of the invention preferably additionally comprises an amphoteric surfactant preferably a betaine surfactant preferably an alkyl amidopropyl betaine surfactant for example cocamidopropyl betaine. In a preferred embodiment, the composition comprises from 0.1 to 10 wt.%, preferably from 0.5 to 8 wt.%, more preferably from 1 to 5 wt.% of a betaine surfactant

To enhance deposition of actives from compositions of the invention especially shampoos, cationic polymers are generally included therein. In the present invention too, it is preferred that the composition additionally includes 0.01 to 2.0% of a cationic polymer. The cationic polymer is preferably guar hydroxypropyl trimonium chloride. Guar polymer predominantly contains galactomannan polymer chains. This polymer is available at various molecular weights and degree of cationic substitutions depending on how much the guar has been hydrolysed and cationised. The cationic polymer is preferably present in 0.04 to 0.5%, more preferably 0.08 to 0.25% by weight of the composition.

The pH of the composition is preferably equal to or higher than 4.0, more preferably in the range of 5.0 to 7.0.

The composition as per the invention especially for anti-dandruff shampoos preferably additionally comprises a zinc compound. The presence of additional zinc compound in the composition is believed to improve the antidandruff efficacy of the metal salt of pyrithione. Suitable zinc compounds are zinc oxide, zinc citrate, zinc malonate, zinc carbonate or combinations thereof. The zinc compound is preferably present in 0.1 to 3%, more preferably 0.1 to 1.5% by weight of the composition.

Shampoo composition as per the invention preferably additionally comprises a conazole fungicide. Preferably the conazole fungicide is selected form ketoconazole, climbazole or mixtures thereof. The azole fungicide is preferably included in 0.01 to 2%, more preferably 0.025 to 0.75% by weight of the composition. The presence of a conazole fungicide is believed to improve the deposition of zinc pyrithione.

Suspending Agent

Preferably the composition of the invention further comprises a suspending agent. Suitable suspending agents are selected from polyacrylic acids, cross-linked polymers of acrylic acid, copolymers of acrylic acid with a hydrophobic monomer, copolymers of carboxylic acid-containing monomers and acrylic esters, cross-linked copolymers of acrylic acid and acrylate esters, heteropolysaccharide gums and crystalline long chain acyl derivatives. The long chain acyl derivative is desirably selected from ethylene glycol stearate, alkanolamides of fatty acids having from 16 to 22 carbon atoms and mixtures thereof. Ethylene glycol distearate and polyethylene glycol 3 distearate are preferred long chain acyl derivatives, since these impart pearlescence to the composition. Polyacrylic acid is available commercially as Carbopol 420, Carbopol 488 or Carbopol 493. Polymers of acrylic acid cross-linked with a polyfunctional agent may also be used; they are available commercially as Carbopol 910, Carbopol 934, Carbopol 941 and Carbopol 980. An example of a suitable copolymer of a carboxylic acid containing monomer and acrylic acid esters is Carbopol 1342. All Carbopol (trademark) materials are available from Goodrich.

Suitable cross-linked polymers of acrylic acid and acrylate esters are Pemulen TR1 or Pemulen TR2. A suitable heteropolysaccharide gum is xanthan gum, for example that available as Kelzan mu.

Mixtures of any of the above suspending agents may be used. Preferred is a mixture of cross-linked polymer of acrylic acid and crystalline long chain acyl derivative.

Suspending agent, if included, will generally be present in a shampoo composition of the invention at levels of from 0.1 to 10%, preferably from 0.5 to 6%, more preferably from 0.5 to 4% by total weight of suspending agent based on the total weight of the composition.

A composition of the invention may contain other ingredients for enhancing performance and/or consumer acceptability. Such ingredients include fragrance, dyes and pigments, pH adjusting agents, pearlescers or opacifiers, viscosity modifiers, preservatives, and natural hair nutrients such as botanicals, fruit extracts, sugar derivatives and amino acids. The composition of the invention is preferably aqueous based. It preferably comprises high amounts of water preferably from 70 to 95% by weight of the composition.

Hair Conditioner When conditioning benefits are to be delivered through the composition of the invention the composition is called a hair conditioner. Typically, the most popular conditioning agents used in hair care compositions are water-insoluble oily materials such as mineral oils, naturally occurring oils such as triglycerides and silicone polymers. Conditioning benefit is achieved by the oily material being deposited onto the hair resulting in the formation of a film, which makes the hair easier to comb when wet and more manageable when dry. An especially useful conditioning agent is a silicone compound, preferably a non-volatile silicone compound. Advantageously compositions herein may include one or more silicones. The silicones are conditioning agents found in dispersed or suspended particulate form. They are intended to deposit onto hair remaining behind after rinsing of the hair with water. Suitable silicone oils may include polyalkyl siloxanes, polyaryl siloxanes, polyalkylaryl siloxanes, polyether siloxane copolymers and mixtures thereof. Amino silicones are often formulated with shampoo compositions. Amino silicones are silicones containing at least one primary amine, secondary amine, tertiary amine or a quaternary ammonium group. High molecular weight silicone gums can also be utilized. Another useful type are the crosslinked silicone elastomers such as Dimethicone/Vinyl/Dimethicone Crosspolymers (e.g. Dow Corning 9040 and 9041 ).

Amounts of the silicone in compositions where present may range from about 0.1 to about 10 wt.%, preferably from about 0.1 to about 8wt.%, more preferably from about 0.3 to about 5wt.% by weight of the hair care compositions.

The pH of the composition is preferably equal to or higher than 4.0, more preferably in the range of 5.0 to 7.0. The hair conditioning composition usually comprises conditioning surfactants selected from cationic surfactants, used singly or in admixture. Suitable cationic surfactants for use in conditioner compositions according to the invention include behenyltnmethylammonium chloride, cetylpyridinium chloride, tetramethylammonium chloride, tetraethylammonium chloride, octyltrimethylammonium chloride, dodecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, octyldimethylbenzylammonium chloride, decyldimethylbenzylammonium chloride, stearyldimethylbenzylammonium chloride, didodecyldimethylammonium chloride, dioctadecyldimethylammonium chloride, tallowtrimethylammonium chloride, dihydrogenated tallow dimethyl ammonium chloride (eg, Arquad 2HT/75 from Akzo Nobel), cocotrimethylammonium chloride, PEG-2-oleammonium chloride and the corresponding hydroxides thereof. Further suitable cationic surfactants include those materials having the CTFA designations Quaternium-5, Quaternium-31 and Quaternium- 18. Mixtures of any of the foregoing materials may also be suitable. A particularly useful cationic surfactant for use in conditioners according to the invention is cetyltrimethylammonium chloride, available commercially, for example as GENAMIN CTAC, ex Hoechst Celanese. Another particularly useful cationic surfactant for use in conditioners according to the invention is behenyltnmethylammonium chloride, available commercially, for example as GENAMIN KDMP, ex Clariant. Yet another preferred cationic surfactant is stearamidopropyl dimethylamine.

The most preferred cationic surfactants for use in the composition are stearamidopropyl dimethylamine, behentrimonium chloride, or stearyl trimethyl ammonium chloride. In conditioners of the invention, the level of cationic surfactant will generally range from 0.1 % to 5%, preferably 0.5 to 2.5% by weight of the composition.

Hair conditioning compositions of the invention preferably may also additionally comprise a fatty alcohol. The combined use of fatty alcohols and cationic surfactants in conditioning compositions is believed to be especially advantageous, because this leads to the formation of a lamellar phase, in which the cationic surfactant is dispersed.

Representative fatty alcohols comprise from 8 to 22 carbon atoms, more preferably 16 to 22. Fatty alcohols are typically compounds containing straight chain alkyl groups. Examples of suitable fatty alcohols include cetyl alcohol, stearyl alcohol and mixtures thereof. The use of these materials is also advantageous in that they contribute to the overall conditioning properties of compositions of the invention. The level of fatty alcohol in conditioners of the invention will generally range from 0.5 to 10%, preferably from 0.1 % to 8%, more preferably from 0.2 % to 7 %, most preferably from 0.3 % to 6 % by weight of the composition. The weight ratio of cationic surfactant to fatty alcohol is suitably from 1 :1 to 1 :10, more preferably from 1 :1.5 to 1 :8, optimally from 1 :2 to 1 :5.

Skin cleansing

The composition of the invention may be used for skin care e.g. body or face wash. The antimicrobial composition may further comprise a surfactant. The preferred surfactants are nonionic surfactants

Thus, in a highly preferred aspect, the antimicrobial compositions include the surfactant selected from the group of anionic surfactant.

When the surfactants are present, the antimicrobial composition preferably comprises 1 to 90% surfactant by weight of the composition When surfactant is used, a particularly preferred surfactant is soap. Soap is a suitable surfactant for personal washing applications of the antimicrobial composition of the invention.

When present, the soap, of the present is preferably present in an amount of 1 to 90%, preferably from 10 to 85%, more preferably 25 to 75% by weight of the composition. Preferred compositions may include other known ingredients such as perfumes, pigments, preservatives, emollients, sunscreens, emulsifiers, gelling agents and thickening agents. Choice of these ingredients will largely depend on the format of the composition.

Water is a preferred carrier. 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 composition comprises 10 to 99.8% by weight water. The liquid antimicrobial composition is useful as a skin antiseptic liquid, for skin cleansing, in particular for hand wash or a face wash. When water is the carrier, a preferred solid composition comprises 5 to 30% by weight water.

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.

According to another aspect, 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.

In another aspect of the present invention, the composition of the present invention is suitable for use in wipes for personal hygiene.

The present invention also relates to a method of inhibiting the growth of Malassezia furfur comprising the step of applying a composition of the first aspect on to the scalp of an individual. Alternately and preferably the above step is followed by rinsing the surface with water. The step of rinsing is generally carried out within 1 to 5 minutes after application of the composition on the skin or hair/ scalp. According to one aspect, the invention provides for non-therapeutic benefits.

When the alcoholic compound is magnalol or honokiol, the method is more effective when the mole ratio of the polyvalent metal salt of pyrithione to the alcoholic compound on the Malassezia furfur cell is in the range of 1 :1500 to 1 :2000. When the alcoholic compound is paeonol, the method is more effective when the mole ratio of the polyvalent metal salt of pyrithione to the alcoholic compound on the Malassezia furfur cell is in the range of 1 :70 to 1 :3000.

The invention will now be illustrated with reference to the following non-limiting Examples. Examples

Examples 1 -3: Synergy between ZPTO and the alcoholic compounds of the invention: The synergistic interaction between the ZPTO and the alcoholic compounds was determined using the∑FIC assay. A brief description of the method is given below.

Methods:∑FIC assay against Malassezia furfur

Microorganism preparation:

M. furfur (CBS 1878) was maintained on MD agar plate (Solution A) was cultured into 20ml of growth medium Pityrosporum Broth (PB, Solution B).

Solution A preparation Modified Dixon Agar (MD)

36g Malt Extract (Oxoid)

6g Mycological Peptone (Oxoid)

10 Purified Agar (Oxoid)

20g Ox-bile (Oxoid)

2ml Oleic acid (Sigma)

2ml Glycerol (Sigma)

10ml Tween 40 (Sigma)

Deionized Water to 1000ml

50mg (1 vial) dissolved in 2ml 95% ethanol Chloramphenicol (Oxoid SR078E) (Ensure stirring thoroughly including after autoclaving)

Solution B preparation

Pityrosporum Broth (PB)

10g Bacteriological Peptone

0.1 g Yeast extract

10g Ox-bile

2.5g Taurocholic acid

10g Glucose

1 L Deionised water 0.5ml Tween60

1 ml Glycerol

Adjust pH to 6.2

After sterilization

0.5ml UHT milk

They are then incubated at 32 °C for 48 hrs with shaking. Then, 1 ml of the first broth culture is transferred into 9ml of fresh PB and incubated at 32 °C for 48 hrs with shaking. The final culture should contain 2 to 6x10 ⁶ cells/ml. This is achieved by diluting using PB to 5 ^* 10 ⁵ cell/ml.

In-vitro susceptibility testing

Zinc pyrithione was serially diluted (2 -fold) to prepare in a range of 2-125 ppm in the growth medium. The alcoholic compound was serially diluted (2-fold) to prepare in a range of 10-10,000 ppm in growth medium.

Binary combinations of ZPTO and the testing compound were prepared in 96-well plate by mixing 20 μΙ of zinc pyrithione solution with 20 μΙ of testing compound. The solution in each well were further mixed with 160 μΙ of PB and 20 μΙ of M. furfur. The final cell density in the testing plate is around 5 ^* 10 ⁴ cell/ml, and the final concentration of actives in each well of the 96-wel plate were shown below.

ZPTO concentrations (in ppm): 12.5, 6.3, 3.1 , 1.6, 0.8, 0.4, 0.2 and 0

Alcoholic compound concentrations (in ppm): 1000, 500, 250, 125, 62.5, 31 .3, 15.6, 7.8, 3.9, 2.0, 1 .0 and 0.

The plate was incubated at 32°C for one day. Then 20 μΙ of alamar blue (0.01 %) was added into each well with a further incubation for one day to observe the color change. Red color indicates microbial growth and the non-changing blue color indicates no growth. In addition, it was also confirmed that the testing compound alone will not cause color change of alamar blue with a control mixture without adding microbes.

Calculation: (1 ) Minimum Inhibition Concentration (MIC):

The MIC is defined as the absolute lowest concentration of active that provides complete microbial growth inhibition as indicated by the blue color of alamar blue under the tested condition. For example, for zinc pyrithione, the MIC was determined to be 1.6-3.2 ppm.

The MIC of honokiol, magnalol and paeonol was determined to be 1000 ppm, 500 ppm and 500 ppm respectively.

(2) Fractional Inhibition Concentration (FIC):

The differing behaviours of inhibitory antimicrobials in isolation and mixtures have been widely explored using the concept of the Fractional Concentration (FC) and Fractional Inhibitory Concentration (FIC). The parameter can be defined as follows:

FIC (component a) = MIC (component a tested in the mixture)

MIC (component a tested as a single active)

(3) Synergy and Additivity

The interactions between antimicrobials can be additive, synergistic or 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 MIC values for all the components present in the mixture to give the "fractional inhibition index". There is no consistent approach in the academic or patent literature in defining precise limiting∑FIC values that differentiate synergy from additivity or antagonism. In this study, we have adopted a liberal approach defining any binary mixture with∑FIC < 0.9 as showing evidence of synergistic behavior.

∑FIC = FIC (component 1 ) + FIC (component 2)

∑FIC = 1 corresponds to additive bactericidal activity

∑FIC > 1 corresponds to antagonistic bactericidal activity

∑FIC < 0.9 corresponds to synergistic bactericidal activity 4. Results:

Based on the concentration range of ZPTO (0 to 12.5 ppm) and the alcoholic compound (0 to 1000 ppm) at which the experiments were carried out, the∑FIC of the combinations is given below.

The data in Table above indicates that each of the three alcoholic compounds in combination with ZPTO exhibit synergistic antimicrobial activity.

Example 4 and 5: Combination of ZPTO with other known anti-dandruff actives climbazole and selenium sulfide:

Invitro assay similar to that used in examples 1-3 was used to carry out experiments to determine if the interaction between zinc pyrithione and other well known anti-dandruff agents like climbazole and selenium sulfide yields synergy. The experiments were carried out over a concentration range of zinc pyrithione of 0 to 12.5 ppm. Climbazole was varied over a concentration of 0 to 500 ppm and selenium sulfide over a concentration of 0 to 100 ppm.

MIC of climbazole was found to be 3.9 ppm and that of selenium sulfide to be 50 ppm. The∑FIC assay was carried out and the data is summarized in the table below:

Compound ∑ Fl C

Selenium sulfide 2

Climbazole 2 The data in the table above indicates that a combination of zinc pyrithione along with well known anti-dandruff active like climbazole and selenium sulphide is not synergistic. Thus one cannot conclude that combinations of known actives will always yield synergies. Surprisingly, the combination of zinc pyrithione with the alcoholic

compounds claimed in the present invention displays synergistic behaviour.