ANTIMICROBIAL MIXTURE CONTAINING 4-(3-ETHOXY-4-HYDROXYPHENYL)BUTAN-2-ONE AND AN AROMATIC ALCOHOL, AND COSMETIC COMPOSITION CONTAINING SAME

Abstract

The invention relates to an antimicrobial mixture containing 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and an aromatic alcohol chosen from a phenoxyalcohol of formula (I) C.sub.6H.sub.6-0-ROH, R denoting a divalent hydrocarbon-based radical having 2 or 3 carbon atoms; phenylethyl alcohol; benzyl alcohol; and a cresol compound of formula (I) in which R.sub.1 denotes H or an isopropyl radical; and also to a cosmetic composition containing such a mixture. Use in caring for, making up and cleansing keratin materials.

##STR00001##

Claims

1. An antimicrobial mixture comprising 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and an aromatic alcohol chosen from: i) a phenoxyalcohol of formula C.sub.6H.sub.5OROH, R denoting a divalent hydrocarbon-based radical having 2 or 3 carbon atoms; ii) phenylethyl alcohol; ii) benzyl alcohol; and iv) a cresol compound of formula (I): ##STR00007## in which R.sub.1 denotes H or an isopropyl radical.

2. The antimicrobial mixture according to claim 1, wherein the aromatic alcohol is a phenoxyalcohol of formula C.sub.6H.sub.5OROH, R denoting a divalent hydrocarbon-based radical having 2 or 3 carbon atoms.

3. The antimicrobial mixture according to claim 2, wherein said phenoxyalcohol is chosen from 2-phenoxyethanol or 1-phenoxy-2-propanol.

4. The antimicrobial mixture according to claim 2, wherein said phenoxyalcohol is 2-phenoxyethanol.

5. The antimicrobial mixture according to claim 2, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and phenoxyalcohol in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/2-phenoxyalcohol weight ratio ranges from 0.15 to 3.5.

6. The antimicrobial mixture according to claim 2, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/phenoxyalcohol weight ratio ranging from 0.3 to 3.2.

7. The antimicrobial mixture according to claim 2, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/phenoxyalcohol weight ratio ranging from 0.4 to 0.8.

8. The antimicrobial mixture according to claim 2, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/phenoxyalcohol weight ratio ranging from 0.15 to 1.3.

9. The antimicrobial mixture according to claim 1, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/phenoxyalcohol weight ratio ranging from 0.6 to 1.6.

10. The antimicrobial mixture according to claim 1, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/phenoxyalcohol weight ratio ranging from 0.2 to 3.2, preferably ranging from 0.4 to 3.2.

11. The antimicrobial mixture as claimed in claim 1, wherein the aromatic alcohol is phenylethyl alcohol.

12. The antimicrobial mixture according to claim 11, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and phenylethyl alcohol in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/phenylethyl alcohol weight ratio ranges from 0.05 to 1.

13. The antimicrobial mixture as claimed in claim 1, wherein the aromatic alcohol is benzyl alcohol.

14. The antimicrobial mixture according to claim 13, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and benzyl alcohol in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/benzyl alcohol weight ratio ranges from 0.1 to 1.5.

15. The antimicrobial mixture as claimed in claim 1, wherein the aromatic alcohol is a cresol compound of formula (I): ##STR00008## in which R.sub.1 denotes H or an isopropyl radical.

16. The antimicrobial mixture according to claim 15, wherein the cresol compound of formula (I) is chosen from 2-methylphenol, 3-methylphenol, 4-methylphenol, and 4-isopropyl-3-methylphenol.

17. The antimicrobial mixture according to claim 15, wherein cresol compound of formula (I) is 4-isopropyl-3-methylphenol.

18. The antimicrobial mixture according to claim 15, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and the cresol compound (I) in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/cresol compound (I) weight ratio ranges from 0.5 to 15.

19. The antimicrobial mixture according to claim 15, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/cresol compound (I) weight ratio ranging from 2 to 15.

20. A composition comprising, in a physiologically acceptable medium, an antimicrobial mixture according to claim 1.

21. The composition according to claim 20, which comprises at least one additional ingredient chosen from water, oils, polyols containing from 2 to 10 carbon atoms, gelling agents, surfactants, film-forming polymers, colorants, fragrances, fillers, UV-screening agents, plant extracts, cosmetic and dermatological active agents, and salts.

22. The composition according to claim 20, wherein the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one is present in a content ranging from 0.01% to 5% by weight relative to the total weight of the composition.

23. A non-therapeutic Non therapeutic cosmetic treatment process for caring for and/or making up and/or cleansing keratin materials, comprising the application to said keratin materials of a composition according to claim 20.

24. A process for conserving a composition comprising a physiologically acceptable medium which comprises incorporating into said composition an antimicrobial mixture as defined in claim 1.

Description

EXAMPLE 1: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0068] The demonstration of a synergistic antimicrobial activity effect with a mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (referred to as substance A) and of aromatic alcohol (referred to as substance B) is performed by calculating the synergy index (or FIC index) according to the following formula:

FIC Index=(MIC of A with B/MIC of A)+(MIC of B with A/MIC of B)

with: [0069] MIC of A with B: minimum concentration of product A in the combination A+B which makes it possible to obtain an inhibitory effect; [0070] MIC of B with A: minimum concentration of product B in the combination A+B which makes it possible to obtain an inhibitory effect; [0071] MIC of A: minimum inhibitory concentration of product A alone; [0072] MIC of B: minimum inhibitory concentration of product B alone.

[0073] This formula was described for the first time in the article by F. C. Kull, P. C. Eisman, H. D. Sylwestrowka, and R. L. Mayer, Applied Microbiology 9:538-541, 1961.

[0074] For each compound tested alone, the MIC is considered as the first concentration which makes it possible to obtain a microbial growth percentage of less than or equal to 20%.

[0075] As regards the combinations tested, MIC of A with B and MIC of B with A are the respective concentrations of A and of B in the combinations which make it possible to obtain a microbial growth percentage of less than or equal to 20%.

[0076] Interpretation of the FIC Index:

[0077] When the FIC index value is less than or equal to 1, it is considered that the combination of test compounds has a synergistic effect.

[0078] The results obtained are summarized in the following tables.

[0079] The combination of compounds A and B was tested on the following strains or a part thereof: Aspergillus niger, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans.

[0080] The microbial strain Aspergillus niger ATCC 6275, and a double-concentration Sabouraud broth liquid culture medium supplemented with polyoxyethylenated (20 OE) sorbitan monopalmitate (Tween 40 from Croda) and Phytagel BioReagent were used (i.e. a mixture of 5 g of Phytagel+0.6 g Tween 40+60 g of Sabouraud broth).

[0081] The microbial strain Enterococcus faecalis ATCC 33186 and a double-concentration BHI (Brain Heart Infusion) broth liquid culture medium were used.

[0082] The microbial strain Staphylococcus aureus ATCC 6538 and a double-concentration nutrient broth liquid culture medium were used.

[0083] The microbial strain Pseudomonas aeruginosa ATCC 9027 and a double-concentration nutrient broth liquid culture medium were used.

[0084] The microbial strain Candida albicans ATCC 10231 and a double-concentration Sabouraud broth liquid culture medium were used.

[0085] A 96-well microplate and an incubation temprature of 32.5 C. are used.

[0086] The incubation time of the microplate is: [0087] from 18 to 24 h under aerobic conditions for Candida albicans ATCC 10231, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538; [0088] from 24 to 30 h under aerobic conditions for microbial Aspergillus niger ATCC 6275; [0089] from 24 to 48 h under aerobic conditions for Enterococcus faecalis ATCC 33186.

[0090] Tests

[0091] For each compound:

[0092] A=4-(3-ethoxy-4-hydroxyphenyl) butan-2-one compound

[0093] B=aromatic alcohol compound

[0094] A 10% (weight/volume) stock solution was prepared by mixing 1 g of compound in 9 ml of aqueous 1 agar solution. Successive dilutions were made with the 1 agar solution.

[0095] Tests of Compounds A and B Alone

50 l of each of the daughter solutions obtained containing compound A or B are added to the microplate wells. 100 l of Sabouraud liquid nutrient broth inoculated at double concentration with the Aspergillus niger strain and 50 l of aqueous 1 agar solution are also added thereto.

[0096] Tests of Compounds A and B as a Mixture

[0097] 50 l of each of the daughter solutions obtained containing compound A and 50 l of each of the daughter solutions obtained containing compound B are added to the microplate wells. 100 l of Sabouraud liquid nutrient broth inoculated at double concentration with the strain Aspergillus niger are also added thereto.

[0098] Microbial Growth Control

[0099] A positive microbial growth control was also prepared. The positive microbial growth control corresponds to a mixture of 100 l of aqueous 1 agar solution with 100 l of Sabouraud liquid nutrient broth inoculated at double concentration with the strain Aspergillus niger in the absence of compounds A and B.

[0100] Absorbance Control for Compounds A and B Alone

[0101] An absorbance control was performed in parallel on compounds A and B alone. This control corresponds to 100 l of double concentration sterile Sabouraud liquid nutrient broth+100 l of double concentration compound A or B.

[0102] In the three cases (absorbance control, growth control and test), the final volume present in each of the microplate wells is 200 l.

[0103] In the two cases (test and control), the inoculum represents the concentration of the Aspergillus niger strain present in the final volume of the wells (200 l) and is between 2 and 610.sup.5 cfu/ml of Aspergillus niger.

[0104] The minimum inhibitory concentration (MIC) of each compound A and B alone and in combination was determined in a known manner by means of optical density measurements at a wavelength of 620 nm.

[0105] The test as described above (tests, absorbance control and growth control) was performed again to test the combination A+B on the following strains Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans.

[0106] The following results were obtained with compound B1=2-phenoxyethanol:

[0107] Aspergillus niger

TABLE-US-00001 Concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0 B1 85 47 7 0.0625 B1 95 76 37 5 0.125 B1 89 34 14 2 (FIC 1) 0.25 B1 8 3 2 2 % MIC % MIC MIC of each compound of A of B1 as a mixture alone alone A % B1 % FIC Index Ratio A/B1 0.25 0.25 0.125 0.125 1 1

[0108] The results obtained show synergistic inhibitory activity for the mixture: 0.125% of A and 0.125% of B1, i.e. ratio A/B1=1

[0109] Enterococcus faecalis

TABLE-US-00002 Concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 B1 82 89 89 75 13 0.0625 B1 87 83 87 97 82 34 0.125 B1 86 90 80 95 73 30 0.25 B1 84 95 75 83 74 30 0.5 B1.sup. 80 79 68 5 (FIC 0.75) 37 2 1 B1 6 6 2 3 2 23 MIC of each % MIC of % MIC of compound A B1 as a mixture FIC Ratio alone alone A % B1% Index A/B1 1 1 0.25 0.5 0.75 0.5

[0110] The results obtained show synergistic inhibitory activity for the mixture: 0.25% of A and 0.5% of B1, i.e. ratio A/B1=0.5

[0111] Staphylococcus aureus

TABLE-US-00003 Concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 0 B1 71 57 52 1 0.0625 B1 66 68 62 38 1 0.125 B1 73 65 55 17 (FIC 2) 1 0.25 B1 58 43 15 (FIC 0.5) 1 (FIC 1) 0 0.5 B1 1 1 0 1 0 MIC of each % MIC of % MIC of compound A B1 as a mixture FIC Ratio alone alone A % B1% Index A/B1 0.5 0.5 0.25 0.25 1 0.5

[0112] The results obtained show synergistic inhibitory activity for the mixtures: [0113] i) 0.25% of A and 0.125% of B1, i.e. ratio A/B1=2 [0114] ii) 0.125% of A and 0.25% of B1, i.e. ratio A/B1=0.5 [0115] iii) 0.25% of A and 0.25% of B1, i.e. ratio A/B1=1

[0116] Pseudomonas aeruginosa

TABLE-US-00004 Concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 B1 49 82 116 67 4 0.0625 B1 45 86 118 92 87 27 0.125 B1 39 89 82 95 78 16 0.25 B1 39 16 (FIC 0.25) 14 (FIC 0.5) 10 (FIC 1) 0 (FIC 2) 20 0.5 B1.sup. 0 0 0 0 0 65 1 B1 0 0 0 0 0 14 MIC of each % MIC of % MIC of compound A B1 as a mixture FIC Ratio alone alone A % B1% Index A/B1 1 0.5 0.5 0.25 1 0.25

[0117] The results obtained show synergistic inhibitory activity for the mixtures: [0118] i) 0.0625% of A and 0.25% of B1, i.e. ratio A/B1=0.25 [0119] ii) 0.125% of A and 0.25% of B1, i.e. ratio A/B1=0.5 [0120] iii) 0.25% of A and 0.25% of B1, i.e. ratio A/B1=1 [0121] iv) 0.5% of A and 0.25% of B1, i.e. ratio A/B1=2

[0122] Candida Albicans

TABLE-US-00005 Concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0 B1 48 29 16 0.0625 B1 44 26 17 8 (FIC 0.75) 0.125 B1 22 10 5 1 (FIC 0.75) (FIC 1) 0.25 B1 2 1 1 1 % MIC % MIC MIC of each compound of A of B1 as a mixture FIC Ratio alone alone A % B1% Index A/B1 0.1 0.25 0.05 0.125 0.75 0.2

[0123] The results obtained show synergistic inhibitory activity for the mixtures: [0124] i) 0.05% of A and 0.0625% of B1, i.e. ratio A/B1=0.8 [0125] ii) 0.05% of A and 0.125% of B1, i.e. ratio A/B1=0.4 [0126] iii) 0.025% of A and 0.125% of B1, i.e. ratio A/B1=0.2

EXAMPLE 2: DETERMINATION OF THE ANTIMICROBIAL ACTIVITY OF THE ANTIMICROBIAL MIXTURE

[0127] The antimicrobial efficacy of the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one+2-phenoxyethanol antimicrobial mixture (respective weight ratio of 0.1) was evaluated by the Challenge Test method.

[0128] Protocol

[0129] The method of the challenge test is constituted of an artificial contamination of the sample with microbial strains from collection (bacteria, yeasts and moulds) and of an evaluation of the number of revivable microorganisms seven days after inoculation.

[0130] In order to demonstrate the effect of the antimicrobial mixture, the antimicrobial activity of a cosmetic formula containing 0.05% of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and 0.5% of 2-phenoxyethanol was compared with the same formula alone (control), after inoculation with about 10.sup.6 cfu (colony-forming units)/gram of cosmetic formulation.

[0131] Cosmetic Formula

[0132] A facial care oil-in-water emulsion having the following composition was prepared (contents in weight percentages):

TABLE-US-00006 Sorbitan tristearate (Span 65 V from Croda) 0.9%.sup. Glyceryl mono/distearate (36/64)/potassium stearate mixture (Tegin Pellets from Goldschmidt) 3% Polyethylene glycol stearate (40 ethylene oxide units) 2% 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one 0.05% 2-Phenoxyethanol 0.5%.sup. Propane-1,3-diol 2% Mineral oil, microcrystalline wax and paraffin mixture (Vaseline Blanche Codex 236 from Aiglon) 4% Liquid fraction of shea butter (Shea Olein from Olvea) 1% Cyclopentadimethylsiloxane 5% Cetyl alcohol 4% Apricot kernel oil 0.3%.sup. Hydrogenated polyisobutene (Parleam from NOF Corporation) 7.2%.sup. Myristyl myristate 2% Stearic acid 1.2%.sup. Caffeine 0.1%.sup. Citric acid 0.2%.sup. Glycerol 3% Sodium hydroxide 0.05% Water qs 100%

[0133] Control Formula A:

[0134] Formulation similar to the preceding one containing 0.05% of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and without 2-phenoxyethanol (0.5% compensated with water).

[0135] Control Formula B:

[0136] Similar formulation containing 0.5% of 2-phenoxyethanol and without 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (0.05% compensated with water).

[0137] Microorganism Cultures

[0138] 5 pure cultures of microorganisms were used.

TABLE-US-00007 MICROORGANISMS Subculturing medium T ATCC Escherichia coli (Ec) Trypto-casein soya 35 C. 8739 Enterococcus faecalis (Ef) Trypto-casein soya 35 C. 33186 Pseudomonas aeruginosa (Pa) Trypto-casein soya 35 C. 19429 Candida albicans (Ca) Sabouraud 35 C. 10231 Aspergillus niger (An) Malt 35 C. 6275 ATCC = American Type Culture Collection

[0139] The strains of gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), gram-positive bacterium (Enterococcus faecalis), yeast (Candida albicans), and mould (Aspergillus niger) are inoculated into subculturing medium, respectively the day before inoculation for the bacteria and the yeast, and 5 days before inoculation for the mould.

[0140] On the day of inoculation: [0141] a suspension in tryptone salt diluent is prepared, respectively, for the bacteria and the yeast, so as to obtain by spectrophotometer a suspension with an optical density of between 35% and 45% of transmitted light at 544 nm; [0142] for the mould, the spores are collected by washing the agar with 6 to 7 ml of harvesting solution and the suspension is recovered in a sterile tube or flask.

[0143] After homogenizing the microbial suspension, 0.2 ml of inoculum is introduced into each pill bottle (the suspensions are used pure: between 110.sup.8 and 310.sup.8 cfu per ml) and the microbial suspension in the 20 g of product (=cosmetic formulation) is homogenized thoroughly using a spatula.

[0144] The content of microorganisms present in the product corresponds after homogenization to a concentration of 10.sup.6 microorganisms per gram of product, i.e. inoculation to 1% of an inoculum containing 10.sup.8 microorganisms per ml.

[0145] After 7 days of contact time between the microorganisms and the product at 22 C.2 C. and in the dark, ten-fold dilutions are carried out and the number of revivable microorganisms remaining in the product is counted.

[0146] Results

TABLE-US-00008 No. of CFU/gram of product at T7 days P. C. E. coli aeruginosa E. faecalis albicans A. niger Antimicrobial <200 <200 <200 <200 <200 mixture <200 CFU: sensitivity threshold of the method

EXAMPLE 3: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY IN MIC ON THE MICROBIAL STRAIN CANDIDA ALBICANS

[0147] The demonstration of a synergistic antimicrobial activity effect with a mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (referred to as substance A) and of phenylethyl alcohol (referred to as substance B2) is performed according to the conditions described in Example 1.

[0148] The following results were obtained:

TABLE-US-00009 concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0.2 A 0 B2 85 64 25 3 0.125 B2 41 23 14 7 0 (FIC 0.75) (FIC 1) 0.25 B2 7 2 1 0 0 MIC of each compound as a mixture % MIC A MIC B2 A % B2% FIC Index 0.2 0.25 0.05 0.125 0.75 Ratio A/ B2 = 0.4

[0149] The results obtained show synergistic inhibitory activity for the mixtures: [0150] i) 0.025% of A and 0.125% of B2, i.e. ratio A/B2=0.2 [0151] ii) 0.05% of A and 0.125% of B2, i.e. ratio A/B2=0.4 [0152] iii) 0.1% of A and 0.125% of B2, i.e. ratio A/B2=0.8

EXAMPLE 4: DETERMINATION OF THE ANTIMICROBIAL ACTIVITY OF THE ANTIMICROBIAL MIXTURE

[0153] The antimicrobial efficacy of the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one+2-phenylethyl alcohol antimicrobial mixture (respective weight ratio of 0.1) was evaluated by the Challenge Test method according to the protocol described in Example 2.

[0154] Cosmetic Formula

[0155] A facial care oil-in-water emulsion having the following composition was prepared (contents in weight percentages):

TABLE-US-00010 Sorbitan tristearate (Span 65 V from Croda) 0.9%.sup. Glyceryl mono/distearate (36/64)/potassium stearate mixture (Tegin Pellets from Goldschmidt) 3% Polyethylene glycol stearate (40 ethylene oxide units) 2% 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one 0.05% Phenylethyl alcohol 0.5%.sup. Propane-1,3-diol 2% Mineral oil, microcrystalline wax and paraffin mixture (Vaseline Blanche Codex 236 from Aiglon) 4% Liquid fraction of shea butter (Shea Olein from Olvea) 1% Cyclopentadimethylsiloxane 5% Cetyl alcohol 4% Apricot kernel oil 0.3%.sup. Hydrogenated polyisobutene (Parleam from NOF Corporation) 7.2%.sup. Myristyl myristate 2% Stearic acid 1.2%.sup. Caffeine 0.1%.sup. Citric acid 0.2%.sup. Glycerol 3% Sodium hydroxide 0.05% Water qs 100%

[0156] Results

TABLE-US-00011 No. of CFU/gram of product at T7 days P. C. E. coli aeruginosa E. faecalis albicans A. niger Antimicrobial <200 <200 <200 <200 <200 mixture <200 CFU: sensitivity threshold of the method

EXAMPLE 5: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0157] The demonstration of a synergistic antimicrobial activity effect with a mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (referred to as substance A) and of benzyl alcohol (referred to as substance B3) is performed according to the conditions described in Example 1.

[0158] The following results were obtained:

[0159] Aspergillus niger

TABLE-US-00012 concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0 B3 86 41 6 0.0125 B3 94 49 24 3 0.25 B3 31 7 3 2 (FIC 0.75) (FIC 1) 0.5 B3 1 2 1 2 % MIC % MIC MIC of each compound of A of B3 as a mixture alone alone A % B3% FIC Index 0.25 0.5 0.0625 0.25 0.75 Ratio A/ B3 = 0.25

[0160] The results obtained show synergistic inhibitory activity for the mixtures: [0161] i) 0.0625% of A and 0.25% of B3, i.e. ratio A/B3=0.25 [0162] ii) 0.125% of A and 0.25% of B3, i.e. ratio A/B3=0.5

[0163] Enteroccoccus faecalis

TABLE-US-00013 concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 B3 78 86 92 72 4 0.25 B3 77 76 79 59 45 6 0.5 B3.sup. 30 6 (FIC 0.56) 3 (FIC 0.63) 2 (FIC 0.75) 3 (FIC 1) 21 1 B3 3 5 2 4 3 2 MIC of each % MIC of % MIC of compound A B3 as a mixture FIC alone alone A % B3% Index 1 1 0.0625 0.5 0.56 Ratio A/B3 = 0.125

[0164] The results obtained show synergistic inhibitory activity for the mixture: [0165] i) 0.0625% of A and 0.5% of B3, i.e. ratio A/B3=0.125 [0166] ii) 0.125% of A and 0.5% of B3, i.e. ratio A/B3=0.25 [0167] iii) 0.25% of A and 0.5% of B3, i.e. ratio A/B3=0.5 [0168] iv) 0.5% of A and 0.5% of B3, i.e. ratio A/B3=1

EXAMPLE 6: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0169] The demonstration of a synergistic antimicrobial activity effect with a mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (referred to as substance A) and of 4-isopropyl-3-methylphenol (referred to as substance B4) is performed according to the method described in Example 1.

[0170] The following results were obtained:

[0171] Candida Albicans

TABLE-US-00014 Concentrations tested (in weight %) 0 de A 0.025 A 0.05 A 0.1 A 0.2 A 0 of B4 69 63 38 4 0.0025 B4 87 77 63 41 6 0.005 B4 82 58 54 28 2 0.01 B4 73 52 41 18 2 (FIC 0.75) 0.02 B4 35 18 8 3 1 (FIC 0.63) (FIC 0.75) (FIC 1) 0.04 B4 1 1 1 1 0 % MIC % MIC MIC of each compound of A of B4 as a mixture Ratio alone alone A % B4% FIC Index A/B4 0.2 0.04 0.025 0.2 0.63 1.25

[0172] The results obtained show synergistic inhibitory activity for the mixtures: [0173] i) 0.025% of A and 0.02% of B4, i.e. ratio A/B4=1.25 [0174] ii) 0.05% of A and 0.02% of B4, i.e. ratio A/B4=2.5 [0175] iii) 0.1% of A and 0.02% of B4, i.e. ratio A/B4=5 [0176] iv) 0.1% of A and 0.01% of B4, i.e. ratio A/B4=10

[0177] Enterococcus Faecalis

TABLE-US-00015 Concentrations tested (in weight %) 0 of A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A .sup.0 of B4 42 42 33 34 24 0.0125 B4 45 41 41 37 40 21 0.025 B4 44 36 38 30 36 19 0.05 B4 27 39 12 (FIC 0.625) 11 (FIC 0.75) 11 (FIC 1) 27 0.1 B4 23 18 7 8 4 90 MIC of each % MIC of % MIC of compound A B4 as a mixture FIC Ratio alone alone A % B4% Index A/B4 1 0.1 0.125 0.05 0.63 1.25

[0178] The results obtained show synergistic inhibitory activity for the mixtures: [0179] i) 0.125% of A and 0.05% of B4, i.e. ratio A/B4=1.25 [0180] ii) 0.25% of A and 0.05% of B4, i.e. ratio A/B4=2.5 [0181] iii) 0.5% of A and 0.05% of B4, i.e. ratio A/B4=10