ANTIMICROBIAL MIXTURE CONTAINING 4-(3-ETHOXY-4-HYDROXYPHENYL)BUTAN-2-ONE AND AN ORGANIC ACID COMPOUND, AND COSMETIC COMPOSITION CONTAINING SAME

Abstract

The invention relates to an antimicrobial mixture containing 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and an organic acid compound chosen from a benzoic acid salt, sorbic acid or a salt thereof, methyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, 5-n-octanoylsalicylic acid, a hydroxamic acid and dehydroacetic acid or a salt thereof, and also to a cosmetic composition containing such a mixture. Use in caring for, making up and cleansing keratin materials.

Claims

1. A synergistic antimicrobial mixture comprising 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and an organic acid compound chosen from: i) a benzoic acid salt in an amount such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/benzoic acid salt weight ratio ranges from 0.1 to 3; ii) sorbic acid or a salt thereof in an amount such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sorbic acid or salt thereof weight ratio ranges from 0.1 to 2.5; iii) 5-n-octanoylsalicylic acid in an amount such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/5-n-octanoylsalicylic acid weight ratio ranges from 0.5 to 60; iv) a hydroxamic acid or a salt thereof, of formula (I):
R—C(═O)—NH—OH  (I) in which R represents a linear or branched, saturated or unsaturated C.sub.5 to C.sub.11 alkyl radical in an amount such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/hydroxamic acid (I) or salt thereof weight ratio ranges from 4 to 150; vi) dehydroacetic acid or a salt thereof in an amount such that the weight ratio of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/dehydroacetic acid or salt thereof ranges from 4 to 60; and wherein the amounts of the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and organic acid compound provide synergistic antimicrobial activity.

2. The antimicrobial mixture according to claim 1, wherein the organic acid compound is a benzoic acid salt.

3. The antimicrobial mixture according to claim 1, wherein the benzoic acid salt is chosen from alkali metal salts, alkaline-earth metal salts, the monoethanolamine salt and the ammonium salt.

4. The antimicrobial mixture according to claim 2, wherein the benzoic acid salt is the sodium salt.

5. The antimicrobial mixture according to claim 2, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and benzoic acid or a salt thereof in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/benzoic acid salt weight ratio ranges from 0.1 to 1.5.

6. The antimicrobial mixture according to claim 1, wherein the organic acid compound is sorbic acid or a salt thereof.

7. The antimicrobial mixture according to claim 6, wherein the sorbic acid salt is chosen from alkali metal salts, alkaline-earth metal salts and the triethanolamine salt.

8. The antimicrobial mixture according to claim 6, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and sorbic acid or a salt thereof in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sorbic acid or a salt thereof weight ratio ranges from 0.1 to 1.5.

9. The antimicrobial mixture according to claim 6, wherein the antimicrobial mixture comprises sorbic acid.

10. The antimicrobial mixture according to claim 9, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sorbic acid weight ratio ranging from 0.1 to 1.5.

11. The antimicrobial mixture according to claim 9, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sorbic acid weight ratio ranging from 0.4 to 1.2.

12. The antimicrobial mixture according to claim 9, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sorbic acid weight ratio ranging from 0.1 to 0.8.

13. The antimicrobial mixture according to claim 9, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sorbic acid weight ratio ranging from 0.15 to 0.4.

14. The antimicrobial mixture according to claim 6, wherein the antimicrobial mixture comprises potassium sorbate.

15. The antimicrobial mixture according to claim 14, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/potassium sorbate weight ratio ranging from 0.1 to 1.5.

16. The antimicrobial mixture according to claim 14, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/potassium sorbate weight ratio ranging from 0.3 to 2.5.

17. The antimicrobial mixture according to claim 1, wherein the organic acid compound is 5-n-octanoylsalicylic acid.

18. The antimicrobial mixture according to claim 17, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and 5-n-octanoylsalicylic acid in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/5-n-octanoylsalicylic acid weight ratio ranges from 0.5 to 50.

19. The antimicrobial mixture according to claim 17, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/5-n-octanoylsalicylic acid weight ratio ranging from 3 to 12.

20. The antimicrobial mixture according to claim 17, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/5-n-octanoylsalicylic acid weight ratio ranging from 0.5 to 12.

21. The antimicrobial mixture according to claim 17, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/5-n-octanoylsalicylic acid weight ratio ranging from 7 to 60.

22. The antimicrobial mixture according to claim 1, wherein the organic acid compound is hydroxamic acid of formula (I).

23. The antimicrobial mixture according to claim 22, wherein said hydroxamic acid is chosen from caprohydroxamic acid, caprylhydroxamic acid, caprihydroxamic acid and laurylhydroxamic acid, and mixtures thereof.

24. The antimicrobial mixture according to claim 22, wherein the hydroxamic acid is caprylhydroxamic acid.

25. The antimicrobial mixture according to claim 22, which comprises 1,3-propanediol.

26. The antimicrobial mixture according to claim 25, wherein the 1,3-propanediol is present according to a 1,3-propanediol/hydroxamic acid (I) weight ratio ranging from 5 to 20.

27. The antimicrobial mixture according to claim 22, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and the hydroxamic acid (I) in amounts such that the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/hydroxamic acid (I) weight ratio ranges from 30 to 130.

28. The antimicrobial mixture according to claim 1, wherein the organic acid compound is dehydroacetic acid or a salt thereof.

29. The antimicrobial mixture according to claim 28, wherein the dehydroacetic acid salt is chosen from alkali metal salts.

30. The antimicrobial mixture according to claim 28, which contains sodium dehydroacetate.

31. The antimicrobial mixture according to claim 28, which comprises 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and dehydroacetic acid or a salt thereof in amounts such that the weight ratio of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/dehydroacetic acid or a salt thereof ranges from 5 to 50.

32. The antimicrobial mixture according to claim 30, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sodium dehydroacetate weight ratio ranging from 10 to 60.

33. The antimicrobial mixture according to claim 30, which has a 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one/sodium dehydroacetate weight ratio ranging from 4 to 40.

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

35. The composition according to claim 34, wherein it 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.

36. The composition according to claim 34, 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.

37. A method for the conservation of a composition comprising a physiologically acceptable medium which comprises including an antimicrobial mixture according to claim 1 in the composition.

Description

EXAMPLE 1: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0102] 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 organic acid compound (referred to as substance B) is performed by calculating the synergy index (or FIC index) according to the following formula:

FIC Index=(MICa with B/MICa)+(MICb with A/MICb)

with: [0103] MICa with B: minimum concentration of product A in the combination A+B which makes it possible to obtain an inhibitory effect [0104] MICb with A: minimum concentration of product B in the combination A+B which makes it possible to obtain an inhibitory effect. [0105] MICa: minimum inhibitory concentration of product A alone. [0106] MICb: minimum inhibitory concentration of product B alone.

[0107] 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.

[0108] 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 25%.

[0109] As regards the combinations tested, MICa with b and MICb 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 25%.

Interpretation of the FIC Index:

[0110] 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.

[0111] The results obtained are summarized in the following tables.

[0112] 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.

[0113] 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).

[0114] The microbial strain Enterococcus faecalis ATCC 33186 and a double-concentration BHI (Brain Heart Infusion) broth liquid culture medium were used.

[0115] The microbial strain Staphylococcus aureus ATCC 6538 and a double-concentration nutritive broth liquid culture medium were used.

[0116] The microbial strain Pseudomonas aeruginosa ATCC 9027 and a double-concentration nutritive broth liquid culture medium were used.

[0117] The microbial strain Candida albicans ATCC 10231 and a double-concentration Sabouraud broth liquid culture medium were used.

[0118] A 96-well microplate and an incubation temperature of 32.5° C. are used.

[0119] The incubation time of the microplate is: [0120] from 18 to 24 h under aerobic conditions for Candida albicans ATCC 10231, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538; [0121] from 24 to 30 h under aerobic conditions for microbial Aspergillus niger ATCC 6275; [0122] from 24 to 48 h under aerobic conditions for Enterococcus faecalis ATCC 33186.

Tests

[0123] For each compound:

[0124] A=4-(3-ethoxy-4-hydroxyphenyl)butan-2-one compound

[0125] B=organic acid compound

[0126] 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.

[0127] Tests of Compounds A and B Alone

[0128] 50 μl of each of the daughter solutions obtained containing compound A or B are added to the microplate wells. 100 μl of Sabouraud liquid nutrient broth inoculated at double concentration with the strain Candida albicans and 50 μl of aqueous 1‰ agar solution are also added thereto.

[0129] Tests of Compounds A and B as a Mixture

[0130] 50 μl of each of the daughter solutions obtained containing compound A and 50 μl of each of the daughter solutions obtained containing compound B are added to the microplate wells. 100 μl of Sabouraud liquid nutrient broth inoculated at double concentration with the strain Candida albicans are also added thereto.

Microbial Growth Control

[0131] A positive microbial growth control was also prepared. The positive microbial growth control corresponds to a mixture of 100 μl of aqueous 1‰ agar solution with 100 μl of Sabouraud liquid nutrient broth seeded at double concentration with the strain Candida albicans in the absence of compounds A and B.

Absorbance Control of Compounds A and B Alone

[0132] An absorbance control was performed in parallel on compounds A and B alone. This control corresponds to 100 μl of double concentration sterile Sabouraud liquid nutrient broth+100 μl of double concentration compound A or B.

[0133] In the three cases (absorbance control, growth control and test), the final volume present in each of the microplate wells is 200 μl.

[0134] In the two cases (test and control), the inoculum represents the concentration of the strain Candida albicans present in the final volume of the wells (200 μl) and is between 2 and 6χ 10.sup.5 cfu/ml of Candida albicans.

[0135] 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.

[0136] The test as described above (tests, absorbance controls and growth control) was performed again to test the combination A+B on the following strains Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans.

[0137] The following results were obtained with compound B1=sodium benzoate:

Staphylococcus aureus

TABLE-US-00001 concentra- tions tested (in weight %) 0 of A 0.0625 A 0.125 A 0.25 A 0.5 A 0 of B1 67 65 55 5 0.0625 B1 51 48 37 41 42 0.125 B1  34 35 35 10 1 (FIC 0.75) 0.25 B1  31 19 15  4 0 (FIC 0.63) (FIC 0.75) (FIC 1) 0.5 B1    5  3  3  0 3 % MIC % MIC MIC of each compound as of A of B1 a mixture FIC alone alone A % B1 % Index 0.5 0.5 0.0625 0.25 0.63 Ratio A/ B1 = 0.25

[0138] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.0625% of A and 0.25% of B1, i.e. ratio A/B1=0.25
ii) 0.125% of A and 0.25% of B1, i.e. ratio A/B1=0.5
iii) 0.25% of A and 0.25% of B1, i.e. ratio A/B1=1
iv) 0.25% of A and 0.125% of B1, i.e. ratio A/B1=2
Candida albicans

TABLE-US-00002 concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0 B1    55 31 19 0.025 B1 55 41 33 17 0.05 B1  33 27 19 10 (FIC 0.5) 0.1 B1  24 21 18 10 (FIC 0.75) (FIC 1) 0.2 B1  11  7  5  2 % MIC % MIC MIC of each compound of A of B1 as a mixture FIC alone alone A % B1 % Index 0.1 0.2 0.025 0.1 0.75 Ratio A/ B1 = 0.25

[0139] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.025% of A and 0.1% of B1, i.e. ratio A/B1=0.25
ii) 0.05% of A and 0.1% of B1, i.e. ratio A/B1=0.5
iii) 0.05% of A and 0.05% of B1, i.e. ratio A/B1=1
Pseudomonas aeruginosa

TABLE-US-00003 concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 B1 133 142 79 74 22 0.0625 B1 93 40 90 91 79 42 0.125 B1 96 117 105 81 79 20 0.25 B1 116 96 80 79 80 24 0.5 B1 89 19 (FIC 0.56) 19 (FIC 0.63) 5 (FIC 0.75) 0 (FIC 1) 12 1 B 0 0 0  0  0 33 % MIC of % MIC of MIC of each compound as a mixture FIC A alone B1 alone A % B1% Index 1 1 0.0625 0.5 0.56 Ratio A/B1 = 0.125 0.25 0.5 0.0625 0.25 0.75 Ratio A/B2 = 0.25 

[0140] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.0625% of A and 0.5% of B1, i.e. ratio A/B1=0.125
ii) 0.125% of A and 0.5% of B1, i.e. ratio A/B1=0.25
iii) 0.25% of A and 0.5% of B1, i.e. ratio A/B1=0.5
iv) 0.5% of A and 0.5% of B1, i.e. ratio A/B1=1

EXAMPLE 2: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0141] 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 sorbic acid (referred to as substance B2) and a mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (referred to as substance A) and of potassium sorbate (referred to as substance B3) is carried out according to the protocol described in Example 1.

[0142] The combination of compounds A and B2 was tested on the following strains: Staphylococcus aureus, Candida albicans, Aspergillus niger, Enterococcus faecalis.

[0143] The combination of compounds A and B3 was tested on the following strains: Aspergillus niger, Enterococcus faecalis.

[0144] The following results were obtained:

A) Mixtures with Compound B2: Sorbic Acid
Staphylococcus aureus

TABLE-US-00004 concentrations tested (in weight %) 0 of A 0.0625 A 0.125 A 0.25 A 0.5 A 0 de B2 63 60 52 1  0.125 B2   52 59 62 47 1 0.25 B2   28 12  6  0 0 (FIC 0.63) (FIC 0.75) (FIC 1) 0.5 B2   −5  0  1  0 0 % MIC MIC of each compound % MIC of of B2 as a mixture FIC A alone alone A % B2 % Index 0.5 0.5 0.0625 0.25 0.63 Ratio A/ B2 = 0.25

[0145] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.0625% of A and 0.25% of B2, i.e. ratio A/B2=0.25
ii) 0.125% of A and 0.25% of B2, i.e. ratio A/B2=0.5
iii) 0.25% of A and 0.25% of B2, i.e. ratio A/B2=1
Candida albicans

TABLE-US-00005 concentrations tested (in weight %) 0 of A 0.025 A 0.05 A 0.1 A 0 of B2 56 41 16  0.025 B2 37 31 24 18 0.05 B2 24 11 12  8 (FIC 0.75) (FIC 1) 0.1 B2  21 20 14  6 % MIC MIC of each compound % MIC of of B2 as a mixture FIC A alone alone A % B2 % Index 0.1 0.1 0.025 0.05 0.75 Ratio A/ B2 = 0.5

[0146] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.025% of A and 0.05% of B2, i.e. ratio A/B2=0.5
ii) 0.05% of A and 0.05% of B2, i.e. ratio A/B2=1
Aspergillus niger

TABLE-US-00006 concentrations tested (in weight %) 0 of A 0.0625 A 0.125 A 0.25 A 0 of B2 84   40   6  0.0125 B2 98 89   37   5 0.25 B2 95  2    3 −2    (0.75) (FIC 1) 0.5 B2   8  0    1 −4 % MIC MIC of each compound % MIC of of B2 as a mixture FIC A alone alone A % B2 % Index 0.25 0.5 0.0625 0.25 0.75 Ratio A/ B2 = 0.25

[0147] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.0625% of A and 0.25% of B2, i.e. ratio A/B2=0.25
ii) 0.125% of A and 0.25% of B2, i.e. ratio A/B2=0.5
Enterococcus faecalis

TABLE-US-00007 concentrations tested (in weight %) 0 of A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 of B2 82 86 105 100 15 0.25 B2 53 91 75 5 100 5 0.5 B2 78 42 0 (FIC 0.63) 90 85 13 1 B2 0 38  0 10 0 −16 % MIC of % MIC of MIC of each compound as a mixture FIC A alone B2 alone A % B2% Index 1 1 0.125 0.5 0.63 Ratio A/B2 = 0.25

[0148] The results obtained show synergistic inhibitory activity for the mixture:

i) 0.125% of A and 0.5% of B2, i.e. ratio A/B2=0.25
B) Mixtures with compound B3: potassium sorbate
Enterococcus faecalis

TABLE-US-00008 concentrations tested (in weight %) 0 A 0.125 A 0.25 A 0.5 A 1 A 2A 0 B3 56 55 52 28 24 1 B3 22 19 (FIC 0.57) 19 (FIC 0.63) 13 (FIC 0.75) 4 (FIC 1) −1 2 B3 10 15 17 11 −3 0 % MIC of % MIC of MIC of each compound as a mixture FIC A alone B3 alone A % B3% Index 2 2 0.125 1 0.57 Ratio A/B3 = 0.125

[0149] The results obtained show synergistic inhibitory activity for the mixtures:

[0150] i) 0.125% of A and 1% of B3, i.e. ratio A/B3=0.125

ii) 0.25% of A and 1% of B3, i.e. ratio A/B3=0.25
iii) 0.5% of A and 1% of B3, i.e. ratio A/B3=0.5
iv) 1% of A and 1% of B3, i.e. ratio A/B3=1
Aspergillus niger

TABLE-US-00009 concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0 B3   65 40 15   0.025 B3 58 44 12 21 (FIC 0.75)  0.05 B3 37 10 16  8 (FIC 0.75) (FIC 1)    0.1 B3  10 10 16  8 % MIC MIC of each compound % MIC of of B3 as a mixture FIC A alone alone A % B3 % Index 0.1 0.1 0.025 0.05 0.75 Ratio A/ B3 = 0.5
The results obtained show synergistic inhibitory activity for the mixtures:
i) 0.025% of A and 0.05% of B3, i.e. ratio A/B3=0.5
ii) 0.05% of A and 0.05% of B3, i.e. ratio A/B3=1
iii) 0.05% of A and 0.025% of B3, i.e. ratio A/B3=2

EXAMPLE 3

[0151] 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% Glyceryl mono/distearate (36/64)/potassium stearate mixture    3% (Tegin Pellets from Goldschmidt) Polyethylene glycol stearate (40 ethylene oxide units)    2% 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one 0.05% Potassium sorbate  0.2% Propane-1,3-diol    3% Mixture of mineral oil, microcrystalline wax and paraffin    4% (Vaseline Blanche Codex 236 from Aiglon) Liquid fraction of shea butter (Shea Olein from Olvea)    1% Cyclopentadimethylsiloxane    5% Cetyl alcohol    4% Apricot kernel oil  0.3% Hydrogenated polyisobutene (Parleam from NOF Corporation)  7.2% Myristyl myristate    2% Stearic acid  1.2% Caffeine  0.1% Citric acid  0.2% Glycerol    3% Sodium hydroxide 0.05% Water qs  100%

EXAMPLE 4: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0152] 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 methyl 4-hydroxybenzoate (referred to as substance B4) and a mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (referred to as substance A) and of propyl 4-hydroxybenzoate (referred to as substance B5) is carried out according to the protocol described in Example 1.

[0153] The combination of compounds A and B4 was tested on the following strains: Staphylococcus aureus, Candida albicans, and Pseudomonas aeruginosa.

[0154] The combination of compounds A and B5 was tested on the following strains: Candida albicans, and Pseudomonas aeruginosa.

[0155] The following results were obtained:

A) Mixtures with Compound B4: Methyl 4-Hydroxybenzoate
Staphylococcus aureus

TABLE-US-00011 Concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 0 B4   66 61 51 1  0.25 B4 51 60 55 51 1 0.5 B4 44 16 14  6 3 (FIC 0.63) (FIC 0.75) (FIC 1) 1 B4   19  1  1  3 2 % MIC MIC of each compound % MIC of of B4 as a mixture FIC A alone alone A % B4 % Index 0.5 1 0.0625 0.5 0.63 Ratio A/ B4 = 0.125

[0156] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.0625% of A and 0.5% of B4, i.e. ratio A/B4=0.125
ii) 0.125% of A and 0.5% of B4, i.e. ratio A/B4=0.25
iii) 0.25% of A and 0.5% of B4, i.e. ratio A/B4=0.5
Candida albicans

TABLE-US-00012 concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0 B4   61 38 18   0.025 B4 67 53 36 19  0.05 B4 40 38 20 12 (FIC 0.75) 0.1 B4 28 23 15 10 (FIC 0.75) (FIC 1)   0.2 B4 12  6  1  1 % MIC MIC of each compound % MIC of of B4 as a mixture FIC A alone alone A % B4 % Index 0.1 0.2 0.05 0.05 0.75 Ratio A/ B4 = 1

[0157] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.025% of A and 0.1% of B4, i.e. ratio A/B4=0.25
ii) 0.05% of A and 0.1% of B4, i.e. ratio A/B4=0.5
iii) 0.05% of A and 0.05% of B4, i.e. ratio A/B4=1
Pseudomonas aeruginosa

TABLE-US-00013 concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 B4 37 52 78 52 24 0.0625 B4 187 135 113 101 66 3 0.125 B4 150 61 166 115 43 24 0.25 B4 142 99 4 (FIC 0.63) 1 (FIC 0.75) 0 (FIC 1) −4 0.5 B4 1 0 0 0  0 34 % MIC of % MIC of MIC of each compound as a mixture FIC A alone B4 alone A % B4% Index 1 0.5 0.125 0.25 0.63 Ratio A/B4 = 0.5

[0158] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.125% of A and 0.25% of B4, i.e. ratio A/B4=0.5
ii) 0.25% of A and 0.25% of B4, i.e. ratio A/B4=1
iii) 0.5% of A and 0.25% of B4, i.e. ratio A/B4=2
B) Mixtures with Compound B5: Propyl 4-Hydroxybenzoate
Candida albicans

TABLE-US-00014 concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0 B5   56 39 18   0.025 B5 52 43 32 19  0.05 B5 33 20 11  7 (FIC 0.75) (FIC 1)  0.1 B5  21 13  6  2 % MIC MIC of each compound % MIC of of B5 as a mixture FIC A alone alone A % B5 % Index 0.1 0.1 0.025 0.05 0.75 Ratio A/ B5 = 0.5

[0159] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.025% of A and 0.05% of B5, i.e. ratio A/B5=0.5
ii) 0.05% of A and 0.05% of B5, i.e. ratio A/B5=1
Pseudomonas aeruginosa

TABLE-US-00015 concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 B5 117 54 87 64 45 0.125 B5 76 58 113 58 65 67 0.25 B5 45 36 31 25 20 (FIC ≤ 0.75) 14 (FIC ≤ 1) 0.5 B5 42 57 38 47 −23 (FIC ≤ 1)     −3 1 B5 24 0 33 −18 −17   8 % MIC of % MIC of MIC of each compound as a mixture FIC A alone B5 alone A % B5% Index ≥1 1 0.5 0.25 ≤0.75 Ratio A/B5 = 2

[0160] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.5% of A and 0.5% of B5, i.e. ratio A/B5=1
ii) 1% of A and 0.5% of B5, i.e. ratio A/B5=2
iii) 0.5% of A and 0.25% of B5, i.e. ratio A/B5=2
iv) 1% of A and 0.25% of B5, i.e. ratio A/B5=4

C)

[0161] It was moreover noted that the following mixtures: [0162] mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one with ethyl 4-hydroxybenzoate, [0163] mixture of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one with butyl 4-hydroxybenzoate,
tested in the same tests described previously have no synergistic antimicrobial activity.

EXAMPLE 5: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0164] 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 5-n-octanoylsalicylic acid (referred to as substance B6) is performed according to the method described in Example 1.

[0165] The following results were obtained:

Candida albicans

TABLE-US-00016 concen- trations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0.2 A 0 B6      74 69   36   6   0.0125 B6 66   48 37   13   1 (FIC 0.625)  0.025 B6 40   31 24    3   1 (FIC 0.5)  (FIC 0.75)  0.05 B6 30    6  5    1   0 (FIC 0.625) (FIC 0.75) (FIC 1)    0.1 B6   1  −3  0  −3 −1 % MIC MIC of each compound % MIC of of B6 as a mixture FIC A alone alone A % B6 % Index 0.2 0.1 0.05 0.025 0.5 Ratio A/ B6 = 2

[0166] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.0625% of A and 0.05% of B6, i.e. ratio A/B6=0.5
ii) 0.05% of A and 0.05% of B6, i.e. ratio A/B6=1
iii) 0.1% of A and 0.05% of B6, i.e. ratio A/B6=2
iv) 0.05% of A and 0.025% of B6, i.e. ratio A/B6=2
v) 0.1% of A and 0.025% of B6, i.e. ratio A/B6=4
vi) 0.1% of A and 0.0125% of B6, i.e. ratio A/B6=8
Aspergillus niger

TABLE-US-00017 concentrations tested (in weight %) 0 A 0.0025 A 0.05 A 0.1 A 0.2 A 0.4 A 0 B6 84 40 6 59 5 0.025 B6 98 111 103 97 10 (FIC 0.75) 4 0.05 B6 97 75 63 68 6 (FIC 1).sup.  4 0.1 B6 17 17 8 5  3 0 % MIC of % MIC of MIC of each compound as a mixture FIC A alone B6 alone A % B6% Index 0.4 0.1 0.2 0.025 0.75 Ratio A/B6 = 8

[0167] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.2% of A and 0.05% of B6, i.e. ratio A/B6=4
ii) 0.2% of A and 0.025% of B6, i.e. ratio A/B6=8
Enterococcus faecalis

TABLE-US-00018 concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0.5 A 1 A 0 B6 35 40 41 36 16 0.0125 B6 28 26 20 (FIC 0.625) 18 (FIC 0.75) 21 (FIC 1) −2 0.025 B6 2 2  1  2  2 −19 % MIC of % MIC of MIC of each compound as a mixture FIC A alone B6 alone A % B6% Index 1 0.025 0.125 0.0125 0.63 Ratio A/B6 = 10

[0168] The results obtained show synergistic inhibitory activity for the mixture:

i) 0.125% of A and 0.0125% of B6, i.e. ratio A/B6=10
ii) 0.25% of A and 0.0125% of B6, i.e. ratio A/B6=20
iii) 0.5% of A and 0.0125% of B6, i.e. ratio A/B6=40

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 caprylhydroxamic acid (referred to as substance B7) is performed according to the method described in Example 1.

[0170] The combination of compounds A and B7 was tested on the Aspergillus niger strain.

[0171] For B7, use was made of B′=caprylhydroxamic acid (compound B7) at 7.5% by weight in 1,3-propanediol (sold under the name Zeastat by the company Inolex Chemical).

[0172] The following results were obtained:

Aspergillus niger

TABLE-US-00019 Concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0.2 A 0 B′    62 32 25   10   0.0125 B′ 79 36 23  8    6 (FIC 0.63)  0.025 B′ 57 22 16 11   10 (FIC 0.63) (FIC 0.75) 0.05 B′ 28 16 14  4  −3 (FIC 0.63) (FIC 0.75) (FIC 1)   0.1 B′  13  8 10  3    6 Ratio % MIC MIC of each compound A/B′ % MIC of of B′ as a mixture FIC (Ratio A alone alone A % B′ % Index A/B7) 0.2 0.1 0.025 0.05 0.63 0.5 (6.7)

[0173] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.025% of A and 0.05% of B′ (3.75 10.sup.−3% of B7), i.e. ratio A/B7=6.7
ii) 0.05% of A and 0.05% of B′ (3.75 10.sup.−3% of B7), i.e. ratio A/B7=13.3
iii) 0.1% of A and 0.05% of B′ (3.75 10.sup.−3% of B7), i.e. ratio A/B7=26.7
iv) 0.05% of A and 0.025% of B′ (1.875 10.sup.−3% of B7), i.e. ratio A/B7=26.7
v) 0.1% of A and 0.025% of B′ (1.875 10.sup.−3% of B7), i.e. ratio A/B7=53.3
vi) 0.1% of A and 0.0125% of B′ (0.94 10.sup.−3% of B7), i.e. ratio A/B7=106

EXAMPLE 7: DETERMINATION OF THE ANTIMICROBIAL ACTIVITY OF THE ANTIMICROBIAL MIXTURE

[0174] The antimicrobial efficacy of the 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one+caprylhydroxamic acid antimicrobial mixture (respective weight ratio of 0.16) was evaluated by the Challenge Test method.

Cosmetic Formula

[0175] A facial care oil-in-water emulsion having the following composition was prepared (contents in weight percentages):

TABLE-US-00020 Sorbitan tristearate (Span 65 V from Croda)  0.9% Glyceryl mono/distearate (36/64)/potassium stearate mixture    3% (Tegin Pellets from Goldschmidt) Polyethylene glycol stearate (40 ethylene oxide units)    2% 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one 0.15% Caprylhydroxamic acid in solution at 7.5% by weight  1.5% in 1,3-propanediol Propane-1,3-diol    2% Mixture of mineral oil, microcrystalline wax and paraffin    4% (Vaseline Blanche Codex 236 from Aiglon) Liquid fraction of shea butter (Shea Olein from Olvea)    1% Cyclopentadimethylsiloxane    5% Cetyl alcohol    4% Apricot kernel oil  0.3% Hydrogenated polyisobutene (Parleam from NOF Corporation)  7.2% Myristyl myristate    2% Stearic acid  1.2% Caffeine  0.1% Citric acid  0.2% Glycerol    3% Sodium hydroxide 0.05% Water qs  100%
Control formula A: Formulation similar to the preceding one containing 0.15% of 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one and without caprylhydroxamic acid (1.5% compensated with water)
Control formula B: Similar formula containing 1.5% of caprylhydroxamic acid (in solution with 1,3-propanediol) and without 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one (0.15% compensated with water)

Microorganism Cultures

[0176] 5 pure cultures of microorganisms were used.

TABLE-US-00021 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

[0177] 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.

[0178] On the day of inoculation: [0179] 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; [0180] 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.

[0181] After homogenizing the microbial suspension, 0.2 ml of inoculum is introduced into each pill bottle (the suspensions are used pure: between 1×10.sup.8 and 3×10.sup.8 cfu per ml) and the microbial suspension in the 20 g of product (=cosmetic formula) is homogenized thoroughly using a spatula.

[0182] 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.

[0183] 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 performed and the number of revivable microorganisms remaining in the product is counted.

Results

[0184]

TABLE-US-00022 No. of CFU/gram of product at T7 days E. coli P. aeruginosa E. faecalis C. albicans A. niger Antimicrobial <200 <200 <200 <200 2 E3 mixture <200 CFU: sensitivity threshold of the method

EXAMPLE 8: DETERMINATION OF THE SYNERGISTIC ANTIMICROBIAL ACTIVITY AS MIC

[0185] 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 sodium dehydroacetate (referred to as substance B8) is performed according to the protocol described in Example 1.

[0186] The following results were obtained:

Candida albicans

TABLE-US-00023 Concentrations tested (in weight %) 0 A 0.025 A 0.05 A 0.1 A 0 B8     53 35 18   0.00125 B8 28 19 13  8 (FIC 0.75) (FIC 1)  0.0025 B8 15  8  6  3 % MIC MIC of each compound % MIC of of B8 as a mixture FIC A alone alone A % B8 % Index 0.1 0.0025 0.025 0.000125 0.75 Ratio A/ B8 = 20

[0187] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.025% of A and 0.00125% of B8, i.e. ratio A/B8=20
ii) 0.05% of A and 0.00125% of B8, i.e. ratio A/B8=40
Aspergillus niger

TABLE-US-00024 Concentrations tested (in weight %) 0 A 0.0625 A 0.125 A 0.25 A 0 B8    96 41 7   0.0025 B8 97 69 29 4  0.005 B8 29 12  3 2 (FIC 0.5)  (FIC 0.75) 0.01 B8  3  2  2 2 (FIC 0.75) (FIC 1)   0.02 B8  3  2  2 2 % MIC MIC of each compound % MIC of of B8 as a mixture FIC A alone alone A % B8 % Index 0.25 0.02 0.0625 0.01 0.75 Ratio A/ B8 = 6.25

[0188] The results obtained show synergistic inhibitory activity for the mixtures:

i) 0.0625% of A and 0.01% of B8, i.e. ratio A/B8=6.25
ii) 0.125% of A and 0.01% of B8, i.e. ratio A/B8=12.5
iii) 0.0625% of A and 0.005% of B8, i.e. ratio A/B8=12.5
iv) 0.125% of A and 0.005% of B8, i.e. ratio A/B8=25