Patent Application
20130156708
The present invention relates to (i) a compound of formula (I) or a (pharmaceutically) acceptable salt thereof or (ii) a mixture comprising one or more of said compounds of formula (I) and/or said salts
wherein
R1 denotes a radical in ortho-, meta, or para-position to the phenolic OH group, the radical being selected from the group consisting of
Herein E/Z-3-methylbut-1-en-1-yl designates —CH═CH—CH(CH3)2, 3-methylbut-2-en-1-yl designates —CH2—CH═C(CH3) and 3-methylbut-3-en-1-yl designates —CH2—CH2—C(CH3)═CH2.
In particular, the present invention relates to certain uses of said compound of formula (I) or said mixture comprising one or more compounds of formula (I). As will be described in more detail below, a primary aspect of the present invention is the use of said compound or mixture as antimicrobial agent for inactivation of microorganisms, in particular for rapid inactivation of microorganisms. Further relevant uses of said compound or mixture relate to the use as fragrance substance, flavoring substance, tingling agent, numbing agent, antioxidant, and agent or agent mixture for synergistically co-operating with a further antimicrobial agent so that a total antimicrobial activity is synergistically increased, wherein the synergistic effect is preferably determined according to Kull.
The present invention furthermore relates to (i) compounds of formula (I), (pharmaceutically) acceptable salts thereof and to (ii) mixtures comprising or consisting of one or more compounds of formula (I) or salts thereof as defined above, for use as antimicrobial agent for inactivation of microorganisms. In relevant aspects of the present invention said compound or mixture is employed for combined use as (a) antimicrobial agent and (b) fragrance substance, flavoring substance, tingling agent, numbing agent, antioxidant, and/or agent or agent mixture for synergistically co-operating with a further antimicrobial agent so that a total antimicrobial activity is synergistically increased, wherein the synergistic effect is preferably determined according to Kull.
The present invention also relates to antimicrobial compositions comprising a compound of formula (I) or an (pharmaceutically) acceptable salt thereof.
Furthermore, the present invention relates to methods for inactivation, preferably rapid inactivation, of microorganisms present on a surface area or in a volume area in which methods said compound of formula (I) or said mixture is used.
As will be explained in more detail below, according to a first aspect the present invention is based on the surprising finding that compounds of formula (I) can be used as antimicrobial agents and show a fast time-kill activity so that they are extremely useful in particular personal care, oral care, home care, and pharmaceutical application.
Furthermore, the invention is based on the finding that compounds of formula (I) (as well as (pharmaceutically) acceptable salts thereof) possess further outstanding properties. In particular, (i) compounds of formula (I) and/or their acceptable salts or (ii) corresponding mixtures can be advantageously used as a fragrance substance, as a flavoring substance, as tingling agents, as numbing agents, as antioxidants, and as agents for synergistically co-operating with further antimicrobial agents so that the total antimicrobial activity is synergistically increased, wherein the synergistic effect is preferably determined according to Kull.
There is a well-documented link between contaminated hands and infectious disease transmission. Hand and body wash is therefore an effective means for preventing spread of pathogens, specifically by the fecal-oral route and from the respiratory tract. It has been shown that the effectiveness of washing procedure can be substantially increased by using soaps containing antimicrobial agents. However, many of the currently used antibacterial agents are not effective, as their mode of action is not fast enough to ensure pathogen inactivation within usually short application periods of below 1 min. For example, Triclosan and Triclocarban do not provide a fast inactivation against gram negative bacteria, and therefore may not increase the clinical benefit of a plain soap against this important bacterial group. In addition, concerns have been raised that widespread use of antiseptics such as Triclosan may lead to multiply-antibiotic resistant pathogens. In consequence, there is an ongoing search for safe broad-spectrum antimicrobials with fast time-kill activity to be used in personal care applications.
Another application, where antimicrobials with a fast inactivation are required, is in home care, i.e. hard surface cleaning with a focus on kitchen, bath and toilet cleaning. Effective cleaning of kitchen equipment and surfaces can help preventing the spread of foodborne pathogens and subsequent food infection. For example, in the US, contamination with Campylobacter jejuni or Salmonella spp. from raw poultry, eggs or dairy products are estimated to be responsible for over 3 million disease cases per year. Part of these infections are transmitted via contaminated hands, tools or surfaces and could potentially be avoided by effective cleaning agents and procedures.
Several disadvantages are connected with antimicrobial compounds and/or preservative helpers currently used:
Thus, according to a first aspect it was an object of the present invention to provide antimicrobial substances (compounds or mixtures) which posses a fast time-kill activity and preferably additionally broad spectrum antimicrobial activity and/or favourable sensoric properties and/or formulation properties that are not detrimental to the final product.
The compounds of formula (I) of the present invention are characterized by broad spectrum of antimicrobial activity and an exceptionally fast time-kill against relevant pathogenic microorganisms.
Thus, a first aspect of the present invention relates to the use of (i) a compound of formula (I) or a (pharmaceutically) acceptable salt thereof or (ii) a mixture comprising one or more or consisting of two or more compounds of formula (I) and/or said salts
wherein
R1 denotes a radical in ortho-, meta, or para-position to the phenolic OH group, the radical being selected from the group consisting of
The term “corresponding (pharmaceutically) acceptable salt thereof” indicates those salts of a compound or of compounds of formula (I) that can be safely used for pharmaceutical purposes. This does not mean that the present invention or any aspect thereof is restricted to the use of a compound of formula (I) or a corresponding mixture for pharmaceutical purposes. Generally, if a salt can be used for pharmaceutical purposes it can likewise be used for cosmetic purposes, or in food or beverage formulations. In particular, the sodium and potassium and ammonium salts of compounds of formula (I) are considered as (pharmaceutically) acceptable salts. In some cases the utilization of the respective ionic compound or solvatecarrier proves to be superior to the unmodified derivative. The (pharmaceutically) acceptable salts (and the corresponding solvates) of compounds of formula (I) can be prepared by standard procedures. Hereinafter, any reference to a compound of formula (I) or a corresponding mixture as defined above is to be understood as comprising an additional reference to corresponding (pharmaceutically) acceptable salts thereof.
A related aspect of the present invention relates to a compound of formula (I) or a pharmaceutically) acceptable salt thereof or a mixture comprising one or more or consisting of two or more compounds of formula (I), and/or said salts
wherein
R1 denotes a radical in ortho-, meta, or para-position to the phenolic OH group, the radical being selected from the group consisting of
While compounds of formula (I) and corresponding mixtures as defined above generally have antimicrobial activity (and additional advantageous properties as listed above and discussed below in more detail) the use of certain specific compounds of formula (I) is preferred. In particular, a use according to the present inventions is preferred wherein the compound of formula (I) or one, more or all compounds of formula (I) in the mixture, are selected from the group consisting of formulae (IIa), (IIb), (IIIa), and (IIIb):
Correspondingly, a compound or mixture for use according to the present invention is preferred, wherein the compound of formula (I) is selected from the group consisting of formulae (IIa), (IIb), (IIIa), and (IIIb) as defined above. The (pharmaceutically) acceptable salts of the compounds of formulae (IIa), (IIb), (IIIa) and (IIIb) are likewise preferred.
Compounds of formula (I) and corresponding mixtures are effective against a wide range of microorganisms. Preferably, in uses and methods of the present invention the microorganisms are selected from the group consisting of Bacteria, Archaea, Fungi, Viruses, Protozoa and other small eukaryotes considered as microorganisms.
Preferably the microorganisms are selected from (i) the phyla consisting of Proteobacteria, Firmicutes, Actinobacteria, Chlamydiae, Spirochaetes, Bacteroidetes, Fusobacteria, (ii) fungi from the phyla consisting of Ascomycota, Zygomycota and Basidiomycota, and (iii) protozoa (in particular protozoa harmful to humans and animals), more preferably selected from the phyla consisting of Proteobacteria, Firmicutes, Actinobacteria, Chlamydiae, Spirochaetes, Bacteroidetes, Fusobacteria, and fungi from the phylum consisting of Ascomycota.
More preferably the microorganisms are selected from the group consisting of Streptococcus mutans, Streptococcus sobrinus, Actinomyces naeslundii, Porphyromonas gingivalis, Fusobacterium nucleatum, Veillonella parvula, Treponema denticola, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Capnocytophagy spp., Corynebacterium xerosis, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes, Bacillus cereus, Clostridium perfringens, Burkholderia cepacia, Escherichia coli, Salmonalla enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa, Yersinia enterocolitica, Shigella flexneri, Campylobacter jejuni, Listeria monocytogenes, Candida albicans, Malassezia furfur, Malassezia globosa, Propionibacterium acnes, Aspergillus flavus, Aspergillus brasiliensis, Aspergillus niger, Cryptosporidium parvum, Cyclospora cayetanenis, Giardia lamblia, Toxoplasma gondii, and Trichomonas vaginalis, more preferably selected from the group consisting of Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Veillonella parvula, Treponema denticola, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Corynebacterium xerosis, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Burkholderia cepacia, Escherichia coli, Salmonalla enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa, Shigella flexneri, Campylobacter jejuni, Candida albicans, Malassezia furfur, Malassezia globosa, Propionibacterium acnes, Aspergillus flavus, Aspergillus brasiliensis, and Aspergillus niger, particularly preferably selected from the group consisting of Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Corynebacterium xerosis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Salmonalla enteritidis, Pseudomonas aeruginosa, Candida albicans, Malassezia furfur, and Aspergillus niger.
The compounds of formula (I) used according to the present invention and in particular the preferred compounds of formulae (IIa), (IIb), (IIIa) and (IIIb), are characterized by an exceptionally fast time-kill against a broad spectrum of microorganisms from different groups, such as gram positive bacteria, gram negative bacteria, and yeast, as shown in the examples below (examples A1 and A2,
Similar results have been obtained for the compounds of formulae (IIb), (IIIa) and (IIIb), i.e. for the compounds (IIb): 4-(3-Methylbut-3-enyl)phenol, (IIIa): 2-(3-Methylbut-2-enyl)phenol, and (IIIb): 2-(3-Methylbut-3-enyl)phenol.
In uses according to the present invention the total amount of compounds of formula (I) in (ii) the mixture is preferably in the range of from 0.001 to 5 wt.-%, preferably in the range of from 0.01 to 0.5 wt.-%, based on the total weight of the mixture. Correspondingly, in preferred mixtures for use according to the present invention the total amount of compounds of formula (I) in the mixture is in the range of from 0.001 to 5 wt.-%, preferably in the range of from 0.01 to 0.5 wt.-%, based on the total weight of the mixture.
When used according to the first aspect of the present invention, i.e. when used as antimicrobial agent for inactivation of microorganisms, (i) the compound of formula (I) or the (pharmaceutically) acceptable salt thereof or (ii) the mixture comprising one or more or consisting or two or more compounds of formula (I), as defined above, is preferably simultaneously used as
With respect to the compounds and mixtures of the present invention a corresponding combined use is preferred.
Surprisingly, compounds of formula (I) as defined above, and in particular the preferred compounds of formulae (IIa), (IIb), (IIIa), and (IIIb) have specific and particularly valuable odor properties. Correspondingly, a further aspect of the present invention is the use of (i) a compound of formula (I) or (ii) a corresponding mixture, as defined above, as a fragrance substance, preferably as a fragrance substance causing one or more odour impressions selected from the group consisting of rosy, green, and clove-like. In particular, the smell of the compounds of formula (IIa) and (IIIa) in own experiments has been described as rosy, green, clove-like, slightly technical.
Furthermore surprisingly, the compounds of formula (I), the (pharmaceutically) acceptable salts thereof and the corresponding mixtures (as defined above) when used in certain concentrations have an absolutely unexpected and favourable taste profile. In particular, compound (IIa) as well as compound (IIIa) have been tested for taste impressions in sugar slurry dependent on different notices. The taste impressions are described as follows:
In particular the numbing and tingling note sensations combined with favorable taste impressions (and of course in combination with the antimicrobial activity and the other properties of the compounds of formula (I)) make these compounds valuable ingredients in a huge number of compositions. For preferred compositions see below.
The compounds of formulae (IIa) and (IIIa) were further tested in oral care formulations. In particular, an alcohol free mouth wash formulation in combination with a standard peppermint flavor (Optamint® Flavor, dosage 0.15%) plus compound of formula (IIa) or (IIIa), respectively, (dosage 0.1%) was compared with a sample without any compound of formula (I), but being otherwise identically composed. The taste impressions of the samples including the respective compound of formula (IIa) or (IIIa) are described as follows:
No impairment of the flavor profile is noticeable, but the mouthwash clearly leaves a numbing/tingling feeling on the tongue in comparison to a mouthwash not containing the respective material.
The compounds of formula (I) and the corresponding mixtures of the present invention do not display a medicinal taste (as e.g. thymol). They blend in nicely with the most commonly used flavor profiles for oral care products.
A silica toothpaste formulation in combination with a standard spearmint flavour (Optamint® Flavor, dosage 1%) plus the respective compound of formula (IIa) or (IIb) dosage 0.1% was compared to a sample without such compound of formula (I). The taste impressions of the sample including the compound of formula (IIa) or (IIb), respectively, are described as follows:
No impairment of the flavor profile is noticeable, but the toothpaste clearly leaves a numbing/tingling feeling on the tongue in comparison to a toothpaste not containing the respective material.
It was particularly surprising and unexpected that compounds of formula (I) as defined above cause a tingling sensation and/or numbing sensation on the tongue in typical taste experiments. In particular with respect to oral care compositions it is often advantageous when the composition causes a tingling and/or numbing sensation. There is a steady demand for oral care compositions and other compositions providing both an antimicrobial effect and a numbing and/or tingling sensation when brought into contact with the oral cavity.
Correspondingly a further aspect of the present invention relates to the use of (i) a compound of formula (I) or a (pharmaceutically) acceptable salt thereof or (ii) a corresponding mixture as defined above as flavouring substance, preferably causing one or more taste impressions selected from the group consisting of green, sweet, camphor-like, rock-candy, floral, as tingling agent and/or numbing agent.
Regarding the inventive uses of compounds of formula (I) or corresponding mixtures, in particular regarding the uses as antimicrobial agent, fragrance substance, flavouring substance, tingling agent, and numbing agent, it is to be noted that these uses correspond to methods of the present invention. In particular, the use of a compound or mixture as antimicrobial agent corresponds to a method for inactivation of microorganisms. For preferred methods see below. Similarly, the use of a compound or mixture as a fragrance substance corresponds to a method for imparting or enhancing an odour (in particular imparting or enhancing an odour impression selected from the group consisting of rosy, green, and clove-like). Similarly, the use of a compound or mixture as flavouring substance according to the present invention corresponds to a method for imparting or enhancing a flavour or taste impression (in particular for imparting or enhancing a taste impression selected from the group consisting of green, sweet, camphor-like, rock-candy, floral). Similarly, the use of a compound or mixture as a tingling agent according to the present invention corresponds to a method for imparting or enhancing a numbing and/or tingling sensation (in particular on the tongue) wherein said compound of formula (I) or a corresponding mixture is contacted with the oral mucosa (especially in the regions of the taste buds of the tongue).
Compounds of formula (I), in particular compounds of formula (IIa) and (IIIa), were tested in basic formulations for personal care and home care. When added at a dosage of 0.5% to bar soap, all purpose cleaner, liquid detergent, detergent powder, fabric softener, body lotion, and hair shampoo, there was no negative sensory impression observed as compared to comparison compositions comprising no compound of formula (I) but being otherwise identically composed.
It was surprising and particularly unexpected that compounds of formula (I) and corresponding mixtures as defined above additionally possess antioxidative properties and correspondingly can be used as an antioxidant, in particular as an antioxidant for delaying on the onset or for slowing the development of rancidity. For example, it is well known that tallow-based soap (and other soap comprising salts of fatty acids) can develop negative sensory characteristics after prolonged storage. This is due to the oxidative degradation of fat and fatty acid components conferring an unpleasant rancid odour to the soap. In own experiments, the incorporation of compounds of formula (I) in tallow-based soap base (and in other soap bases on the basis of fatty acid salts) showed a very favourable effect on the sensory properties and stability of tallow-based soap after storage. The development of unpleasant odours typically connected with the development of rancidity was in many cases completely inhibited. In those cases the odour remained identical to the unstored original sample. Correspondingly, a further aspect of the present invention relates to the use of (i) a compound of formula (I) or a (pharmaceutically) acceptable salt thereof or (ii) a corresponding mixture as defined above as antioxidant, in particular as an antioxidant for delaying the onset or slowing the development of rancidity.
Regarding the use of a compound, salt or mixture as defined above as an antioxidant, a particular focus is on the use in soap formulations (in particular bar soap) comprising salts of fatty acids, preferably sodium, or potassium salts, (ammonium salts being also preferred), preferably from tallow, or vegetable oils (fish oils being also preferred).
Use of a compound, salt or mixture as defined above as an antioxidant is also useful and therefore preferred for vegetable oils, which are prone to oxidation and subsequent development of rancid odour. Vegetable oils are frequently included in soap bar, liquid soap and in other cosmetic formulations, e.g. creams and lotions for improved skin care.
Examples of vegetables oil, which are effectively protected from oxidation and development of rancid odour are sunflower, yoyoba, avocado, palm, palm kernel, corn, olive, cocoa, nut, rape seed, soya, groundnut, almond, castor, rice bran, babassu, cotton seed, shea butter, and coconut oil.
Because of the presence of several advantageous properties compounds of formula (I) or corresponding mixtures can be favorably used as ingredients in a considerable number of compositions. In particular, the compounds of formula (I) and the corresponding mixtures can be used as an ingredient of compositions selected from the group consisting of bar soaps, liquid soaps, cleaners, fabric softeners, washing powders with or without perborate, fabric conditioners, liquid detergents, shampoos with or without anti-dandruff agents, shower gels, shaving formulations, depilatory formulations, perfume oils, aerosol formulations, scented candle formulations, air freshener formulations, deodorant formulations, antiperspirant formulations, creams, anti-acne wash, O/W-lotions, body lotions, body care creams, face care creams, hair conditioner with or without UV protection agents, microemulsion gels, suspension sticks, toilet rim formulations, toilet cleaners, dishwashing formulations, bleaching formulations, odour masking formulations with or without action against urine odour, wet cleansing wipes, flavor formulations, toothpaste formulations, dental cleaning formulations with or without anti-plaque agents and with or without agents effective against sensitive teeth, mouth rinse formulations, mouth wash formulations, chewing gums with or without non-caloric sweeteners, gelatine capsules, compressed tablets, drink formulations with or without alcohol, instant beverage formulations, candies with or without core filling and with or without effect against coughing, sore throat and/or hoarseness, cosmetic sun protection compositions, after sun lotions, soothing powders, hair care products, hair styling products, silicone emulsions, and disinfection compositions with or without ethanol.
When used in one of the stated compositions the compound of formula (I), the (pharmaceutically) acceptable salt thereof or the corresponding mixture typically acts as an antimicrobial agent, a fragrance substance, a flavoring substance, a tingling agent, a numbing agent, an antioxidant and/or or as an agent or agent mixture synergistically co-operating with a further antimicrobial agent so that the total antimicrobial activity is synergistically increased, wherein the synergistic effect is preferably determined according to Kull, in the manner stated above.
Preferably the use as an antimicrobial agent is combined with one or more of the other preferred uses (fragrance, flavor, tingling agent, numbing agent, antioxidant, agent or agent mixture for synergistically co-operating with a further antimicrobial agent so that the total antimicrobial activity is synergistically increased, wherein the synergistic effect is preferably determined according to Kull).
The present invention also relates to an antimicrobial composition comprising
and/or one, two or more (pharmaceutically) acceptable salts thereof wherein
R1 denotes a radical in ortho-, meta, or para-position to the phenolic OH group, the radical being selected from the group consisting of
Preferably, in the antimicrobial composition of the present invention component (b) comprises one, two or more further antimicrobially active compounds selected from the group consisting of
More preferably, in the antimicrobial composition of the present invention component (b) comprises one, two or more further antimicrobially active compounds selected from the group consisting of
The present inventions also relates to an antimicrobial composition wherein the total concentrations of said (a) compounds of formula (I) and (b) further antimicrobially active compounds is adjusted so that the composition has a kill activity of 99.9% (corresponding to 3 log-units reduction) or more after 1 min against one, two or all microorganisms selected from the group consisting of E. coli, Salmonella spp., P. aeruginosa, S. aureus, and C. albicans, with the proviso that
An antimicrobial composition according to the present invention is preferably selected from the group consisting of bar soaps, liquid soaps, cleaners, fabric softeners, washing powders with or without perborate, fabric conditioners, liquid detergents, shampoos with or without anti-dandruff agents, shower gels, shaving formulations, depilatory formulations, perfume oils, aerosol formulations, scented candle formulations, air freshener formulations, deodorant formulations, antiperspirant formulations, creams, O/W-lotions, body lotions, body care creams, face care creams, hair conditioner with or without UV protection agents, microemulsion gels, suspension sticks, toilet rim formulations, toilet cleaners, dishwashing formulations, bleaching formulations, odour masking formulations with or without action against urine odour, wet cleansing wipes, flavor formulations, toothpaste formulations, dental cleaning formulations with or without anti-plaque agents and with or without agents effective against sensitive teeth, mouth rinse formulations, mouth wash formulations, chewing gums with or without non-caloric sweeteners, gelatine capsules, compressed tablets, drink formulations with or without alcohol, instant beverage formulations, candies with or without core filling and with or without effect against coughing, sore throat and/or hoarseness, cosmetic sun protection compositions, after sun lotions, soothing powders, hair care products, hair styling products, silicone emulsions, and disinfection compositions with or without ethanol.
An antimicrobial composition of the present invention preferably comprises a total amount of compounds of formula (I) in the range of from 0.01 wt.-% to 0.5 wt.-%, based on the total mass of the composition.
Regarding the antimicrobial compositions according to the present invention the explanations given above regarding the uses of compounds or mixtures of the present invention apply mutatis mutandis.
In the context of the present invention specific antimicrobial compositions are preferred which comprise specific ingredients. Hereinafter preferred types of antimicrobial compositions and corresponding preferred ingredients are listed. By way of example, “oral (care) formulations: flavour materials e.g. carvone, menthol, cinnamic aldehyde, frambinone, capsaicin” indicates that a preferred antimicrobial composition is an oral (care) formulation which preferably comprises one or more flavour materials, wherein preferably one or more of the flavour materials are selected from the group consisting of carvone, menthol, cinnamic aldehyde, frambinone, and capsaicin. For the other preferred types of antimicrobial compositions the following list has to be interpreted in an analogous way:
Powder detergent, dish washing tabs, rim bar, soap bar, deo stick, cat litter, wet wipes: soap (saponified fatty acid), primary alkyl sulphates, secondary alkyl sulphonates, alkyl benzene sulphonates, ethoxylated alkyl sulphates, alkyl ether sulphate, alkyl polyglucosides, enzymes (e.g. lipase, protease, cellulase, alpha-amylase), brightener (Tinopal CBS-X), pH adjuster (sodium hydrogen carbonate), zeolithes (Zeolite 4A), matrix material (sodium sulfate), capsules as carrier for other components (e.g. perfume oil).
Preferably, in an antimicrobial composition of the present invention one, two or more compounds of formula (I) of component (a) cooperate with one, two or more of the antimicrobially active compounds of component (b) as defined above so that the antimicrobial effect of the composition is synergistically, wherein the synergistic effect is preferably determined according to Kull, increased.
Surprisingly, compounds of formula (I) as defined above (and the (pharmaceutically) acceptable salts thereof), in particular the compound of formula (IIa) (i.e. 4-(3-Methylbut-2-enyl)phenol), synergistically co-operate both with phenoxyethanol and methylparaben.
Corresponding hereto, the present invention also relates to the use of (i) a compound of formula (I) or (ii) a corresponding mixture as defined above for synergistically co-operating with a further antimicrobial agent so that the total antimicrobial activity is synergistically increased, wherein the synergistic effect is preferably determined according to Kull. The further antimicrobial agent is preferably selected from the group consisting of phenoxyethanol and methylparaben.
In a preferred antimicrobial composition according to the present invention the (i) total concentration of compounds of formula (I) and salts thereof is adjusted so that
Particularly preferred are antimicrobial compositions of the present invention wherein the composition comprises compounds (IIa)
with the proviso that
More particularly preferred are antimicrobial compositions of the present invention wherein the composition comprises compounds (IIa)
with the proviso that
The present invention also relates to an antimicrobial composition wherein the composition comprises (i) one to or more compounds of formula (I) or (pharmaceutically) acceptable salts thereof different from compound (IIa) wherein
An antimicrobial composition is preferred, wherein the composition comprises (i) one to or more compounds of formula (I) or (pharmaceutically) acceptable salts thereof different from compound (IIa) wherein
This antimicrobial composition is preferably an antimicrobial composition as defined above, i.e. an antimicrobial composition comprising components (b) and (c). Insofar, the statements and explanations presented above apply mutatis mutandis.
Antimicrobial compositions of the present invention are preferred, wherein the total amount of compounds of formula (I) in (ii) the composition is in the range of from 0.001 to 5 wt.-%, preferably in the range of from 0.01 to 0.5 wt.-%, based on the total weight of the composition. This is likewise the case for uses, mixtures and methods of the present invention.
Within the given range the compounds of formula (I) are particularly effective against the microorganisms stated above.
Compositions disclosed in WO 03/082233 A1 (Ghisalberti, Carlo) are not antimicrobial compositions of the present invention. Correspondingly, antimicrobial compositions comprising
(i) both butylhydroxytoluene and ximenynic acid
(ii) both alpha-bisabolol and aluminium oxychloride
(iii) both erythromycin base and L-ascorbic acid
(iv) both alpha-bisabolol and benzoyl peroxide
(v) both triethanolamine and hops glycolic extract, or
(vi) both sodium laurylsulfate and betaine lauryldimethylaminoacetate
are not preferred.
The present invention also relates to a method for inactivation, preferably rapid inactivation, of microorganisms present on a surface area or in a volume area, with the following step:
Preferred is a method for inactivation according to the present invention, wherein said surface area or volume area is contaminated with one or more microorganisms are selected from the group consisting of Bacteria, Archaea, Fungi, Viruses, Protozoa and other small eukaryotes considered as microorganisms.
Preferably the microorganisms are selected from the phyla consisting of Proteobacteria, Firmicutes, Actinobacteria, Chlamydiae, Spirochaetes, Bacteroidetes, Fusobacteria, and fungi from the phylum consisting of Ascomycota, Zygomycota and Basidiomycota and protozoa harmful to humans and animals, more preferably selected from the phyla consisting of Proteobacteria, Firmicutes, Actinobacteria, Chlamydiae, Spirochaetes, Bacteroidetes, Fusobacteria, and fungi from the phylum consisting of Ascomycota.
More preferably the microorganisms are selected from the group consisting of Streptococcus mutans, Streptococcus sobrinus, Actinomyces naeslundii, Porphyromonas gingivalis, Fusobacterium nucleatum, Veillonella parvula, Treponema denticola, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Capnocytophaga spp., Corynebacterium xerosis, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes, Bacillus cereus, Clostridium perfringens, Burkholderia cepacia, Escherichia coli, Salmonalla enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa, Yersinia enterocolitica, Shigella flexneri, Campylobacter jejuni, Listeria monocytogenes, Candida albicans, Malassezia furfur, Malassezia globosa, Propionibacterium acnes, Aspergillus flavus, Aspergillus brasiliensis, Aspergillus niger, Cryptosporidium parvum, Cyclospora cayetanenis, Giardia lamblia, Toxoplasma gondii, and Trichomonas vaginalis, particularly selected from the group consisting of Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Veillonella parvula, Treponema denticola, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Corynebacterium xerosis, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Burkholderia cepacia, Escherichia coli, Salmonalla enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa, Shigella flexneri, Campylobacter jejuni, Candida albicans, Malassezia furfur, Malassezia globosa, Propionibacterium acnes, Aspergillus flavus, Aspergillus brasiliensis, and Aspergillus niger, particularly preferably selected from the group consisting of Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Corynebacterium xerosis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Salmonalla enteritidis, Pseudomonas aeruginosa, Candida albicans, Malassezia furfur, and Aspergillus niger.
The present invention also relates to a method for imparting or enhancing an odor (in particular imparting or enhancing an odor impression selected from the group consisting of rosy, green, and clove-like), comprising the step of a contacting or mixing an effective amount of a compound of formula (I) or a (pharmaceutically) acceptable salt thereof or (ii) a mixture comprising one or more or consisting of two or more compounds of formula (I) and/or said salts, with a product, wherein the product preferably is a fragrance composition.
The present invention also relates to a method for imparting or enhancing a flavor or taste impression (in particular for imparting or enhancing a taste impression selected from the group consisting of green, sweet, camphor-like, rock-candy, floral) and/or a tingling sensation (in particular on the tongue) and/or a numbing sensation, comprising the step of contacting or mixing a compound of formula (I) or a (pharmaceutically) acceptable salt thereof or (ii) a mixture comprising one or more or consisting of two or more compounds of formula (I) and/or said salts, with a product, wherein the product preferably is a pharmaceutical product, an oral care product, a food product, or a beverage. Preferred resulting products comprise a total amount of said compounds of formula (I) and said salts in the range of from 0.001% to 1%, based on the total mass of the resulting product.
A corresponding method of producing an antimicrobial composition or another article comprising (i) a compound of formula (I) or a (pharmaceutically) acceptable salt thereof or (ii) a mixture comprising one or more or consisting of two or more compounds of formula (I) and/or said salts comprises the step of contacting or mixing said compound(s) of formula (I) and/or said salt(s) thereof or said mixture with a product.
Hereinafter, related art is described that might be considered relevant in a discussion of certain or all aspects of the present invention:
CA 1 213 613 (Fritzsche Dodge and Olcott, Inc., USA) discloses alicyclic ketone and alcohol derivatives as odor-modifying ingredients in perfumes and flavor-modifying ingredients in foodstuffs and tobacco products. As intermediates for the synthesis of these derivatives, prenylated phenols are described (in the present text the term “prenylated phenols” depending on context is used to indicate compounds of formula (I) and/or structurally similar compounds). The prenylated phenols are produced by reacting substituted phenols with isoprene or 3-methyl-3-buten-2-ol. The document does not disclose any evaluation regarding the taste or odor or antimicrobial activity of the prenylated phenols.
WO 2006/035010 (Symrise AG) discloses the preparation of the perfuming agent 4-isoamylcyclohexanol. As an intermediate of the synthesis process, 4-(3-methyl-2-butenyl)phenol is mentioned, which is the compound of formula (IIa) as defined above.
Hoarau and Pettus (2003) published methods for the chemical synthesis of orthoprenylated phenols (Strategies for the preparation of differentially protected orthoprenylated phenols, Synlett, 1, 127-137). No data on antimicrobial activity are disclosed for any of the presented compounds.
WO 2003/082233 (Ghisalberti, Carlo) discloses allylphenol compounds for treatment of androgenic disorders. The compounds are used for the manufacture of pharmaceutical and cosmetic compositions against male-pattern alopecia, acne, seborrhea, and dandruff by inhibiting 5α-reductase. 4-(3-Methylbut-2-enyl)phenol is disclosed and covered by the general formula disclosed in this document. However, 4-(3-Methylbut-2-enyl)phenol is not addressed as having antimicrobial or sensoric or antioxidant properties.
In order to further illustrate the object of the present invention, the compound thymol may serve as one (but not the only) very informative example:
Thymol is widely used in various formulations as preservative helper and/or antimicrobial, as well as an oral care product for antimicrobial activity.
Thymol exhibits a very distinct odor, which is not perceived as pleasant by many consumers. Employing thymol as a preservative helper and/or antimicrobial in a product formulation commonly necessitates the use of a corresponding perfume oil to mask the unpleasant odor or even limits the amount used. Otherwise the product would be rejected by the consumer.
It was correspondingly an object of the present invention to overcome the drawbacks associated with the use of thymol (as well as other antimicrobial agents) and to provide antimicrobial substances with the pleasant sensory profile.
Furthermore, thymol is a crystalline substance at room temperature, which is not readily incorporated into any given product formulation. In case the molten thymol can be incorporated into the formulation matrix, sometimes solubilizers are necessary to guarantee overall stability. Over time precipitation can occur which is especially harmful once the final product has reached its destination for the intended purpose.
Compounds of formula (I) as well as the corresponding mixtures are clear liquids with the lowest viscosity at room temperature which blend well with a wide range of raw materials. Correspondingly, incorporation into a vast spectrum of standard consumer good products (see examples) is possible without any difficulties. For incorporation standard procedures known to someone skilled in the art can be followed.
In case thymol is used for an oral care application or another product which is applied to the oral cavity, its distinct flavour profile and lingering taste becomes very apparent. Most consumers dislike this very strong and medicinal taste and therefore the use of thymol in comparable applications is sharply limited by its sensory profile.
Thus, as similarly explained above, it was an object of the present invention to provide an antimicrobial agent having favourable flavor properties.
The compounds of formula (I) and the corresponding mixtures exhibit a considerable tingling and numbing sensation when applied to the oral cavity. This trigeminal effect is highly desired for certain flavor applications, mostly for oral care products such as toothpaste, mouthwash, chewing gum and candies. The numbing effect can also be used to generate/mimic the typical sharpness or pungency of ethanol, in particular in case a low-alcohol or reduced alcohol-content beverage lacks this sensory impression. Thus, as discussed above, an aspect of the present invention is the use of a compound of formula (I) a (pharmaceutically) acceptable salt thereof, or a corresponding mixture of the present invention as a tingling agent for oral care products or beverages. For corresponding flavor applications the compound of formula (IIa) is particularly preferred.
The substances of formula (I) (and the (pharmaceutically) acceptable salts thereof) can be incorporated in different ways into a final formulation. One way is to add them as single ingredient into the formulation base, which is in most cases easily possible due to the favourable formulation properties of these substances. Another way to incorporate substances of formula (I) (and their salts) into a final formulation is via a perfume oil or a flavour as shown below. This is favourable due to the beneficial sensory profile substances of formula (I) (and their salts) are conferring to perfume oils and flavours.
The present invention is further illustrated by the following examples which relate to perfume oils as well as preferred antimicrobial compositions. If not indicated otherwise a reference to a “substance of formula (I)” relates to para-prenylphenol, i.e. 4-(3-Methylbut-2-enyl)phenol (compound of formula (IIa)). However, the “substance of formula (I)” can be replaced by any other compound of formula (I), in particular para-prenylphenol can be replaced by a compound of formula (IIb), (IIIa), or a compound of formula (IIIb). If, in some examples, not para-phenylphenol (compound of formula (IIIa)) was used, the compound of formula (IIa) or any other compound of formula (I) can be used instead of the compound used in the respective example.
The addition of 3% of para-prenylphenol to this floriental-citrus-musk composition produces a perceptible harmonization of the base notes, so that the entire composition seems more rosy, sweet and valuable.
The addition of 2% para-prenylphenol produces, while interacting with Evernyl®, a very distinct woody-mossy effect, which makes the entire composition seem more valuable.
The addition of 3% of ortho-prenylphenol emphasizes the spicy and rosy aspects of this composition and the accompanying harmonization of the base notes makes the entire composition seem more natural and silkier.
The perfume oils P1, P2, P3, P4, P5, P5, and P6 from the above perfume oil examples P1 to P6 were worked separately in each case into the here presented formulations.
The olfactory effects which have been described above for the respective perfume oil were also observed in each case in the here presented formulations.
Furthermore it is possible to incorporate mixtures of prenylphenols in much higher concentrations without affecting the sensory profile negatively, as exemplified below:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Persea Gratissima (Avocado)
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Rosmarinus Officinalis (Rosemary)
Advantage resulting from use of 4-Prenylphenol in this formulation:
Actinidia Chinensis
Citrus, Aurantium
Dulcis (Orange),
Pyrus Malus (Apple)
PrunusAmygdalus
Dulcis (Sweet
Bambusa Vulgaris
Advantage resulting from use of 4-Prenylphenol in this formulation:
Officinalis, (Rosemary)
Advantage resulting from use of 4-Prenylphenol in this formulation:
Triticum Vulgare
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Sensory benefit as fragrance substance (rosy, green, and clove-like)
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Butyrospermum
parkii (Shea Butter)
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
The following components are mixed to form a composition or mixture: 25 parts by weight of Substance of formula (I), 10 parts by weight of isomenthyl acetate, 20 parts by weight of isopropyl myristate, 30 parts by weight of triethyl citrate and 15 parts by weight of benzyl benzoate. This composition is brought into a microencapsulated form (average particle size: 78 micrometres) with starch sodium octenyl succinate (E 1450) and a sugar alcohol by means of spray drying, such that the charging of the microcapsules with the above mentioned composition is 50% by weight. The microcapsules are placed between two layers of paper bonded at the edges and incorporated into a paper nappy. On contact with moisture, the odour-preventing composition is released and significantly helps to reduce the urine odour which develops, compared to untreated nappies.
Advantage resulting from use of 4-Prenylphenol in this formulation:
1 part by weight of a mixture, consisting of 70 parts by weight of perfume oil containing 64% by weight of odorous substances and 30% by weight of Substance of formula (I) are sprayed, with mixing, onto 3000 parts by weight of cat litter (bentonite). For comparison, the same perfume oil without Substance of formula (I) is applied in the same quantity. After the addition of cat urine, it was found that the cat litter which was only treated with perfume oil only reduced the unpleasant odour of cat urine to an insufficient extent, while the cat litter which also contained the Substance of formula (I) had a relatively neutral smell and thus greatly reduced the unpleasant odour by the strong odour of cat urine.
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Preparation of a Composition or Mixture for Neutralising Unpleasant Odours and/or Disinfecting with Wet Cleansing Wipes:
The following components are mixed to form a composition or mixture: 30 parts by weight of dipropylene glycol, 25 parts by weight of Substance of formula (I), 15 parts by weight of isopropyl myristate, 15 parts by weight of triethyl citrate and 15 parts by weight of benzyl benzoate. Using an emulsifier (Dracorin GOC), a 0.05% aqueous solution is produced from this composition and is used to manufacture wet cleansing wipes.
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Aroma here had the following composition (data in each case in wt. %):
0.1% neotame powder, 0.05% aspartame, 29.3% peppermint oil arvensis, 29.3% peppermint piperita oil Willamette, 2.97% sucralose, 2.28% triacetin, 5.4% diethyl tartrate, 12.1% peppermint oil yakima, 0.7% ethanol, 3.36% 2-hydroxyethyl menthyl carbonate, 3.0% 2-hydroxypropyl menthyl carbonate, 0.27% vanillin, 5.5% D-limonene, 5.67% L-menthyl acetate.
The gelatine capsule, which is suitable for direct consumption, had a diameter of 5 mm, and the weight ratio of core material to shell material was 90:10. The capsules opened in the mouth within less than 10 seconds and dissolved completely within less than 50 seconds.
Advantage resulting from use of 4-Prenylphenol in this formulation:
Candies with a liquid/viscous core were produced on the basis of the methods described in U.S. Pat. No. 6,432,441 and those described in U.S. Pat. No. 5,458,894 or U.S. Pat. No. 5,002,791. The two mixtures A and B were separately processed to form bases for the shell (mixture A) or core (mixture B). When consumed by affected individuals, the filled throat candies obtained by means of coextrusion were effective against coughing, sore throat and hoarseness.
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Advantage resulting from use of 4-Prenylphenol in this formulation:
Make up to 100 l with demineralized water
Total amount 100 l
Make up to 100 l with demineralized water
Total amount 100 l
Advantage resulting from use of 4-Prenylphenol in this formulation:
This protocol gives an exact description on how to perform a fast time-kill experiment. Fast time-kill experiments serve to determine the antimicrobial kill activity of a specific substance or substance mixture against a particular microorganism or a mixture of microorganisms. To kill (synonymous: to inactivate) a microorganism means that after the treatment, the microorganism has permanently lost its capacity to grow or to replicate.
Microorganisms are grown in suitable growth medium allowing for exponential growth. For E. coli, Salmonella spp., S. aureus, P. aeruginosa, and for C. albicans tryptic soy broth medium is used. One hundred ml Erlenmeyer flasks containing 20 ml growth medium are inoculated with 105 to 108 microbial cells/ml and incubated overnight under continuous shaking (120 rpm) at a temperature allowing for exponential growth, usually between 20 and 37° C. The temperature is chosen in such a way to ensure the exponential growth for the used microorganisms, for E. coli, Salmonella spp., S. aureus, P. aeruginosa, and for C. albicans 37° C. is used.
After reaching the stationary growth phase, 100 μl of this culture are transferred in a second Erlenmeyer flask containing 20 ml of the same growth medium. This culture is shaken for further (preferably to up to 7 hours) under identical conditions as described above. Subsequently, this culture is diluted in the same growth medium to 105 to 109, preferably to 106 to 108 cells/ml. With this suspension of microorganism (named “microorganism working suspension”) the time-kill test is performed.
Test substances are dissolved in DMSO, EtOH and/or H2O, preferably in H2O, to prepare a 100× concentrated stock solution. In case the test substance is not completely soluble in H2O, the solvents are chosen in to ensure a complete dissolution of the test substance, the chosen concentration of DMSO and EtOH being as low as possible. For solid substances, complete dissolution has to be ensured. The concentration of the test substances in the 100× concentrated stock solution can be variable and is adjusted in order to reach the required concentration in the final test (but is limited to the maximal solubility in the above solvents for test substances). For example, if the concentration of the test substances in the final test should be 0.1%, the concentration in the 100× concentrated stock solution has to be 10%. Preferably, the concentrations of the test substances in the 100× concentrated stock solution are between 1 and 100%, corresponding to 0.01 to 1% in the final test. The amount of the 100× concentrated stock solution in the final test should not exceed 1% in order to limit the concentration of DMSO or EtOH to 1% in the final test.
Fast time-kill tests are performed in 1000 μl volumes in 1.5 ml plastic reaction tubes (Eppendorf). The test is started by mixing (in this particular order) 100 μl microorganism working suspension with 890 μl tryptic soy broth growth medium containing 0.1% Tween® 80 and 10 μl 100× concentrated stock solution of test substances. Controls are included that are prepared as described above, but using 100× stock solution without test substances. During the test, tubes are incubated at the same temperature as the microorganisms were grown, usually 20 to 37° C. (for E. coli, Salmonella spp., S. aureus, P. aeruginosa, and for C. albicans 37° C. is used) in an Eppendorf Thermomixer and are vigorously shaken at 1400 rpm.
Samples are taken from reaction tubes after defined time intervals (preferably 1, 2 and 5 min) to determine the number of surviving microorganisms. At time 0 (immediately after starting the test) samples are taken from control tubes to determine the initial number of microorganisms in the test. The number of surviving microorganisms is determined by aspiring 100 μl volumes from reaction tubes and mixing it immediately with 900 μl growth medium to prepare a 10−1 dilution. Further dilutions are prepared in this way from 10−2 to 10−4. One hundred μl volumes of the different dilutions are plated on tryptic soy broth agar plates and incubated for up to 48 h at the same temperature as the microorganisms were grown, usually 20 to 37° C. (for E. coli, Salmonella spp., S. aureus, P. aeruginosa, and for C. albicans 37° C. is used). Colonies grown on plates are counted and the numbers of surviving microorganisms are calculated for the different treatments and time points. For substances with low minimum inhibitory concentrations (MIC), sufficient dilution below the concentration of the MIC value has to be ensured.
A graphical representation of a time-kill experiment is obtained by plotting the surviving cell number as a function of time in a xy-diagram. For most microorganisms and test substances, the decrease of microorganisms follows a so-called pseudo first order kinetics described by the equation
C(t)=C0×e−kt (I)
where
t is the time in minutes
C0 is the number of microorganisms at time 0 (at start of the experiment)
C(t) is the number of (surviving) microorganisms at time t
k is the pseudo first order rate constant or inactivation rate
e is the Euler's number (≈2.718).
The equation (I) can be rearranged to describe the decrease of microorganisms as a straight line
ln C(t)=−kt+ln C0 (II)
where
Thereby, the inactivation rate k can be obtained by plotting ln C(t) versus t in a xy-diagram and subsequent linear regression of data points. The inactivation rate k is given by the slope of the straight line.
The inactivation times needed to kill 99.9% of all microorganisms can be calculated with the following equation from inactivation rates k
T
99.9%=ln 1000/k (III)
where
T99.9% is the time needed for a 99.9% kill of the microorganisms
ln 1000 is the natural logarithm of 1000 (≈6.908)
and k is as defined above.
The main advantage of calculating inactivation rates k or inactivation times T99.9% is that they allow for direct comparison of different experiments, irrespective of experimental parameters such as C0 or the exact time intervals for sampling. The parameters k or T99.9% can also be combined from different experiments to calculate averages and standard deviations leading to statistically valid data.
To calculate the synergism between compounds of formula (I) and other antimicrobial actives, the Kull equation was used
SI═(Rmixture×PA)/RA+(Rmixture×PB) (IV)
where
SI is the Synergy Index according to Kull1), 2) 1) Kull, F. C., Eismann, P. C., Sylvestrowicz, H. D., and R. L. Mayer (1961). Mixtures of Quaternary Ammonium Compounds and Long-chain Fatty Acids as Antifungal Agents. Applied Microbiology 9, 538-541.2) Steinberg, D. C. (2000). Measuring Synergy. Cosmetics & Toiletries 115 (11), 59-62.
RA is the reduction of surviving microorganisms by compound A
RB is the reduction of surviving microorganisms by compound B
Rmixture is the reduction of surviving microorganisms by the mixture of compounds A and B
PA is the proportion of the substance A in the mixture
PB is the proportion of the substance B in the mixture.
The reduction of surviving microorganisms in equation (IV) can be calculated by rearrangement of equation (I) as follows
R=C(t)/C0=e−kt (V)
where
C(t), C0, t, k, and e are as defined above.
The activation times of thymol and 4-prenylphenol against the microorganisms A) E. coli, B) S. aureus and C) C. albicans were determined.
The results are shown in
Inactivation times for a 99.9% kill for 4-prenylphenol are between 1.2 and 1.4 min. at 0.1% concentration. In contrast 0.1% thymol needs between 4.1 and 4.8 min. to achieve the same reduction in microbial counts.
In a further comparison experiment triclosan at a concentration of 0.1% showed an even weaker activity with inactivation times of >100 min. for all investigated microorganisms.
The results for triclosan are not shown in
In each case, values are averages of four independent experiments.
Escherichia
coli
Staphylococcus
aureus
Candiada
albicans
Assessment of minimum inhibitory concentrations of thymol and 4-prenylphenol against versus microorganisms relevant for home and personal care:
The results of comparative assessment of minimum inhibitor concentration for both thymol and 4-prenylphenol are listed in Table 2.
Streptococcus
mutans
Porphyromonas
gingivalis
Fusobacterium
nucleatum
Corynebacterium
xerosis
Staphylococcus
epidermidis
Staphylococcus
aureus
Escherichia
coli
Salmonalla
enteritidis
Pseudomonas
aeruginosa
Candida
albicans
Malassezia
furfur
Aspergillus
niger
In particular, the strong antimicrobial activity of 4-prenylphenol against the mould Aspergillus niger, which is a frequent contaminant of indoor environments as well as food and which is dangerous to humans due to the production of potent mycotoxins, is absolutely surprising and unexpected.
The following examples demonstrate the synergistic action of compounds of formula (I) and other antimicrobial actives in fast time-kill experiments against E. coli and S. aureus. The synergism holds true for any of the combinations of compounds of formula (I) and other antimicrobial actives and was observed against a wide range of microorganisms as listed above.
All synergism data were obtained from experiments that were independently repeated at least three times. Values shown in examples below are averages from these experimental repetitions. If the calculated Synergy Index (SI) value is smaller than 1, synergistic activity between mixtures of compounds is indicated versus the activity of the single compounds.
1) not applicable
The Synergy Index SI for the combination of 4-Prenylphenol and Phenoxyethanol against E. coli, calculated according to Kull's equation (IV), is 0.008, indicating a very strong synergism between these two compounds.
1) not applicable
The Synergy Index SI for the combination of 4-Prenylphenol and Phenoxyethanol against S. aureus, calculated according to Kull's equation (IV), is 0.53, indicating a strong synergism between these two compounds.
1) not applicable
The Synergy Index SI for the combination of 4-Prenylphenol and Methylparaben against E. coli, calculated according to Kull's equation (IV), is 0.01, indicating a very strong synergism between these two compounds.
1) not applicable
The Synergy Index SI for the combination of 4-Prenylphenol and Methylparaben against S. aureus, calculated according to Kull's equation (IV), is 0.55, indicating a strong synergism between these two compounds.
| Number | Date | Country | |
|---|---|---|---|
| 61578048 | Dec 2011 | US |
