GB2408933A

GB2408933A - Antimicrobial composition

Info

Publication number: GB2408933A
Application number: GB0328845A
Authority: GB
Inventors: Steve Aljawhiri; Stephen Denyer; Peter William Dettmar; Frank Chadwick Hampson; Geoffrey William Hanlon; David Norman Payne
Original assignee: Reckitt Benckiser Healthcare UK Ltd
Current assignee: Reckitt Benckiser Healthcare UK Ltd
Priority date: 2003-12-12
Filing date: 2003-12-12
Publication date: 2005-06-15
Also published as: WO2005058364A2; EP1742666A2; GB0328845D0; WO2005058364A3

Abstract

The present invention relates to an antimicrobial composition comprising a) a biocide such as an ester of p-hydroxy benzoic acid, bronopol, chlorocresol or an isothiazole, and b) an essential oil which has a specific gravity of at least 0.89 and a dielectric constant of 1-20. The essential oil may be selected from wintergreen oil, carvone, clove oil, spearmint oil, methyl salicylate and eugenol. The antimicrobial composition of the invention may be used as a preservative, as a pharmaceutical active ingredient, as a disinfectant or in a cleaning composition, inter alia.

Description

Antimicrobial Composition The present invention relates to an

antimicrobial composition.

Antimicrobial compositions are well known. Typically, such compositions include biocides, which inhibit the growth of microorganisms, such as bacteria. Known biocides include phenolic compounds, such as esters of phydroxy benzoic acid. Such esters are known as parabens, examples of which are methyl paraben (methyl ester of p-hydroxy benzoic acid), ethyl paraben (ethyl ester of p-hydroxy benzoic acid), propyl paraben (propyl ester of p-hydroxy benzoic acid) and butyl paraben (butyl ester of p- hydroxy benzoic acid).

In WO 99/09836, it was found that certain plant oils could be used to enhance the antimicrobial activity of parabens. In particular, it was found that the antimicrobial activity of methyl paraben could be synergistically enhanced in the presence of fennel oil, anise oil and basil oil. Estragol, trans-anethole and fenchone were also found to synergistically enhance the antimicrobial activity of methyl paraben.

Without wishing to be bound by any theory, the present inventors have discovered a relationship between the physical properties of an oil and its ability to potentiate the antimicrobial activity of a biocide. Specifically, it is believed that oil having a relatively high specific gravity and a relatively low dielectric constant can be used to potentiate the antimicrobial activity of a biocide, such as a paraben.

According to the present invention, there is provided an antimicrobial composition comprising: a) a biocide, and b) an essential oil, wherein the essential oil has a specific gravity of at least 0.89 and a dielectric constant of 1 to 20, and wherein the essential oil does not consist of a single essential oil selected from fennel oil, anise oil, basil oil, fenchone, trans-anethole and estragol.

It is believed that the antimicrobial activity of the biocide may be enhanced by the presence of an essential oil having a specific gravity of at least 0.89 and a dielectric constant of 1 to 20. Thus, a further aspect of the present invention provides the use of an essential oil to potentiate the activity of a biocide, wherein the essential oil has a specific gravity of at least 0.89 and a dielectric constant of 1 to 20, and wherein the essential oil does not consist of a single essential oil selected from fennel oil, anise oil, basil oil, fenchone, trans-anethole and estragol.

In a preferred embodiment, the antimicrobial activity of the biocide is synergistically enhanced by the essential oil. Thus, in this embodiment, the antimicrobial activity of the combination of the biocide and the oil is greater than the sum of the antimicrobial activity of the biocide in the absence of the oil and the antimicrobial activity of the oil in the absence of the biocide. For example, if a culture of bacteria is treated with the combination of the biocide and oil of the present invention, the number of bacteria eliminated in the sample after a predetermined period of time is preferably greater than the sum of i) the number of bacteria eliminated after the same predetermined period in an identical culture treated with the biocide in the absence of the oil and ii) the number of bacteria eliminated after the same predetermined period in another identical culture treated with the oil in the absence of the biocide.

Preferably, the essential oil has a specific gravity of greater than 0. 890, more preferably, at least 0.990. The essential oil may have a specific gravity of 0.990 to 2.000, for example, 1.000 to 1.700, preferably, 1.010 to 1.500. In one embodiment, the specific gravity of the essential oil is 1.020 to 1.300, preferably, 1.030 to 1.250, most preferably, 1.170 to 1.200. Specific gravity may be measured using any suitable method. For example, conventional instruments, such as a pycnometer may be used. Gravimetric analysis may also be employed.

The essential oil may have a dielectric constant of 2 to 18, preferably 3 to 12, more preferably 5 to 11, for example, 6 to 9. Dielectric constant is measured using any suitable method. For example, a parallel plate capacitor may be employed.

Any suitable essential oil may be employed in the composition of the claimed invention. For example, essential plant oils may be employed. For the avoidance of doubt, the term "essential oil" covers essential oils and the oily components of essential oils. A mixture of two or more oils may also be employed, particularly if the mixture has a specific gravity of at least 0.89 and a dielectric constant of 1 to 20.

Preferably, the oil has an aromatic component content of at least 30 %, more preferably at least 50\, even more preferably, at least 60 %, for example, at least 80. The aromatic components are preferably waterinsoluble.

Preferably, the essential oil comprises at least one oil selected from wintergreen oil, clove oil, spearmint oil, carvone, methyl salicylate and eugenol. The oil may be a mixture of spearmint oil and wintergreen oil or a mixture of spearmint oil and clove oil or eugenol. When a mixture of spearmint oil and wintergreen oil is employed, the weight ratio of spearmint oil to wintergreen oil may be 10 - 90: 90 - 10, preferably, 30 70: 70 - 30, for example, 40 - 60: 60 - 40. When a mixture of spearmint oil and clove oil or eugenol is employed, the ratio of spearmint oil to clove oil or eugenol may be 10 - 90: 90 - 10, preferably, 30 - 70: 70 30, for example, 40 - 60: 60 - 40.

In a further embodiment, the oil is a mixture of wintergreen oil and eugenol. The weight ratio of wintergreen oil to eugenol may be 10 - 90: 90 - 10, preferably, 30 - 70: 70 - 30, for example, 40 - 60: 60 - 40.

As mentioned above, oils consisting of one of fennel oil, basil oil and anise oil are excluded from the scope of the invention. Similarly, oils consisting of one of estragol, trans-anethole and fenchone are also excluded.

For the avoidance of doubt, therefore, oils formed of 100% of one of estragol, trans-anethole and fenchone are excluded. Preferably, oils formed of at least 70 weight %, preferably, at least 80 weight %, more preferably, at least 90 weight %, for example, at least 95 weight % of one of estragol, trans- anethole and fenchone are also excluded.

According to a further embodiment of the invention, oils consisting of peppermint oil may also be excluded from the scope of the invention.

The oil may be present in an amount of from 0.01 to 5 volume % of the composition, preferably, from 0.05 to 2 volume %, more preferably, from 0. 07 to 1 volume %. In one embodiment, the oil is present in an amount of 0. 01 to 0.5, for example, 0.01 to 0.1 volume %.

The biocide employed in the composition may be an ester of p-hydroxy benzoic acid. Such esters are known as parabens. Examples of suitable parabens include methyl paraben, ethyl paraben, propyl paraben and butyl paraben (or their salts [e.g. soluble salts]). Mixtures of parabens may be employed. Preferably, methyl paraben and/or propyl paraben are employed. Alternatively, the biocide may be bronopol, chlorocresol or an isothiazole.

The amount of biocide present in the composition may be 0.001 to 50 weight % of the composition, preferably, from 0.01 to 30 weight %.

The ratio of essential oil to biocide in the composition may be 1:20 to 20:1, preferably, from 1:10 to 10:1, and more preferably, from 1:5 to 5:1.

Typically, the composition is an aqueous composition comprising up to 99. 99% water. For example, the aqueous composition may comprise from 60 to up to 99.99 weight % water, for example, from 70 to 98 weight % water. It may, however, be possible to provide the composition in the form of a concentrate. The concentrate may be substantially anhydrous or may contain water in an amount of 0.1 to 50 weight %, preferably, 10 to 30 weight %. Such a concentrate may be diluted with water or an aqueous composition before use.

The composition may also comprise a chelating agent.

The chelating agent may enhance the interactions between the biocide and the bacterial wall by exposing more lipophilic surfaces on the bacterium. Suitable chelating agents include ethylene diaminetetraacetic acid (EDTA) or its derivatives (e.g. salts).

The composition is in the form of a liquid.

Preferably, the liquid is pourable or in a form suitable for administration in the form of droplets or a spray.

The composition may be suitable for oral administration. Thus, the composition may be in the form of a mouthwash, a toothpaste or chewing gum.

The composition may also be used as a preservative.

Preservatives are intended to preserve a product, such as a food or a pharmaceutical, against microbial growth and product spoilage. Preservatives usually have good selective toxicity. Thus, they are typically safe for topical application and/or ingestion.

For example, the composition may be used as a preservative in foodstuffs or pharmaceutical compositions.

In one embodiment, the composition is in the form of a composition that comprises a pharmaceutically active agent.

For example, the pharmaceutical composition may be suitable for the treatment of gingitis, sore throat, cough, reflux esophagitis, gastritis, dyspepsia or peptic ulceration, or for use as a sustained release or targeted delivery composition. The pharmaceutical composition may be in the form of an aqueous pourable liquid comprising an alginate and a bicarbonate and/or carbonate. When forming the pharmaceutical composition, the biocide and oil may be added to the pharmaceutical composition separately or in combination.

For preservative purposes, the biocide may be present in an amount from 0. 001 to 5 weight % of the composition, preferably, from 0.01 to 2 weight %, more preferably, from 0.07 to 1 weight %, for example, from 0.1 to 0.2 weight %.

Alternatively, the composition may be a used as a disinfectant. For disinfectant purposes, the biocide may be present in an amount from 0.2 to 30 weight % of the composition, preferably, from 2 to 25 weight %, more preferably, from 8 to 20 weight %, yet more preferably, from to 15 weight %, for example, from 11 to 12 weight %.

Disinfectants generally offer better antimicrobial action than preservatives. Thus, disinfectants typically provide rapid kill, usually in the region of 51Og1o cycle reductions (100,000 reduction in bacterial numbers) within 5 minutes of exposure. Some disinfectants do not show selective toxicity and therefore may not be safe for ingestion and/or topical application.

The composition may also be used in a hard surface cleaning composition. For example, the composition may be used as a disinfectant in a hard surface cleaning composition. The biocide and oil may be added to the hard surface cleaning composition separately or in combination.

Examples of hard surface cleaning compositions include kitchen, bathroom and lavatory cleaning compositions.

The composition may be used to inhibit the growth of a number of different bacteria. For example, the composition may be useful against Ps aeruginosa, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumonia and Citobacter freundii.

Example 1

In this example, compositions comprising methyl paraben, propyl paraben and a number of different oils were tested for their antimicrobial activity against the Ps aeruginosa. The following oils were tested: i) spearmint oil, ii) spearmint oil and eugenol (63.5:36.5)and iii) eugenol. A control sample C1) of spearmint oil and eugenol was also prepared (63. 5:36.5). However, no parabens were included in the control.

To carry out the tests, the following procedure was adopted.

A culture of Ps aeruginosa was grown in Tryptone Soy Broth (TSB Oxoid) for 6 hours and spread onto Tryptone Soy Agar (TSA Oxoid). The bacteria were left to multiply overnight at 37 C. The bacteria were then harvested, washed and re-suspended in HEPES buffer (pH=7.9).

9ml of parabens stock containing 0.150%w/v methyl paraben and propyl paraben was measured into a test tube.

Specifically, 0.130%w/vmethyl paraben and 0.020tw/v propyl paraben was employed.

7.81 of the oil under test was added (using SGE 101 micro-syringe) to the 9ml parabens stock. The test-tube was vortexed for 30 seconds and left to stand in a stirred water bath at 25 C for 20 minutes.

lml of the bacteria suspended in HEPES buffer [N-2 hydroxyethylpiperazineN'-2-ethanesulfonic acid] was then added to the test tube. The test tube was then vortexed for seconds and placed in the stirred water bath at 25 C.

At predetermined time intervals, the test tube was vortexed for 5 seconds and a lml sample removed. This sample was added to 9ml of a neutralizer comprising HEPES, Tween [l.ll9w/v Tween (R) 80 (polysorbate 80); laboratory grade, Product #560234H, Cas# 9005-65-6 (BDH, Poole, Dorset, UK)] and Lecithin [0.085\ w/v Lechitin Soya Bean; Laboratory grade, Product # 298632A, Cas#8030-76-0]. The test tube was then returned to the 25 C stirred water bath for the next sample.

Each neutralized sample (201) was spread out onto a plate containing Tryptone Soy Agar (Oxoid) and incubated overnight at 30 C.

Figure 1 shows how the number of bacteria decreased over time. As can be seen from the Figure, the samples containing ii) spearmint oil and eugenol (63.5:36.5) and iii) eugenol were very effective at reducing the number of bacteria. The oils in these samples had specific gravities of 0. 9895 (ii) and 1.0775 (iii), respectively. Although the sample containing i) spearmint oil had some antimicrobial activity, this sample was less effective than samples containing oils ii) and iii). The specific gravity of spearmint oil (i) was 0.9508. The results for the Control show that the oil combination has little or no antimicrobial activity on its own.

Example 2

Example 1 was repeated with the following oils: iv) spearmint oil and wintergreen oil(49:51) and v) wintergreen oil. A control C2) of wintergreen oil only was also prepared. However, no parabens were included in the control.

The results are also shown in Figure 1. As can be seen from the Figure, samples containing iv) wintergreen oil and spearmint oil and v) wintergreen oil had excellent antimicrobial activity. The oils in these samples had specific gravities of iv) 1.067 and v) 1.186, respectively.

Example 3

In this example, the procedure of Example 1 was repeated to test the activity of a composition comprising methyl paraben, propyl paraben and wintergreen oil against Enterobacter cloacae. A composition comprising methyl paraben, propyl paraben and fennel oil was also tested. In each case, 0.117%w/v methyl paraben and 0.018%w/v propyl paraben were employed. The amount of oil in each case was 0.078%v/v.

The results are shown in Figure 2. As can be seen from the Figure, the wintergreen oil composition had better antimicrobial activity than the fennel oil composition.

Wintergreen oil has a specific gravity of 1.1864, whereas fennel oil has a specific gravity of 1.001.

Example 4

The procedure of Example 3 was repeated with a culture of Klebsiella oxytoca. As can be seen from Figure 3, the wintergreen oil composition had better antimicrobial activity than the fennel oil composition.

Example 5

The procedure of Example 4 was repeated with a culture of Ps. Aeruginosa. As can be seen from Figure 4, the wintergreen oil composition had better antimicrobial activity than the fennel oil composition.

Comparative Example

In this Example, mixtures of an essential oil and methyl paraben were inoculated with a suspension of Ps.aeruginosa to give a final cell concentration of 2.3 x 108 CFU/ml. As a control, methyl paraben was also inoculated with a suspension of as. aeruginosa to give a final cell concentration of 2.3 x 108 CFU/ml. The methyl paraben concentration in each case was 0.46 weight %. The oils tested were orange oil and lemon oil.

As can be seen from the Table below, orange oil and lemon oil hardly potentiate the antimicrobial activity of the methyl paraben. Lemon oil has a specific gravity of 0.842 to 0.851. Orange oil has a specific gravity of 0.842 to 0.852.

Time i 10 30 60 120 (%v.v, 1 Orange 1.65 x 108 8.17 x 10' 6 x 107 1.38 x 10' 5.58 x 106 oil (0.05) Orange oil 2.05 x 108 1.0' x 108 6.17 x 10' 1. 5' x 10' 1.87 x 106 Lemon Oil 1.875 x 108 9.5 x 10' 4.07 x 107 1.2 x 10' 2.23 x 105 Le c n Ail 2 23 x 105 1 9.17 x 107 6.83 x 10' 1.9 x 10' S s

Claims

1. An antimicrobial composition comprising: a) a biocide, and b) an essential oil, wherein the essential oil has a specific gravity of at least 0.89 and a dielectric constant of 1 to 20, and wherein the essential oil does not consist of a single essential oil selected from fennel oil, anise oil, basil oil, fenchone, trans-anethole and estragol.

2. A composition as claimed in claim 2, wherein the essential oil has a specific gravity of at least 0.990.

3. A composition as claimed in claim 3, wherein the essential oil has a specific gravity of 1.000 to 1.700.

4. A composition as claimed in any one of the preceding claims, wherein the essential oil has a dielectric constant of 2 to 13.

5. A composition as claimed in claim 4, wherein the essential oil has a dielectric constant of 3 to 12.

6. A composition as claimed in any one of the preceding claims, wherein the biocide is at least one biocide selected from esters of p-hydroxy benzoic acid, bronopol, chlorocresol and an isothiazole.

7. A composition as claimed in any one of the preceding claims, wherein the essential oil is selected from at least one of wintergreen oil, carvone, clove oil, spearmint oil, methyl salicylate and eugenol.

8. A composition as claimed in claim 7, which comprises a mixture of wintergreen oil and eugenol.

9. A composition as claimed in claim 7, which comprises a mixture of wintergreen oil and spearmint oil.

10. A composition as claimed in claim 7, which comprises a mixture of eugenol and spearmint oil.

11. A composition as claimed in any one of the preceding claims, which also comprises water.

12. A composition as claimed in any one of the preceding claims, wherein the biocide is present in an amount of 0.001 to 50 weight %.

13. A composition as claimed in any one of the preceding claims, wherein the essential oil is present in an amount of 0.01 to 5 weight %.

14. A composition as claimed in any one of the preceding claims, which is a hard surface cleaning composition.

15. A composition as claimed in claim 14, which is a kitchen/bathroom cleaning composition or a lavatory cleaning composition.

16. A composition as claimed in any one of claims 1 to 14, which is suitable for oral administration.

17. A composition as claimed in claim 16, which is a pharmaceutical composition and also comprises a pharmaceutically active agent.

18. A composition as claimed in claim 16 or 17, which is in the form of a mouthwash, toothpaste or chewing gum.

19. A composition as claimed in any one of the preceding claims, which is a disinfectant composition.

20. An pharmaceutical composition comprising: a) an alginate, b) a bicarbonate and/or a carbonate, and c) a composition as defined in claim 16.

21. Use of an essential oil to potentiate the activity of a biocide, wherein the essential oil has a specific gravity of at least 0.89 and a dielectric constant of 1 to 20, and wherein the essential oil does not consist of a single essential oil selected from fennel oil, anise oil, basil oil, fenchone, trans-anethole and estragol.