WO1997030685A1

WO1997030685A1 - Reduced alcohol mouthwash

Info

Publication number: WO1997030685A1
Application number: PCT/US1996/002480
Authority: WO
Inventors: Pauline Pan; R. Michael Buch; Frank Volpe; Michael Rubin
Original assignee: Warner-Lambert Company
Priority date: 1996-02-23
Filing date: 1996-02-23
Publication date: 1997-08-28
Also published as: AU5355296A

Abstract

An antimicrobial mouthwash composition that is useful in the prevention and reduction of bad breath, plaque and related gum diseases comprises an antimicrobially effective amount of one or more active essential oils; a reduced level, up to about 22 % w/v, of alcohol; increased levels of at least about 18 % w/v of one or more polyols which enhance the efficacy of the actives in the oral cavity; and water. The actives not only provide enhanced efficacy but are completely solubilized in lower ethanol concentrations providing for an aesthetically appealing product.

Description

TITLE

REDUCED ALCOHOL MOUTHWASH

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates generally to mouthwashes for the prevention and elimination of bad breath as well as for the reduction of oral microorganisms responsible for the development of dental plaque and tooth decay.

Dental plaque can lead to the formation of calculus, gingivitis and other related gum diseases. In

particular, the present invention relates to a reduced alcohol mouthwash that is effective in preventing those problems.

Description of Related Art

Oral rinse and mouthwash compositions have been used by people for many years for the prevention of bad breath and for the elimination of bacteria and other oral microorganisms that are responsible not only for bad breath but also tooth decay, plaque and gum diseases such as gingivitis and periodontitis. To this end, antiseptic mouthwashes in the past have been designed to clean the oral cavity, provide fresh breath and kill these pathogenic microbes.

The leading antiseptic mouthwashes have always

contained alcohol (i.e., ethanol) at fairly high levels, ranging from approximately 25 up to about 30% by volume, based on the total mouthwash volume

(hereinafter referred to as "% v/v"). Alcohol is used both as a vehicle and as a solvent in which the active ingredients, and additives such as astringents,

fluorides, color additives, flavor oils, and the like, can be dissolved and then dispersed into solution.

Alcohol also provides a preservative role for the mouthwash during storage and use, and enhances the flavor oil organoleptic cues.

However, the use of high levels of alcohol has been recently challenged from an overall health standpoint. Some people cannot tolerate alcohol in any form, and there is concern that young children in particular may be adversely affected by ingesting or inadvertently swallowing mouthwash compositions containing alcohol. Senior citizens have also complained about problems related to gargling with such mouthwashes, and chronic exposure has been found to result in gum "burn" by the alcohol. It has also been reported that alcoholic mouthwashes can result in an unpleasant "dry mouth" sensation. However, merely reducing the levels of alcohol in these mouthwash compositions has significant disadvantages. It has been found that lower alcohol levels result not only in a loss in the solubility of the actives and other ingredients in the composition, but there is also a noticeable decrease in the ability of the composition to kill the oral microorganisms responsible for bad breath, plaque and gum disease. This loss in antimicrobial activity is not only due to the reduction of alcohol as a vehicle, but is also related to the bioavailability of the solubilized actives. Thymol, for example, is a well known antiseptic

compound, also known as an essential oil, which is utilized for its antimicrobial activity in a variety of mouthwash preparations. In particular, thymol can be utilized in oral hygiene compositions such as mouth rinses in sufficient quantities to provide desired beneficial therapeutic effects. Listerine^®-brand mouthwash is a well-known antiseptic mouthwash that has been used by millions of people for over one hundred years and has been proven effective in killing microbes in the oral cavity that are responsible for plaque, gingivitis and bad breath. Thymol, together with other essential oils such as methyl salicylate, menthol and eucalyptol, are active ingredients in antiseptic mouth rinses such as Listerine^®. These oils achieve their efficacy although present in small amounts. Without being restricted to any specific theory, it is now believed that the efficacy and taste of antiseptic mouthwashes such as Listerine^® may be due to the dissolution of these four active ingredients.

Dissolution is also important from an aesthetic point of view since a clear mouthwash solution is certainly preferred by consumers to one that is cloudy, turbid or heterogeneous. Obviously then, there is a substantial need for the development of a reduced alcohol mouthwash in which the essential oils are completely dissolved and that continues to be effective in the prevention of bad breath, the killing of oral microbes and the resultant reduction or elimination of plaque. Several attempts at formulating reduced alcohol and alcohol-free mouthwash compositions can be found in the prior art. For example, United States Patent No.

5,292,527 to Konopa discloses a non-alcoholic, aqueous mouthwash composition which contains a water- insoluble flavoring oil and an effective amount of a dispersion system comprising a non-ionic surfactant selected from a hydrogenated castor oil and a polyoxyethylene- polyoxypropylene block copolymer having about 50% to about 90% ethylene oxide, a humectant and a

disinfecting amount of a cationic antimicrobial agent such as cetylpyridinium chloride. The humectant, which is preferably glycerin, but which may also be sorbitol or mannitol, is generally present in amounts from about 1% to 20% by weight. The composition allegedly

exhibits a homogeneous, uniform appearance and a high degree of bactericidal efficacy.

United States Patent No. 5,284,648 to White et al.

discloses an alcohol-free oral rinse and pre-rinse consisting of emulsion-containing cleaning and coating compositions that are non-irritating and low foaming for maximum plaque disruption and removal. The

cleaning effect is achieved using an aqueous system containing a mouth conditioner such as

polydimethylsiloxane emulsified in a surfactant such as a block copolymer of polyoxyethylene and

polyoxybutylene. United States Patent No. 5,185,153 to Pollock discloses a dental rinse formulation that allegedly lyses the bacterial cells responsible for plaque formation and bad breath. The composition comprises (a) a humectant (such as glycerol, sorbitol and the like) in an amount of about 20-80%, preferably about 30-50%, by weight, based on the entire composition volume (hereinafter referred to as "% w/v"); (b) from about 0.5 to about 2% w/v of a bicarbonate; (c) from about 0.05 to about 1% w/v of an anionic detergent; and (d) from about 0.01 to about 3% v/v of a non-ionic detergent such as Tween 20. Alcohol may be added up to a level of 15% v/v, with water comprising the remainder up to 100%. It is postulated that the "high" levels of humectant cause the bacterial cell walls to swell causing separation of the cell wall and membrane, which enables the

surfactants to enter the cell and deregulate autolysin enzymes which attack the cell wall causing holes and eventual cell lysis.

United States Patent No. 5,236,699 to Libin teaches an antimicrobial mouthwash composition comprising two active agents, triclosan and cetylpyridinium chloride. Ethanol comprises about 15% -20% w/v of the

water/alcohol carrier system and sorbitol is added in amounts of about 10% -15% by weight as a humectant. United States Patent No. 4,945,087 to Talwar et al. discloses an oral antiseptic composition containing thymol, eucalyptol, menthol and methyl salicylate wherein the unpleasant, medicinal taste of thymol is masked by the combined presence of an effective amount of one or more sugar alcohols and an effective amount of anethole. The sugar alcohol may be present in amounts of about 20 to about 55% w/v, preferably about 25-50% w/v, most preferably about 28-32% w/v. The amount of anethole may be about 0.01-0.035% w/v, most preferably about 0.015-0.025% w/v. The amount of ethanol is about 5-35% w/v and a level of 22.8% w/v is specifically disclosed.

The assignee of the present inventors has, since about mid-1992, sold Cool Mint Listerine^®-brand mouthwash product containing thymol and other essential oils, 21.6% v/v ethanol, 17.5% w/v sorbitol, and 0.16- 0.20% w/v anethole.

United States Patent No. 5,256,401 to Duckenfield et al. discloses a reduced alcohol antibacterial mouthwash in which a substantially water-insoluble non-cationic antibacterial/anti-plaque agent such as triclosan is dissolved in a solvent whose water/alcohol ratio is greater than 10:1. Sorbitol, glycerin and other sugar alcohols may be incorporated as humectants but nothing is attributed to their functioning as solubilizers or active enhancement compounds.

United States Patent No. 5,100,650 to Carlin et al. discloses an oral mouthwash containing an antibacterial active such as chlorhexidine with non-ionic surfactants and high levels of sorbitol which, it is asserted, increase the antibacterial activity of the

chlorhexidine compounds. Sorbitol solution (70%) is present in amounts of 25-75% w/v, preferably 50-60% w/v, and the water/alcohol weight ratio ranges from about 1:1 to about 20:1, preferably from about 3:1 to about 10:1. Clearly, there is still a need for a reduced alcohol composition that is highly efficacious in the

prevention of bad breath, plaque and gum disease.

Moreover, there is a need for such oral compositions that both kill the oral microflora responsible for these problems and clean the oral cavity leaving a fresh, lubricous mouthfeel.

SUMMARY OF THE INVENTION The present invention is a reduced alcohol,

antimicrobial mouthwash composition with a high level of efficacy in the prevention of plaque, gum disease and bad breath. In addition, the oral mouthwash compositions of this invention are clear, aesthetically appealing products. The reduced alcohol, antimicrobial mouthwash

composition of the present invention comprises (a) an antimicrobially effective amount of one or more

essential oils; (b) a reduced level, up to about 22% v/v, of ethanol; (c) at least about 18% w/v of one or more polyols sufficient to provide enhanced

antimicrobial activity of said essential oils at said reduced level of ethanol; and (d) water.

At ethanol levels of about 22% v/v and below, it has been found that relatively high levels of one or more polyols, such as the sugar alcohol, sorbitol, or propylene glycol or glycerin, surprisingly enhance the antimicrobial activity of the essential oil actives even at these reduced ethanol levels. According to the present invention, the amount of polyols employed is generally inversely proportional to the amount of ethanol; that is, as the level of ethanol is reduced below about 22% v/v, it is generally necessary to increase the concentration of polyols beyond about 18% w/v to maintain good antimicrobial efficacy. The specific level of polyols employed will vary depending upon the reduced level of ethanol used, the particular polyols employed, and the antimicrobial efficacy desired. For example, in comparison to Listerine^®- brand antiseptic mouthwashes containing about 27% v/v ethanol, the mouthwashes of the present invention are at least as, or more, efficacious even at ethanol levels of 19% v/v, or lower. In one embodiment of the present invention, a reduced alcohol, antimicrobial mouthwash composition is provided which comprises (a) an antimicrobially effective amount of one or more essential oils; (b) a reduced level, up to about 22% v/v, of ethanol; (c) at least about 18% w/v of one or more polyols sufficient to provide an enhanced antimicrobial activity of said essential oils at said reduced level of ethanol, such that the R-Factor (defined below) of said mouthwash composition is less than about 2.0, preferably less than about 1.2, and most preferably less than about 1.0; and (d) water.

According to another embodiment of the present

invention, a reduced alcohol, antimicrobial mouthwash composition is provided which comprises (a) an

antimicrobially effective amount of one or more

essential oils; (b) a reduced level, from about 9% v/v up to about 22% v/v, of ethanol; (c) at least about 18% w/v of one or more polyols sufficient to provide enhanced antimicrobial activity of said essential oils at said reduced level of ethanol; and (d) water.

In another embodiment of the present invention, a reduced alcohol, antimicrobial mouthwash composition is provided which comprises (a) antimicrobially effective amounts of thymol and one or more other essential oils; (b) a reduced level, up to about 19% v/v, of ethanol; (c) at least about 18% w/v of one or more polyols sufficient to provide enhanced antimicrobial activity of said essential oils at said reduced level of

ethanol; and (d) water.

In another embodiment of the present invention, a reduced alcohol, antimicrobial mouthwash composition is provided which comprises (a) antimicrobially effective amounts of thymol and one or more other essential oils; (b) a reduced level, from about 12% v/v up to about 19% v/v, of ethanol; (c) at least about 18% w/v of one or more polyols selected from the group consisting of a sugar alcohol, propylene glycol and glycerin sufficient to provide enhanced antimicrobial activity of said essential oils at said reduced level of ethanol; and (d) water.

The mouthwash compositions of this invention may also contain flavoring oils, buffers, surfactants, colorants and other conventional additives. For example, minor amounts of anethole or other flavorants may be

included. When anethole is present in the compositions of the present invention, it may be added in a minor amount or it may be introduced as part of a natural flavor blend. According to another embodiment of the present

invention, a mouthwash product is provided which is essentially free of anethole (i.e., there is no

detectable level of anethole or at least the amount of any anethole present is so small that it performs no substantial taste or flavor function). This particular embodiment contemplates that the desired taste or flavor attributes of the mouthwash are achieved through the use of flavorants and additives other than

anethole.

DETAILED DESCRIPTION OF THE INVENTION

The reduced alcohol mouthwash compositions of the present invention provide an unexpected high degree of antiseptic efficacy towards oral microorganisms

responsible for oral malodor and the build-up of plaque and calculus and the resulting tooth and gum diseases that may follow. The enhanced antimicrobial efficacy of the reduced alcohol mouthwash compositions of the present invention is attributed to the presence of minor amounts of one or more essential oils (i.e., thymol, eucalyptol, menthol and methyl salicylate) combined with high levels (at least about 18% w/v) of one or more polyols such as sorbitol which synergistically enhances the antimicrobial efficacy of the essential oils.

Thymol, (CH₃)₂CHC₆H₃(CH₃)OH (isopropyl-m-cresol), is only slightly soluble in water but is soluble in alcohol, and its presence is one of the reasons alcohol was necessary in the well-established, high alcohol

commercial mouthwashes such as Listerine^® in the past. Methyl salicylate, (C₆H₄OHCOOCH₃), also known as

wintergreen oil, additionally provides flavoring to the mouthwash together with its antimicrobial function.

Eucalyptol (C₁₀H₁₈O; cineol) is a terpene ether and provides a cooling, spicy taste. Eucalyptol may be used in place of thymol in certain formulations in the same amount if desired. Menthol (CH₃C₆H₉(C₃H₇)OH;

hexahydrothymol) also is only slightly soluble in alcohol, is fairly volatile, and in addition to any antiseptic properties provides a cooling, tingling sensation.

The essential oils are used in amounts effective to provide antimicrobial activity in the oral cavity.

Generally, the total amount of essential oils present in a composition of this invention can be from about 0.001% to about 0.35% w/v, with about 0.16% to about 0.28% w/v being preferred. The compositions of the present invention preferably contain thymol and

additionally eucalyptol, menthol, or methyl salicylate, or mixtures thereof. Most preferably, the composition contains all four of these essential oils. Thymol is preferably employed in amounts of from about 0.001% to about 0.25% w/v, and most preferably from about 0.04% to about 0.07% w/v. Eucalyptol may be employed in amounts of from about 0.001% to about 0.11% w/v, and preferably from about 0.085% to about 0.10% w/v. Menthol is preferably employed in amounts of from about 0.001% to about 0.25% w/v and most preferably from about 0.035 to about 0.05% w/v. Methyl salicylate is preferably employed in amounts of from about 0.001% to about 0.08% w/v, and most preferably from about 0.04% to about 0.07% w/v. The carrier for the essential oils (i.e., the active ingredients) is a water-alcohol mixture, generally water- ethanol. In the past, most antiseptic oral mouthwash compositions, such as Listerine^®-brand mouthwashes, required high ethanol levels of up to about 27% v/v. These high levels were necessary to assist the actives in providing the necessary

antimicrobial functionality as well as providing a clear, aesthetically attractive liquid medium. Merely reducing the alcohol levels, without more, results in a cloudy, less efficacious product.

Without being bound to any theory, it is believed that in these high alcohol level oral compositions, the alcohol solubilizes the antimicrobial actives and in so doing acts as an active enhancement mechanism. The actives are more readily dispersed throughout the solution and can attack pathogenic microbes throughout the oral cavity. Reducing the alcohol levels was believed to adversely affect this enhancement

mechanism.

In accordance with the present invention, however, it was surprisingly and unexpectedly found that the level of alcohol can be reduced without sacrificing

antimicrobial efficacy or clarity if the mouthwash composition contains an increased amount of one or more polyols. By "reduced level" of alcohol is meant an amount of ethanol up to about 22% v/v, such as an amount of from about 9% v/v to about 22% v/v,

preferably no more than about 19% v/v (e.g., from about 12% to about 19% v/v), and most preferably about 19% v/v. By "increased amount" of one or more polyols is meant a total amount of one or more polyols, at least about 18% w/v, sufficient to enhance the antimicrobial activity of the essential oils from that activity which exists at a given reduced alcohol level to an activity comparable to that exhibited by an antiseptic mouthwash having high alcohol levels on the order of about 27% v/v. A convenient reference point is the antimicrobial activity exhibited by a high alcohol level Listerine^®- brand mouthwash; i.e., one which contains about 27% v/v ethanol. Expressed in terms of an "R-Factor, " which, as explained below, represents the time necessary to effectively kill typical oral cavity microbes in an in vitro biofilm, normalized against the kill time

exhibited in vitro by a 27% v/v Listerine^®-brand mouthwash, the mouthwash compositions of the present invention exhibit an R-Factor of less than about 2, preferably less than about 1.2, and most preferably less than about 1.0. The amount of polyol(s) employed in the mouthwash compositions of the present invention is directly and inversely proportional to the amount of ethanol in the carrier solution. As the concentration of ethanol is reduced below about 22% v/v, the amount of polyol(s) must be correspondingly increased above about 18% w/v to recapture efficacy lost by decreasing the alcohol level. What is most surprising is that the microbial kill rate of these reduced alcohol mouthwash

compositions is substantially increased at particular levels of polyol(s). The particular amount of

polyol(s) required will depend on a number of factors including the particular reduced ethanol level involved, the particular polyol(s) employed, the antimicrobial efficacy required, the presence of other ingredients which may tend to improve efficacy, the effect of polyol addition on other characteristics of the composition such as consistency and clarity, etc. In general, the amount of any given polyol, and the total amount, will be selected to provide the desired R-Factor. By way of example, for a reduced ethanol level of about 19% v/v, sorbitol used in amounts within the range of from about 25% w/v to about 30% w/v will provide a mouthwash composition with substantially improved antimicrobial activity (i.e., R- Factors as low as about 0.82). At 19% v/v ethanol, the amount of sorbitol preferably is within the range of from about 18% w/v to about 30% w/v, most preferably from about 25% w/v to about 30% w/v. Alternatively, at the same 19% v/v ethanol level, when propylene glycol is used as the polyol in amounts within a range of from about 15% w/v to about 25% w/v, the resulting mouthwash

composition has generally acceptable antimicrobial activity (i.e., R-Factors as low as about 1.35).

The polyol(s) used in the present invention may be selected from those multi-hydroxy-functional compounds that are conventionally used in oral and ingestible products. By way of example, the polyol may be

selected from sugar alcohols such as sorbitol, xylitol, mannitol, maltitol, inositol, allitol, altritol, dulcitol, galactitol, glucitol, hexitol, iditol, pentitol, ribitol, erythritol and mixtures thereof;

hydrogenated starch hydrolysates; (poly) alkylene glycols such as propylene glycol and polyethylene glycols; diols such as 1,3-butanediol; glycerin; and mixtures of them or mixtures containing them. The preferred polyols are the sugar alcohols, propylene glycol and glycerin. Sorbitol is the most preferred polyol. In general, the polyol(s) should be non- metabolized and non-fermentable polyol(s).

The ratio of water to ethanol is in the range of from about 3:1 to about 25:1, preferably from about 3.2:1 to about 10:1, by volume. The total amount of

ethanol/water mixture in a mouthwash preparation according to this invention depends, of course, on the amount of polyol(s) present, but is typically in the range of from about 60% to about 82%, preferably from about 70% to about 82%, by volume of the total

composition.

Surface active agents (surfactants) may be used if desired in the compositions of the present invention. They are organic materials which aid in the complete dispersion of the ingredients throughout the solution as well as dispersing the preparation throughout the oral cavity. Preferably, the surfactant used in the compositions of the present invention is a non- ionic surfactant employed in an amount sufficient to help solubilize the actives. The preferred non-ionic surfactants are selected from the group known as poly(oxyethylene)-poly(oxypropylene) block copolymers. Such copolymers are known commercially as poloxamers and are produced in a wide range of structures and molecular weights with varying contents of ethylene oxide. These non-ionic poloxamers are non-toxic and acceptable as direct food additives. They are stable and readily dispersible in aqueous systems and are compatible with a wide variety of formulations and other ingredients for oral preparations. These surfactants should have an HLB (Hydrophilic-Lipophilic Balance) of between about 10 and about 30 and

preferably between about 10 and about 25. By way of example, non- ionic surfactants useful in this invention include the following poloxamers:

Generally these polymers when present should be used in amounts of from about 0.01% w/v to about 4.0% w/v, and preferably from about 0.25% to about 0.75% w/v. A particularly preferred poloxamer is poloxamer 407 which is incorporated in an amount of about 0.5% w/v. The poloxamer or other surfactant is used to help solubilize the essential oils and flavor oils which may otherwise not be soluble in these aqueous systems due to their reduced ethanol content. The surfactant(s) also act to disperse the actives and flavors throughout the solution and enable the compositions to provide a clear, uniform appearance that is aesthetically more appealing.

In one embodiment of the present invention, three types of peppermint oil -- natural, Far West (redistilled, terpeneless) and Rose Mitchum -- are combined to provide a triple blend. This unique blend of flavor oils not only provides a pleasant tasting mouthwash but also serves to taste mask the bitter tasting essential oil actives discussed above. Each of these peppermint oils is present in substantially the same amount, from about 0.01% w/v to about 1.0% w/v, and preferably in an amount of from about 0.2% w/v to about 0.3% w/v.

Combined, the triple blend is incorporated in the mouthwash composition in an amount of approximately 0.1% w/v to about 2.0 w/v, preferably in an amount of from about 0.5% w/v to about 0.9% w/v. The essential oil methyl salicylate not only provides antimicrobial action but, being a wintergreen flavor oil, also adds to the organoleptic flavor tones and complements the taste masking function of the

peppermint oil blend.

Other flavor oils may also be added to further modify or magnify the cooling minty taste of the peppermint, or reduce or mask the sharp "bite" or "burn" of

ingredients such as thymol and ethanol. Suitable flavors (and typical amounts) include oil of anise (about 0.01% w/v to about 0.2% w/v) and benzyl alcohol (about 0.001% w/v to about 0.1% w/v). In one

particular embodiment of the present invention, the mouthwash is a clear, green color and further includes spearmint oil (about 0.01% w/v to about 2.0% w/v) as an additional flavorant. Other flavors such as citrus oils, vanillin and the like may be incorporated to provide further taste variations.

In another embodiment, the flavorant anethole may be employed in amounts up to about 0.01% w/v. However, it is specifically within the scope of the present

invention to provide a mouthwash composition that is totally free of anethole or at least essentially free of anethole (i.e., trace amounts or amounts which are so small as to not contribute functionally to the taste or flavor properties of the composition). The particular flavor oils and other taste-improving ingredients employed will vary depending upon the particular taste and feel desired. Those skilled in the art can select and customize these types of ingredients to provide the desired results.

Additional conventional components may be added as in mouthwashes of the prior art. Whereas some alcohol containing mouthwashes have a pH of about 7.0,

reduction of the alcohol level requires the addition of acidic preservatives, such as sorbic or benzoic acid, which reduce pH levels. Buffer systems are then necessary to control the pH of the composition at optimal levels. This is generally accomplished through the addition of a weak acid and its salt or a weak base and its salt. Useful systems have been found to be sodium benzoate and benzoic acid in amounts of from about 0.01% to about 4.0% w/v, and sodium citrate and citric acid in amounts of from about 0.001% to about 4.0% w/v. Preferably the buffers are incorporated in amounts that maintain the pH at levels of from about 3.5 to about 6.5, and more preferably from about 4.0 to 5.0. Without being bound to any theory, it is believed that these pH levels provide the essential oils with an environment that also maximizes their antimicrobial activity. Other conventional ingredients may be used in the mouthwash compositions of this invention, including those known and used in the art. For example,

humectants such as polyethylene glycol may be added as an additional solubilizer for the flavor oils and to also provide texture to the composition. These are incorporated in amounts of from about 0.3% w/v to about 0.6% w/v, and preferably about 0.5% w/v. Softeners such as glycerin may be added to enhance the lubricous mouthfeel of the mouthwash as it is used and to provide a refreshing, moist, organoleptic feeling thereafter. Glycerin (when not used as the polyol, as discussed above) may be incorporated in amounts of from about 1.0% w/v to about 10.0% w/v, and preferably in an amount of about 7.5% w/v. Sweeteners such as aspartame or sodium saccharin and the like may be added for better taste in amounts of from about 0.005% w/v to about 1.0% w/v, and preferably in an amount of

approximately 0.05% w/v.

Zinc chloride may be added as an astringent for an "antiseptic cleaning" feeling in an amount of from about 0.0025% w/v to about 0.0075% w/v. And although the mouthwash formulations of the present invention may be formulated to be substantially clear and colorless, acceptably approved food dyes are preferably used to provide a pleasing color to the formulations of the invention. These may be selected from the long list of acceptable food dyes and in particular may be

incorporated to provide the spearmint green formulation discussed infra. Suitable dyes for this purpose include FD&C yellow #5, FD&C yellow #10 and FD&C green #3. These are added in conventional amounts, typically in individual amounts of from about 0.0003% w/v to about 0.0005% w/v, and preferably from about 0.000035% w/v to about 0.00045% w/v.

Water is added to q.s. and the formulation may then be bottled and packaged for shipping. The mouthwash composition of the present invention may also be formulated, if desired, as gels, foams, pastes,

aerosols or tablets using standard formulations known in the art as appropriate.

Alternatively, the mouthwash compositions of the present invention may be formulated in a dry powder, chewing gum, semi-solid, solid or liquid concentrate form. In such embodiments, for example, water is added to q.s. the volume to the necessary total for a liquid concentrate, or water may be removed using standard evaporation procedures known in the art to prepare a composition in dry powder form. Both may then have water added at a later date when ready for use. Such forms are advantageous for storage and shipping. R- Factor

Biofilms of the microorganism Streptococcus mutans

(ATCC #25175) grown on stainless steel wires simulate thick, semipermeable dental plaque. For purposes of the present invention, an "R-Factor" is a convenient measure of the antimicrobial efficacy of the mouthwash compositions of the present invention, as measured by their ability to kill those biofilms. The R-Factor is defined as the ratio of (1) the time, in minutes, necessary for a mouthwash composition to kill S. mutans microorganism biofilms grown in vitro on stainless steel wires, to (2) the time, in minutes, necessary for a standard high alcohol mouthwash composition to kill similar biofilms of the same microorganism grown in vitro on other, identical stainless steel wires. Those kill times are obtained by a plaque penetration assay developed by the present inventors. Plaque Penetration Assay

The plaque penetration assay employed by the present inventors to obtain their biofilm critical kill times is a modification of the well-known procedure of

Tanzer, et al., described or referenced in, e.g.,

Tanzer, et al., "Structural requirements of Guanide, Biguanide and Bisbiguanide Agents for Antiplaque

Activity," Antimicrobial Agents and Chemotherapy, Dec. 1977, pp. 721-729; and Tanzer, et al., "In Vitro

Evaluation of Seven Cationic Detergents as Antiplaque

Agents," Antimicrobial Agents and Chemotherapy, Mar. 1979, pp. 408-414.

The high alcohol mouthwash composition employed by the present inventors as their standard for their plaque penetration assay contains 27% v/v ethanol and has the composition shown in the following Table 1:

The media required for the plaque penetration assay include sterile deionized water; Letheen Broth (DIFCO); and Jordan's complex medium (with and without

bromocresol purple pH indicator) [see Jordan, H.V. et al. J. Dent. Res. 39: 116-123 (1960)]. The equipment requirements for the assay include a large number of sterile glass test tubes (e.g., 13 x 100 millimeters); test tube racks to hold multiple rows of sample tubes; an autoclave; and stainless steel Nichrome wires (1.5 x 85 mm). It is convenient to attach each plaque wire to a Morton Closure by any suitable means, such as

welding.

Jordan's medium may be prepared by blending the

following ingredients with heating as necessary:

The pH is adjusted to 7.1 with 5N HCl followed by cooling to room temperature, if necessary. 50 ml is removed from the batch and placed in a 100 milliter flask. Jordan's medium with pH indicator ("recovery medium") is prepared by adding 1 ml of a 1% bromocresol purple stock solution (i.e., 0.1 gram bromocresol purple in 10 mis distilled water) to 1 liter of

Jordan's medium.

The culture for the assay is prepared as follows. Upon receipt, the ATCC culture is rehydrated and subcultured according to the directions supplied by the American Type Culture Collection. The subculture is streaked for purity on Brain-Heart Infusion Agar (DIFCO) and inoculated into 100 ml of sterile BHI. The agar plates are examined for purity after 14-18 hours. If

acceptable, 11 ml of sterile glycerin are added to the BHI culture, vortexed and then subdivided into 1.8 ml cryogenic tubes. The cultures are then stored at - 80°C. Four days prior to an assay, a frozen vial is thawed and added to the small, 100 ml flask containing 50 ml of Jordan's medium to start the cultures used for the biofilm assay. After 14-18 hours, the contents of the small flask are decanted aseptically into 2 liters of Jordan's medium. The resulting inoculated medium is then aseptically dispensed, in 5.0 ml portions, into a number of empty sterile test tubes, each tube having a plaque wire-equipped Morton cap. The inoculated tubes are then incubated anaerobically overnight (i.e., 14-16 hours) at 37°C. The number of test tubes will vary depending upon the number of different mouthwash samples being tested, but it will be convenient to describe an assay of a

standard high alcohol mouthwash and four reduced alcohol mouthwash samples, which requires racks each holding 75 test tubes (i.e. , five rows of fifteen tubes, each row comprising five sets of three tubes each). The first set of three tubes in each row is usually reserved for the standard high alcohol

mouthwash, with the succeeding four sets of three tubes each in that row being reserved for the four reduced alcohol samples. There are three tubes in each set because each assay is performed in triplicate.

After the overnight incubation, the plaque wires are then transferred into fresh Jordan's medium in 75 tubes (in a second rack) and again incubated anaerobically for 24 hours at 37°C. This procedure is repeated once more. Thus, the plaque wires are cultured for 3 days, with two transfers after initial inoculation.

On the third day, just prior to the assay, five

additional racks (each containing 75 sterile test tubes) are prepared: a first (assay) rack whose test tubes each contain 6 ml of the sample mouthwashes; a second (water) rack whose test tubes each contain 6 ml of sterile deionized water; third and fourth racks whose test tubes each contain 6 ml of Letheen broth rinse; and a fifth rack whose test tubes each contain 5 ml of Jordan's recovery medium. For convenience, the racks may be marked for test series identity and time (by row). Assay Procedure: Each assay will involve triplicate testing at time points separated by one minute

intervals, e.g., at 2, 3, 4, 5 and 6 minutes of

mouthwash treatment. The first (or bottom) row of each rack corresponds to the first test time and the

succeeding four rows correspond, respectively, to the next four test times. The exact time of exposure of the plaque wires to the sample mouthwashes can be varied according to the thickness of the "plaques;" ideally, the exposure period will result in positive microorganism growth in the first one or two sampling intervals of the high alcohol control group, (i.e., the first and second row) and no growth thereafter.

Establishing lower and upper limits of exposure

required for complete kill by the control mouthwash permits an accurate comparison of the four sample mouthwashes to this control. Mouthwash exposure takes place in a 37° New Brunswick shaking water bath (shake speed 3) and may be staggered so that the 5 time points are run concurrently, but with sufficient time to permit accurate timing and handling.

(1) To start assay, transfer one row of plaque wires to the first (bottom) row of tubes in the rack containing 6 ml of sterile water. Leave in place 2 minutes. Repeat for the next four rows of plaque wires.

(2) After the water rinse, transfer each row of plaque wires into the appropriate, corresponding, row of tubes in the rack containing 6 ml of test

mouthwash. Leave each row of plaque wires in place, with shaking in the 37° water bath, for its treatment (exposure) period; i.e., remove the rows of plaque wires sequentially at 5 preset time points so that each succeeding row of plaque wires is exposed to a mouthwash for successively longer periods of time (e.g.. 2, 3, 4, 5 and 6 minutes; individual timing can vary according to estimated "plaque" thickness. (3) For each row of plaque wires, at the end of its treatment period, immediately remove the row and place it in the appropriate corresponding row of the first rack of 6 ml Letheen Broth

neutralization/rinse tubes. Leave each row of wires in that broth for 5 minutes and then

transfer it to the appropriate corresponding row of the second rack of 6 ml Letheen Broth rinse tubes. (4) At the end of the second Letheen Broth rinse,

remove each row of plaque wires and place it in the appropriate corresponding row of the rack of 6 ml Jordan's recovery medium (with bromocresol purple). Incubate anaerobically for 48 hours at 37°C.

(5) Read for growth (+) or no growth (-) at 48 hours.

Positive growth is indicated by a color change from purple to yellow (i.e., if the microorganism is still viable, it will produce an acid which causes the color change); positive growth is often accompanied by an increase in broth turbidity.

Determination of Critical Kill Times and R-Factor:

Since each mouthwash sample is located in the same set of three tubes in each row of the rack, the critical time necessary for the sample to completely kill the microorganism can be determined by observing the point (front to back or bottom to top, as the case may be) at which the Jordan recovery medium color changed from yellow to purple. The critical kill time for any sample, divided by the critical kill time for the control mouthwash in that same rack, gives the R-Factor for that sample.

Table 2 below summarizes a statistical scale developed by the present inventors which relates the observed change from growth (+) to no growth (-) to critical kill times. For example, as shown in the first row of Table 2, where the observed condition changes from growth (continuous +'s) to no growth (continuous -'s) ("no anomaly"), the critical kill time is determined by adding 0.50 minute to the time at which the last growth observation (+) was made. The balance of Table 2 sets forth how critical kill times are determined for different observed growth/no growth intervals between continuous growth segments and continuous no growth segments.

By way of further example, consider the examples of growth/no growth sequences, and their associated critical kill times, in Table 3. In the first row of Table 3, there was no anomaly between continuous +'s and continuous -'s; therefore, CKT (per Table 2) =

4.0 + 0.5 = 4.5 minutes (i.e., kill occurred somewhere between 4.0 and 5.0 minutes). In the second row of Table 3, the interval between continuous +'s and continuous -'s is -+; therefore, CKT (per Table 2) = 2.0 + 1.5 = 3.5 minutes.

In the case of rows 3 and 4 of Table 3, clearly no end point was reached. It is assumed here that kill will occur at some point in excess of 6.5 minutes (>6.5) or much below 2.0 minutes (<2.0), respectively.

Row 5 (Table 3) is an example where the kill scale is dependent on the values which are located to the left of the last + and to the right of the first -. For that particular example, CKT = 2.0 + 1.1 = 3.1 minutes (per Table 2).

The following examples are provided both to illustrate the present invention and its advantages and to provide additional comparison information.

Example 1

A mouthwash composition containing 19% v/v ethanol and 18% w/v sorbitol was prepared as follows. First, an alcohol solution of active essential oils and flavor oils was prepared by dissolving the ingredients listed in Table 4 in 200 mls of ethanol (USP):

To the resulting alcohol solution were then added the additional ingredients shown in Table 5 (along with suitable dyes to obtain the desired color):

The pH was adjusted to 4.2. Approximately 600 mls of deionized water was then added, and the solution was mixed until all solids were dissolved. Additional deionized water was added with mixing to bring the total solution to 1 liter. A clear mouthwash solution was obtained.

Example 2 A number of different mouthwash compositions A-J were prepared. The composition ingredients and amounts used (except for the dyes) are shown in Table 6 below.

The mouthwashes were prepared as follows. An alcohol solution was prepared by dissolving the flavor oils, part of the thymol and part of the methyl salicylate in 227 or 200 milliters of ethanol (USP). Into the resulting solution was then dissolved the remaining methyl salicylate, the remaining thymol, the benzoic acid and the Pluronic surfactant. 300 mis of the deionized water was then slowly added to the resulting solution, with mixing. The sorbitol (USP) solution, or propylene glycol, was then added, with continued mixing, followed by the sodium saccharin, the sodium citrate and the citric acid, with mixing until

dissolved. The dyes were then added, with mixing, followed by the balance of the deionized water.

Additional sodium citrate or citric acid was added, if necessary, to adjust the pH of the resulting clear solution within the range of from about 3.9 to about 4.5, preferably 4.2. Each of the mouthwash compositions of Table 6 was evaluated in vitro for its ability to kill

microorganisms in a biofilm, using the protocol

described above. The results are shown below in Table 7, which also shows the percentages of ethanol and polyol in those compositions.

The above data support the inverse relationship between the ethanol and polyol amounts. Thus, with lower amounts of ethanol, one should employ even higher concentrations of polyol(s) above 18% w/v to maintain similar efficacy. Example 3

The mouthwash formulations shown in Table 8 below, containing 21.6% v/v ethanol and a varying amount of sorbitol, were prepared in a manner similar to the procedure shown in Example 2, and tested for their antimicrobial efficacy as measured by the amount of endotoxin extracted from in vitro cultures of three different Gram negative bacteria known to be plaque- associated microorganisms, Fusobacterium nucleatum

(ATCC #10953), Veillonella parvula (ATCC #10790), and Actinobacillus actinomycetemcomitans (ATCC #33384):

The same proprietary blend of flavor oils shown in Table 6 was used.

Endotoxin is a component of the outer wall of Gram negative bacteria and if shed by the bacteria at or near infection sites about the teeth and gums will elicit inflammation and gingivitis. Removal of

endotoxin from the plaque through use of a mouthwash composition will consequently reduce these problems.

The cultures were treated with successive dilutions of the mouthwash composition of Table 8 with no sorbitol versus those with sorbitol (17.5% w/v), as the data reflect in Table 9. Plus (+) signs indicate the presence of endotoxin while negative (-) signs indicate its absence. Clearly, the presence of sorbitol

enhances the removal of endotoxin. The cultures were also treated with successive

dilutions of the mouthwash composition of Table 8 with varying sorbitol concentrations. The results are shown in Tables 10 and 11 below. In both tables, the

"Control" mouthwash was the high alcohol (27% v/v) standard described above.

As tables 9-11 clearly show, the addition of sorbitol clearly enhances the endotoxin removal ability of the mouthwash composition. A greater degree of endotoxin is extracted from cultures treated with antispectic mouthwash compositions containing sorbitol than those that do not. Moreover, as Tables 10 and 11

demonstrate, increasing sorbitol levels in the

mouthwash clearly decrease the degree of endotoxin remaining in the culture, supporting its value in preventing inflammation and subsequent gum disease.

However, endotoxin measurements are at best a semi- quantitative prescreen, and therefore a more stringent method for assessing plaque and bacterial kill efficacy -- known as the plaque penetration assay (described above) -- was employed in this invention. The results of this assay, reported as "Critical Kill Times" (or "CKT"), are more predictive of the in vivo effect of oral mouthrinses than endotoxin measurements.

Claims

WHAT IS CLAIMED IS:

1. A reduced alcohol antimicrobial mouthwash

composition with enhanced antimicrobial activity for the reduction of calculus, plaque and gum disease comprising (a) an antimicrobial effective amount of one or more essential oils; (b) a reduced level, up to about 22% v/v, of ethanol; (c) at least about 18% w/v of one or more polyols sufficient to provide enhanced antimicrobial activity of said essential oils at said reduced level of ethanol; and (d) water.

2. The reduced alcohol antimicrobial mouthwash composition of Claim 1 wherein said essential oil ingredient (a) comprises thymol and one or more of eucalyptol, methyl salicylate and menthol.

3. The reduced alcohol antimicrobial mouthwash composition of Claim 2 wherein said polyol is selected from the group consisting of multi-hydroxy-functional ingestible compounds.

4. The reduced alcohol antimicrobial mouthwash composition of Claim 3 wherein said polyol is selected from the group consisting of sugar alcohols,

hydrogenated starch hydrolysates, alkylene glycols, diols, glycerin and mixtures thereof.

5. The reduced alcohol antimicrobial mouthwash composition of Claim 4 wherein said polyol is a sugar alcohol.

6. The reduced alcohol antimicrobial mouthwash composition of Claim 5 wherein said sugar alcohol is sorbitol.

7. The reduced alcohol antimicrobial mouthwash composition of Claim 6 wherein the amount of sorbitol in said composition is from about 18% w/v to about 30% w/v.

8. The reduced alcohol antimicrobial mouthwash composition of Claim 6 wherein the amount of sorbitol in said composition is from about 25% w/v to about 30% w/v.

9. The reduced alcohol antimicrobial mouthwash composition of Claim 7 wherein the amount of ethanol in said composition is no more than about 19% v/v.

10. The reduced alcohol antimicrobial mouthwash composition of Claim 9 further comprising (e) a non- ionic surfactant.

11. The reduced alcohol antimicrobial mouthwash composition of Claim 10 wherein said non-ionic

surfactant is selected from the group consisting of poloxamers.

12. The reduced alcohol antimicrobial mouthwash composition of Claim 11 wherein the amount of poloxamer in said composition is from about 0.01% to about 1.0% w/v.

13. The reduced alcohol antimicrobial mouthwash composition of Claim 12 wherein said poloxamer is

Poloxamer 407.

14. The reduced alcohol antimicrobial mouthwash composition of Claim 2 wherein the amount of thymol in said composition is from about 0.001% to about

0.25% w/v.

15. The reduced alcohol antimicrobial mouthwash composition of Claim 2 containing eucalyptol in an amount of from about 0.001% to about 0.11% w/v, containing menthol in an amount of from about 0.001% to about 0.25% w/v, and containing methyl salicylate in an amount of from about 0.001% to about 0.08% w/v.

16. The reduced alcohol antimicrobial mouthwash composition of Claim 2 further comprising flavor oils, buffers, and dyes.

17. A reduced alcohol antimicrobial mouthwash

composition with enhanced antimicrobial activity for the reduction of calculus, plaque and gum disease comprising: (a) an antimicrobially effective amount of one or more essential oils; (b) a reduced level, up to about 22% v/v, of ethanol; (c) at least about 18% w/v of one or more polyols sufficient to provide an

enhanced antimicrobial activity of said essential oils at said reduced level of ethanol, such that the R- Factor of said composition is less than about 2.0; and (d) water.

18. The reduced alcohol antimicrobial mouthwash composition of Claim 17 wherein the amount of ethanol in said composition is no more than about 19% v/v.

19. The reduced alcohol antimicrobial mouthwash composition of Claim 18 wherein the R-Factor of said composition is less than about 1.2.

20. The reduced alcohol antimicrobial mouthwash composition of Claim 18 wherein the R-Factor of said composition is less than about 1.0.

21. The reduced alcohol antimicrobial mouthwash composition of Claim 17 wherein said essential oil ingredient (a) comprises thymol and one or more of eucalyptol, menthol and methyl salicylate.

22. The reduced alcohol antimicrobial mouthwash composition of Claim 17 wherein said polyol is selected from the group consisting of multi-hydroxy-functional ingestible compounds.

23. The reduced alcohol antimicrobial mouthwash composition of Claim 19 wherein said polyol is

sorbitol.

24. The reduced alcohol antimicrobial mouthwash composition of Claim 23 wherein the amount of sorbitol in said composition is from about 18% w/v to about 30% w/v.

25. The reduced alcohol antimicrobial mouthwash composition of Claim 23 wherein the amount of sorbitol in said composition is from about 25% w/v to about 30% w/v.

26. The reduced alcohol antimicrobial mouthwash composition of Claim 17 further comprising flavorants and wherein said composition is essentially free of anethole.

27. A reduced alcohol antimicrobial mouthwash

composition with enhanced antimicrobial activity for the reduction of calculus, plaque and gum disease comprising (a) antimicrobially effective amounts of essential oils comprising thymol and one or more of eucalyptol, menthol and methyl salicylate; (b) a reduced level, no more than about 19% v/v, of ethanol; (c) at least about 13% w/v of one or more polyols sufficient to provide an enhanced antimicrobial activity of said essential oils at said reduced level of ethanol; and (d) water.

28. The reduced alcohol antimicrobial mouthwash composition of Claim 27 wherein said polyol is selected from the group consisting of multi-hydroxy-functional ingestible compounds.

29. The reduced alcohol antimicrobial mouthwash composition of Claim 27 wherein the amount of said polyol is from about 18% w/v to about 30% w/v.

30. The reduced alcohol antimicrobial mouthwash composition of Claim 28 wherein said polyol is

sorbitol.

31. The reduced alcohol antimicrobial mouthwash composition of Claim 30 wherein the amount of sorbitol is said composition is from about 18% w/v to about 30% w/v.

32. The reduced alcohol antimicrobial mouthwash composition of Claim 30 wherein the amount of sorbitol in said composition is from about 25% w/v to about 30% w/v.

33. The reduced alcohol antimicrobial mouthwash composition of Claim 27 further comprising flavorants and wherein said composition is essentially free of anethole.

34. A reduced alcohol antimicrobial mouthwash

composition with enhanced antimicrobial activity for the reduction of calculus, plaque and gum disease comprising (a) antimicrobially effective amounts of essential oils comprising thymol and one or more of eucalyptol, menthol and methyl salicylate; (b) a reduced level, from about 9% to about 22% v/v, of ethanol; (c) a polyol selected from the group

consisting of a sugar alcohol, propylene glycol, glycerin and mixtures thereof in an amount of at least about 18% w/v sufficient to provide an enhanced antimicrobial activity of said essential oils at said reduced level of ethanol; and (d) water.

35. The reduced alcohol antimicrobial mouthwash composition of Claim 34 wherein said polyol is

sorbitol.

36. The reduced alcohol antimicrobial mouthwash composition of Claim 35 wherein the amount of sorbitol in said composition is from about 18% w/v to about 25% w/v.

37. The reduced alcohol antimicrobial mouthwash composition of Claim 35 wherein the amount of sorbitol in said composition is from about 25% w/v to about 30% w/v.

38. The reduced alcohol antimicrobial mouthwash composition of Claim 34 further comprising flavorants and wherein said composition is essentially free of anethole.

39. The reduced alcohol antimicrobial mouthwash composition of Claim 34 wherein the R-Factor of said composition is less than about 2.

40. The reduced alcohol antimicrobial mouthwash composition of Claim 34 wherein the R-Factor of said composition is less than about 1.2.

41. The reduced alcohol antimicrobial mouthwash composition of Claim 34 wherein the R-Factor of said composition is less than about 1.0.