Patent Application
20230329983
The present invention relates to dentifrice compositions comprising bicarbonate salt and dual sweetener. The dentifrice compositions have improved efficacy to help inhibit biofilm formation or help disrupt biofilm and improved sensorial experience to improve compliance and consumer experience.
Dental plaque (also known as dental biofilm) is a sticky, colorless deposit of bacteria that is constantly forming on the tooth surface. Dental plaque is generally made up of bacteria and extracellular polymer substances (so called “EPS”). EPS are biopolymers of microbial origin in which biofilm microorganisms are embedded. J. Bacteriol. 2007, 189(22): 7945. Saliva, food and fluids combine to produce these deposits that collect where the teeth and gums meet. Plaque buildup is the primary factor in poor oral health that can lead to caries and periodontal (gum) disease, including gingivitis. One-way dentifrice compositions help prevent and control plaque is by leveraging anti-bacterial agents; however, the disadvantage and formulation challenge is the unintended reactivity of anti-bacterial agents with formulation ingredients. This may include oxidative degradation, hydrolysis, adsorption or precipitation of oxy-hydroxide species, any of which can impact the bio-availability of the anti-bacterial agent. There is a continuing consumer need to provide such formulations that help prevent plaque formation on teeth and/or minimize the use of antimicrobial agents.
One solution to help inhibit biofilm formation or help disrupt biofilm is the use of baking soda (i.e., sodium bicarbonate). Baking soda mechanism of action against biofilm is likely at least two-fold. Baking soda can displace calcium ions so as to help disrupt or reduce the biofilm. Calcium ions act as a “glue” or “scaffold” of EPS components of dental biofilm. Baking soda may also act as an abrasive to aid in the physical removal of oral biofilm. Some levels of baking soda in dentifrice are reported at over 30 wt. %, and sometimes higher than 60 wt. %.
However, some users report an unpleasant taste experience (attributable to the relatively high level of baking soda). The use of high-level baking soda compositions brings stability challenges around decomposition of the baking soda and consumer acceptance challenges due to poor sensorial experience resulting from an unacceptable saltiness taste.
An approach used to overcome such stability challenges has been to formulate baking soda composition in anhydrous or low water compositions. However, this approach can lead to poor rheology properties and impairs the dispersion and delivery. Furthermore, dental plaque is particularly problematic in developing markets. And thus, dentifrice solutions are best cost effective (e.g., containing a relatively high level of water). Accordingly, there is a need for baking soda containing aqueous dentifrice compositions, maximizing the bicarbonate salt associated therapeutic benefits while minimizing the level of bicarbonate salt (e.g., <60 wt. %) as to help consumer compliance with improved flavor profile, improved dispersion and delivery. There is also a need for a composition that is cost effective for developing markets where arguably the need for such compositions are the greatest.
While addressing the stability challenges, there further remains a need to improve the overall sensorial experience of such compositions by reducing the saltiness taste, while not making the taste of the composition too sweet.
The present invention relates to a dentifrice compositions comprising: (a) water; (b) from about 1% to about 60%, by weight of the composition, of bicarbonate salt; (c) fluoride ion source; (d) medium intensity sweetener having Sweetness Index from about 150 to 350; (e) high intensity sweetener, having Sweetness Index from about 500 to 10,000; and wherein a weight ratio of medium intensity sweetener to high intensity sweetener is from about 1:0.15 to about 1:1.2.
The present invention further relates to dentifrice compositions comprising: (a) from about 15% to about 55%, by weight of the composition, of water; (b) from about 1% to about 60%, by weight of the composition, of bicarbonate salt; (c) from about 0.01% to about 1%, by weight of the composition, of medium intensity sweetener having a Sweetness Index from about 150 to about 350; (d) from about 0.01% to about 0.5%, by weight of the composition, of high intensity sweetener having a Sweetness Index from about 500 to about 10,000; and (e) from about 0.0025% to about 2%, by weight of the composition, of a fluoride ion source.
Significant disruption and destabilization of dental biofilm can be achieved by an aqueous dentifrice composition comprising bicarbonate salt (e.g. sodium bicarbonate) and a combination of medium intensity sweetener (having a Sweetness Index from about 150 to about 350 vs. reference of sucrose with a Sweetness Index of 1) and high intensity sweetener (having a Sweetness Index from about 500 to about 10,000 vs. reference of sucrose with a Sweetness Index of 1), which surprisingly provides improved sensorial profile of masking the known saltiness associated with bicarbonate salt dentifrice compositions both during and after brushing.
While commercial dentifrice compositions containing over 65% sodium bicarbonate claim to provide a clinical benefit in treating gingivitis, users report an unpleasant sensory experience (e.g. taste, foaming, dispersion) associated at this higher a level of sodium bicarbonate. Another aspect of the present invention is lowering the level of bicarbonate salt, while providing the therapeutic benefit in destabilizing the biofilm and having an improved sensorial experience.
The present invention further provides an aqueous dentifrice containing relatively high content of both bicarbonate salt and water that has a good chemical and physical stability over the product shelf life, allowing for an improved dispersion and delivery of bicarbonate salt into the plaque matrix, that maximizes the bicarbonate salt associated therapeutic benefits.
The present invention further encompasses a method of treating dental biofilm comprising the step of brushing teeth with a dentifrice composition of the present invention.
The present invention further encompasses a method of preventing or mitigating plaque formation on tooth enamel comprising the step of brushing teeth with a dentifrice composition of the present invention.
The term “dentifrice” as used herein means paste, gel, powder, tablets, or liquid formulations, unless otherwise specified, that are used to clean the surfaces of the oral cavity. Preferably the dentifrice compositions of the present invention are single phase compositions, although the compositions can be dual phase or multi-phase compositions. One example of a dentifrice composition is toothpaste (for brushing teeth). The term “teeth” as used herein refers to natural teeth as well as artificial teeth or dental prosthesis.
All percentages, parts and ratios are based upon the total weight of the compositions of the present invention, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified. The term “weight percent” may be denoted as “wt. %” herein. All molecular weights as used herein are weight average molecular weights expressed as grams/mole, unless otherwise specified.
The dentifrice compositions of the present invention comprise herein from about 5% to about 55%, preferably from about 15% to about 45%, preferably from about 20% to about 35%, by weight of the composition, of water. The dentifrice composition can comprise from about 15% to about 55%, preferably from about 20% to about 45%, preferably from 25% to 35%, by weight of the composition, of water. The water may be added to the formulation and/or may come into the composition from the inclusion of other ingredients provided in aqueous solution. Preferably, the water is USP water.
The compositions of the present invention comprise from about 1% to about 60%, by weight of the composition, of bicarbonate salt. Preferably, the bicarbonate salt is selected from sodium bicarbonate, calcium bicarbonate, or mixtures thereof; more preferably sodium bicarbonate. The dentifrice compositions can comprise from about 1% to about 60%, preferably from about 10% to about 50%, and more preferably 20% to about 40%, by weight of the composition, of the bicarbonate salt (preferably sodium bicarbonate).
The compositions may optionally, but preferably, include an effective amount of an anti-caries agent, such as a fluoride ion source. The fluoride ion may be present in an amount sufficient to give a fluoride ion concentration in the composition at 25° C., and/or in one embodiment can be used at levels of from about 0.0025% to about 5% by weight of the composition, preferably from about 0.005% to about 2.0%, preferably from about 0.5% to about 1.5%, by weight of the composition, to provide anti-caries effectiveness. Representative fluoride ion sources include stannous fluoride, sodium fluoride, potassium fluoride, amine fluoride, sodium monofluorophosphate, and zinc fluoride, or mixtures thereof. In one aspect, the dentifrice compositions of the present invention may have a dual fluoride ion source, specifically sodium monofluorophosphate and an alkaline metal fluoride. Without wishing to be bound by theory, such an approach may provide an improvement in mean fluoride uptake.
In one aspect, the fluoride ion source is selected from sodium monofluorophosphate, amine fluoride, sodium fluoride, or combinations thereof.
The dentifrice compositions of the present invention comprise a combination of a medium intensity sweetener and a high intensity sweetener, based on the Sweetness Index of the sweetener vs. sucrose as a reference, which has a Sweetness Index of 1.
The term “medium intensity sweetener” as used herein includes natural and synthetic sweeteners that have a Sweetness Index range of from about 150 to about 350 (vs. sucrose as a reference which has a Sweetness Index of 1).
Accordingly, medium intensity sweeteners suitable for use in the invention include, but are not limited to, saccharin, aspartame, stevia, acesulfame, salts thereof, or mixtures thereof. Preferably, medium intensity sweeteners used in the composition of the present invention may include saccharin, stevia, salts thereof, or mixtures thereof.
The dentifrice compositions of the present invention will typically comprise medium intensity sweetener at a level of from about 0.01% to about 1%, preferably from about 0.1% to about 0.7%, preferably from about 0.2% to about 0.6%, or preferably from about 0.3% to about 0.5%, by weight of the dentifrice composition.
The term “high intensity sweetener” as used herein includes natural and synthetic sweeteners that have a Sweetness Index range of from about 400 to about 10,000, preferably from about 500 to about 10,000, preferably from about 500 to 5,000, or preferably from about 500 to about 1,000 (vs. sucrose as a reference which has a Sweetness Index of 1).
Accordingly, high intensity sweeteners suitable for use in the invention include, but are not limited to, sucralose, brazzein, pentadin, thaumatin, monellin, dihydrochalcones, neotame, salts thereof, or mixtures thereof.
The dentifrice compositions of the present invention will typically comprise high intensity sweetener at a level of from about 0.01% to about 0.5%, preferably from about 0.05% to about 0.7%, preferably from about 0.1% to about 0.7%, preferably from about 0.1% to about 0.5%, or preferably from about 0.2% to about 0.3%, by weight of the dentifrice composition.
Preferably, the compositions of the present invention comprise medium intensity sweetener and high intensity sweetener in a weight ratio of medium intensity sweetener to high intensity sweetener of from about 1:0.15 to about 1:1.2, preferably from about 1:0.25 to about 1:0.75, or preferably from about 1:0.4 to about 1:0.65.
Sweetness Index, also referred to as Sweetness Power, is a property well-known in the literature and is measured in reference to sucrose, which has a Sweetness Index of 1. Sweetness Index/Power values and further examples of medium intensity sweeteners and high intensity sweetners are provided in H. Mitchell, Sweeteners and Sugar Alternatives in Food Technology (Blackwell Publishing 2006) and in M. Carocho et al., Sweeteners as food alternatives in the XXI century: A review of what is known, and what is to come, 107 Food and Chemical Technology 302-317, 303 (2017) (citing the H. Mitchell reference indicated above).
The compositions of the present invention can optionally, and in some aspects preferably, comprise from about 5% to about 50%, preferably from about 10% to about 50%, by weight of the composition, of a calcium-containing abrasive, wherein preferably the calcium-containing abrasive is selected from the group consisting of calcium carbonate, calcium glycerophosphate, dicalcium phosphate, tricalcium phosphate, calcium orthophosphate, calcium metaphosphate, calcium polyphosphate, calcium oxyapatite, sodium carbonate, and mixtures thereof; wherein more preferably the calcium-containing abrasive is calcium carbonate. Preferably, the composition comprises from about 15% to about 50%, preferably from about 15% to about 40%, preferably from about 15% to about 25%, by weight of the composition, of a calcium-containing abrasive.
Preferably, the calcium-containing abrasive is calcium carbonate. More preferably, the calcium-containing abrasive is selected from the group consisting of fine ground natural chalk, ground calcium carbonate, precipitated calcium carbonate, and combinations thereof. Non-limiting examples of the weight percentages of the calcium-containing abrasive include: about 15%, about 20%, about 25%, or about 30%, by weight of the composition, preferably wherein the calcium-containing abrasive is calcium carbonate.
Silica dental abrasives of various types are preferred because of their unique benefits of exceptional dental cleaning and polishing performance without unduly abrading tooth enamel or dentine. The silica abrasive polishing materials herein, as well as other abrasives, generally have an average particle size ranging between about 0.1 to about 30 microns, and preferably from about to about 15 microns. The abrasive can be precipitated silica or silica gels such as the silica xerogels describe in Pader et al., U.S. Pat. No. 3,538,230, issued Mar. 2, 1970, and DiGiulio, U.S. Pat. No. 3,862,307, issued Jan. 21, 1975. Preferred are the silica xerogels marketed under the trade name “Syloid” by the W. R Grace & Company, Davison Chemical Division. Also preferred are the precipitated silica materials such as those marketed by the J. M. Huber Corporation under the trade name, Zeodent®, particularly the silicas carrying the designation Zeodent® 119, Zeodent® 118, Zeodent® 109 and Zeodent® 129. The types of silica dental abrasives useful in the toothpastes of the present invention are described in more detail in Wason, U.S. Pat. No. 4,340,583, issued Jul. 29, 1982; and in commonly-assigned U.S. Pat. No. 5,603,920, issued on Feb. 18, 1997; U.S. Pat. No. 5,589,160, issued Dec. 31, 1996; U.S. Pat. No. 5,658,553, issued Aug. 19, 1997; U.S. Pat. No. 5,651,958, issued Jul. 29, 1997, and U.S. Provisional Application Ser. No. 60/300,766, filed Jun. 25, 2001.
In one aspect, the composition comprises from 0% to about 15%, preferably 0% to about 10%, more preferably 0% to about 5%, by weight of the composition, of silica abrasive. Preferably, the silica abrasive is thickening silica.
pH
The pH of the dentifrice composition may be greater than about 7.3, preferably greater than about 8, preferably from about 8 to about 11, preferably from about 8.5 to about 11, preferably about 8.5 to about 10.5, preferably from about 8.5 to about 10. The relatively high pH of the present inventive composition is for fluoride stability. Thus, it is desirable to have the dentifrice composition have a greater than pH 8.0 to maximize the stability of the fluoride ion source. A method for assessing pH of dentifrice is described is below. For purposes of clarification, although the analytical method describes testing the dentifrice composition when freshly prepared, for purposes of claiming the present invention, the pH may be taken at any time during the product's reasonable lifecycle (including but not limited to the time the product is purchased from a store and brought to the user's home).
The dentifrice compositions herein may include an effective amount of a pH modifying agent, preferably wherein the pH modifying agent is a pH buffering agent. The pH modifying agents, as used herein, refer to agents that can be used to adjust the pH of the dentifrice compositions to the above-identified pH range. The compositions can comprise from about 0.001% to about 5%, by weight of the composition, of pH modifying agent. The pH modifying agents may include alkali metal hydroxides, ammonium hydroxide, organic ammonium compounds, carbonates, sesquicarbonates, borates, silicates, phosphates, imidazole, and mixtures thereof. Specific pH agents include monosodium phosphate (monobasic sodium phosphate or “MSP”), trisodium phosphate (sodium phosphate tribasic dodecahydrate or “TSP”), sodium benzoate, benzoic acid, sodium hydroxide, potassium hydroxide, alkali metal carbonate salts, sodium carbonate, imidazole, pyrophosphate salts, tripolyphoshpate salts, sodium gluconate, lactic acid, sodium lactate, citric acid, sodium citrate, phosphoric acid. Without wishing to be bound by theory, phosphate may also have calcium ion chelating activity and therefore provide some monofluorophosphate stabilization (in those formulations containing monofluorophosphate).
A method for assessing pH of dentifrice is described. The pH is measured by a pH Meter with Automatic Temperature Compensating (ATC) probe. The pH Meter is capable of reading to 0.001 pH unit. The pH electrode may be selected from one of the following (i) Orion Ross Sure-Flow combination: Glass body—VWR #34104-834/Orion #8172BN or VWR #10010-772/Orion #8172BNWP; Epoxy body—VWR #34104-830/Orion #8165BN or VWR #10010-770/Orion #8165BNWP; Semi-micro, epoxy body—VWR #34104-837/Orion #8175BN or VWR #10010-774/Orion #3175BNWP; or (ii) Orion PerpHect combination:VWR #34104-843/Orion #8203BN semi-micro, glass body; or (iii) suitable equivalent. The automatic temperature compensating probe is Fisher Scientific, Cat #13-620-16.
A 25% by weight slurry of dentifrice is prepared with deionized water, and thereafter is centrifuged for 10 minutes at 15000 rotations-per-minute using a SORVALL RC 28S centrifuge and SS-34 rotor (or equivalent gravitational force, at 24149 g force). The pH is assessed in supernatant after one minute or the taking reading is stabilized. After each pH assessment, the electrode is washed with deionized water. Any excess water is wiped with a laboratory grade tissue. When not in issue, the electrode is kept immersed in a pH 7 buffer solution or an appropriate electrode storage solution.
The compositions herein contains relatively low amount, or even be substantially free or free, of humectants. Non-limiting examples of humectant levels, by weight of the dentifrice composition, include about 0.1%, about 0.5%, about 1%, about 5%, no more than 10%, or 0%. Without wishing to be bound by theory, the presence of humectant (e.g., sorbitol/glyercol) may have a negative role in fluoride uptake in dental plaque in the high water and high carbonate containing dentifrice formulations of the present invention. Reduced levels of sorbitol/glycerol in these dentifrice compositions provide superior fluoride uptake results. Preferably the dentifrice compositions of the present invention comprise from 0% to about 10%, by weight of the composition, of a humectant, wherein the humectant is selected from sorbitol, glycerol, or mixtures thereof; more preferably the composition comprises from 0% to about 7%, preferably 0% to about 5%, by weight of the composition of said humectant. In one aspect, the composition is substantially free of humectant.
The term “humectant,” for the purposes of present invention, include edible polyhydric alcohols such as glycerin, sorbitol, xylitol, butylene glycol, propylene glycol, or mixtures thereof. In one aspect, the humectant is a polyol, preferably wherein the polyol is selected from sorbitol, glycerin, or mixtures thereof. In yet another example, the humectant is sorbitol. A potential advantage of having a dentifrice composition that contains low levels of humectant (i.e., at or less than about 2 wt. %), without wishing to be bound by theory, is those dentifrice compositions that are free of humectants such as glyercol or sorbitol may provide better fluoride uptake as compared to those compositions having the high levels of such humectants. In one aspect, the dentifrice compositions of the present invention comprise from 0% to about 2%, preferably 0% to about 1%, more preferably 0% to about 0.5%, by weight of the composition, of glycerin and/or sorbitol. In one aspect, the composition is preferably substantially free of both glycerin and sorbitol.
The dentifrice compositions of the present invention may comprise a thickening system. Preferably the dentifrice composition comprises from about 0.5% to about 10%, preferably from about 0.8% to about 5%, more preferably from about 1% to about 5%, by weight of the composition, of the thickening system. More preferably the thickening system comprises a thickening polymer, a thickening silica, or mixtures thereof. Yet more preferably, when the thickening system comprises a thickening polymer, the thickening polymer is selected from a carboxymethyl cellulose, a linear sulfated polysaccharide, a natural gum, or mixtures thereof. Yet still more preferably, when the thickening system comprises a thickening polymer, the thickening polymer is selected from the group consisting of: (a) from about 0.01% to about 3% of a carboxymethyl cellulose (“CMC”) by weight of the composition, preferably from about 0.1% to about 2.5%, more preferably from about 0.5% to about 1.7%, by weight of the composition, of CMC; (b) from about 0.01% to about 2.5%, preferably from about 0.05% to about 2%, more preferably from about 0.1% to about 1.5%, by weight of the composition, of a linear sulfated polysaccharide, preferably wherein the linear sulfated polysaccharide is a carrageenan; (c) from about 0.01% to about 3%, preferably from about 0.1% to about 2%, more preferably from about 0.2% to about 1.8%, by weight of the composition, of a natural gum; or (d) mixtures thereof.
Preferably, when the thickening system comprises a thickening silica, the thickening silica is from about 0.01% to about 8%, preferably from about 0.1% to about 5%, preferably about 1% to about 3%, by weight of the composition.
Preferably, the linear sulfated polysaccharide is a carrageenan (also known as carrageenan). Examples of carrageenan include Kappa-carrageenan, Iota-carrageenan, Lambda-carrageenan, or mixtures thereof.
In one aspect the thickening silica is obtained from sodium silicate solution by destabilizing with acid as to yield very fine particles. One commercially available example is ZEODENT® branded silicas from Huber Engineered Materials (e.g., ZEODENT® 103, 124, 113 115, 163, 165, 167).
In one aspect, the CMC is prepared from cellulose by treatment with alkali and monochloro-acetic acid or its sodium salt. Different varieties are commercially characterized by viscosity. One commercially available example is Aqualon™ branded CMC from Ashland Special Ingredients (e.g., Aqualon™ 7H3SF; Aqualon™ 9M3SF Aqualon™ TM9A; Aqualon™ TM12A).
Preferably, a natural gum is selected from the group consisting of gum karaya, gum arabic (also known as acacia gum), gum tragacanth, xanthan gum, and mixtures thereof. More preferably the natural gum is xanthan gum. Xanthan gum is a polysaccharide secreted by the bacterium Xanthomonas camestris. Generally, xanthan gum is composed of a pentasaccharide repeat units, comprising glucose, mannose, and glucuronic acid in a molar ratio of 2:2:1, respectively. The chemical formula (of the monomer) is C35H49029. In one aspect, the xanthan gum is from CP Kelco Inc (Okmulgee, US).
In one aspect, the thickening polymer is selected from group consisting of a carboxymethyl cellulose, a linear sulfated polysaccharide, a natural gum, and mixtures thereof.
Preferably the dentifrice compositions of the present invention have a viscosity range from about 200,000 centipoise to about 850,000 centipoise (“cP”). A method for assessing viscosity is described. The viscometer is Brookfield® viscometer, Model DV-I Prime with a Brookfield “Helipath” stand. The viscometer is placed on the Helipath stand and leveled via spirit levels. The E spindle is attached, and the viscometer is set to 2.5 RPM. Detach the spindle, zero the viscometer and install the E spindle. Then, lower the spindle until the crosspiece is partially submerged in the paste before starting the measurement. Simultaneously turn on the power switch on the viscometer and the helipath to start rotation of the spindle downward. Set a timer for 48 seconds and turn the timer on at the same time as the motor and helipath. Take a reading after the 48 seconds. The reading is in cP.
The compositions of the present invention may optionally comprise polyethylene glycol (“PEG”), of various weight percentages of the composition as well as various ranges of average molecular weights. In one aspect of the invention, the compositions have from about 0.1% to about 5%, preferably from about 0.5% to about 4%, more preferably from about 1% to about 3%, by weight of the composition, of PEG. In another aspect of the invention, the PEG is one having a range of average molecular weight from about 100 to about 1600, preferably from about 200 to about 1000, preferably from about 400 to about 800, preferably from about 500 to about 700 Daltons. PEG is a water soluble linear polymer formed by the addition reaction of ethylene oxide to an ethylene glycol equivalent having the general formula is: H—(OCH2CH2)n—OH. One supplier of PEG is Dow Chemical Company under the brandname of CARBOWAX™. Without wishing to be bound by theory, having some PEG in the dentifrice composition may help with physical stability.
The dentifrice compositions herein may include a surfactant. The surfactant may be selected from anionic, nonionic, amphoteric, zwitterionic, cationic surfactants, or mixtures thereof. The composition may include a surfactant at a level of from about 0.01% to about 10%, from about 0.025% to about 9%, from about 0.05% to about 5%, from about 0.1% to about 2.5%, from about 0.5% to about 2%, or from about 0.1% to about 1%, by weight of the composition. Non-limiting examples of anionic surfactants may include those described at US 2012/0082630 A1 at paragraphs 32, 33, 34, and 35. Non-limiting examples of zwitterionic or amphoteric surfactants may include those described at US 2012/0082630 A1 at paragraph 36; cationic surfactants may include those described at paragraphs 37 of the reference; and nonionic surfactants may include those described at paragraph 38 of the reference. Preferred surfactants include sodium lauryl sulfate, cocamidopropyl betaine, or mixtures thereof. In one aspect, the composition comprises from about 0.1% to about 5%, preferably about 0.1% to about 3%, preferably from about 0.3% to about 3%, preferably from about 1.2% to about 2.4%, preferably from about 1.2% to about 1.8%, preferably from about 1.5% to about 1.8%, by weight of the composition, of an anionic surfactant, e.g. sodium lauryl sulfate (SLS).
The compositions herein may include a colorant. Titanium dioxide is one example of a colorant. Titanium dioxide is a white powder which adds opacity to the compositions. Colorant, e.g. titanium dioxide, generally can comprise from about 0.25% to about 5%, by weight of the composition.
The compositions herein may include from about 0.001% to about 5%, preferably from about 0.01% to about 4%, preferably from about 0.1% to about 3%, preferably from about 0.5% to about 2%, preferably about 1% to about 1.5%, preferably about 0.5% to about 1%, by weight of the composition, of a flavorant. The term flavorant is used in the broadest sense to include flavor ingredients, sensates, or combinations thereof. Flavor ingredients may include those described in US 2012/0082630 A1 at paragraph 39; sensates may include those described at paragraphs 40-45, incorporated herein by reference. Excluded from the definition of flavorant is “sweetener” (as described above).
The following assay is used an in situ plaque biofilm for inventive oral care compositions of the present invention in order to assess the biofilm Extracellular Polymeric Substances (EPS) matrix destabilization and thickness of the dental biofilm by measuring fluorescent light emitted from the labeled calcium & EPS within the biofilm.
The term “biofilm” refers to the layer(s) of cells attached to a surface. A biofilm can be a bacterial biofilm that includes both alive and growing microbe cells as well as dead microbe cells. The biofilm can be composed of one cell type or it may be composed of two or more cell types, for example, a biofilm complex that is a multispecies bacterial community. A specific type of biofilm is “dental biofilm” (also known as “plaque biofilm,” used herein interchangeably) which is biofilm that typically forms on tooth surfaces in the human mouth). Bacteria in a plaque biofilm have significantly different physiological characteristics, e.g. increased resistance to detergents and antibiotics, making biofilm research highly important. A non-limiting list of oral bacterial species is described at U.S. Pat. No. 6,309,835 B1, column 7, lines 12-30. These adherent microbe cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (“EPS”). EPS are biopolymers of microbial origin in which biofilm microorganisms are embedded. J. Bacteria 2007, 189(22):7945. Biofilm extracellular polymeric substance is a polymeric conglomeration generally composed of calcium, extracellular DNA, proteins, and polysaccharides.
Details of the assay are described below.
(a) Substrate for Biofilm Growth
Hydroxyapatite (“HA”) disks are used for in situ growth of biofilms. The HA disks are designed having three parallel grooves (i.e., 200 μm wide; 200 μm deep for two sides' grooves; while 500 μm wide and 500 μm deep for the middle groove) in each disk. When attaching disks to subject's mouth, keep these grooves vertical, to mimic interproximal gap between teeth, which is the hard-to-clean area where plaque generally tends to accumulate. This model allows the collection of undisturbed plaque from the grooves. HA disks are manufactured by Shanghai Bei′erkang biomedicine limited company (Shanghai, China).
(b) Wearing the Splint
Human subjects wear the splint. Each subject wears up to 12 HA disks on the splint to ensure that, at least, 9 HA disks are available after 48 hours. A non-limiting example of such a splint and HA disks are shown in
Although not shown in
(c) In-situ Biofilms Release from HA Desk
All HA disks are removed from the splint at 48 hours by tweezers. Tweezers are used to hold the edge of HA chips and transfer the HA disk to a 2 mL centrifuge tube containing PBS (phosphate buffered saline) solution. Tweezers are washed thoroughly (water; 75% alcohol; and then deionized water) before every disk transfer.
(d) Preparation of Toothpaste Supernatant
15 grams of deionized water is added to 5 grams toothpaste (using any one of the Examples 1-5). After stirring thoroughly, the mixture is centrifuge at 12,000 RPM for 20 minutes. The supernatant is prepared one day before usage and stored at 4° C.
(e) Confocal Laser Scanning Microscopy
After the HA disks are removed from the splint. The HA disks are used for ex vivo treatment by the different inventive and comparative compositions. After being treated with the subject supernatant and labeled with microbial fluorescent probe and stannous fluorescent probe (such as described in US2018/0072944A1; Shi et al.), the biofilms in the grooves are measured by confocal laser scanning microscopy (“CLSM”) (as described below).
(f) Disk Preparation
The HA disks are rinsed in PBS solution and each HA disk is divided into two halves by tweezers. Thereafter, each half-disk is placed into 500-1000 μL of PBS solution statically for 1 minute. Each disk is treated for two minutes by either PBS solution or toothpaste supernatant. Each disk is washed by holding each disk with tweezers, shaken for ten rounds of back and forth in 1 mL of PBS solution, and then this washing cycle is repeated. Then each disk is immersed into 500-1000 μL PBS solution statically for 5 minutes.
After being treated with PBS and/or the oral care composition (e.g., toothpaste) supernatant and labeled with specific fluorescent probes, the biofilm in the grooves is measured by confocal laser scanning microscopy (CLSM).
(g) Fluorescence Probe Staining and Microscopy
“Ion fluorescent probe” means a fluorescent probe that specifically binds to one kind of ions and emit fluorescence at a certain wavelength. In recent years, significant emphasis has been placed on the development of new, highly selective fluorescent probes of ions because of their potential applications in biochemistry and environmental research. Many kinds of signaling mechanisms have been proposed and utilized for optical detection of ions, including photo-induced electron/energy transfer (PET), intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), and so on. Some of these fluorescent probes can also be applied in fluorescence bioimaging, which causes little cell damage and is highly sensitive with high-speed spatial analysis of living cells. Specifically, FRET imaging that affords simultaneous recording of two emission intensities at different wavelengths in the presence and absence of analytes has provided a facile method for visualizing complex biological processes at the molecular level. This technique appears to be suited to the study of physiological functions or pathogenesis of ions in biofilm and human body.
Fluorescence labeled calcium probes are molecules that exhibit an increase in fluorescence upon binding Ca2+. The biofilm is labeled with a calcium fluorescent probe. Examples of a calcium fluorescent probe suitable for labeling the biofilm may be any one or more of the following compounds:
Fluo-3™ is used to image the spatial dynamics of Ca2+ signaling. Biofilm may be treated with the AM™ ester forms of calcium probes by adding the dissolved probe directly to biofilm. Fluo-3™ AM™, cell permeant fluorescent probes are used for intracellular and extracellular calcium staining using confocal microscopy, flow cytometry, and microplate screening applications (absorption/emission maxima ˜506/526 nm). It is reported that the Concanavalin A™ (Con A), Alexa Fluor® 594 Conjugate is a reliable alternative to stain EPS of biofilm. Alexa Fluor® 594 conjugate of Con A exhibits the bright, red fluorescence of the Alexa Fluor® 594 dye (absorption/emission maxima ˜590/617 nm). Concanavalin A™, Alexa Fluor® 594 Conjugate selectively binds to α-mannopyranosyl and α-glucopyranosyl residues which are rich in EPS part of biofilm.
One specific example is Concanavalin A™, Fluorescein Conjugate™, wherein excitation is 494 nm, and maximum light emission is detected at 518 nm. These EPS fluorescent probes are widely available as well as the procedure details in how to use them to quantitatively determine the location and/or amount of EPS.
Examples of an EPS fluorescent probe suitable for labeling the biofilm may be any one of the following compounds:
After treatment and immersing, each half-disk specimen is stained with a dye mixture solution of the Fluo-3™, AM™, cell permeant fluorescent probe together with Concanavalin A™, Alexa Fluor® 594 Conjugate probe (containing 5 uM Fluo-3™+5 uM Con-A™) for 30 minutes in the dark. After staining, each specimen is immersed into 500-1000 ul PBS solution statically for 2 minutes. The Fluo-3™, AM™/Concanavalin A™, Alexa Fluor® 594 Conjugate dye stained samples, the following parameters are used: λex=506 nm/590 nm, λem=526/617 nm, 20λ objective lens, and scanning from bottom of surface bacteria for 60 μm with step size=3 μm.
(h) Confocal Laser Scanning Microscopy
The Leica™ TCS SP8 AOBS spectral confocal microscope is used. The confocal system consists of a Leica™ DM6000B upright microscope and a Leica™ DMIRE2 inverted microscope. An upright stand is used for applications involving slide-mounted specimens; whereas the inverted stand, having a 37° C. incubation chamber and CO2 enrichment accessories, provides for live cell applications. The microscopes share an exchangeable laser scan head and, in addition to their own electromotor-driven stages, a galvanometer-driven high precision Z-stage which facilitates rapid imaging in the focal (Z) plane. In addition to epifluorescence, the microscopes support a variety of transmitted light contrast methods including bright field, polarizing light and differential interference contrast, and are equipped with 5×, 20×, 40×, 63× (oil and dry) and 100× (oil) Leica™ objective lenses.
The laser scanning and detection system is described. The TCS SP8 AOBS confocal system is supplied with four lasers (one diode, one argon, and two helium neon lasers) thus allowing excitation of a broad range of fluorochromes within the UV, visible and far red ranges of the electromagnetic spectrum. The design of the laser scan head, which incorporates acousto-optical tunable filters (“AOTF”), an acousto-optical beam splitter (“AOBS”) and four prism spectrophotometer detectors, permits simultaneous excitation and detection of three fluorochromes. The upright microscope also has a transmission light detector making it possible to overlay a transmitted light image upon a fluorescence recording.
Leica™ Confocal software LAS AF3.3.0 is used. The confocal is controlled via a standard Pentium PC equipped with dual monitors and running Leica™ Confocal Software. The Leica Confocal Software LAS AF3.3.0 (available from Leica Lasertechnik GmbH, Heidelberg, Germany) provides an interface for multi-dimensional image series acquisition, processing and analysis, that includes 3D reconstruction and measurement, physiological recording and analysis, time-lapse, fluorochrome co-localization, photo-bleaching techniques such as FRAP and FRET, spectral immixing, and multicolour restoration.
(i) Image Analysis
Ca:EPS; The Fluo-3™/Con-A™ stained specimens, both fluorescence channels are chosen to quantify fluorescence intensity ratio of green pixels (Calcium) to red pixels (EPS) and Con-A™ fluorescence channel is chosen to measure the biofilm thickness. Whereby, six selected fields of Con-A™ fluorescence channel of each specimen are evaluated. These fields are considered as representative of the whole sample after the observer's general examination. The distance is measured from the surface of the biofilm to its base, measuring the thickness of the field, and subsequently the mean thickness of the biofilm of the corresponding specimen is calculated.
Provided below are non-limited examples of the dentifrice compositions of the present invention (Examples 5-7), as well as comparative examples (Examples 2-4, and Commercial Examples A and B) and a control example (Example 1), in the form of toothpaste compositions.
The following Table 2 provides data regarding Fluorescence Ratio of Ca/EPS and Biofilm Thickness, according to the test methods described above, for Control Example 1, Comparative Examples 2-4, Commercial Examples A-B, and Example 6 of the present invention.
1Commercial Example A: Parodontax Original ™ (LOT 190796KWA), having the ingredients: Sodium Bicarbonate, Aqua, Glycerin, Alcohol, Cocamidopropyl Betaine, Mentha Oil, Mentha Piperita Oil, Xanthan Gum, Echinacea Purpurea Flower/Leaf/Stem Juice, Krameria Trianda Extract, Sodium Fluoride, Chamomilla Recutita Extract, Salvia Officinalis Oil, Commiphora Myrrha Extract, Limonene, Sodium Saccharin, Linalool, CI 77491.
2Commercial Example B: Arm & Hammer Sensitive Pro Repair ™ (LOT FE9092D 2022/03), having the ingredients: Sodium Bicarbonate, PEG/PPG-38/8 Copolymer, Calcium Sulfate, PEG/PPG-116/66 Copolymer, Silica, Sodium Lauryl Sulfate, Aroma, Sodium Saccharin, Dipotassium Phosphate, PEG-8, Chondrus Crispus Powder, Sodium Fluoride, Limonene, CI 77891.
In addition, Comparative Examples 2 and 4, and Examples 5-6 of the present invention, as referenced in Table 1, along with Commercial Examples A and B, are evaluated for saltiness taste, foam amount and in use dispersion using a method which is a paired comparison study with randomised brushing order using 18 trained sensory panellists. Each assigned dentifrice composition is dispensed on an Oral B Navigator brush and brushed for a controlled 2 minutes, with each panellist recording the sensorial attributes on a standard questionnaire using a 0 to 5 grading score, with score of 0 being at least sensorial preferred and 5 being the most sensorial preferred according to the Attribute Grading Scales indicated below. The results are shown in Table 3 below.
The Saltiness Taste data shown in Table 3 above shows that Comparative Example 2 (which has no bicarbonate) exhibits no saltiness taste, while Comparative Example 4 (which has 20% bicarbonate and 0.5% medium intensity sweetener) exhibits unpleasant saltiness taste. Also Commercial Examples A and B (which have high bicarbonate levels and only medium intensity sweetener) also exhibit unpleasant saltiness taste. Examples 5 and 6 of the present invention (which have 40% bicarbonate along with a combination of high intensity sweetener and medium intensity sweetener) exhibit almost no saltiness taste and improved taste.
The data presented in
Surprisingly, the data presented in
TABLES 2 and 3 show the synergistic benefits of combining medium intensity sweeteners and high intensity sweeteners with sodium bicarbonate to reduce both Ca:EPS ratio and the biofilm thickness, whilst allowing for a more preferred sensorial experience.
The Control Example 1 in TABLE 1 represents the salivary salts of a phosphate buffer solution, that led to a final biofilm thickness of 72.5 μm and a Ca:EPS biofilm ratio of 2.39. Comparative Examples 2 and 3 as shown in TABLE 1 contain calcium carbonate or silica abrasives, respectively, with the absence of sodium bicarbonate. Comparative Examples 2 and 3 led to a final biofilm thickness of 37.7 μm and 35.4 μm, and a Ca:EPS biofilm ratio of 2.13 and 2.05, respectively. Comparative Example 4 as shown in TABLE 1 contains calcium carbonate and silica abrasives with presence of sodium bicarbonate and only sodium saccharin sweetener. Comparative Example 4 led to a final biofilm thickness of 27.8 μm and a Ca:EPS biofilm ratio of 1.81. The inclusion of 20%, by weight, of sodium bicarbonate in Comparative Example 4 vs. Comparative Example 2 (which contains no sodium bicarbonate) leads to an improved reduction in biofilm thickness and reduced Ca:EPS ratio; however, Comparative Example 4 has poor sensorial grading (1) for saltiness taste as a result of the sodium bicarbonate.
Commercial Example A is a commercial dentifrice composition sold under the trade name Parodontax Original™, with 67% sodium bicarbonate and only a medium intensity sweetener (sodium saccharin). Commercial Example A led to a final biofilm thickness of 26.9 μm and a Ca:EPS biofilm ratio of 1.76. The high sodium bicarbonate content of Commercial Example A led to a reduction in biofilm thickness and Ca:EPS ratio. However, this high sodium carbonate formulation of Commercial Example A also led to poor sensorial grading for high saltiness taste (0) and poor/low foam (1) as shown in TABLE 2. Commercial Example B is a commercial dentifrice composition sold under the trade name Arm & Hammer Sensitive Pro Repair™, containing approximately 35% to 45% sodium bicarbonate formulated within an anhydrous composition, led to a final biofilm thickness of 28.7 μm and a Ca:EPS biofilm ratio of 1.81. However, this anhydrous composition of Commercial Example B, also led to poor sensorial grading for saltiness taste (1), poor/low foam (2) and dispersion (1) as shown in TABLE 2.
Unexpectedly, the synergy in combining high intensity sweetener and medium intensity sweetener with sodium bicarbonate, as in Example 6 of the present invention, led to a final biofilm thickness of 23.8 μm, a Ca:EPS biofilm ratio of 1.56, and good sensorial experiences. The inclusion of sodium bicarbonate in Example 6 significantly improved the therapeutic benefits in reducing the biofilm thickness and Ca:EPS ratio vs. Comparative Example 2, though surprisingly did not have any sensorial trades offs associated with high levels of sodium bicarbonate, with acceptable saltiness taste (4), good foaming (3), and good dispersion (4).
In other words, the synergy of medium intensity sweeteners and high intensity sweeteners with a relatively high level of sodium bicarbonate (e.g. at least about 20%, preferably about 40%) within water-based dentifrice compositions had improved sensorial experience vs. commercial high sodium bicarbonate dentifrice compositions such as Commercial Examples A and B, and surprisingly had an improved reduction in biofilm thickness.
The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.”
Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/085779 | Apr 2021 | US |
| Child | 18337506 | US |
