Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ANTIMICROBIAL AGENTS FOR ORAL HYGIENE PRODUCTS
Field of the Invention
The present invention relates to antimicrobial agents for use in oral hygiene
products and methods for using such agents.
Back~round of the Invention
Periodontal disease and dental caries are of major public health and economic
interest worldwide. It is now widely recognized that both of these oral diseases are
caused by bacteria which grow in masses on the teeth and in the gingival area. Acommonly used descriptive term for these bacterial masses is "dental plaque". In the
case of periodontal disease, Schluger et al. (Schluger, Yuodelis, Page & Johnson,
Periodontal Diseases, second edition, pp. 153-262, Lea & Febiger, l'J90) report that
dental plaque bacteria, growing in the area where the teeth and gingival tissues meet,
cause an infl~mm~tion of the gingiva called "gingivitis". This is characterized by
swollen, edematous gingiva ("gums") which are reddened and bleed easily. If plaque
removal is inadequate, gingivitis may progress to "periodontitis" or periodontaldisease in many individuals. Periodontitis generally is a characterized by a chronic
infl~mm~tion of the tissues around the teeth, which leads to a resorption of
supporting bone. Periodontal disease is the leading cause of tooth loss among adults.
Dental caries (cavities) are also caused by bacteria, with Streptococcus mutans being
~ 20 the principal etiologic agent (McGhee, Michalek & Cassell, Dental Microbiology,
p. 279, Harper & Row, 1982).
- The prevention of dental plaque or the removal thereof has long been the
focus of development with the ultimate goal of inhibiting both caries and periodontal
diseases. While the formation of dental plaque can be inhibited to a certain extent by
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brushing the teeth at frequent intervals, brushing alone is not sufficient to effectively
prevent the formation of dental plaque or remove substantially all of the dentalplaque that has formed on the teeth.
Since brushing alone is not suI'i'icient to prevent and remove plaque, chemical
methods using antibacterials such as chlorhexidine, benzalkonium chloride, and
cetylpyridinium chloride have been proposed. In addition, the use of natural
products for the treatment of teeth and gums is old in the art, having been practiced
and documented since the mid-1880s. Since thcn, numerous patents have disclosed
compositions of oral products containing natural product extracts. There are
numerous natural essential oils available. Many of these oils are described in KIRK
OTHMER ENCYCLOPEDIA OF CHEMICAL TECHNOLOC~Y, 4th ed., vol. 17, pp. 603-674,
John Wiley & Sons, Inc. Morton Pader, in "Oral l-Iygiene Products and Practice,"Cosmetic Science and Technology Series. vol. 6, at pp. 356-373, Marcel Dekker, Inc.,
describes sanguinaria extract as an anti-plaque agent with antimicrobial properties.
Pader also describes that volatile oils such as eucalyptol, menthol, thymol,
methylsalicylate have varying degrees of antimicrobial activity, and antiplaque
activity has been reported under appropriate test conditions. Pader describes that
cinnamon oil is a very weak antiseptic, and that eucalyptus oil and eucalyptol are
antiseptic. Padcr notes that some essential oils are used in othcr products primarily
for flavor. Among these are cinnamon, cassia, clove, thyme, peppermint, anise and
anethol. Pader also describes that these essential oils have detectable antimicrobial
activity.
For example, it is known that cocamidopropyl betaine, hinokitiol, and
berberine and the essential oils, citral, geraniol, and 3uniper berries oil individually
exhibit antimicrobial properties against certain bacteria.
U.S. Patent No. 3,940,476 describes a method for inhibiting the formation of
dental plaque, which comprises topically applying to the teeth as an active ingredient
an amount of either one or a combination of (a) allyl isothiocyanate, (b) uranine,
(c) obtusastyrene, (d) citral, (e) citronellol, (f) nerol, or (g) geraniol.
U.S. Patent No. 4,913,895 describes an oral composition including a linear
polyphosphate or a cyclic polyphosphate and menthol, anethol, or mixtures thereof in
an aqueous medium. The composition is reported to have antibacterial effects andprevent the development of calculus and periodontal diseases.
U.S. Patent No. 4,966,754 describes that certain essential oils and
combinations thereof possess antimicrobial properties against Aspergillus niger,
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Candic~a albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, and
therefore are suitable as preservatives in cosmetic compositions. A blend of 14
essential oils is described as providing desirable antimicrobial properties against the
notcd microorg~ni~.m~.
U.S. Patent No. 4,999,184 describes oil compositions containing certain
pyrophosphate salts which are reported to provide an anticalculus benefit.
U.S. Patent No. 5,316,760 describes a mouth care product that contains a
combination of Urtica dioica extract and an extract of Juniperus communis. The
combination of these extracts is described as leading to a synergistic reduction of
both dental plaque and bleeding or inflamm~tion of the gingiva. Achillaea
millefolium extract is also described as being a suitable additive to the combination
of the Urtica dioica and Juniperu~ communis extracts.
U.S. Patent No. 5,472,684 describes a composition including thymol and
eugenol, and optionally a sesquiterpene alcohol, such as farnesol, that reportedly has
antiplaque and antigingivitis effects. Australian tea tree oil, sage oil, and eucalyptol
are described as enhancing the antiplaque and antigingivitis activity of mouth rinses
formulated from the disclosed compositions.
One property that characterizes the effectiveness of an antimicrobial agent as
an antiplaque and anticalculus agent is the minimum inhibitory concentration, orMIC, of the agent. The MIC is the minimum concentration in micrograms per
milliliter of an antimicrobial agent at which no bacterial growth are observed. At
concentrations below the MIC, an antimicrobial agent is ineffective at killing or
inhibiting the growth and reproduction of bacteria. At concentrations above the
MIC, an antimicrobial agent is effective at killing or inhibiting the growth andreproduction of bacteria.
Typicaliy, antimicrobial agents are introduced into the oral cavity at an initial
concentration. Almost immediately, the initial concentration begins to decrease
because of the dynamics of the oral cavity. Eventually, the concentration of theantimicrobial agent within the oral cavity will fall below the MIC. Thus, it has been
a goal of those working to develop antiplaque and anticalculus formulations to use
antimicrobial agents that ha~e low MICs.
Chlorhexidine has a MIC of about one ~lg/ml and is the standard against
which other antimicrobial agents are measured. While chlorhexidine has a desirable
MIC, it also exhibits undesirable taste and has the undesirable side effect of staining
3 5 teeth.
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Summar~/ of the Invention
In one aspect, the present invention is an oral hygiene composition that
includes an antimicrobial agent selected from cedarwood oil, chloramphenicol,
citronella oil, Glycyrrhiza glabra extract, juicy fiuit basil oil, lemon basil oil, and
5 Rosmarinus of,~icinalis oil. According to the present invention, oral hygiene
compositions including these antimicrobial agents are effective at inhibiting and
preventing the growth of bacteria present in oral cavities such as Actinomyces
viscosus, Fusohacterium nucleatum, Porphyromonas gingivali~, Streptococcus
mutans, and Streptococcus sanguis. Oral hygiene compositions formed in
10 accordance with the present invention are characterized in that the antimicrobial
agent is present in an amount sufficient to retard the growth of bacteria or kill
bacteria.
Thc antimicrobial agents can be combined with components typically found
in oral hygiene products such as dentifrices. These components include abrasives,
15 humectants, binders, and surfactants. Other dentifrice components include flavoring
agents and thickening agents.
In accordance with the present invention, the oral hygiene compositions and
products of the present invention can be used in the method of practicing oral
hygiene that includes the step of contacting an oral cavity with an antimicrobial agent
20 selected from cedarwood oil, chloramphenicol, citronella oil, Glycyrrhiza glabra
extract, juicy fruit basil oil, lemon basil oil, lemon oil, or Rosmarinus officinali.s oil.
Detailed Descri~tion of the Preferred Embodiment
As used herein, the following terms have the following meanings.
"Cedar~,vood oil" refers to volatile whole oil extracts from the heartwood of
25 Juniperus virginiana or Juniperis ashei. Constituents of the whole oil extract include
thujopsene, cedrol, alpha-copaene, alpha-cedrene, beta-cedrene and widdrol. The
CAS number for cedarwood oil is 8000-27-9.
"Chloramphenicol" refers to 2,2-dich]oro-N-[2-hydroxyl-1-(hydroxymethyl)-
2-(4-nitrophenyl) ethyl] acetamide. Chloramphenicol is derived from Streptomyces30 vene~uelae or by organic synthesis. The CAS number for chloramphenicol is
56-75-7.
"C~itronella oil" refers to a commercially available oil produced by steam
distillation of either Cymbopogon nardu.s or Cymhopogon winterianus. The CAS
number for citronella oil is 8000-29-1.
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"Glycyrrhiza glabra extract", also known as licorice root extract, refers to thecrude powder extract from Glycyrrhiza glabraL. Several varieties including
G. typica and G. ~landuli,fera exist. Glycyrrhiza glabra extract contains glycyrrhizic
acid and glycyrrhetinic acid. The whole extract is available from commercial sources
or may be collected by solvent extraction, such as ethanol extraction described
below.
"Juicy fruit basil oil" refers to the whole extract of a selected variety of basil
with a juicy fruit component. Juicy fruit basil is a cultivar of Ocimum bascilicum L.
"Lemon basil oil" refers to the volatile whole oil extract from a selected
variety of basil with a citral component. Lemon basil is a cultivar of Ocimum
bascilicum L. with a high content of citral.
"Lemon oil" refers to the volatile whole oil extract from the fresh peel of
Citrus limon. Lemon oil is also known as oil of lemon or citrus limon oil. The CAS
number for lemon oil is 8008-56-8.
"Rosmarinus officinalis oil" refers to the whole oil extract from the flowering
tops of Rosmarinus officinalis. Rosmarinu~s o,,fficinali.s oil is also known as the
extract of Rosemary, or the extract of Rosmarinus officinalis oil. The CAS number
for Rosmarinus officinalis oil is 84604-14-8.
All of the foregoing are available from commercial sources.
"Minimal inhibitory concentration or MIC" refers to the minimum
concentration in micrograms per milliliter of an antimicrobial agent at which nobacterial growth are observed. At concentrations below the MIC, the antimicrobial
agent is ineffective at killing or inhibiting the growth and reproduction of bacteria.
At concentrations above the MIC, the antimicrobial agent is effective at killing or
inhibiting the growth and reproduction of bacteria.
An oral hygiene composition formed in accordance with the present invention
includes an antimicrobial agent selected from cedarwood oil, chloramphenicol,
citronella oil, Glycyrrhiza glabra extract, juicy fruit basil oil, lemon basil oil, and
Rosmarinus officinalis oil. These oral hygiene compositions can be incorporated into
oral hygiene products formulated in accordance with the present invention, such as
~ dentifrices, mouth washes, and mouth rinses.
Preferred oral hygiene compositions include an antimicrobial agent selected
from cedarwood oil, chloramphenicol, and Glycyrrhiza glabra extract. Particularly
preferred f'or incorporation into an oral hygiene composition formulated in
accordance with the present invention are antimicrobial agents selected from
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cedarwood oil and Glycyrrhiza glabra extract. The preferred antimicrobial agentsare selected because these agents are surprisingly eff'ective at retarding the growth of
and/or preventing the growth of representative gram-positive and gram-negative oral
pathogenic bacteria such as ~l ctinomyces vlscosus, Fusobacterium nucleal tlm,
5 Porphyro~lonas gingivalis, Strept(lcoccus mutan~, and Streptococcus sanguis. The
examples that follow illustrate the effectiveness that the antimicrobial agents of the
present invention have against these particular bacteria. Pref'erably, the antimicrobial
a~ent is effectivc against more than one of the bacteria noted above, and preferably
all of the bacteria noted above.
The particular amount of antimicrobial agent present in compositions formed
in accordance with the present invention is not limited to any particular value,provided that the amount present is effective at retarding the gro~vth of bacteria
and/or preventing the growth of bacteria, i.e., an amount that is greater than the MIC
of the antimicrobial agent with respect to the particular bacteria.
As illustrated in the examples that follow, the antimicrobial agents of the
present invention exhibit MICs that range from about 3.1 to about 156 against the
representative oral pathogens Actinomyces viscosus, Fllsobacterium nucleatum,
Porphyromona3 gingivalis, ~Slrept-)coccus mutans, and Streplococcu.s sanguis.
Dentifrices, or toothpastes, are generally a thickened slurry of an abrasive
polishing material in an aqueous humectant system. Typically, dentifrices include an
abrasive to remove stained pellicle, humectant(s) to provide a vehicle for the flavor,
abrasive, thickening agent(s) to structure and stabilize the dentifrice, surfactant
mainly to supply foam during use, fluoride to prevent dental caries, and flavor to
make the product taste pleasant.
Numerous abrasives are available for use in dentifrices, examples include
silica xerogel, silica precipitates, dicalcium phosphate, dicalcium phosphate
dihydrate, alumina trihydrate, calcium pyrophosphate, calcium carbonate, and
insoluble sodium metaphosphate.
Examples of suitable humectants include sorbitols, glycerin, and polyethylene
glycols.
Silica aerogels, pyrogenic silica, silica precipitates, carboxymethylcellulose,
carboxyvinyl polymers, xanthan gum, and carrageenan are examples of materials that
are suitable as thickeners.
Exemplary surfactants include sodium lauryl sulfate and dodecylbenzene
3 5 sulfonate.
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Numerous flavoring agents are commercially available with those providing
mint or other refreshing flavors such as cinnamon being commonly used.
Oral rinses or mouth washes are generally, aqueous, pourable emulsions of
flavors into which, in most instances, an antimicrobial has been incorporated.
Typical components of an oral rinse include flavoring agent to make the product
pleasant to use and to emphasize therapeutic or freshness qualities, surfactant(s) to
maintain flavor in stable dispersion, humectant(s) to improve mouth feel, thickening
agent, and an active agent. Often times, a surfactant is used to impart light foaming
properties to the oral rinse.
Dentifrices and oral rinses incorporating the antimicrobial agents for
inhibiting the growth of bacteria in the oral cavity in accordance with the present
invention are forrnulated in a conventional manner with the antimicrobial agentsbeing present in an amount ranging from about 0.001 wt. % to about 5.0 wt. % based
on total weight of dentifrice. Preferably the antimicrobial agents are present in an
amount ranging from about 0.01 wt. % to about 2.5 wt. %. The specific componentsused in the dentifrices and oral rinses incorporating the antimicrobial agents of the
present invention are not limited to those set forth above; however, the components
selected preferably do not have an antagonistic effect on the antimicrobial properties
of the selected antimicrobial agent.
The following examples illustrate the effectiveness of oral hygiene
compositions of the present invention against bacteria present in the oral cavity, the
MIC for the antimicrobial agents in the compositions against such bacteria, dentifrice
forrnulations including antimicrobial compositions of the present invention,
consumer preference for such dentifrice formulations, and in vivo effectiveness data
for such dentifrice formulations.
Example 1
Determination of Minimum Inhibitory Concentration of Antimicrobial A,eents
The following example illustrates how antimicrobial agents useful in
accordance with the present invention retard or prevent the growth of dental plaque
bacteria present in the oral cavity. In addition, the example illustrates the lowest
~ concentration of various antimicrobial agents that will inhibit visible in vitro growth
of a particular bacteria.
The assay used a microtiter plate to dilute the antimicrobial agent to varying
concentrations in order to determine the MIC.
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Table 1 below provides a listing of antimicrobial agents used in this example
and abbreviations therefor.
Table I
Antimicrobial A-Jents and Abbreviations Therefor
cedarwood oil (RCl)
chloramphenicol (CRI)
citronella oil (CTRI )
C~lycyrrhiza glabr~l extract (GLY)
juicy fruit basil oil (JFB I )
lemon basil oil (LMB I )
Ro.smari~us of~icinalis oil (ROFI)
A bacteria culture was incubated overnight at 37~C. Prior to dilution of the
antimicrobial agents as described below, the bacterial culture was spun down at
2000 rpm into a pellet and resuspended in a solution of buffered phosphate. The
innoculum was normalized with a spectraphotometer to an optical density at
550 nanometers of between 0.18-0.22, equivalent to 5.0 x 107 colony-forming units
(CFU per milliliter). The innoculum was set aside until the completion of the
antimicrobial agcnt dilution.
A sterile polystyrene 96-well plate was used to dilute the antimicrobial
agents. Usiny aseptic technique, 100 microliters of distilled water was placed in each
test well. In the first well in each column, 100 microliters of antimicrobial agent was
added. Stock solutions of antimicrobial agents were prepared with methanol as a
solvent to bring the agents into solution. This resulted in a one-half dilution of the
stock solution. 100 microliters from these wells was then transferred to the next well
in the column, and so on, down each column. Each transl'er accomplished a one-half
dilution of the concentration in the prece-ling well. After the dilution of the
antimicrobial agent was completed, 80 microliters of growth media specific for the
bacteria under study was added to each well. The specific growth media for a given
bacteria are set forth in Table 2.
Next, 20 microliters of innoculum was added to each well. This resulted in
the first well of each column having a final dilution of one quarter of the stock
solution. The remaining wells were a one-half dilution of the preceding well for each
transfer.
The 96-well plate was incubated under conditions that varied depending on
the particular microorganism. Thc incubation conditions are set forth in Table 2.
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The aerobic bacteria were incubated under normal room conditions and the anaerobic
bacteria were incubated under an atmospherc of 10% hydrogen gas, 5% carbon
dioxide gas and the balance nitrogen gas. Following 48 hours of incubation, the
incubated plate was read for microbial gro~-vth with a spcctraphotometer by optical
5 density (OD). The welJ containing the lowest dilution achievable with a
spectraphotometer reading below 0.05 OD (i.e., no detectable microbial growth) was
considered representative for the antimicrobial agcnt. The MIC for the agent wasdetermined by accounting for the starting stock solution concentration and the
resulting dilutions in the 96-well plate.
The specific bacteria inoculated into the 96-well plate are set t'orth in Table 2
below, along with the growth media and incubation conditions for that
microorganism.
Table 2
Microor~ni~m~lGrowth Media/Incubation Conditions
ATCC Growth
Microorganism No. Media Incubation Conditions
Campylohacter rectus 33238 CR media4 48 hrs/37~C/anaerobic
Actinomyces l~iscosus (AV) 19246 TSB~ 48 hrs/37 C/aerobic
Fusobacterium nucleahlm (FN)10933FN media3 48 hrs/37~C/anaerobic
l'orphyromonas gingivalis (PG) 33277 PG media2 48 hrs/37~ClanaerobicStreptococcus mutans (SM) 25175 TSBl 48 hrs/37 Claerobic
Streptococcus sanguis (SS) 49295 TSB' 48 hrsl37 Claerobic
Tryptic Soy Broth 3.0% wt. to vol., yeast extract 0.1%, and 999 milliliters distilled water.
2 Tryptic Soy Broth 3.0% wt. to vol., yeast extract 0.5%, L-cystein 0.05%, Hemin 0.0005%,
Menadione 0.00002%, and 990 milliliters distilled water.
3 Tryptic Soy Broth 3.0% wt. to vol., yeast extract 0.5%, Peptone 1.0%, L-cystein extract,
20 Hemin 0.0005%, Menadione 0.00002%, and 990 milliliters distilled water.
4 Brain Heart Infusion Broth 0.74% wt. to vol., yeast extract 0.01%, sodium formate 0.2%,
sodium fumerate 0.03%, hemin 0.005% and 990 millileters distilled water.
In the following tables, the antimicrobial agents are identified with respect tothe abbreviations set forth in Table 1. In addition, the five microor~ni~m~ set forth
25 in Table 2 are referred to by the abbreviations set forth in Table 2.
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Table 3
MTC (!lg/ml) for Antimicrobial A~ents for lndicated Bacteria
Agent AV CR FN PG SM SS
RCl 31.331.3 31.3 7.8 31.3 15.6
CR1 3.1 - 3.1 6.3 3.1 3.1
CTRl 62.5 125 31.3 31.3 62.5 31.3
GLY 15.615.6 15.6 7.8 15.6 7.8
JFB1 156.3156.3 156.3 62.5 156.3 156.3
LMOI 312.5125 62.5 31.3 125 125
LCCI 312.5125 156.3 78.1 156.3 156.3
ROF1 125 125 62.5 62.5 125 125
This example illustrates the minimum inhibitory concentration of the noted
S antimicrobial agents and the ability of thc antimicrobial agents to inhibit growth of
the specific bacteria.
Example 2
Dentifrice Formulations
This example describes dentifrice formulations comprising antimicrobial
10 agents of the present invention. Formulation 18-88 does not include antimicrobial
agents in accordance with the present invention.
Table 4
Formulation 5-82: 0.1% Cedarwood Oil (W/W)
Component Weight Percent
Sorbitol 70% 37 00
Poloxamer 407 (PLURONIC(~ F127) 9.50
Deionized Water 24.50
Carbomer g40 (CARBOPOL(~) 940)0.30
Sodium Hydroxide 0.20
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Component Weight Percent
Xanthan Gum 0.40
Glycerin 4.75
Sodium Fluoride 0.25
Sodium Saccharin 0.30
SYLODENT~' 750 (silica) 9.50
SYLODENT~' 15 (silica) 9.50
Cedarwood oil 0.50
Flavor 0.90
FD&C Blue #l 1% solution 0.10
Titanium Dioxide 0.90
Sodium Lauryl Sulfate l 40
Table 5
Formulation 5-81: 0.1 % Cedarwood Oil (W/W)
Component Weight Percent
Sorbitol 70% 58.63
Sodium Carboxymethylcellulose 0.35
Deionized Water 3.00
Polyethylene Glycol 600 5.00
Glycerin 10.00
Sodium Fluoride 0.22
Sodium Saccharin 0.30
Sodium Benzoate 0.50
SYLODENT~ 700 (silica) 15.00
AEROSIL~3 200 (silica) 3.00
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Component Weight Percent
Flavor 0.80
Ethanol 1 .50
Cedarwood 0. 10
I;D&C Blue #1 1% solution 0.10
Sodium Lauryl Sulfate 1.50
Table 6
Formulation 5-1()8: 0.4% Cedarwood Oil (W/W)
Component Weight Percent
Sorbitol 70% 50.00
Deionized water 24.63
Carbomer 940 (CARBOPOL(~ 940)0.30
Sodium hydroxide 0.20
Xanthan gum 0.500
Sodium fluoride 0.22
Sodium saccharin 0.55
SYLODENT(~) 750 (silica) 10.00
SYLODENT(~) 15 (silica) 10.00
Cedarwood oil 0.40
Flavoring agents 0.80
Titanium dioxide 1.00
Sodium lauryl sulfate 1.40
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Table 7
Formulation 1 X-87: 0.5% Glvcyrrhiza ~labra Extract (W/W)
Component Weight Percent
Sorbitol 70% 50 00
Deionized water 24.53
Carbomer 940 (CARBOPOL(~) 940) 0.30
Sodium hydroxide 0.20
Xanthan gum 0 50
Sodium fluoride 0.22
Sodium saccharin 0.55
SYLODENT(~) 750 (silica) 10.00
SYLODENT(fi) 15 (silica) 10.00
Glycyrrhiza glabra extract 0.50
Flavoring agents 0.80
Titanium dioxide I .00
Sodium lauryl sulfate 1.40
Table 8
Formulation 18-90: ] % Cedarwood Oil (W/W)
Component Weight Percent
Sorbitol 70% 50 00
Deionized water 24.03
Carbomer 940 (CARBOPOL6) 940) 0.30
Sodium hydroxide 0.20
Xanthan gum 0.50
Sodium fluoride 0.22
Sodium saccharin 0.55
SYLODENT~' 750 (silica) 10.00
SYLODENT~) 15 (silica) 10.00
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Component Weight Pcrcent
Cedarwood oil 1.00
Flavoring agents 0.80
Titanium dioxide 1.00
Sodium lauryl sulfate 1.40
Table 9
Formulation 18-25: 0.1% Hinokitiol
Component Weight Percent
Deionized Water 92.02
Carhomer 940 (CARF~OI'OI ('~ 940) 1.00
Sodium Hydroxide 0.75
Sodium Saccharin 0.50
Sodium Fluoride 0.23
Flavor 0. 80
Hinokitiol 0 10
Ethanol 3 50
Sodium Lauryl Sulf'ate 1.10
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Table 10
Formulation 18-88: Control
Component Weight Percent
Sorbitol 70% 37.70
Poloxamer 407 (PLURONIC(~ F 127) 9.50
Deionized Water 25.50
Carbomer 940 (CARBOPOL(~) 940) 0.30
Sodium Hydroxide 0.20
Xanthan Gum 0.40
Glycerin 4.75
Sodium Fluoride 0.25
Sodium Saccharin 0.40
SYLODENT~' 750 (silica) 9.50
SYLODENT~) 15 (silica) 9.50
Titanium Dioxide 1.00
Sodium Lauryl Sulfate 1.00
"Carbomer" refers to a polymer composed of acrylic acid crosslinked with
allyl sucrose available as CARBOPOL(~)940. CARBOPOL(}'940is commercially
available from B.F. Goodrich. SYLODENT~) 750 and 700 are silica gels (silicon
dioxide), and SYLODENT(~lSis a silica gel (silicon dioxide). SYLODENT(E~is
available commercially from W.R. Grace & Co. Conn. Davison Chemical Division.
The formulations described above are formed in a vacuum mixer by adding
deionized water and dispersing the Carbomer while pulling a vacuum. When the
Carbomer was well dispersed, the sodium hydroxide was added. The 70% sorbitol
and Poloxamer were heated and mixed in another vessel. The Poloxamer mixture
was then added to the vacuum mixer and blended with the Carbomer mixture.
Xanthan gum was mixed with glycerin and then added to the solution in the vacuummixer and incorporated therein. The salts were then added to the vacuum mixer
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followed by the silicas. These components were slowly mixed in. Next7 the activeagents flavoring agents, sodium lauryl sulfate and coloring agents were added
followed by mixing until well incorporated.
This exarnple illustrates several dentifrice formulations incorporating
5 antimicrobial agents of the present invention.
Example 3
Customer l~cccptance
This example illustrates customer acceptance of dentifrice formulations
incorporating an antimicrobial agent in accordance with the present invention.
The i'ormulations set forth above in Tables 4-9 were evaluated for customer
acceptance. For comparison purposes, several commercially available dentifrices
were also e~Jaluated. These included Viadent Gel (Formulation ~-35), availablc from
Viadent, Inc.; Crest Regular Blue Paste (Formulation 5-97), available *om Proctor &
Gamble Company; and Listerine Teal Gel (Formulation 5-99), available from Warner1 5 Wellcomc.
The preference study was carried out with a group of approximately
20 peoplc. One tube of dentifrice formulation was given to cach person to use over
the course of a weck. At the end of the week, participants filled out a questionnaire,
the results of which are summarized in Table 1 I below. The scoring range was 1-10,~0 with 1 bein~ "poor" and 10 being "good." The scores were tallied and averaged.
Table 1 1
Consumer Evaluation
Formulation 5-82 5-81 5-108 5-35 5-97 5-99
Color 6.61 8.00 6.75 2.72 6.64 7.25
Appearance 6.42 7.90 6.90 3.16 6.62 6.75
Taste 5.80~ 6.20 7.15 3.72 6.27 4.00
After Taste 6.80 5.85 6.05 3.38 6.09 3.85
Aroma 7.19 6.79 7.05 4.33 6.32 4.5S
Cleaning 7.38 5.95 7.00 3.94 6.41 6.10
Foam 6.42 5.10 7.55 3.66 6.05 5.85
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Fonnulation 5-82 5-81 5- 108 5-35 5-97 5-99
Consistency 7.23 5.95 7.70 4.00 6.14 5.75
Dry Mouth 8.14 6.90 7.10 3.89 7.91 5.70
Next Morning Feel 7.00 6.16 7.05 3.39 5.50 5.15
Overall Mouth Feel 7.33 6.16 7.05 3.11 6.09 4.45
Overall Satisfaction 6.90 6.10 6.90 3.05 5.95 4.20
This example illustrates how the overall consumer satisfaction for the
formulations with active agents in accordance with the present invention is greater
than the overall consumer satisfaction for certain commercially available products.
Example 4
S In Vivo Gin~ival ~argin Bacterial Reduction
This example illustrates how dentifrice formulations including compositions
of the present invention reduce bacteria levels in the gingival margin.
In this example, the subject's brushing habits remained constant. On the day
of sampling, subjects had not brushed their teeth for 16-18 hours (overnight) prior to
testing. The level of bacteria was enumerated by recovering in vivo samples and
counting the number of bacterial colonies forrned on agar after 48 hours.
Subjects were 18-65 years old, had a minimum of 20 teeth, and had normal
salivary flow. Subjects were rejected if they had systemic diseases that affect the
oral tissues, such as diabetes, blood cell abnormalities, Down's syndrome, or known
HIV-infection; had glossitis, moderate to severe gingivitis, periodontitis or other oral
infections, or were taking a systemic antibiotic or had done so in the two weekspreceding the initiation of the test.
Each of the formulations was assayed for antimicrobial efficacy on
approximately 20 subjects. The subjects were requested to have overnight plaque the
morning they were sampled for a "single brushing effect" of a formulation.
At baseline, a gingival margin plaque sample was taken with a sterile cotton
swab and placed in I milliliter of sterile phosphate buffered saline (PBS). Swabs
were gently rubbed along the gingival margin of the mandibular buccal surface
between the incisors. Subjects then brushed immediately after the baseline sample
with a dentifrice formulation using an electric toothbrush for two minutes. At
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intervals of one and two hours after completion of the brushing, gingival plaquesamples were recovered with swabs as described above.
The clinical design was a multi-period crossover study. By multi-period
crossovcr study is meant an experiment in whicll subjects are ~lministered first one
treatment, then "crossed over" to receive a second, then third and finally a fourth
treatment. The experimental design enables each subject to serve as his or her own
control. A "wash-out" period of 2 days was used between testing different
antibacterial f'ormulations.
The gingival samples were placed in test tubes and remained at room
temperature until processed within one hour. The test tube samples were seriallydiluted in sterile PBS with a Spiral Plater (Spiral Systems, Cincinnati, Ohio) and
plated on blood agar supplemented with 5% sheep blood (BBL, Becton Dickinson,
Cockeysville, Maryland). The plated samples were then incubated aerobically at
37~C for 48 hours. Bacteria were counted for total aerobic colony forming units.The bacterial counts were transformed into logl0 units. Mean and standard
deviations for each test condition were generated. Data was paired by subject for
relevant comparisons and analyzed by the nonparametric Wilcoxon sign-rank test for
significance level. Hypothesis testing relied on statistics utilizing the multiperiod
crossover design of the clinical study.
The data are presented below in Table 12. Formulations 18-25, 18-87, and
18-90 included components set forth in Tables 9, 7 and 8, respectively.
Formulatioll 18-88 included the components set forth in Table 10 (no antibacterial
agent) and was used as a control for comparison purposes against
formulations 18-25, 18-87 and 18-90.
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Table 12
Effect of Antimicrobial A~ent Containin~ Dentifrice
Formulations on In Vivo Bacterial Levels
% ~/o Number
Conc.(Ix10 C~U/ml)Bacterial . . of
Formul- Active Slgnlficance
wt./ . Reduction Subjects
ation Ingredient Tlme: Level
wt. vs. Control in Trial
Baseline 2 hrs.
18-25 Hinokitiol 0.1 33.11 4 47 49.8% ** 17
18-87 Glycyrrh~za 0.5 39.81 2.14 76.0% *** 17
gl~bra ext.
18-90 Cedarwood1.0 31.62 3.1664.5% *** 17
18-88 Con~ol -- 33.88 8.91NA NA 17
Statistical Results: Significance testing was against Control (formulation 18-88) at 2 hours.
Wilcoxon sign-rank matched pairs with log,0 transfonned data.
***p<o ol, **p<0.05, *p<O.l
NA = Not Applicable
10This example illustrates how the dentifrice formulations including
antimicrobial agents of the present invention reduce gingival in vivo bacterial levels.
Example 5
Ethanol Extraction of Glvcyrrhiza glabra
25 grams of powdered plant material from Glycyrrhiza glabra was combined
15with 250 grams of a 95:5 ethanol/water mixture. The mixture was stirred overnight
at room temperature. Solids were removed from the stirred mixture with a No. 4
Whatman filter in a Buchner funnel. Further removal of solids was achieved with a
No. 5 Whatman filter in a Buchner funnel. Additional solids were removed with a
Whatman I micrometer filter in a Buchner funnel. A vacuum filtration apparatus and
a 0.2 micrometer filter was employed to clean the solution a final time. The clean
solution was then concentrated down to a solid using a rotovaporizer.
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Approximately, 2.5 grams of a rust colored solid was collected as the crude extract of
Glycyrrhiza glcrbra.
While the preferred embodiment of the invention has been illustrated and
described, it will be appreciated that various changes can be made therein without
5 departing from the spirit and scope of the invention.
