wjd
WJD

Original research

10.5005/jp-journals-10015-1536
Comparative Evaluation of Antimicrobial
Efficacy of Theobromine Toothpaste

Comparative Evaluation of Antimicrobial Efficacyof Theobromine Toothpaste and a Low-fluoridated
Toothpaste against Plaque Streptococcus mutans
in Children
1

Manjusha B Ranju, 2Madarahalli S Girish, 3Sadashivamurthy Prashanth

ABSTRACTIntroduction: Cocoa bean extracts contain certain anticariogenic substances and studies have proven that it has a rolein preventing dental caries. Theobromine, a white crystalline
powder, is an alkaloid, readily available in cocoa and chocolate
has been considered responsible for its anticariogenic activity.
Aim: The aim of the present study was to evaluate the in vivo
anticaries activity of theobromine toothpaste and a commercially available low-fluoridated toothpaste against plaqueStreptococcus mutans in children of age group 6 to 9 years
and to evaluate the efficacy of both the toothpastes on supragingival plaque.
Materials and methods: A total of 14 subjects were selected
and randomly divided into experimental and control groups(n=7) in which theobromine toothpaste and a low-fluoridated
toothpaste were provided. The subjects were instructedto carry out toothbrushing twice daily for 1 minute and theplaque samples were collected at baseline and after 21 daysof intervention. Microbial analysis was done using selective
media, mitis salivarius bacitracin (MSB) agar. The plaque index(Turesky modification of Quigley–Hein index) and gingivalindex (Loe and Silness) were recorded at baseline and after
21 days. The values were statistically analyzed using paired
and unpaired t test.
Results: There was a significant reduction in the S. mutans
count and plaque scores from baseline to postintervention
in both the groups (p = 0.001). When both the groups werecompared, there was no significant difference in the mean
S. mutans count and plaque scores (p = 0.254).
Conclusion: Theobromine toothpaste is highly effective in
reducing S. mutans count and plaque formation in children.
Clinical significance: In the future, theobromine toothpaste
will prove to be beneficial as a safe anticariogenic agent, which
could open a new era of approach to caries prevention.
Keywords: Anticariogenic, Fluoride, Streptococcus mutans,
Theobromine, Toothpaste.

1-3

Department of Pedodontics and Preventive Dentistry, JSS
Dental College and Hospital (Constituent College), JSS Academyof Higher Education and Research, Mysuru, Karnataka, India
Corresponding Author: Manjusha B Ranju, Department of
Pedodontics and Preventive Dentistry, JSS Dental College andHospital (Constituent College), JSS Academy of Higher
Education and Research, Mysuru, Karnataka, India, Phone:+919497408595, e-mail: drranjumb@gmail.com

World Journal of Dentistry, May-June 2018;9(3):215-219

How to cite this article: Ranju MB, Girish MS, Prashanth S.
Comparative Evaluation of Antimicrobial Efficacy of Theobromine Toothpaste and a Low-fluoridated Toothpaste against
Plaque Streptococcus mutans in Children. World J Dent
2018;9(3):215-219.
Source of support: Nil
Conflict of interest: None

INTRODUCTION
Dental caries is a major health problem and the increasing prevalence of dental caries signals a pending publichealth crisis.1 Prevention and treatment efforts concernedwith dental caries problems in children require further
and special attention. It is because dental caries not only
cause pain and discomfort but also have a significant
impact on the children’s quality of life and daily activities.2 Streptococcus mutans, an acidogenic and aciduric
microorganism colonizing the oral cavity, is considered tobe the main cause of dental caries.3 A shift in the dynamic
relationship among the dental plaque microbiota, dietarycarbohydrate, saliva, and the pH-lowering and cariogenic
potential of dental plaque results in localized dissolution
and destruction of the calcified tissues often resulting incavitation.4
The prevention and control of dental caries as wellas periodontal disease are dependent greatly on optimalplaque control. Mechanical plaque removal using a toothbrush twice daily, together with daily interdental cleaning, has been recommended as an effective oral hygiene
regimen.5 For better plaque control, chemical inhibitorsof plaque and calculus are incorporated in dentifrices andmouthwashes to enhance the effectiveness of mechanicalaids employed.6 The success of any dentifrice in part lies
on its ability to eliminate pathogenic oral microflora.7
Toothpaste containing fluoride are widely popular andfluorides have been historically reported to have effects
against dental caries.8-11 However, it has certain drawbacks, such as toxicity and fluorosis.12,13 It is also well
documented that young children who brush their teeth
often ingest fluoride-containing dentifrices.14 Preventive
measures which are safe for long-term use without any
adverse effects should be promoted. Hence, researchers215

Manjusha B Ranju et al

are keen to identify indigenous agents with enhanced
anticariogenic property and reduced ill effects.
Cocoa bean extracts have been incorporated inpreventing caries incidence and plaque formation. A
series of studies suggested that pure cocoa powder
inhibited dental caries.15-20 Theobromine is one of the
major constituents in cocoa bean and studies have foundthat cocoa bean husk extract (CBHE) mouthwash ishighly effective in reducing mutans streptococci counts
and plaque deposition when used as a mouthrinse by
children.6,21 The purine derivatives of cocoa powder(theobromine and caffeine) have been studied (animalstudies) for its major anticariogenic property and they
have found that caffeine probably had no appreciable
effect.22 This focused more on theobromine, while theresearch on the effect of theobromine to rebuild thehydroxyapatite in enamel is proven.23,24 Even though
the effect of theobromine toothpaste on cariogenic
microflora like S. mutans has been proven in vitro,7 thein vivo efficacy against S. mutans has yet to be investigated and definitely proven.
The aim of the study was to evaluate and compare thein vivo antimicrobial effect of theobromine toothpaste anda low-fluoridated toothpaste against plaque S. mutans ina pediatric population aged 6 to 9 years and to evaluate
its efficacy on supragingival plaque.

MATERIALS AND METHODSTheobromine toothpaste (Theodent™, United States)
and a low-fluoridated commercially available kids toothpaste (Kidodent, Indoco Remedies Ltd, Mumbai) wereused in the study. Pure strain of S. mutans [Microbial
Type Culture Collection and Gene Bank (MTCC) 890]
was obtained from MTCC, Chandigarh. The bacterium(lyophilized culture) was revived by suspending in brain
heart infusion broth. The strains were cultured on MSB
agar plates.25
A double-blinded study (subject and the examiner
were blinded during allocation of toothpastes and plaquescoring) was conducted in 14 children of age group 6 to
9 years who came to the Department of Pedodontics andPreventive Dentistry, JSS Dental College, Mysuru, India,
for their various dental needs. The study protocol wasapproved and ethical clearance was obtained from theinstitutional ethical committee. Informed consent was
obtained from the parents and the study was in compliance with the code of ethics (Declaration of Helsinki).

Inclusion Criteria• Children of age group 6 to 9 years with fully erupted
first permanent molars who gave their assent andparental consent

216• Children with early-stage enamel decay according to
the International Caries Detection and Assessment
System (ICDAS) II criteria• Cooperative children having behavioral ratings “positive” or “definitively positive” according to Frankel’s
behavior rating scale• Children who are not on any medication prior to or
during the study period• Children without any overretained deciduous teeth
and mobile teeth

Exclusion Criteria• Children with systemic diseases and medically compromising conditions• Children undergoing orthodontic treatment or withan intraoral prosthesis• Children who could not brush their teeth or rinse on
their own
• Children who had any intraoral soft tissue pathology
After obtaining the necessary consent, the toothbrushing technique (Fones Technique) was demonstrated
to the subjects chosen to be a part of the study. The children were put on a washout period for 1 week wherein
they were provided a common nonfluoridated toothpasteand new soft bristled, age-specific toothbrush. They wereasked to refrain from the use of any oral hygiene aids
other than the materials allotted in the study and were
instructed to brush before breakfast and after dinner for
1 minute by using a pea-sized amount of toothpaste. The
study commenced with the recording of ICDAS criteria,
plaque index (Turesky modification of Quigley–Hein
Index, 1970), and gingival index (Loe and Sillness 1967).26
Disclosing solution (Alpha Plac, two-tone disclosing
solution) was used and the index teeth recorded were 16,11, 64, 36, 41, and 84.27 The examination for the plaqueindex always preceded the examination for the gingivalindex.28 After recording the baseline values, the subjects
were divided into two groups randomly (lottery method);group I was given theobromine toothpaste and group
II was given commercially available low-fluoridated
toothpaste. They were instructed to use the assigned
toothpaste for 21 days and instructed to refrain frombrushing the night before the dental visit in order to allow
sufficient plaque accumulation.
On day 1, baseline plaque samples were collectedprior to brushing without inducing bleeding. Collectionof plaque samples was done from the buccal and lingualsurface of 16, 11, 64, 36, 41, and 84. Plaque collection was
done using an autoclaved scaler and placed in a screwcapped vial. Plaque samples were standardized using
microbalance; 100 µL of saline was added to each 1 mgof plaque with the ratio being constant and vortexed. The

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Comparative Evaluation of Antimicrobial Efficacy of Theobromine Toothpaste

plaque samples were serially diluted and spread plate
method was used to disperse the samples on MSB agar
plates.25 Plates were stored in an anaerobic jar and incubated at 37°C. Streptococcus mutans levels were assessed
after 72 hours of incubation. Gram staining was done
to confirm the presence of S. mutans. Estimation of thegrowth of S. mutans in colony-forming units (CFU) was
carried to evaluate the cariogenic potential of the plaque.
The microbial levels were checked after 21 days of usageof the intervention to test the antibacterial effect of the
products. Postintervention plaque scores and gingivalindex scores were checked in both the groups and compared with the baseline.

Counting Bacteria by Dilution and Plating
Colonies of S. mutans appear with the following morphologic characteristics: 0.5 mm raised convex undulatedcolonies of light blue color with rough margins, granular
frosted glass appearance.29 Plates were examined andthe bacterial colonies were counted. Duplicate for eachsample was used for counting the mean. The standard
formula for CFU is:
CFU
Colony count on agar plate=
Total dilution of the tube (used to make plate formLcolony count ) × amount plated

Statistical Analysis
Data were analyzed using Statistical Package for theSocial Sciences (version 22.0; SPSS Inc., Chicago, Illinois,
USA). Paired t test and independent samples t test wereused for statistical analysis.

RESULTS
The mean age of children in group I (theobromine toothpaste) was 7.5 and in group I (low-fluoridated toothpaste),
it was 7.28.

Table 1: Colony-forming units (CFU/mL) of S. mutans at baseline
Toothpaste Mean ± SD
groups(CFU/mL)
Mean diff ± SE
p-valueI
1.39×106 ± 0.09×106 3.42×104 ± 4.88×104 0.496aII
1.43×106 ± 0.08×106
n = 7, SE: Standard error of mean, aNonsignificant

The results of the microbial analysis showed that thebaseline S. mutans count was similar in both the groups,
p = 0.496 (Table 1). Table 2 shows the mean S. mutans
count (CFU/mL) in both the groups at baseline and after
intervention. At baseline, the mean S. mutans CFU/mL
was 1.39×106 with a standard deviation (SD) of 0.09×106
and the mean S. mutans CFU/mL after intervention was
7.05×105 with an SD of 0.41×105 in theobromine group.
The mean CFU/mL of S. mutans in low-fluoride groupwas 1.43×106 with an SD of 0.08×106 at baseline and after
intervention, it was 7.38×105 with an SD of 0.59×105. The
intragroup comparison showed a statistically significant reduction of mean S. mutans CFU count in both thegroups. This proves that both the toothpastes are highly
effective against plaque S. mutans.
After 21 days of toothpaste use, no significant difference was found in the mean S. mutans count in both thegroups, p = 0.254 (Table 3). This proves that theobromine
toothpaste is as effective as low-fluoridated toothpaste inits anti-S. mutans activity. In fact, theobromine toothpaste
showed slightly more reduction in S. mutans count after
a period of 21 days.

Plaque Scores
Plaque index scores significantly reduced from baseline topostintervention in both the toothpaste groups (Table 4).
When both the toothpastes were compared based on the
antiplaque action, there was no statistically significant
difference, p = 0.552 (Table 5). This proves that both thetoothpastes are similar in their antiplaque action.

Table 2: Intragroup comparison of CFU/mL of S. mutans
Toothpaste groups
I
II

Baseline
Postintervention
Baseline
Postintervention

Mean±SD (CFU/mL)
1.39×106 ± 0.09×106
7.05×105 ± 0.41×105
1.43×106 ± 0.08×106
7.38×105 ± 0.59×105

Mean diff ± SD
6.92×105 ± 0.72×105

p-value0.001a

6.94×105 ± 0.32×105

0.001an = 7, aHighly significant
Table 4: Intragroup comparison of plaque index
Table 3: Intergroup comparison of CFU/mL of S. mutans
Toothpaste Mean ± SD
groups(CFU/mL)
Mean diff ± SE
p-valueI
7.05×105 ± 0.41×105 3.28 ×104 ± 2.74×104 0.254aII
7.38×105 ± 0.59×105
n = 7, SE: Standard error of mean, aNonsignificant

World Journal of Dentistry, May-June 2018;9(3):215-219

Toothpaste
groupsI
Baseline
Postintervention
II
Baseline
Postintervention
n = 7, aHighly significant

Mean
Mean ± SD diff ± SD
p-value1.24±0.11 0.21±100.001a1.02±0.04
1.19±0.08 0.17±0.06 0.001a
1.01±0.03

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Manjusha B Ranju et al
Table 5: Intergroup comparison of plaque index
Toothpaste
groupsMean ± SD
Mean diff ± SE
I
1.02 ± 0.04
0.011 ± 0.02
II
1.01 ± 0.03
n = 7, SE: Standard error of mean, aNonsignificant

p-value
0.552aGingival index scores were taken and no significant
difference was found in the mean gingival scores between
day 1 and day 22 in both the groups. Gingivitis was absent
in most of the children included in the study group andhence, the gingival index scores did not show any variation after 21 days of intervention.

DISCUSSION
The effect of natural compounds on the resident oral
microflora, both in terms of their ability to promote thegrowth of beneficial organisms and by their inhibitionof the growth and metabolism of species associated withdisease, has been studied many times. The evolution ofsome innovative strategies that act against microorganisms involved in the causation of dental caries and periodontal diseases without the development of resistantmicroorganisms and without any adverse effects is achallenging task.
Cocoa bean husk is separated from the pre-roasted
beans of Theobroma cacao. Cocoa bean naturally contains
approximately 1 to 4% of theobromine. Cocoa bean husk
extract has been shown to possess two types of cariostatic
substances, one showing anti-GTF activity and the other,
antibacterial activity.22,30 An in vitro study compared theantimicrobial activity of theobromine-containing toothpaste and two commercially available kids toothpasteand has proved that toothpaste containing theobromine
showed higher antimicrobial effect against S. mutans,
Lactobacillus acidophilus, and Enterococcus faecalis.7 A study
done by Amaechi et al23 to identify the anticariogenic
effect of theobromine by examining its ability to cause
remineralization of enamel lesion concluded that theobromine in an apatite-forming medium can enhance
the remineralization potential of the tooth. Sadeghpour
and Carey found that theobromine protected teeth fromdecay better than fluoride. They found that “the amountof theobromine in an ounce dark chocolate bar has a better
effect on tooth hardness than a 1.1% prescription sodium
fluoride treatment,” and Carey showed that at a concentration 142 times less than that of fluoride, the theobromine
activity had twice the protective effect on teeth.31
The effectiveness of CBHE rinse in reducing S. mutans
and plaque accumulation when used as a mouthwash bychildren has been proven.6,21 Studies have reported thatcocoa bean extract mouthrinse can be considered as analternative to chlorhexidine mouthrinse, as it has similar

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antimicrobial properties and evades the side effects of thelatter.32 Another study compared chlorhexidine mouthrinse and CBHE mouth rinse with the addition of xylitol
and proved that both mouth rinses are equally effective
in reducing the S. mutans counts in saliva and reducing
the plaque scores in children.33
Theobromine toothpaste contains other inactive agents,
such as hydrated silica, sorbitol, xylitol, titanium dioxide,
stevia extract, and sodium bicarbonate. Theobromine ismore active in hydrated silica, glycerin, and xylitol. Xylitoland stevia are nonsugar sweeteners. Sodium bicarbonate
is known to neutralize bacterial acids in the mouth, andtitanium dioxide as a whitening agent and slight abrasive
to help clean the surface of the tooth. The titanium dioxide
removes the bacteria and proteins that stick to the toothand prepares the tooth to work more effectively. Lowfluoride toothpaste composed of sodium monofluorophosphate (495 ppm) along with xylitol and its antimicrobial
action is mainly attributed due to the presence of fluoride.7
Theobromine toothpaste used in this study is completely
fluoride-free and the active ingredient theobromine is
responsible for its superior anticariogenic action. It has
been suggested that theobromine in dentifrices has noadverse effects, even if swallowed accidentally.14

CONCLUSION
With the limitations (less number of samples) of thepresent study, it firmly establishes the anticariogenic
effect of theobromine toothpaste by the reduction of
S. mutans count in dental plaque. The anticariogenic effectof theobromine has to be proven effectively with further
clinical trials in vitro and in vivo.

Clinical Significance
Theobromine could be used in future as a safe andeffective alternative to fluoride in the war against dental
caries and acid attack. So, it can be incorporated in oral
care products other than toothpastes, such as mouthwash
and chewing gum for prevention of dental caries and it
probably creates a revolution in oral care.

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Comparative Evaluation of Antimicrobial Efficacy of Theobromine Toothpaste
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