Efficacy of Extrinsic Stain Removal by Novel Dentifrice Containing Papain and Bromelain Extracts

Abstract

To evaluate the stain removal efficacy of a novel dentifrice containing papain and bromelain extracts (Glodent) in comparison with a control dentifrice (Colgate regular). This was a randomized, positive-controlled, double-blinded, clinical study. Subjects were randomly divided into one of the two study groups. Pre-treatment and post treatment photographs of the 4 anterior teeth were recorded under standardized conditions and analyzed for lightness or luminosity values using Adobe Photoshop. The difference between the mean pre-treatment luminosity of test and control groups was not statistically significant. In both test and control groups, the post-treatment luminosity was significantly higher than pre- treatment luminosity (P < 0.001 and P = 0.003 respectively). The mean post-treatment luminosity for test group was found to be significantly higher than control group. The mean percentage removal of stains for test group was significantly higher than control group. The test dentifrice showed significant stain removal when compared to control which could be attributed to the role of proteolytic enzymes in the test dentifrice.

Full text

Journal of Young Pharmacists Vol 4 / No 4 245
Pharmacology
areca nut chewing, dietary intake of colored foods (e.g.
red wine, coffee and tea consumption), subject’s age and
the use of certain cationic agents such as chlorhexidine or
metal salts like tin and iron.[1,3,4]
Consumers and patients have always had a strong
desire for white teeth which has given rise to a growing
trend in the increased use of ‘over-the-counter’ tooth
whitening products. Manufacturers of oral care products
are constantly developing new approaches for tooth
whitening in order to meet the expectations of patients and
consumers. Thus, today there is a huge range of products
and technologies available that are self-applied and require
no professional involvement.[5]
The majority of these products work in two possible
INTRODUCTION
Teeth color is a combination of intrinsic color of the teeth
and the presence of extrinsic stains that might accumulate
on the tooth surface.[1,2] Extrinsic stains are linked with
the adsorption of materials into the acquired pellicle on
the enamel surface.[3] Factors inuencing extrinsic stain
formation include poor tooth brushing technique, smoking,
Efcacy of Extrinsic Stain Removal by Novel Dentifrice Containing
Papain and Bromelain Extracts
Chakravarthy PK, Acharya S
Department of Public Health Dentistry, Manipal College of Dental Sciences, Manipal University,
Manipal, India
Address for correspondence: Dr. P. Kalyana Chakravarthy, E-mail: drkalyan81@gmail.com
Access this article online
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DOI:
10.4103/0975-1483.104368
ABSTRACT
To evaluate the stain removal efcacy of a novel dentifrice containing papain and bromelain extracts (Glodent)
in comparison with a control dentifrice (Colgate regular). This was a randomized, positive-controlled, double-
blinded, clinical study. Subjects were randomly divided into one of the two study groups. Pre-treatment and
post treatment photographs of the 4 anterior teeth were recorded under standardized conditions and analyzed
for lightness or luminosity values using Adobe Photoshop. The difference between the mean pre-treatment
luminosity of test and control groups was not statistically signicant. In both test and control groups, the
post-treatment luminosity was signicantly higher than pre- treatment luminosity (P < 0.001 and P = 0.003
respectively). The mean post-treatment luminosity for test group was found to be signicantly higher than
control group. The mean percentage removal of stains for test group was signicantly higher than control
group. The test dentifrice showed signicant stain removal when compared to control which could be attributed
to the role of proteolytic enzymes in the test dentifrice.
Key words: Bromelain, extrinsic stains, image analysis, papain, proteolytic enzymes, stain removal

Chakravarthy and Acharya: Efcacy of extrinsic stain removal by novel dentifrice
246 Journal of Young Pharmacists Vol 4 / No 4
ways. It can be by bleaching of the teeth, or by addition
of specic abrasives or chemical agents to toothpaste for
the removal and control of extrinsic stain. Abrasives have
been shown to effectively remove extrinsic stains along
with food debris and plaque and also help in preventing
extrinsic teeth stains from reforming.[5]
Toothpaste abrasion of dental hard tissues is an important
factor in terms of its trade off with cleaning efcacy during
the formulation of whitening toothpastes. Hence whitening
toothpastes contain additional chemical agents which augment
the abrasive cleaning by aiding the removal and/or prevention
of extrinsic stains. Many substances like surfactants, peroxides,
enzymes, citrate, pyrophosphates and hexametaphosphate
were studied previously for their stain removal efcacy.[5]
Since extrinsic stains are primarily incorporated into the
pellicle, it is possible that enzymes such as proteases could
help degrade the stained lms and potentiate their removal.
Early clinical evidence demonstrated that a highly proteolytic
mixture of enzymes of fungal origin formulated into
toothpaste were effective at reducing extrinsic stain levels as
compared to a negative control toothpaste after 6-months of
use.[6] Clinical studies have demonstrated the stain removal
efcacy of dentifrice containing papain, alumina and sodium
citrate.[7-9] Recently an in vitro study reported that a papain and
bromelain (proteolytic enzymes) containing dentifrice was
more effective in removing stains than the control dentifrice.[10]
The methods used to measure extrinsic stain levels in
clinical studies include subjective clinician determinations
based on indices and objective instrumental methods.
The objective instrumental methods used in the past were
Vita shade guides,[11] colorimeters,[12] and image analysis of
digital photographs of teeth.[13] Digital image analysis can
be utilized for assessing stains and overcomes some of
the problems associated with some of the subjective and
instrumental approaches.[13]
Hence the present study aimed to evaluate the stain removal
efficacy of a novel commercially available dentifrice
containing papain and bromelain (Glodent, Group
Pharmaceuticals Ltd, Mumbai, India) in comparison with
a control dentifrice (Colgate Regular, Colgate Palmolive
India Ltd, Mumbai, India) using a customized digital image
analysis system.
MATERIALS AND METHODS
Subjects and study design
This study was randomized and double blinded (participants
and investigators) who compared the novel dentifrice
containing papain and bromelain (Glodent) with control
dentifrice (Colgate regular). The composition of test
dentifrice was Papain, Bromelain, Miswak, Neem and 1000
ppm uoride and while that of control was calcium carbonate,
sorbitol, titanium dioxide, sodium silicate, sodium saccharin
and 1000 ppm uoride dentifrice. Papain and Bromelain are
proteolytic enzymes derived from Papaya (Carica papaya) and
Pineapple (Ananas comosus).
Before the start of the study, the protocol was approved
by the Institute Ethics Committee of Manipal University,
Manipal and subjects gave informed consent prior to their
participation. For inclusion in the study, the subjects were
required to have visible stains on maxillary incisors with
Lobene score of >1; at least 4 maxillary anterior teeth
without restorations; no oral prostheses; no untreated caries.
Lobene stain index (1968) involves assessment of extent
and intensity of stain on the gingival and body regions of
the labial surface of incisor teeth. Intensity scores were:
0- no stain, 1- light stain, 2- moderate stain, 3- heavy stain.
Extent scores were: 0- no stain, 1- stain covering up to 1/3rd
of the region, 2- stain covering >1/3rd to 2/3rd of the region
and 3- stain covering >2/3rd of the region. Many stain
indices proposed recently were mainly to determine the
efcacy of anti-stain agents, or to investigate the interaction
between chlorhexidine and dietary constituents. Subjects
were excluded from participating in the study if there
were generalized recession of the gingiva, or generalized
malocclusion, or overlapping/spacing of anterior teeth,
inability to comply with brushing instructions (e.g,dexterity
or comprehension issues), obvious periodontal disease, or
facial calculus on the anterior teeth. A total of 100 subjects
were screened in out-reach centers of Manipal College of
Dental Sciences, Manipal out of whom 29 subjects satised
the inclusion criteria. Out of these 24 subjects consented
to participate in the study. This study was conducted for
2-months (November-December 2010).
Eligible subjects were randomly assigned to one of the
two treatment groups and called to record the baseline
photographs of their 4 maxillary anterior teeth. After
brieng the purpose of the study to the participants,
oral hygiene and tooth brushing instructions were given.
Subjects who were divided into two groups were provided
with the assigned products (either of the toothpaste and
Colgate classic toothbrush with soft bristles) and were
required to brush in front of a mirror for 2 minutes
twice a day, covering the entire toothbrush head with the
dentifrice each time. Test and control dentifrices tubes
were painted in white color and delivered by a person not
involved in examination. All investigators and participants

Chakravarthy and Acharya: Efcacy of extrinsic stain removal by novel dentifrice
Journal of Young Pharmacists Vol 4 / No 4 247
were unaware of the identity of the tubes given. Tubes
were identied as either the “Group 1” or the “Group
2” tubes. Identity of each tube was revealed only after
2-weeks when the subjects were recalled for the follow-up
examination. During this visit, standardized photographs
of the 4 maxillary anterior teeth were repeated. Subjects
were given a printed timetable for 2-weeks and were asked
to check at the respective area after tooth brushing to
ensure the compliance of brushing frequency. At the end
of 2-weeks, participants were offered free treatment for
scaling and polishing [Figure 1].
Photographs of the teeth were captured in a customized
wooden box with standardized lighting conditions, a chin
rest and high resolution digital camera mounted on a
platform.
Wooden box
A customized wooden box was made with plywood in
dimensions of 18 × 18 × 18 inches. One end of the box
was kept open for the subject to keep the head and the
other end for the recorder to capture the image of the
area of interest. The open ends of the box were covered
with black drapes suspended with closed coiled springs
at the upper and lower margins of the box [Figure 2].
These drapes had a slit opening for the subjects to place
their head inside the box, for the investigator to check
for positioning of the head, focusing, and adjustments
needed for image capture. The inner surface of the box
was painted matt black to prevent reection of light
[Figure 3]. For the box to be portable, a handle was tted
on the top. The oor of the box was mounted with a
rigid customized chin cup fabricated with acrylic on one
end and a rigid acrylic platform for mounting the digital
camera on the other end. Both these units had provisions
for adjusting height.
Lighting conditions
To avoid variation in lighting and possibly affect the stain
intensity measurement, a standard light source was kept on
the oor between the chin rest and the platform for the
camera. The light was articially powered with operational
specications of 220-240V, 5W power and 6500K. (Phillips
India Ltd., India).
Camera
Digital images were captured using a high resolution camera
(DSC – S2000, 10.1 megapixels Sony® India,) tted inside
the wooden box in front of the patient’s chin rest on a
platform with xed base. Images were captured without
ash and were transferred to a computer.
Figure 1: Consort ow chart showing the various steps in the study
Figure 2: Inside view of the box
Figure 3: Outside view of the box

Chakravarthy and Acharya: Efcacy of extrinsic stain removal by novel dentifrice
248 Journal of Young Pharmacists Vol 4 / No 4
Figure 4: Pre-treatment and post-treatment photographs of subjects
Image capture
Before capturing the images all the subjects were instructed
to place their head inside the wooden box and place the
chin on the chin rest in order to capture the image of their
4 anterior teeth. Cheek retractor was used to visualize the
teeth during photography. After focusing on the area of
interest, the digital image was captured. Subjects were asked
not to move during the image capture.
Scoring
The images were opened in Adobe Photoshop (version 8.0)
and the area of interest was outlined using a magnetic lasso
tool in the toolbar option of Adobe Photoshop software.
Once the outline was selected, mean L-value of all the pixels
within the area of interest was shown in the histogram
window provided in the software.[10] The amount of stain
was graded digitally using only the Luminosity or Lightness
(L value of Adobe Photoshop version 8.0. L that represents
the lightness of the stain, with L = 255 being white, and
L = 0 being black). This procedure was repeated for 5 subjects
randomly to check the reliability in obtaining the L values. The
coefcient of reliability (Pearson correlation) of the image
analysis system was found to be 0.98.
Statistical analysis
The Lightness values were entered in SPSS version 14
(SPSS Inc., Chicago, IL, USA). Independent sample t-test
was performed to compare the pre-treatment Luminosity,
post-treatment Luminosity and percent removal of stains.
Paired t-test was used to compare the pre-treatment and
post-treatment Luminosity between the test and control
groups. A P-value of 0.05 was considered statistically
signicant. Percentage removal stains was calculated by
[(Post-treatment Luminosity – Pre-treatment Luminosity)/
Pre-treatment Luminosity] ×100.
RESULTS
The mean age of the study population was 32.09 ± 5.05
with a range of 22-40 years. Out of 24 participants, 23
completed the follow-up (12 in Test and 11 in Control). The
difference between the mean pre-treatment Luminosity
of test (157.95 ± 16.93) and control groups (143.82 ±
17.48) was not statistically signicant (P = 0.062). In both
test and control groups, the post-treatment Luminosity
was signicantly higher than pre-treatment Luminosity
(P < 0.001 and P = 0.003 respectively). The mean post-
treatment Luminosity for test group (170.76 ± 14.14) was
found to be signicantly higher than control group (147.81
± 16.47) (P = 0.002) [Table 1]. The mean percentage
removal of stains for test group (8.44 ± 4.83) was
signicantly higher than control group (2.92 ± 2.89) (P =
0.004) [Table 2 and Figure 4].
DISCUSSION
The current study evaluated the clinical stain removal
efficacy of a novel dentifrice containing papain and
bromelain in comparison with a control dentifrice. In
this study an economical and portable customized digital
image analysis system was developed and used to evaluate
stain removal.
A number of studies have reported the use of clinical indices
for stain evaluation. These indices are quick and easy to use,
but they face reliability issues because of their subjective
nature. To overcome such problems, image analysis has
been used as an objective method in the past to assess stain
build up in vitro on acrylic and enamel slabs. [14,15] It was also
shown to be an effective tool for assessing plaque[16] and
stains[13] clinically. However, the methodology used in this
study was different although the underlying construct for
color assessment was same.
Image analysis is very sensitive even for minor changes
in stain removal. This might facilitate in reduction of the
Table 2: Mean percent removal of stains among test
and control groups
Percent
removal of
stains
Test group
(Glodent)
Mean±SD
Control Group
(Colgate regular)
Mean±SD
P-value
8.44±4.83 2.92±2.89 0.004
Table 1: Comparison of luminosity values among test
and control groups
Group N Pre-treatment
luminosity
Mean±SD
Post-treatment
luminosity
Mean±SD
P-value
Test group (Glodent) 12 157.95±16.93 170.76±14.14 <0.001†
Control Group (Colgate
regular)
11 143.82±17.48 147.81±16.47 0.003†
P-value 0.062‡0.002‡
†P-value for paired t-test, ‡P-value for independent sample t-test

Chakravarthy and Acharya: Efcacy of extrinsic stain removal by novel dentifrice
Journal of Young Pharmacists Vol 4 / No 4 249
duration of the clinical trials involving stain removal to
as short as 2-weeks. This short duration of the study can
reduce the costs of conducting a clinical trial, improve
patient compliance, reduce attrition of subjects while
eliminating subjective bias. The apparatus developed for
our study was light in weight and could easily be carried to
different eld situations and could be replicated elsewhere.
The stain removal efcacy of novel dentifrice was assessed
in terms of Lightness values only. The mean pre-treatment
Lightness value for test and control group did not differ
signicantly. But the mean post-treatment Lightness value
was signicantly higher for test than control dentifrice.
Similar results were reported by a previous in-vitro study [10]
using this test dentifrice. Clinical studies using toothpaste
containing a mixture of papain, alumina and sodium
citrate (Rembrandt) have also reported a significant
stain removal.
[7-9] The test dentifrice in the present study
contained extracts of papain and bromelain, which are
proteolytic enzymes. They disrupt and ⁄ or remove the
protein portion of the pellicle ⁄ plaque layer that forms on
the surface of teeth over time, thus removing the stains
that are bound to these proteins. The control dentifrice was
chosen as Colgate regular since the relative dentine abrasive
value was nearly the same as test dentifrice [67 (test) and
70 (control) respectively]. Hence, signicant increase in the
mean Lightness values for the test dentifrice group could
partly be attributed to these enzymes.
Limitations of the study were small sample size and habits
like tobacco and areca nut usage was not accounted. Habits
like tobacco and areca nut usage can have a direct effect on
staining and are widely prevalent in this area. Although our
study did not account for habits, the result remains valid
in terms of maintenance of stain removal on a daily usage
of the dentifrice whilst using these stain causing agents.
In this particular geographic area where chewing areca
nut is culturally acceptable, it is less likely to nd subjects
not having habits and but had stains on their teeth. Hence
we could not warrant that subjects have not used these
substances during the study period. Also, the shelf life
of test dentifrice might be shorter than usual dentifrices
because of papain and bromelain being proteolytic in
nature. Overall, our study can be considered as a pilot study
to evaluate the stain removal of this novel dentifrice and a
feasibility study of digital image analysis in the assessment
of stains. The results of this study can be useful to plan
the needed clinical trials with large number of participants.
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How to cite this article: Chakravarthy PK, Acharya S. Efcacy of extrinsic
stain removal by novel dentifrice containing papain and bromelain extracts. J
Young Pharmacists 2012;4:245-9.
Source of Support: Nil, Conict of Interest: None declared.