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119]

Original Article

Effect of bleaching with two different concentrations

of hydrogen peroxide containing sweet potato
extract as an additive on human enamel: An
in vitro spectrophotometric and scanning electron
microscopy analysis
Sarath Gopinath, Vandana James, Sampath Vidhya, Kittappa Karthikeyan, Sanjeev Kavitha, Sekar Mahalaxmi
Department of Conservative Dentistry and Endodontics, SRM Dental College, Bharathi Salai, Ramapuram, Chennai, India

Abstract
Objectives: To evaluate the color change in teeth bleached with two different concentrations of hydrogen peroxide, containing
sweet potato extract as an additive, using a spectrophotometer, and to evaluate the surface changes in enamel using a
scanning electron microscope (SEM).
Materials and Methods: Baseline color values of 24 artificially stained incisors were obtained using a spectrophotometer. The
specimens were divided into two groups of 12 teeth, each based on the concentration of hydrogen peroxide (H2O2) as follows:
Group I — 35% H2O2 and Group II — 10% H2O2. One‑half of the tooth was bleached with H2O2 alone (Subgroup A) and the
other half was bleached with a combination of H2O2 and sweet potato extract (Subgroup B). Post bleaching the Commission
Internationale de l’ Eclairage L*, a*, b* (CIEL*a*b*) values were obtained and ΔE was calculated. The surfaces of the samples
were examined using SEM.
Results: The mean ΔE values of groups IB (72.52  ±  2.03) and IIB (71.50  ±  1.81) were significantly higher than those of
groups IA (65.24 ± 1.02) and IIA (64.19 ± 1.88), respectively, (P < 0.05). The SEM images of groups IB and IIB showed lesser
surface irregularities and morphological alterations in enamel.
Conclusion: The addition of sweet potato extract to hydrogen peroxide not only resulted in the restoration of the natural tooth
color, but also decreased the effects of bleaching on the enamel morphology, compared to the use of hydrogen peroxide
alone.
Keywords: Catalase, CIEL*a*b*, enamel, hydrogen peroxide, sweet potato, tooth bleaching

INTRODUCTION bleaching with less or no deleterious effects on enamel is

Vital bleaching represents a conservative treatment option
in the treatment of discolored teeth.[1] The development
of bleaching agents containing hydrogen peroxide or its
precursor, carbamide peroxide, in varying concentrations,
ranging from 10 to 38%, has made in‑office bleaching
effective.[2] Although effective bleaching has been achieved
in terms of esthetics, the deleterious effects of bleaching
agents on the tooth surface have been an area of concern.[3,4]
Various scientific reports have demonstrated that changes
occur in the surface texture, composition, and microhardness
of the enamel, when teeth are bleached with hydrogen
peroxide.[5,6] Any attempt to achieve high efficiency dental
Address for correspondence:
Dr. Sampath Vidhya, Department of Conservative Dentistry and
Endodontics, SRM Dental College, Bharathi Salai, Ramapuram,
Chennai ‑ 600 089, India. E‑mail: drvidhyas@yahoo.co.in
Date of submission : 14.05.2012
Review completed : 15.06.2012
Date of acceptance : 05.08.2012
a welcome change in dentistry. One such attempt is the
addition of vegetative enzymes to the bleaching agent that
may promote and accelerate the dissociation of the latter.[7,8]
The present study aims at providing a tooth bleaching
formulation containing vegetative enzyme extract obtained
from plant tubers. Sweet potato (Ipomoea batatas L) is one
such plant tuber, which has a high content of enzymes
such as polyphenol peroxidase (PO), catalase (CAT), and
superoxide dismutase (SOD).[9] Hence, the aim of this in vitro
study is to evaluate the color change in teeth bleached
with two different concentrations of hydrogen peroxide,
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DOI:
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Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1 45

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Sarath, et al.: Effect of hydrogen peroxide containing vegetative enzymes on enamel
using sweet potato extract as an additive, with the help
of a spectrophotometer, and furthermore, to evaluate the
surface changes in the enamel post bleaching, using a
scanning electron microscope (SEM).
MATERIALS AND METHODS
Preparation of sweet potato extract
Two hundred grams of sweet potato, with purple color flesh,
were cleaned, peeled, and cut into cubes. The cubes were
smashed to a concentrate with 25 ml of deionized water in a
blender and the concentrate was filtered out. One hundred
milliliters of the filtered sweet potato concentrate was
centrifuged at 2000 rpm for two minutes at a temperature
of 4°C. The clear liquid thus collected is the sweet potato
extract. This extract was refrigerated at 4°C until use.
Specimen collection
Twenty‑four maxillary central incisor teeth, extracted due
to periodontal disease, and free of visible cracks, caries,
defects, or decalcification were collected for the study.
The teeth were cleaned of calculus and the remaining soft
tissue using an ultrasonic scaler (Satelec/Acteon Suprasson
Newtron Ultrasonic Scaler, New Jersey, USA). The teeth were
stored in 0.2% thymol and refrigerated under 4°C until use.
Staining of the specimens
Artificial staining of the teeth as established by Suleiman M
et al.,[10] was followed in this study. A tea bag (2 g) (Ranfer
Tea, Colombo, Sri Lanka) was suspended in 100 ml of
boiling deionized water for five minutes, and the solution
was cooled to room temperature. The solution was strained
and the clear tea solution was used for staining. The teeth
were then immersed in tea solution for 24 hours. After
24 hours, the teeth were washed and stored under 100%
humidity at 37°C, until use. The baseline color values of the
specimens were measured over a white background using
a reflectance spectrophotometer (X‑Rite Gretag Macbeth,
Berlin, Germany), which recorded the color variables L*, a*,
b* according to the CIEL*a*b* color system.
The teeth were randomly divided into two groups of
12 teeth each, based on the concentration of hydrogen
peroxide, as follows:
Group I (n = 12) ‑ 35% (H2O2)
Group II (n = 12) ‑ 10% (H2O2)
The roots of all the teeth were sectioned at the cementoenamel
junction using a rotating diamond disk under water cooling.
Then the crowns were sectioned labio‑palatally to obtain
equal mesial and distal halves. Each half was mounted on
a self‑curing acrylic resin block, leaving the labial surface
exposed. One‑half of the tooth was bleached with hydrogen
peroxide alone (Subgroup A) and the other half of the same
tooth was bleached with a combination of hydrogen peroxide
and sweet potato extract (Subgroup B), as follows:
46 Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1
Group IA: (n = 12) 35% hydrogen peroxide solution.
Group  IB: (n = 12) 1 ml of sweet potato extract was blended
with 28 ml of (100 mM) phosphate buffered
saline (PBS) and 1 ml of 35% (1M) hydrogen
peroxide to prepare a solution of 30 ml.
Group  IIA: (n = 12) 10% hydrogen peroxide solution.
Group  IIB: (n  =  12) 1 ml of sweet potato extract was
blended with 28 ml of PBS (100 mM) and 1 ml
of 10% hydrogen peroxide (1M) to prepare a
solution of 30 ml.
Bleaching protocol
The specimens in each subgroup were covered with a piece
of gauze, saturated with 3 ml of the experimental solution,
for a period of 30 minutes, with the bleaching agent being
changed every 10 minutes. This bleaching procedure was
repeated twice, with a one‑week interval, during which
time the specimens were stored in artificial saliva at 37°C.
After carrying out the bleaching protocols, the teeth were
rinsed and stored in artificial saliva at 37°C for 24 hours.
The color of the bleached specimens were then measured
over a white background employing a spectrophotometer,
which recorded the color variables L*, a*, b* according to
the CIEL*a*b* color system.
The color change (ΔE) was calculated from the L*, a*, and b*
values employing the following formula: [11]
ΔE = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2
The color change before and after bleaching was taken as
an index to evaluate the whiteness after bleaching.
Statistical analysis
Data pertaining to color change was analyzed using the
student’s t test at a 5% significance level.
Scanning electron microscopic analysis
To evaluate the effect of bleaching solutions on the surface
morphology of enamel, all the samples were subjected to
scanning electron microscopic analysis (SEM JEOL model,
JSE‑5610 LV). The specimens were vacuum desiccated
first in alcohol and subsequently in acetone, followed by
sputter‑coating with gold. Micrographs were taken at a
magnification of 2000x. The entire exposed labial surface
was scanned and the most critical areas were selected for
SEM photomicrographs.
RESULTS
The results of this in vitro study are given in Table 1. There
was no significant difference between the mean ΔE values
of group IA (35% H2O2 ‑ 65.24 ± 1.02) and group IIA (10%
H2O2 ‑ 64.19  ±  1.88) (P  >  0.05). The mean ΔE values of
group IB (35% H2O2 + sweet potato extract ‑ 72.52 ± 2.03) and
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Table 1: Mean ΔE values and standard deviation of the
study groups
Groups Mean ΔE values
After staining Following bleaching
Baseline ΔE Subgroup A
(Mean±SD) (Mean±SD)
Group I (35% H2O2) 60.70±1.30a 65.24±1.02b
Group II (10% H2O2) 59.52±1.89a 64.19±1.88b
*For each column, the same superscript letters indicate no statistically significant
difference (P>0.05) between the groups
group IIB (10%H2O2 + sweet potato extract ‑ 71.50 ± 1.81)
were significantly higher compared to their respective
baseline values (P < 0.001). The mean ΔE values of groups IB
and IIB were significantly higher than those of groups IA
(35% H2O2 ‑ 65.24 ± 1.02) and IIA (10% H2O2 ‑ 64.19 ± 1.88),
respectively, (P < 0.05). There was no statistically significant
difference between the mean ΔE values of groups IB and
group IIB (P > 0.05).
The SEM micrographs of group IA [Figure 1a] and
group IIA [Figure 1c] showed a roughened enamel surface,
with loss of aprismatic enamel. A presence of cracks and
craters was also evident. The SEM micrographs of group IB
[Figure 1b] and group IIB [Figure 1d] showed a lesser
number of morphological surface irregularities, with an
absence of cracks and craters.
DISCUSSION
Treatment of discoloration varies from bleaching to even
a full coverage restoration, depending on the severity of
the stains, with bleaching serving as a more conservative,
cost‑effective and time‑saving procedure.[1] The theory of
bleaching involves the release of free radicals from the
dissociation of the bleaching agent, which attacks the
organic molecules (stains) in the teeth to achieve stability.
This is followed by a further release of free radicals, which
in turn react with the unsaturated bonds of the stains,
disrupting the electron conjugation and providing a change
in the absorption energy of the organic molecules in the
enamel. These disrupted molecules reflect less light, thus
making the tooth appear lighter in shade.[2]
Hydrogen peroxide readily decomposes when it
encounters substances with which it can react and results
in the release of free radicals. If the bleaching procedure
is continued beyond the saturation point, in the course
of decomposition, hydrogen peroxide also interacts with
the organic components of the tooth, such as proteins
and lipids, resulting in their removal.[5] This makes
the surface rough. Hydrogen peroxide also dissolves
the inorganic components of enamel by penetrating
the intra‑ and interprismatic regions. An increased
exposure to hydrogen peroxide leads to an increase in its
penetration into the enamel. This leads to a deepening of
Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1
Sarath, et al.: Effect of hydrogen peroxide containing vegetative enzymes on enamel
Subgroup B
(Mean±SD)
72.52±2.03c
a b
71.50±1.81c
c d
Figure 1: (a) SEM micrograph of group  IA (35% H2O2).
Loss of the aprismatic layer, presence of crater and surface
irregularities, (b) SEM micrograph of group IB (35% H2O2+
sweet potato extract). Lesser number of surface irregularities,
with absence of craters and cracks, (c) SEM micrograph of
group  IIA (10% H2O2). Loss of aprismatic layer, surface
irregularities, cracks and depressions, (d) SEM micrograph of
group IIB (10% H2O2+ sweet potato extract). Lesser number
of surface irregularities and depressions, with absence of
cracks
the grooves in an already roughened enamel surface.[4,12]
Hence, this in vitro study aims at achieving high efficiency
dental bleaching, with less or no deleterious effects on
the enamel surface.
Sweet potato contains a plurality of antioxidant molecules,
which are either enzymatic or non‑enzymatic in nature such
as catalase (CAT), superoxide dismutase (SOD), carotenoids,
phenolic compounds, and ascorbic acid.[9] Several studies
in the field of food chemistry have indicated that these
phytochemicals have a high free‑radical scavenging
activity.[9,13]
In the present study, the CIE L*a*b* values were used to
assess the color change of the bleached enamel. In 1976,
CIE recommended the CIE L*a*b* color scale for use. This
scale defined the color more closely to human perception,
and thus, provided a standard scale for comparison of color
values.[11,14] A spectrophotometer was selected for the study
over a calorimeter, as the latter was designed to measure the
color of flat surfaces of objects rather than tooth surfaces,
which were not usually flat.[15] Also the inter‑instrument
repeatability with a calorimeter was comparatively poor
when compared with intra‑instrument repeatability.[16]
Okubo et al., (1998)[17] and Tung et al., (2002)[18] inferred
that the colors assessed by the colorimeter and the
shade guides were inconsistent. On the other hand, the
spectrophotometer not only demonstrated high accuracy
and reproducibility similar to the colorimeter,[19] it even
performed better than the shade guide assessment.[20]
47
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Sarath, et al.: Effect of hydrogen peroxide containing vegetative enzymes on enamel
The results of this in vitro study indicated that there was
no significant difference in the mean ΔE values of group IA
and group IIA. Leonard et al.,[21] in 1998, and Matis et al.,[22]
in 2000, showed that lower concentrations of carbamide
peroxide achieved the same result in whitening teeth,
as compared to the higher concentrations, when shade
assessment was done after a period of two to six weeks.
The addition of sweet potato extract to both 35%
(group IB) and 10% (group IIB) of hydrogen peroxide yields
significantly higher mean ΔE values, when compared to
groups IA and IIA, in which no sweet potato extract is
added. This can be attributed to the enzymatic action of
the catalase present in sweet potato, which, when added to
hydrogen peroxide, reduces its high activation energy and
increases the rate of release of the free radicals. Activation
energy is critical to any intended chemical reaction. In
order to initiate the reaction, this minimum energy barrier
has to be overcome by the reactant molecules, so that the
reaction can be activated, whereby, subsequent reaction
products can be produced. The activation energy of
hydrogen peroxide is 75 kJ/mol.[23] Certain enzymes, when
added to the reactant molecules, can reduce the needed
level of activation energy and increase the efficiency or
rate of the chemical reaction.[8,23]
When catalase is added to hydrogen peroxide in specific
concentrations, the enzyme catalase combines temporarily
with the hydrogen peroxide molecule forming a
catalase‑hydrogen peroxide complex.[23] This combination
places added stress on the intermolecular bonds holding
the substrate molecules together, thereby lowering the
activation energy (21 kJ/mol) needed to initiate the chemical
reaction. Owing to this, large quantities of free radicals of
hydrogen peroxide are released within a shorter period of
time.[8,23] This in turn reduces the time the bleaching agent
is in contact with the tooth, and thus, lessens the possibility
of creating grooves or micropores across the surface of the
bleached enamel.
The effectiveness of bleaching is directly related to
exposure time and concentration of the active ingredient.
With an increase in the exposure time and concentration,
the oxidative process becomes stronger, and hence, the
resultant complications become greater — the most
common being weakening of the enamel.[4,24] This has been
demonstrated in the SEM micrographs of the present study.
The SEM micrographs of group IB [Figure 1b] and group IIB
[Figure 1d] show lesser number of surface irregularities,
with absence of cracks and craters, when compared to
group IA [Figure 1a] and group IIA [Figure 1c]. The lesser
enamel surface changes in groups IB and IIB may be
attributed to the presence of enzymatic and non‑enzymatic
antioxidants in sweet potato, which exert a scavenging
action on free radicals, thus limiting their deleterious
effects on the enamel. Achieving a smooth enamel surface
48 Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1
post bleaching reduces the possibility of stain or pigment
deposition within a certain period of time, after bleaching,
and the addition of sweet potato extract to hydrogen
peroxide enables one to achieve the same.
CONCLUSIONS
Under the limitations of this in vitro study, it can be concluded
that the use of hydrogen peroxide (both 35 and 10%)
containing sweet potato extract, as a bleaching agent, not
only results in the restoration of natural tooth color, but also
decreases the effects of bleaching on the enamel morphology,
compared to the use of hydrogen peroxide alone.
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How to cite this article: Gopinath S, James V, Vidhya S, Karthikeyan
K, Kavitha S, Mahalaxmi S. Effect of bleaching with two different
concentrations of hydrogen peroxide containing sweet potato extract
as an additive on human enamel: An in vitro spectrophotometric and
scanning electron microscopy analysis. J Conserv Dent 2013;16:45-9.
Source of Support: Nil, Conflict of Interest: None declared.

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Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1 49