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102]

Original Article

Anti-microbial efficacy of green tea and chlorhexidine

mouth rinses against Streptococcus mutans,
Lactobacilli spp. and Candida albicans in children
with severe early childhood caries: A randomized
clinical study
Ann Thomas, Sneha R Thakur, Sowmya B Shetty
Department of Pedodontics and Preventive Dentistry, A. J. Institute of Dental Sciences, Mangalore, Karnataka, India

ABSTRACT Address for correspondence:

Introduction: Green tea is a beverage which is consumed
worldwide and is reported to have anti-cariogenic effect.
So, if it was as effective as chlorhexidine (CHX) mouth
rinse against cariogenic microbes it could be considered
a natural, economical alternative. The purpose of this
study was to evaluate and compare the anti-microbial
efficacy of 0.5% green tea and 0.2% CHX mouth rinses
against Streptococcus Mutans, Lactobacilli spp. and
Candida Albicans. Materials and Methods: 30 children
aged 4-6 years with S-ECC (based ondefs score) were
selected. Children were divided randomly into 2 equal
groups and were asked to rinse with the prescribed
mouth rinse once daily for 2 weeks after breakfast under
supervision. A base-line and post rinsing non-stimulated
whole salivary sample (2 ml) was collected and tested
for the number of colony forming units. The data was
statistically analyzed using SPSS v16.0 software with
one-way ANOVA and Tukey’sPOSTHOC test. Results:
A statistically significant fall in colony count was found
with both the mouth rinses in Streptococcus Mutans (P
< 0.001, P < 0.001) and lactobacilli (P < 0.001, P < 0.001)
but not against Candida albicans (P = 0.264, P = 0.264).
Against Streptococcus Mutans, green tea mouth rinse
was found to be significantly better than CHX mouth
rinse (P = 0.005). Against lactobacilli spp, CHX mouth
rinse was significantly better than green tea mouth rinse
(P < 0.001). Conclusion: Green tea mouth rinse can be
considered safe, economical and used without much
concern. However, further studies are recommended.
KEYWORDS: Herbal mouth wash, severe early
childhood caries, home oral hygiene, caries prevention
Introduction
The use of mouth rinses as an aide to other oral hygiene
measures was first propagated by ancient Egyptians
Dr. Sneha R Thakur,
Department of Pedodontics and Preventive Dentistry, A. J.
Institute of Dental Sciences, Kuntikana, Mangalore - 575 004,
Karnataka, India.
E-mail: snehathakur68@gmail.com
Access this article online
Quick response code Website:
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DOI:
10.4103/0970-4388.175518
PMID:
26838151
and Romans. As early as 40-90 A. D, Pedanius
Dioscorides, a Greek physician suggested a concoction
of olive juice, pomegranate fillings, wine, and gum
myrrh to treat halitosis, while Hippocrates is known
to have advocated mouth rinsing with a mixture of
alum, salt, and vinegar in the ancient times.[1] Mouth
rinses are used generally for their analgesic, anti-
inflammatory, anti-microbial, and anti-cariogenic
This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
allows others to remix, tweak, and build upon the work non-commercially,
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For reprints contact: reprints@medknow.com
How to cite this article: Thomas A, Thakur SR, Shetty SB.
Anti-microbial efficacy of green tea and chlorhexidine mouth
rinses against Streptococcus mutans, Lactobacilli spp. and
Candida albicans in children with severe early childhood
caries: A randomized clinical study. J Indian Soc Pedod Prev
Dent 2016;34:65-70.

© 2016 Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow 65
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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes
activities. Currently, a wide range of mouth rinses such
as chlorhexidine (CHX), sodium fluoride, and essential
oils are available commercially. The American Dental
Association (ADA) recommends that mouth rinses
should be effective at modifying the micro-biota by
selectively eliminating pathogens without negatively
affecting the normal commensals of oral cavity.[2]
Evidence illustrates that the long-term twice daily
use of anti-plaque and anti-gingivitis mouth rinses,
namely 0.12% CHX gluconate and essential oils with
methyl salicylate which are approved by the Council
on Dental Therapeutics of the ADA, do not have any
negative effect on the oral microbial flora.[2] Studies
have also demonstrated that the use of mouth rinses
in children provided a significant reduction in the
decayed extracted filled surface (defs) index.[2,3]
The most common plaque-mediated disease in children
is dental caries, which is also one of the most common
childhood diseases. It is a preventable, localized
infectious, multi-factorial disease resulting from the
interaction among host, diet, and microflora on the
tooth surface over a period of time, resulting in localized
de-mineralization of hard tissues. The most implicated
bacteria in its etiology are Streptococcus mutans for its
initiation and Lactobacillus acidophilus for its progression.
Recently, a number of reports in the scientific literature
show an association between Candida albicans, a fungi,
which is an opportunistic pathogen and a normal
commensal of the oral cavity and progression of dental
caries.[4,5] Dental caries in children below 6 years is
called early childhood caries (ECC), and its severe
debilitating form is called severe-ECC (S-ECC).
Of the commercially available mouth rinses, CHX
mouth rinse is widely used and is considered to be
the “gold standard” due to its broad spectrum anti-
microbial activity. CHX de-stabilizes the bacterial
cell wall and interferes with osmosis. In addition, the
bacterial uptake of CHX is very rapid, which facilitates
rupture of the cell wall and finally the cytoplasmic
membrane causing cell death.[6-8] However, Lindhe
et al., reported a number of local side-effects with
CHX use such as discoloration of teeth, altered taste
sensation, mucosal irritation, parotid swelling, and
enhanced supra-gingival calculus formation due to
precipitation of salivary proteins and organic salts,
which limits its use as a therapeutic agent.[9]
The ease of availability and affordability of commercial
mouth rinses is a major hurdle faced by the low
socioeconomic communities. Thus, to achieve better
oral health for masses, there is always a constant quest
to identify and promote newer, economical products
that can be easily procured even by the people living in
remote areas where advanced healthcare facilities are
inadequate. Identification of an effective mouth rinse
formulation containing a commonly used condiment
would prove highly beneficial and currently a number
of them are reported in the scientific literature.
66 Journal of Indian Society of Pedodontics and Preventive Dentistry | Jan-Mar 2016 | Vol 34 | Issue 1 |
Worldwide, tea is consumed as a beverage for centuries
and has shown many health benefits. It is also a
condiment available in most homes. Some reports in
the Japanese literature have quoted that “those who
continuously drink a large amount of green tea have
less tooth decay”[10] and “drinking green tea makes
the mouth clean.”[11] Green tea is reported to be very
rich in fluoride and catechin, a bioactive component,
which exerts an anti-cariogenic effect by inhibiting
the proliferation of the streptococcal agent, interfering
with the process of bacterial adhesion to tooth enamel
and also by inhibiting glucosyltransferase.[12-14] It also
mediates protective salivary components such as
secretory immunoglobulins, lysozymes, lactoferrin,
oral peroxidases histatins, mucins, or others, thus
exhibiting an indirect anti-cariogenic effect.[15]
Pharmacokinetic studies have shown that after rinsing
with tea, catechins can be found in the saliva for up
to 60 min and that the enzymatic breakdown of starch
on food particles trapped in the mouth is noticeably
reduced.[14] Studies on the anti-cariogenic effect of green
tea polyphenols have revealed that these compounds
have a high inhibitory effect against S. mutans and
the acids produced by it.[14] The cariogenic activity of
catechins present in green tea was found to be related
to its role in the depletion of thiol group which in turn
exerted bactericidal effect.[14]
To our knowledge, there is a paucity of reports on
comparing the anti-microbial efficacy of green tea
mouth rinse to that of CHX, the “gold standard,” and
also no reports regarding its antifungal effect against
C. albicans, which is recently being implicated in the
etiology of caries. Thus, the main purpose of this study
was to evaluate and compare the anti-microbial efficacy
of green tea (0.5%) mouth rinse to that of CHX (0.2%)
against S. mutans, Lactobacilli spp., and C. albicans.
Materials and Methods
A randomized, double-blind, active, controlled clinical
trial was conducted at a local preschool between
November 2013 and February 2014. The study protocol
was reviewed and approved by the Ethical Committee
of the Institution and the study was in compliance
with The Code of Ethics of the World Medication
(Declaration of Helsinki) for experiments involving
human subjects. An informed consent was obtained
from authorities of the preschool and the parents of the
subjects before the onset of the study.
To conduct the study, 30 children aged 4-6 years were
randomly selected from the preschool. Physically
fit children with S-ECC as per the definition given
by the American Academy of Pediatric Dentistry[16]
were included in the study. Children who were
unable to expectorate completely or brush their
teeth on their own, had positive history of taking
antibiotics 3 months prior to and during the study
period, undergoing orthodontic treatment or with an
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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes
intraoral prosthesis, and had any intraoral pathology
were medically compromised or for whom parental
consent was not given were also not included
in the study.
The defs of the children was recorded using visible
light, mouth mirror, and community periodontal
index probe. The sum total of defs was considered
and based on the caries experience, children were
selected for the study. All the study participants were
given a tube of nonfluoridated tooth paste and a tooth
brush. The brushing and mouth rinsing technique
was demonstrated to all and were advised to brush
twice daily. The participants were randomly divided
into two groups of 15 each by lottery method, i.e.,
they were asked to pick up chits with the name of
the mouth rinse written on them. The children were
given prenumbered identical mouth rinse bottles and
advised to rinse the mouth for 1 min using 5 ml of the
respective mouth rinse daily for 2 weeks in the school.
Rinsing was carried out under the supervision of the
investigator for six working days of the week and on
Sundays under parental supervision.
Preparation of mouth rinses
Green tea mouth rinse was custom prepared by the
pharmacist. To prepare green tea mouth rinse, dried
green tea leaves (obtained by open air drying) were
grounded to a desirable size using an electrical mill,
and then extracted by percolation using distilled
water as solvent. Green tea which is rich in phenolic
compounds (6%) was diluted to obtain a concentration
of 0.5% phenolic compound using double distilled
water. Authorized additive, peppermint flavor
(1 g/l), and sodium saccharine (1 g/l), a sweetening
agent, were used to formulate the mouth rinse.[17]
Commercially available CHX (0.2%) mouth rinse
(CLOHEX, Dr. Reddy’s) was used in this study.
Saliva samples
A volume of 2 ml saliva samples were collected
prior to the commencement of mouth rinsing, i.e., at
baseline and after 2 weeks rinsing, i.e., postrinsing.
Un-stimulated whole saliva samples were collected
by asking the children to drool into a sterile container
for 3-5 min, sitting in an upright position in a well
lit room with good ventilation. They were collected
in the morning between 10.00 and 11.00 a.m. to
prevent any bias in the concentration of saliva due
to circadian rhythm.[18] Children were also informed
not to eat or drink anything (except water) 1 h before
saliva collection to minimize possible food debris and
stimulation of saliva.
Microbial evaluation
The samples were transported to the microbiological
laboratory in box with ice-packs. They were tested
for the colony forming units (CFU) of S. mutans,
Lactobacilli spp., and C. albicans using mitis-salivarius-
bacitracin agar, Rogosa agar, and HiCrome agar,
Journal of Indian Society of Pedodontics and Preventive Dentistry | Jan-Mar 2016 | Vol 34 | Issue 1 |
respectively (HiMedia Laboratories, Mumbai). All the
saliva samples were serially diluted using physiological
saline to obtain −3 concentration. A volume of 0.1 ml
saliva sample was spread on the selective agar plates
using a sterile glass spreader. The plates were incubated
for 48 h at 37°C in the incubation chamber (ROTEK)
to obtain maximum growth of microbial colonies.
The CFU were identified by morphology, size, and
color, and counting was done with a handheld digital
colony counter (HiMedia, Mumbai). The colony count
was expressed as the number of CFUs per millimeter
(CFU/ml) of saliva. Semi-quantification of the number
of colonies was done by multiplying the actual colony
count with 1 × 103 to adjust for the dilution factor. The
colony counting was done by the same observer under
the same conditions and at the same time of the day to
avoid the intraobserver variability in the results.
Evaluation of acceptability
After completion of the study, the study participants
were given a self-administered, close-ended
dichotomous questionnaire regarding the acceptability
of mouth rinse prescribed to each of them. The
questionnaire consisted of three questions with two
mutually exclusive options (YES/NO) to answer.
The questions were based on the acceptability of the
mouth rinses in terms of flavor, smell, and willingness
to continue using the mouth rinse. A paper and pencil
method was used to administer the questionnaire. With
the help of the caretaker, the participants’ response
was elicited and the questionnaire was completed.
Outcome measures and statistical methods
The study had a single end point at 2 weeks. The data were
statistically analyzed using one-way variance ANOVA
and Tukey’s post-hoc honestly significant difference
(HSD) test in SPSS Software 16.0 (SPSS Inc., Chicago).
The results were considered statistically significant at
0.05 probability level. One-way variance ANOVA test
was used to compare the mean of differential colony
counts in the two mouth rinse groups and to assess
the anti-microbial efficacy of green tea mouth rinse,
which was the primary outcome of the study. Tukey’s
post-hoc HSD test was used for comparative analysis of
two mouth rinse groups and to measure the secondary
outcome, i.e., whether the newly formulated green tea
mouth rinse was better than CHX mouth rinse.
Results
The mean age of the participants was 5 (standard
deviation [SD]: 0.69) years and the mean defs was 16.20
(SD: 8.58). Green tea showed a statistically significant
fall in the colony counts of S. mutans and Lactobacilli
spp. (P < 0.001, P < 0.001); whereas only a numerical
fall in C. albicans colony count was found, which was
not statistically significant (P = 0.264) [Table 1]. CHX
showed a statistically significant fall in the colony
counts of S. mutans and Lactobacilli spp. (P < 0.001,
P < 0.001); whereas a numerical increase was observed
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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes

in the colony count of C. albicans, which was also not
statistically significant (P = 0.264) [Table 1].
On comparison against S. mutans, green tea mouth rinse
was found to be significantly better than CHX mouth
rinse (P = 0.005). Against Lactobacilli spp., CHX mouth
rinse was found to be significantly more effective than
green tea mouth rinse (P < 0.001). Whereas against
C. albicans, no statistically significant difference was
found (P = 0.081) [Table 2].
Majority of the study participants in the green tea group
had a positive response to flavor (n = 10; 66.7%); smell
(n = 8; 55.3%); and willingness to rinse (n = 10; 66.7%).
However, the response of the study participants in
the CHX group was varying. Only 40% (n = 6) were
positive to flavor, 66.7% (n = 10) were positive to smell,
and 46.7% (n = 7) were positive to willingness to rinse.
Discussion
The purpose of this study was to evaluate and compare
the anti-microbial efficacy of green tea mouth rinse
to that of CHX mouth rinse on the level of salivary
S. mutans, Lactobacilli spp., and C. albicans in children.
The present study was carried out under real life
conditions without altering the subjects’ routine oral
hygiene practices except that they were advised to use
a nonfluoridated dentifrice for cleaning their teeth.
This was done to eliminate the bias resulting due
to additional anti-microbial effect of fluoride from
the dentifrice. To standardize rinsing, all the study
participants were asked to rinse under monitoring at
school. Thus, rinsing was carried out after breakfast
and this could have also contributed toward reducing
the microbial challenge.
According to the results of this study, green tea was
found to be a very effective anti-bacterial mouth rinse
against S. mutans and Lactobacilli spp. with some anti-
fungal activity against C. albicans. The antibacterial
effect of green tea mouth rinse is in accordance to the
previous reports where the authors stated that rinsing
with green tea extract had valuable anti-cariogenic
activities including inhibitory effect on cariogenic
bacteria by inhibiting the adherence of bacterial cells to
the tooth surfaces.[14,17,19,20] And also, green tea catechins
maintain the salivary pH at a normal range, which is
not a favorable condition for cariogenic bacteria to
flourish.[21,22] The anti-fungal activity is in agreement to
the earlier in vitro studies that have reported that green
tea polyphenols and catechins inhibit the growth of
C. albicans by 40% and 75%, respectively.[21,22]
CHX mouth rinse is one of the best proven
commercially available mouth rinses due to its broad
spectrum anti-microbial activity and no systemic side
effects[23] Scientific studies have reported that CHX
mouth rinse was effective in reducing the colony
counts of S. mutans in plaque biofilm;[18,23,24] Lactobacilli
spp. colony counts,[23,25] and culturable microbes of
tongue and saliva.[24] The present study also confirmed
the anti-bacterial activity of CHX mouth rinse against
S. mutans and Lactobacilli spp. However, no anti-
candida effect was found; in fact, a numerical increase
was observed which was not statistically significant.
This increase in C. albicans count could be assigned to
various factors such as a direct trigger of C. albicans by
CHX or due to depression of the bacterial counts aiding
in the proliferation of this opportunistic pathogen.
Another probable explanation is the basic differences
in outer cell structure between bacteria and fungi, with
the latter having a rigid outer chitin wall making it
less amenable for lysis.[26] Bioavailability of CHX may
also have been compromised in this study because
rinsing was carried out only once daily and also, only
5 ml of mouth rinse was used, for reports have stated
it to be about 12 h with 10 ml of 0.2% of CHX mouth
rinse.[7,8] Most scientific reports have shown fungicidal

Table 1: Mean differential colony counts of Streptococcus mutans, Lactobacilli spp., and Candida albicans
in both the mouth rinse groups
Mouth rinse group n Microorganism Mean baseline (CFU/ml) Mean postrinse (CFU/ml) Mean difference (CFU/ml) Significance (P)
CHX 15 S. mutans 4.7×107 5.9×103 4.7×103 <0.001**
Lactobacilli spp. 5.2×105 3.9×105 1.3×105 <0.001**
C. albicans 1.7×105 3.03×105 −1.33×103 0.264
Green tea 15 S. mutans 2.9×108 1.9×108 1×108 <0.001**
Lactobacilli spp. 4.9×104 2.2×104 2.7×104 <0.001**
C. albicans 6.4×10 4
4.1×104
2.3×104 0.264
**Very highly significant (P < 0.001). S. mutans = Streptococcus mutans; C. albicans = Candida albicans; CFU/ml = Colony forming units per millimeter

Table 2: Inter comparison of the mean difference of colony counts of both the mouth rinse groups for
Streptococcus mutans, Lactobacilli spp., and Candida albicans
Dependent variable Group (I) Group (J) Mean difference (I–J) ×103 (CFU/ml) SE P
S. mutans CHX Green tea −208.412 59,868.01 0.005*
Lactobacilli spp. CHX Green tea 232.067 50.431 <0.001**
C. albicans CHX Green tea −176.333 72.176 0.081
*Significant (P = 0.005); **Very highly significant (P < 0.001). SE = Standard error; CHX = Chlorhexidine; S. mutans = Streptococcus mutans; C. albicans = Candida albicans

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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes
and fungi static action of CHX where it was used in
combination with other chemical solutions such as
cetylpyridinium chloride, fluconazole, and chitosan
due to their synergistic action.[27-29]
On comparison of the anti-microbial efficacy of green
tea mouth rinse to that of 0.2% CHX, we found that it was
significantly better against S. mutans, but less effective
against Lactobacilli spp. and comparable against C.
albicans. There is a paucity of reports comparing the
anti-bacterial effect of green tea and CHX mouth rinses
whereas the anti-plaque property of these mouth rinses
has been reported to be similar.[19] While in another
report, 0.5% green tea mouth rinse was reported to be
better than 0.2% CHX mouth rinse.[30] Kaur et al., have
suggested that green tea catechins helps to maintain
the normal plaque pH, which in turn is an unfavorable
condition for S. mutans to colonize and multiply.[19]
In our study, the green tea mouth rinse was widely
accepted by the study participants, whereas CHX
mouth rinse had poor acceptance rate and this is a
documented drawback of CHX.[6,8,31]
Conclusion
From the results of our study, it can be concluded
that green tea mouth rinse could be very good cost-
effective mouth rinse. However, further studies would
be beneficial to evaluate any potential adverse effects
with long-term use of this mouth rinse.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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70 Journal of Indian Society of Pedodontics and Preventive Dentistry | Jan-Mar 2016 | Vol 34 | Issue 1 |