Original Article
Effect of green tea mouth rinse on
Streptococcus mutans in plaque and saliva in children:
An in vivo study
Anil Kumar Goyal, Manohar Bhat1, Meenakshi Sharma, Mamta Garg2, Abhishek Khairwa1,
Rakesh Garg3
Department of Pedodontics and Preventive Dentistry, Government Dental College, 1Department of Pedodontics and Preventive Dentistry,
Jaipur Dental College, 2Department of Dentistry, Goyal Child and Dental Clinic, Jaipur, 3Department of General Dentistry, S.P. Medical
College, Bikaner, Rajasthan, India
ABSTRACT
Aims: This study was conducted to evaluate
and compare the antimicrobial efficacy of
green tea catechin as a mouth wash on colony
count of Streptococcus mutans in children.
Materials and Methods: A  sample size of thirty
children was selected out of screened 290 children
by simple random sampling between the age group
of 7 and 12 years. The study was conducted over a
period of 2  weeks. After 24  h of oral prophylaxis,
the baseline samples were collected and each group
was subjected to mouth rinsing with green tea
mouth wash for 2  weeks and further plaque and
saliva samples were collected at 1‑  and 2‑week
intervals from baseline. Microbiological analysis of
plaque and saliva samples was done by Dentocult
SM strip kit  (Orion Diagnostica, Finland), and the
results were statistically analyzed and tabulated.
Results: Statistically, there was highly significant
reduction in S. mutans count in plaque as well as in
saliva for after 1‑ and 2‑week intervals from baseline.
Conclusion: Hence, finally, our study showed that
green tea catechin is effective as a mouth wash
against S. mutans and having better action in plaque
as compared to saliva. It can be used as an adjunct to
commercially available mouthwashes.
KEYWORDS: Antimicrobial efficacy, green tea
catechin, mouth wash, Streptococcus mutans plaque,
saliva
Introduction
Healthy diet and lifestyle are the best weapons to fight
disease. Hence, diet and lifestyle of an individual have
been studied extensively.[1]
Tea after water is the most popularly consumed
beverage worldwide, with a per capita consumption
© 2017 Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow
Address for correspondence:
Dr. Anil Kumar Goyal,
Department of Pedodontics and Preventive Dentistry,
Government Dental College, Jaipur, Rajasthan, India.
E‑mail: goyaldr_anil@yahoo.co.in
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DOI:
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of 120  ml/day.[1] Tea has had a significant role in
human history, from its legendary discovery in the
year 2737 BC by the Emperor Shen Nung when leaves
fell into a pot of boiling water to the present day when
tea is a universally enjoyed beverage. Green tea has
been used in China as a healthy, medicinal drink for
approximately 5000 years. Green tea was the drink of
choice among the leaders and the wealthy. However,
following the fall of the Mongolian Empire back in
1368 AD, the whole population of China began to
experience the wonderful consumption of tea. The
medicinal uses of tea were first reported by Chinese
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How to cite this article: Goyal AK, Bhat M, Sharma M,
Garg M, Khairwa A, Garg R. Effect of green tea mouth rinse on
Streptococcus mutans in plaque and saliva in children: An in vivo
study. J Indian Soc Pedod Prev Dent 2017;35:41-6.
41
Goyal, et al.: Effect of green tea mouth rinse on S. mutans in plaque and saliva in children

scholars in a text written by Pen T’Sao circa 24‑221 AD, • Patient labels (10)
and the physiological effects of tea continue to challenge • Selective culture vials (10)
scientists.[2] • Manufacturer model chart (1)
• Incubator (BOD Incubator – Super Deluxe) [Table 1].
The flavonoids present in green tea include
catechins  (flavan‑2‑ols), such as epicatechin  (EC), Preparation of green tea catechin mouth wash
epicatechin‑3‑gallate  (ECG), epigallocatechin  (EGC), Green tea extract  (50%) powder was prepared by
and epigallocatechin‑3‑gallate  (EGCG). Green tea Lakshyam Herbs Pvt. Ltd., Noida, Uttar Pradesh,
also contains carotenoids, tocopherols, ascorbic which was sent to Guru Nanak Institute of Pharmacy,
acid (Vitamin C), minerals, such as Cr, Mn, Se, or Zn, Hyderabad, for preparation of catechin mouth
and certain phytochemical compounds.[1,3] wash (0.25%w/v).
Numerous studies in a variety of experimental
Method
animal models have demonstrated that catechin
First, the 0.25%  w/v catechin solution was prepared
possesses antioxidant, antimutagenic, antidiabetic,
by dissolving 250 mg of catechin in 25 ml of distilled
anti‑inflammatory, antibacterial and antiviral, and above
water in a 100 ml beaker.
all, cancer‑preventive properties. It also contributes to
control the body weight, rise the bone density, as well as
stimulate the immune system. Its potential for decreasing In another beaker, 25  ml of water was mixed and
the risk of skin cancer is under investigation.[1,3,4] dissolved completely with sodium chloride, sodium
benzoate, and sodium bicarbonate.
Increasing scientific and consumer interest in health
benefits of tea has led to the inclusion of tea extracts in The second solution is transferred in 0.25% catechin
oral nutritional supplements and topical preparations. solution and mixed with amaranth solution which
Green tea contains a number of bioactive chemicals; it was then filtered and collected in a 100 ml measuring
is particularly rich in flavonoids, including catechins cylinder, followed by addition of distilled water to
and their derivatives. The most abundant polyphenolic make the volume 100 ml.
compound EGCG is thought to contribute to the
beneficial effects of tea.[4] The solution was transferred to 100 ml plastic bottles
which were sealed under sterile conditions for
Green tea  (Camellia sinensis) contains polyphenolic dispensing to children [Figure 2 and Table 2].
catechins reported to exhibit significant antimicrobial
properties. Green tea, at a concentration of 40 mg/mL, Table 1: Selective culture media
brewed at 90°C at 5, 20, and 40 min, was determined to
Typical formulation
be moderately effective against Streptococcus mutans.[5]
Tryptone 10 g/L
Peptone 10 g/L
Therefore, keeping in mind these facts about green tea,
Glucose 1 g/L
this study was conducted to evaluate the antimicrobial
efficacy of green tea catechin as a mouth wash on Saccharose 300 g/L
colony count S. mutans in children. K2HPO4 5 g/L
Trypan blue 12 mg/L
K‑tellurite (1%) 1 ml/L
Materials and Methods
This study was conducted in S.S. Public Higher
Secondary School, Triveni Nagar, Jaipur, to
evaluate and compare the antimicrobial efficacy of
conventionally prepared green tea mouth wash. The
materials and equipment used for the study [Figure 1]
are as follows.
• Mouth mirror, tweezers, and explorer
• Sterile gloves
• Ultrasonic scaler
• Green tea extract (50%)
• Green tea catechin mouth wash (0.25% w/v)
• Dentocult SM Strip Mutans Kit of Orion
Diagnostica, Finland, containing:
• Square tip plaque strip (10)
• Round tip salivary strip (10)
• Paraffin tablets (10)
• Bacitracin disc (50) Figure 1: Armamentarium

42 Journal of Indian Society of Pedodontics and Preventive Dentistry | January-March 2017 | Vol 35 | Issue 1 |
 Goyal, et al.: Effect of green tea mouth rinse on S. mutans in plaque and saliva in children

Selection criteria
A sample size of thirty children was selected out of
screened 290 children by simple random sampling
between the age group of 7–12  years. The study was
conducted over a period of 2 weeks.
The subjects volunteered to participate after verbal and
written information. Ethical clearance was taken from
Ethical Clearance Committee, and informed consent
was taken from the parents of all the children. The
selected sampling was done taking into consideration
the following inclusion and exclusion criteria.
Plaque samples were collected with a sterile probe tip
from four specific sites including [Figure 3]:[6]
• Buccal surface of right maxillary first molar
• Labial surface of maxillary incisor
• Lingual surface of mandibular incisor
• Lingual surface of the left mandibular first molar.
Collected plaque samples were evenly and thoroughly
distributed on square tip plaque strip  (Dentocult SM
Strip Mutans Kit of Orion Diagnostica, Finland) using
sterile ear buds. The same procedure was repeated
after 1 week and 2 weeks.

Inclusion criteria Saliva sample collection

• Systemically healthy patients Before collection of salivary samples, the subjects were
• No fixed or removable orthodontic appliances or asked to chew paraffin tablets for 1 min and then spit
removable prosthesis the remaining saliva. Saliva samples were collected by
• No use of green tea catechin as oral rinse earlier pressing the round tip salivary strips against the dorsal
• No history of oral prophylaxis done for at least surface of tongue and removed with gently closed lips
3 months before the study [Figure 4]. The same sample collection procedure was
• Mixed dentition period repeated after 1 and 2 weeks.
• Decay‑Missing‑Filled/decay‑missing‑filled score
>4.
After collection of both saliva and plaque samples,
the strips were placed in the labeled culture vials and
Exclusion criteria incubated at 37°C for 48 h.
• History of fluoride treatment in the past 2 weeks
• Eating before 1–2 h before sample collection After incubation, the colony counts of S. mutans
• Use of antimicrobial mouth rinse before several were interpreted using model chart provided by
hours of sample collection manufacturer and score were given from 0 to 3
• History of antibiotic therapy in the subjects within, after comparing the incubated strips with model
previous 3 months. chart [Figures 5 and 6].
After selection of the children according to inclusion
and exclusion criteria, oral prophylaxis of all the Table 2: Ingredients of mouth wash
subjects was done using an ultrasonic scaler. Then, Name of the ingredient Quantity Purpose
the subjects were instructed to abstain from any oral Catechin 250 mg Medicament
hygiene measure for next 24 h. Sodium chloride 100 mg Antiseptic
Sodium benzoate 50 mg Preservative
Plaque sample collection Sodium bicarbonate 50 mg Alkaliser
After 24 h of oral prophylaxis, baseline samples were Amaranth solution 0.012 ml Colouring agent
collected from subjects. Culture vials were taken at Distilled water q.s. 100 ml Vehicle
room temperature 1  h before the sample collection.
Bacitracin discs were placed in the culture vials 15 min
before the sample collection and vials were shacked
thoroughly.

Figure 2: Green tea catechin mouth wash Figure 3: Plaque sample collection

Journal of Indian Society of Pedodontics and Preventive Dentistry | January-March 2017 | Vol 35 | Issue 1 | 43
Goyal, et al.: Effect of green tea mouth rinse on S. mutans in plaque and saliva in children

After collecting baseline samples, the subjects were
given the green tea catechin mouth rinses and were
asked to use 10 ml of mouth wash for 3 min twice in a
day to rinse under supervision for 2 weeks.
The subjects were then asked to start maintaining their
regular oral hygiene measures.
Statistical analysis
The values recorded and tabulated separately and
compared. The results were statistically analyzed
using Student’s paired t‑test.
After 1  week, the mean change from baseline value
was 0.67 ± 0.47, and after 2 weeks, it was 1.13 ± 0.49,
which is statistically highly significant in relation to
P < 0.001 for both 1‑ and 2‑week intervals from baseline
[Table 6 and Graph 4].
Discussion
Green tea which has been considered to have medicinal
benefits and is a healthful beverage since ancient times
consisting mainly of polyphenols (catechins) which
constitute up to 30% of the dry weight. The major

Results
Mean change and standard deviation of S. mutans
count in plaque followed by mouth rinsing with green
tea catechin mouth wash subjects were baseline value
2.67 ± 0.47 that was reduced after rinsing with green tea
catechin mouth wash to 1.80 ± 0.74 and 1.20 ± 0.54 after
1 week and 2 weeks, respectively [Table 3 and Graph 1].

After 1  week, the mean change from baseline value

was 0.87  ±  0.62, and after 2  weeks, it was 1.47  ±  0.50
which is statistically highly significant in relation to
P < 0.001 for both 1‑ and 2‑week intervals from baseline
[Table 4 and Graph 2].

In subjects, baseline value of S. mutans in saliva was

2.14 ± 0.72 that was reduced after rinsing with green tea Figure 4: Saliva sample collection
catechin mouth wash to 1.47 ± 0.72 and 1.00 ± 0.73 after
1 week and 2 weeks, respectively [Table 5 and Graph 3].

Table 3: Mean ± standard deviation of Streptococcus

mutans in green tea catechin subjects from baseline
to various intervals in plaque
Group Interval
Baseline At 1st week At 2nd week
Green tea catechin 2.67±0.47 1.80±0.74 1.20±0.54
(plaque)

Figure 5: Dentocult SM Strip mutans Model chart

Table 4: Mean change ± standard deviation of
Streptococcus mutans in green tea catechin subjects
from baseline to various intervals in plaque a b c
Interval
At 1st week At 2nd week
Mean change±SD 0.87±0.62 1.47±0.50
P <0.001 <0.001
Significance HS HS
SD=Standard deviation; HS=Highly significant

Table 5: Mean ± standard deviation of

Streptococcus mutans in green tea catechin subjects
from baseline to various intervals in saliva
Group Interval
Figure  6: Slide showing Streptococcus mutans CFU/ml in plaque
Baseline At 1st week At 2nd week
and saliva at (a) baseline  (Class‑3),  (b) 1  week  (Class‑1) and
Green tea catechin (saliva) 2.14±0.72 1.47±0.72 1.00±0.73 (c) 2 weeks (Class‑0) intervals

44 Journal of Indian Society of Pedodontics and Preventive Dentistry | January-March 2017 | Vol 35 | Issue 1 |
 Goyal, et al.: Effect of green tea mouth rinse on S. mutans in plaque and saliva in children
Graph 1: Mean ± standard deviation of Streptococcus mutans green tea
catechin subjects from baseline to various intervals in plaque
Graph 3: Mean ± standard deviation of Streptococcus mutans in green
tea catechin subjects from baseline to various intervals in saliva
catechins in green tea are  (+)‑catechin  (C), (‑)‑EC,
(+)‑gallocatechin, (‑)‑ECG, (‑)‑EGC, and (‑)‑EGCG.[6]
Numerous health benefits of green tea and its
constituents have been reported. Green tea is a
powerful antioxidant and has anti‑inflammatory
properties.[7]
In subjects, baseline value in plaque was 2.67  ±  0.47
that was reduced after rinsing with green tea catechin
mouth wash to 1.80 ± 0.74 and 1.20 ± 0.54 after 1 week
and 2 weeks, respectively [Table 3 and Graph 1].
Otake et al. showed that a mixture of simple catechins
[8]
extracted from green tea (consisting mainly of EGCG,
EGC, and its epimer gallocatechin, EC and ECG), at
100  mg/L  (i.e.,  less than “cup of tea” concentration),
caused very substantial inhibition of adherence of
S. mutans to saliva‑coated hydroxyapatite.
Several workers have demonstrated that the enzymatic
activity of glucosyl transferase from S. mutans and
Streptococcus sobrinus is inhibited by tea catechins.
Otake et al.[8] and Hattori et al.[9] both found that EGCG
and ECG were more active in inhibition of glucosyl
transferase than other catechins, but the former group
reported greater potency (EGCG at 167 mg/L caused
91% inhibition) than that found by the latter (EGCG at
450 mg/L caused 50% inhibition).
Journal of Indian Society of Pedodontics and Preventive Dentistry | January-March 2017 | Vol 35 | Issue 1 |
Graph 2: Mean change ± standard deviation of Streptococcus mutans in
green tea catechin subjects from baseline to various intervals in plaque
Graph 4: Mean change ± standard deviation of Streptococcus mutans
in green tea catechin subjects from baseline to various intervals in
saliva
Table 6: Mean change ± standard deviation of
Streptococcus mutans in green tea catechin subjects
from baseline to various intervals in saliva
Interval
At 1st week At 2nd week
Mean change±SD 0.67±0.47 1.13±0.49
P <0.001 <0.001
Significance HS HS
SD=Standard deviation; HS=Highly significant
Mean change and standard deviation of S. mutans
count in plaque followed by mouth rinsing with
green tea catechin mouth wash from baseline
to various intervals in subjects are shown in
Table 4 and Graph 2.
After 1 week, the mean change from baseline value was
0.87 ± 0.62, and after 2 weeks, it was 1.47 ± 0.50 which
is statistically highly significant in relation to P < 0.001
for both 1‑ and 2‑week intervals from baseline.
EGCG and EC have been reported to disrupt
reconstituted bacterial membranes in a model
system.[10] Tea and its components show broad‑spectrum
antibacterial activity.[11]
45
Goyal, et al.: Effect of green tea mouth rinse on S. mutans in plaque and saliva in children
A study done by Awadalla et al.[12] 2009 to assess the
possible protective properties of green tea on oral
health, using the following measurements: S. mutans
count in saliva and plaque, salivary and plaque pH
values, gingival bleeding index (GBI). The results of this
study showed that there was a statistically significant
difference among subjects pre‑  and post‑rinsing with
2% green tea for 5  min concerning S. mutans count
in saliva and plaque, salivary and plaque pH values,
and GBI. This study supports the effectiveness of
local application of green tea as antibacterial and
anticarcinogenic material as it decreases the acidity of
the saliva and plaque.
Tehrani et al. compared green tea with sodium fluoride
mouth rinse on salivary S. mutans. Green tea mouth
rinse resulted in significant reduction of colony
number of salivary S. mutans and Lactobacillus, which
is comparable with sodium fluoride mouth rinse.[13]
Neturi et  al. compared green tea with chlorhexidine
(CHX) and plain water on plaque and found that green
tea and CHX were equally effective against S. mutans.[14]
In some other study, green tea and honey mouth rinse
were assessed on salivary S. mutans. A  statistically
significant reduction in the average number of
S. mutans at baseline and postintervention in the
children who were assigned to the honey  (P  =  0.001)
and green tea (P = 0.001) groups was found.[15]
In a recent study against salivary S. mutans, green tea
mouth rinse was found to be significantly better than
CHX mouth rinse (P = 0.005).[16]
Conclusion
Hence, finally, our study showed that green tea
catechin is effective as a mouth wash against S. mutans
and having better action in plaque as compared to
saliva.
Green tea catechin can be used as an adjunct to
commercially available mouthwashes because:
• Taste of green tea is well accepted by the children
• Effective in reducing the number of S. mutans in
plaque as well as in saliva
• Cost‑effective when compared to commercially
available mouthwashes.
Since it is just a beginning, further studies can be
conducted to evaluate systemic effects, long‑term trials,
shelf life, and other oral microorganism antibacterial
efficacy with a larger sample size.
Financial support and sponsorship
Nil.
46 Journal of Indian Society of Pedodontics and Preventive Dentistry | January-March 2017 | Vol 35 | Issue 1 |
Conflicts of interest
There are no conflicts of interest.
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