Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

“Comparative Evaluation of Effects of Rinsing with

Green Tea on Streptococcus Mutans Count in Plaque
with that of Chlorhexidine Mouth Wash and Plain
Water: A Randomized Controlled Trial”
Abstract:- I. INTRODUCTION
Introduction: Chlorhexidine mouthwash is
conventionally used antibacterial agent but it has Streptococcus mutans is the major etiological agent
reported side-effects on its long term use. Herbal responsible for initiation of dental caries , a gram negative
mouthwash can be an effective alternative to gold facultative anaerobic bacteria. [1] Hence reducing the
standard chlorhexidine mouth wash on long term use. Streptococcus Mutans count in the oral cavity is the best
way to reduce the incidence of dental caries . Chlorhexidine
Materials and Methods: It was a single centre multiple has a broad antibacterial activity, very low toxicity, and
arm parallel placebo controlled randomized controlled more substantivity. Chorhexidine is a bis- biguanide that
trial. A total of 90 participants were selected for the significantly reduces the total number /count of bacteria in
study and were divided in 3 groups ie Group A(2% the oral cavity. It interferes with the adherence factors of
Green Tea ), Group B(0,2% Chlorhexidine) , Group these cariogenic micro-organisms that are present on the
C(Distilled water) by envelope method. The participants tooth surface.[3] But Chlorhexidine when used for a
were stratified according to gender to ensure that equal longer time period causes various side effects like staining
number of males and females participate in the study. of tooth, alterations in taste and mucosal erosions and
The study was conducted in two phases. In Phase 1 lesions instead of its beneficial antiplaque effects . [1]
Baseline plaque samples were collected before rinsing
with mouthwash In 2nd phase plaque samples were Herbal products are considered a part of Indian
collected after 7 days of using the rinse . Baseline culture since decades and is used for the treatment of
plaque samples and plaque samples collected after 7 days various ailments affecting systemic health and oral health
of rinsing were subjected to microbial analysis to assess . Green tea is one of the herbal products with medicinal
the effect of intervention in reducing and antioxidant properties which has a positive effect on
Streptococcusmutans count in plaque after using the overall systemic health . [2]
rinse . Paired –t test was used for within group
comparison and One Way ANOVA test was used for It also has anticarcinogenic effects and is considered
between group comparison. A p-value < 0.05 was as one of the most efficient and powerful antimicrobial and
considered statistically significant . antiviral agent. Polyphenols is one of the major component
Results: The mean reduction in Streptococcus mutans in green tea which inhibits the growth of pathogenic
count in plaque sample was more in green tea and bacteria that cause oral and periodontal diseases and
Chlorhexidine group in comparison to plain water group thereby directs positive impact and improves oral and
and the values were statistically significant(p<0.05).On gingival health. [3] The polyphenols target the virulence
comparing mean reduction in Streptococcus mutans factors of the gram negative Streptococcus mutans and also
count in plaque sample among Chlorhexidine and Green inhibit the adherence of micro-organisms to the tooth
tea group . Chlorhexidine group showed more reduction surface [4] thereby decreasing the acid production in the
in Streptococcus mutans count but there was no oral cavity and increase the pH of the saliva . These
statistically significant difference between both the activities confers anticariogenic properties to green tea .[5]
groups (p>0.05). Epidemiological studies done in the past are more
Conclusion: Chlorhexidine mouthwash is still the most centered towards evaluation of the systemic health
superior antibacterial agent , but side effects on its long benefits of green tea. Studies done pertaining to its
term use cannot be neglected . Green tea mouthwash , a antimicrobial effect on Streptococcus mutans count as
herbal antibacterial agent is equally efficacious and mouth rinse and further comparative evaluation of its
effective as conventional chlorhexidine mouth-wash with efficacy with that of chlorhexidine mouth wash are very
few . The aim of the study is to evaluate the effects of
pleasant taste and no side –effects .Hence , it can be
used as an alternative to conventional chlorhexidine rinsing with Green tea on Streptococcus mutans count in
mouth-wash on routine basis as an adjunct to plaque.
mechanical plaque removal for a longer period of time.

Keywords:- Mouthwash , Green Tea , Chlorhexidine , Plain

Water , Plaque , Streptococcus Mutans.

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II. MATERIALS AND METHODS microbiologist and statistician remained blinded to the
mouthrinse which were given to the study participants .To
The study was conducted in the Department of ensure this the interventions were kept in brown colored
Public Health Dentistry , BBDCODS BBDU, Lucknow opaque containers. They were each labeled as either code
city, Uttar Pradesh in collaboration with Department of “A”, “B” or “C”. The codes were broken only after the
Microbiology , Ram Manohar Lohiya Institute of Medical clinical trial was completed. The ethical clearance was
Sciences, Lucknow for microbial analysis.The study is a, obtained fromInstitutional Review Board and ethical
parallel multiple arm , in vivo comparative experimental clearance was obtained and permission to conduct the pilot
trial designed to assess the effect of rinsing with Green tea study and the main study in the colleges was obtained by the
on Streptococcus mutans count in plaque.Simple random Institutional Ethical Committee of BabuBanarasi Das
sampling technique was used to select the subjects for the College of Dental Sciences ,BabuBanarasi Das University ,
study .Inclusion Criteria of the study included both genders Lucknow ,Uttar Pradesh.The purpose of the study was
,subjects of 18-25 years of age, subjects without known explained in detail using simple and comprehensible
systemic illness, subjects with plaque score of 1 according language with the help of the patient information letter.
to Silness and Loe Plaque Index .Pilot study was conducted Informed consent was obtained from all the research
in BabuBanarasi Das College of Dental Sciences participants.Fresh green tea (packing date less than one
,BBDU,Lucknow in the month of December 2020 . A total month) was procured from local market which is available
of 24 participants 8 in each group were recruited for the in the form of green tea dip bags(LIPTON GREEN TEA ,
pilot study to check for the feasibility of the study and Manufacturer: Lipton, Country of Origin : India).Two
subjects included in the pilot study were excluded from the percent green tea was prepared with 2 grams of green tea dip
main study. Practical issues like the acceptability of the bag dipped in 100 ml hot water for five
mouthrinsesby the subjects, plaque collection, the time taken minutes.Commercially available 0.2% Chlorhexidine
to transport the plaque samples to the lab, microbiological gluconate mouthwash (Hexidine , ICPA Health Products Ltd
analyses of the plaque samples were all tested during this India)) was used as positive control . Chlorhexidine was
pilot study. Appropriate adjustments were made in the chosen as it is hailed as the “gold standard” mouthwash.
methodology and implemented in the main study.The Chlorhexidine exhibits both bactericidal and bacteriostatic
sample size for the present study was determined effects depending on the concentration. However, the
scientifically. The data required for determining the sample property of substantivity is a unique characteristic of this
size was obtained from a previous literature. [6] [7] chemical plaque control agent. Previous researches have
documented the initial suppression of Streptococcusmutans
Sample size was calculated using G-power analysis in the plaque after administration of chlorhexidine.Plain
software .Making an allowance of 10% for attrition, the water was used as placebo or negative control .The study is
required sample size for each group is 30. There are 3 a single centre parallel placebo controlled experimental trial
groups hence taking 30 subjects in each group 90 healthy .After obtaining informed consent from the study
volunteers were involved in the present study.Prior to the participants , eligible participants fulfilling the inclusion
study , investigator [1] was trained and calibrated in the criteria were allocated one among the 4 groups( via
Department of Public Health Dentistry, BabuBanarasi Das envelope method).The selected 90 participants were divided
College of Dental Sciences . Training of the investigator was into four groups:30*3) namely Group A: 2% Green
carried out on the out-patients under the guidance of a tea(Treatment group),Group B: 0.2% Chlorhexidine
trained person. About eight subjects were examined to gluconate , Group C: Plain water . All the study participants
assess the consistency of intra-examiner reproducibility and were given 10 ml of three different mouth rinses depending
intra-examiner reliability . The agreement for most upon the group assigned to them (via enveloped method).All
assessments was expected to be 90%.ADA specification the subjects were instructed to rinse with the given mouth
type III examination was followed.A study specific rinses for 30 seconds twice daily for 7 days after 30 minutes
proformawas used to record and evaluate demographic of brushing . The study participants were refrained from
details ,medical and dental history of each using any other medicated prophylactic tooth paste ,
participant.Recording clerk was assigned to record the data interdental cleaning aids and other mouth-rinses except the
of study participants. The Recording clerk was well versed intervention(three different mouth rinses) during the study
pertaining to terms and codes of the survey prior to survey phase and were instructed to avoid drinking tea , coffee 1
to avoid any error while recording data of study hour after using the mouthwash. The subjects were
participants.Participants were allocated to groups on the reminded at regular intervals about the instructions through
basis of envelope method. A box containing concealed phone calls.The study was done in two phases.In the first
envelopes was used for this purpose. Each envelope carried phase plaque sample of all the 90 study participants were
either code “A”. “B” or code “C”. The enrollment was collected from lingual surface of lower molars using sterile
stratified by gender to ensure that equal number of males cotton bud before they were subjected to three different
and females were allocated to each group (n=15 males and interventions .The collected plaque sample was transferred
15 females in each group) .Participants who were selected to to sterile test tube containing transport media
participate in the study were each asked to pick a concealed (thioglycolate-broth) and was send to the laboratory for
envelope from the box. Participants were allocated to the microbial analysis within half an hour of sample collection
respective group.The study was a triple blind clinical trial .This was done to obtain the base line analysis .In the
.The study subjects remained blind to the type of second phase all the 90 subjects were instructed to rinse
mouthrinse they were given. The investigator , with the 10 ml of given mouth rinses for 30 seconds twice

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Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
daily for 7 days . After 7 days they were recalled and plaque subjected to statistical analyses to observe the efficacy of
sample was collected in the same manner as described in Group A ( 2% green tea) mouthrinse on Streptococcus
first phase and was send to the laboratory for microbial mutans count and further to compare its efficacy with gold
analysis within half an hour of sample collection. standard mouthrinse or positive control Group B (0.2%
chlorhexidine) mouthrinse and placebo or negative
III. MICROBIAL ANALYSIS control Group C (Plain water) rinse.
The plaque samples collected from subjects assigned IV. DATA ANALYSIS
to three different groups that is Group A ( 2% green tea ),
Group B(0.2% chlorhexidine ) and Group C(Plain water) Data was entered in Microsoft Excel and the analysis
were diluted with sterile saline in a ratio of 1:1000 . The was done using SPSS Software version 20(IBM
plaque samples were streaked on blood agar plates .The Corporation). Paired t test was used for intragroup
plates were incubated for 48 hours at 37 degree Celsius and comparison between baseline and test values and analysis of
bacterial colony count was done as Colony forming Unit variance (ANOVA) was used for multiple intergroup
(CFU/ml).Total Colony count was calculated manually comparison . Level of significance (p value <0.05) was
using the formulae: Total Colony forming unit/ml=total considered as statistically significant for all of the above
colony count * 1000. Further the colony count was mentioned statistical tests.

Assessed for eligibility

n=300

Excluded n=180

Eligible Subjects =120

Randomization n=90

Group A Group B Group A

(Green tea) (Chlorhexidine) (Plain Water)
N=30 N=30 N=30

Data collection , Analysis and Interpretation

Fig. 1: Schematic representation of methodology

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Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology
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V. RESULTS rinsing with Plain Water Mean Streptococcus Mutans
count (CFU/ml) was 31.97+1.95 and after 7 days of rinsing
Demographic details of the study participants it was 31.08+ 1.63. There was no statistically significant
represents that 90 study subjects participated in the study reduction in the mean streptococcus count before and after 7
among which 30 study subjects were included in Group A( days of rinse with plain water (p>0.05).(Table 2)
2% Green Tea ) , Group B (0.2% Chlorhexidine) , Group C
(Plain(Group A( 2% Green Tea ) , Group B (0.2% Pairwise comparison of mean streptococcus mutans
Chlorhexidine) , Group C (Plain Water) respectively. 15 count (CFU/ml) between groups (Group A(2% Green Tea)
males and 15 females were included in each of the three , Group B (0.2% Chlorhexidine) and Group C(Plain Water)
groups.(Table 1) over two phases (Phase 1(Before Rinse)) , Phase 2 (7 days
after rinse).On pairwise comparison between Group A (2%
On Intragroup comparison of Mean Streptococcus Green Tea) and Group B(0.2% Chlorhexidine ) there was
Mutans Count (CFU/ml) among Group A(2% Green Tea) , no statistically significant difference in mean streptococcus
Group B (0.2% Chlorhexidine ) and Group C(Plain Water) mutans count(CFU/ml before and after 7 days of rinse
over two phases(Phase 1(Before Rinse)) , Phase 2 (7 days (p<0.05) .Between Group A(2% Green Tea) and Group
after rinse).In Group A(2% Green Tea) before rinsing with 2 C(Plain Water) there was no statistically significant
% Green Tea Mean Streptococcus Mutans count (CFU/ml) difference in mean streptococcus mutans count(CFU/ml)
was 32.07+1.83 and after 7 days of rinsing it was before rinse(p>0.05) but after 7 days of rinse(p<0.05) there
24.54+1.92. There was a statistically significant reduction in was statistically significant difference between
the Mean Streptococcus mutans count before and after 7 them.Between Group B(0.2% Chlorhexidine ) and Group
days of rinse (p<0.05).In Group B(0.2%Chlorhexidine) C(Plain Water) there was no statistically significant
before rinsing with 0.2% Chlorhexidine mean difference in mean streptococcus mutans count(CFU/ml)
streptococcus mutans count (CFU/ml) was 32.25+1.87 and before rinse(p>0.05) but after 7 days of rinse(p<0.05) there
after 7 days of rinsing it was 24.09+ 1.94. There was a was no statistically significant difference between
statistically significant reduction in the Mean Streptococcus them.(Table 3)
mutans count before and after 7 days of rinse with 0.2%
Chlorhexidine (p<0.05).In Group C (Plain Water) before

Age Group A Group B Group C

(2% Green Tea ) (0.2% Chlorhexidine ) (Plain Water)
No % No % No %
<=20 years 11 36.7 17 56.7 16 53.3
>20 years 19 63.3 13 43.3 14 46.7
Total 30 100 30 100 30 100
Mean Age 21.3 years 20.7 years 20.6 years
Standard Deviation 2.0 years 2.1 years 1.8 years
** p-value 0.251
Table 1: Demographic details of the study Participants

*Chi -Square test ** p-value<0.05 is statistically significant

Gender Group A Group B Group C

(Green Tea ) (Chlorhexidine ) (Plain Water)

No % No % No %
Male 15 50 15 50 15 50
Female 15 50 15 50 15 50
Total 30 100 30 100 30 100
** p-value >0.05

*Chi -Square test ** p-value<0.05 is statistically significant

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Phases Group A Group B Group C *F **p-

( 2% Green Tea) (0.2% Chlorhexidine) (Plain Water) value value
#Streptococcus mutans #Streptococcus mutans #Streptococcus mutans
count (CFU/ml) count (CFU/ml) count (CFU/ml)
Mean Standard Mean Standard Mean Standard
Deviation Deviation Deviation
Phase 1(Before Rinse) 32.07 1.83 32.25 1.87 31.97 1.95 0.165 >0.05
Phase 2 (7 days after rinse) 24.54 1.92 24.09 1.94 31.08 1.63 135.8 <0.05
Table 2: Intergroup comparison of Mean Streptococcus mutans count (CFU/ml) among Group A(2% Green Tea) , Group B
(0.2% Chlorhexidine ) and Group C(Plain Water) over two phases (Phase 1(Before Rinse)) , Phase 2 (7 days after rinse)

* One -way ANOVA test

**p-value<0.05 is statistically significant
#
Mean Streptococcus mutans count = Colony Forming Unit/ml=Total Colony Count *103

Before rinse After rinse

Groups *p-value *p -value
Group A(2% Green Tea) : Group B(0.2% Chlorhexidine) p>0.05 p>0.05
Group A(2% Green Tea) : Group C(Plain Water) p>0.05 p<0.05
Group B(0.2% Chlorhexidine) : Group C(Plain Water p>0.05 p<0.05
Table 3: Pairwise comparison between groups (Group A(2% Green Tea) , Group B (0.2% Chlorhexidine ) and Group C(Plain
Water) over two phases (Phase 1(Before Rinse)) , Phase 2 (7 days after rinse)

** Tukey Post Hoc test

*p-value<0.05 is Statistically significant

35
Mean reduction in Sreptococcus Mutans Count

30

25

20 Before Rinse
CFU/ml)

After 7 days of Rinse

15

10

0
Group 1 (2% Group 2( 0.2% Group 3 (Plain
Green tea) Chlorhexidine) Water)

Graph 1: Mean reduction in the Streptococcus mutans(CFU/ml) count before and after 7 days of rinse in Group A(2% Green
Tea) , Group B(0.2% Chlorhexidine) , Group C(Plain Water)

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[21]
VI. DISCUSSION conducted by Axelsson P et al . Chlorhexidine has been
proven to be particularly efficient against Streptococcus
Dental plaque is made up of a diverse microbial mutans in plaque by Menendez et al. [22] In a study
community that grows as biofilm. It is embedded in a matrix conducted by Salehi et al antibacterial effects of persica
of host and bacterial polymers .[8][27] Mature dental plaque mouthwash was compared with that of standard
with a high microbial density community containing a chlorhexidine on Streptocccusmutans count . [23] Taking a
variety and diverse group of microorganisms. [9][28]Hence queue from these studies the gold standard mouthwash was
,nowdays an increased focus is given to the use of herbal used as positive control in the present study to assess the
products obtained from natural sources like plants with effect of green tea mouth rinse on Streptococcus mutans
antibacterial and anti-inflammatory activities . Green tea count in plaque.In the present study a significant reduction
(Camellia sinensis) is one of the many herbal extracts with on intragroup comparison in the Streptococcus mutans
numerous medicinal benefits due to its antibacterial and count in plaque after 7 days of rinsing with green tea and
antioxidant properties. [10],[11]Green tea comprise of a larger chlorhexidine mouth rinse was observed . With plain water
number of bioactive chemicals that is rich in flavonoids least and statistically non-significant reduction in
which includes catechins . [12][36]Catechins in green tea is Streptococcus mutans count was observed . The findings
a mixture of epigallocatechin gallate (EGCG), were similar to the study conducted by Neturi RS et al. [6]
epicatechingallate, epicatechin, and eppigallocatechin. Singh O et al [14] ,Nandan N et al [17]in whichsimilar effect
[13][37] Considering this thepresent study was conducted to was observed on rinsing with green tea and chlorhexidine
evaluate the effects of rinsing with green tea on the mouth rinse. But in these studies the time duration for
Streptococcus mutans count in plaque and to compare its which the participants were given intervention differed
efficacy with that of chlorhexidine mouth wash and plain which was 5 minutes , 14 and 21 days . Not many studies
water.In the present study students of age group 18-25 years are mentioned in the literature on the efficacy of green tea
were selected for the study The participants were randomly mouth rinse on Streptococcus mutans count in plaque
allocated to 2% green tea mouth rinse group , 0.2 % considering the same concentration of green tea mouthrinse
chlorhexidine mouth wash and plain water group . The used in the present study .On intergroup comparison
three intervention mouth rinse used in the present study was chlorhexidine showed greater reduction in the Streptococcus
similar to the intervention mouth rinse used in the study mutans count in plaque in comparison to green tea and it
conducted by Neturi RS et al [6] and Singh O et al [14]with was least with plain water after 7 days of rinse . The
difference in the concentration of chlorhexidine mouth wash reduction in Streptococcusmutans count in plaque was
which was taken as 0.12% in both studies . In the study statistically significant on intergroup comparison after 7
conducted by Singh O et al [14] Colgate Plax green tea days of rinse. . The findings was similar to the study
mouth wash which is readily available in the market was conducted by Neturi RS et al. [6] Singh O et al [14] ,Nandan
used which was different from the green tea mouth wash N et al [17], Jennifer F et al [24] , which showed the similar
used in the present study which was prepared manually by effect on Streptococcus mutans count in plaque on rinsing
boiling 2g green tea bag in 100 ml of hot water for which with green tea , chlorhexidine and plain water as in the
green tea bags(Lipton green tea ) that is readily available in present study .
the market was used.In the present study participants were
included in the study on the basis of plaque index given by On pairwise comparison between green tea mouth
Silness and Loe . [15] Participants with plaque score 1 were rinse and plain water , chlorhexidine mouth wash and plain
selected for the intervention .Green tea mouthrinse water a statistically significant reduction in streptococcus
andchorhexidinemouthrinse decreased plaque score when mutans count was observed after 7 days of rinse with the
used regularly for 7 days . [5] Considering this in the present mouthwashes .
study the time period for which the participants were given
intervention was 7 days. It was similar to the study Considering the findings of the present study green tea
conducted by Kaur H et al [16] The study conducted by mouthrinse and chlorhexidine mouthwash are highly
Kaur H et al [5].further concluded that participants were effective in reducing Streptococcus mutans count in plaque
kept on a follow-up period of 7 days. In a similar study in comparison to plain water. The findings of the present
conducted by Kamath N et al [15] , Nandan N et al [17]the study was similar to the study conducted by Neturi RS et al
[6]
time period for which the participants were subjected to Singh O et al [14] ,Nandan N et al [17]which showed
intervention mouth rinse group that is green tea group and statistically significant reduction in Streptococcus count in
chlorhexidine mouth rinse group was 15 [18] days and 21 plaque on pairwise comparison between green tea mouth
days respectively [19] and showed similar effects on rinse and plain water , chlorhexidine mouthwash and
cariogenic bacterias in plaque similar to the study conducted plain water .
with 7 days[20] .The participants were instructed to rinse for
30 seconds with the allocated mouth rinse after 30 minutes On pairwise comparison between green tea mouthrinse
of brushing The participants were further refrained from and chlorhexidine mouth wash chlorhexidine showed more
using medicated prophylactic tooth paste and from drinking reduction in Streptococcus mutans count in comparison to
coffee and tea 1hour after the use of mouth -rinse as it may green tea mouthrinse but there was no statistically
neutralize the beneficial effect of mouth rinses which was significant difference between them which showed that
similar to the study conducted by Kaur H et al green tea mouthrinse is equally effective in reducing
[20].
Chlorhexidine mouthwash has been found to be Streptococcus mutans count as chlorhexidine mouth wash .
beneficial in decreasing plaque and gingivitis in a study

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The findings of the study was similar to the study conducted IX. RECOMMENDATIONS
by Neturi RS et al. [6] Singh O et al [14] ,Nandan N et al [17].
More extensive studies should be done involving
Considering the findings of the present study and more number of participants from different age groups with
herbal properties of green tea mouthrinse , it can be used longer follow-up period .Studies should also be done
as an alternative to chlorhexidine mouth wash due to its including Decayed , Missing , Filled teeth Score of the
beneficial antibacterial effect on cariogenic bacteria that is participants to evaluate the antibacterial effect of green tea
Streptococcus mutans in plaque without any side effects mouthwash on incidence of dental caries .Comparative
which is commonly seen with conventional chemical Studies should be done to evaluate the efficacy of green tea
containing chlorhexidine mouth wash. mouthrinse at different concentration and its effect of green
tea on different oral microbes.
VII. CONCLUSION
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