People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023

Comparative Evaluation of the Effects of Black Tea

Extract Mouthrinse and Chlorhexidine Mouthwash on

Salivary Streptococcus Mutans Load

Ruchita Khade1, Swati Saawarn1, Shubhangi D Mishra2, Einstein A3, Anushree Rathore1,

Tarjani Shankar1

1Department of Oral Pathology and Microbiology, Bhabha College of Dental Sciences, Bhopal, ,

2Department of Oral Pathology and Microbiology, NIMS Dental College and Hospital, NIMS University

Rajasthan,3Department of Oral Pathology, Thai Moogambigai Dental College and Hospital, Chennai.

ABSTRACT:

Background- Dental caries is one of the most frequent oral health problems. The present study shows

the antibacterial effect of black tea extract on salivary Sterptococcus Mutans load.

Materials & Methods- The study was conducted on 125 individuals. The differences in the Colony

Forming Units and count-scores of S.mutans were analyzed in salivary samples collected from

individuals before and after administration of 2% black tea extract mouth-rinse and chlorhexidine

mouthwash(CM).

Results- There was a statistical difference in mean salivary S. mutans colony count and mean count-

score before and after administration of black tea extract mouth-rinse (p = 0.0003) and chlorhexidine

mouthwash (p = 0.0002) respectively. Hence, it was found that there is no statistically signiﬁcant

difference in the fall of S.mutans load due to black tea mouth-rinse and chlorhexidine mouthwash.

Conclusions- A 2% black tea extract mouth-rinse signiﬁcantly reduces salivary S.mutans load,

irrespective of age and gender. Also, it is an effective natural anti-cariogenic agent with no known

implicated side effects.

KEYWORDS: dental caries; oral health; streptococcus mutans; black tea extract mouth-rinse;

chlorhexidine mouthwash.

Address for correspondence : Dr Ruchita Khade, Department of Oral Pathology and Microbiology, Bhabha College of Dental Sciences,

Bhopal-462026 , E-mail: ruchitakhade1@gmail.com

Submitted: 23.05.2023, Accepted: 14.06.2023, Published: 26.06.2023

INTRODUCTION:

Dental caries is a public health problem

throughout the world. In the western world, the

factors, socio-economic and nutritional status

influence the likelihood of caries developing and its

speed of progression, so that caries is truly a

prevalence of caries has declined, but 5-20% multifactorial disease.[ 2 ] The association of

population still remain at high risk.[1] Many factors,

both local like diet, tooth structure and anatomy, saliva,

plaque, crevicular fluid, bacteria and systemic like age,

gender, race, religion, culture, familial and genetic

Streptococcus mutans (S.mutans) and dental caries

was first reported by Clarke (1924). Since then, the

experiments with gnotobiotic animals have revealed

mutans streptococci to be the main etiological

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How to cite this article: Khade R, Saawarn S, Mishra SD, Einstein A,

Rathore A, Shankar T. Comparative Evaluation of the Effects of Black

Tea Extract Mouthrinse and Chlorhexidine Mouthwash on Salivary

Streptococcus Mutans Load. PJSR. 2023;16(1):14-19.

Research Article

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People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023

Khade R et al. Effects of Black Tea & Chlorhexidine on S.mutan

microorganisms in causation of dental caries.

The S.mutan is a facultatively anaerobic, gram-

positive coccus. The mutans streptococci comprise of a

group of seven species (mutans, sobrinus, cricetus,

rattus, downeii, ferus and maccae). S.mutans and

Streptococcus sobrinus are the predominant species

isolated from human saliva and dental plaque.[3] These

organisms are unique in their cariogenic potential. They

are acidogenic and aciduric and once established, they

can survive even in unfavourable conditions. Besides,

the microorganisms in dental plaque degrade the dietary

carbohydrates producing lactic acid leading to localized

demineralization and the eventual formation of dental

caries. S.mutans also encourage the accumulation and

adherence of plaque biofilm by metabolizing sucrose

into sticky glucan. It is now confirmed that S. mutans

are the major bacteria responsible for the initiation of a

carious lesion followed by Lactobacillus species which

may be responsible for caries progression.[4]Thus, the

obvious role of S mutans that was reflected all the way

long in the causation of dental caries, justifies the need

for its elimination or reduction in order to prevent

occurrence of carious lesions.

Drug resistance and side effects encountered

with the use of synthetic drugs has led to the surge for

novel and safe alternatives. Since ancient times, plants

have proven to be an archetypal source of medicine.

One such plant of high medicinal use is the black tea

(Camellia sinensis). The leaves of this plant are usually

handpicked and based on the processing of the leaves

three different types of tea are produced, namely green

tea (non-fermented), Oolong tea (semi-fermented) and

Black tea (fermented). Black tea being the fermented

type possesses more raw nutrients and health effective

compounds than the other two types.[5]Data available

also enumerates some of the anticariogenic actions of

certain components of black tea like bringing about

remineralization of the dental hard tissues due to the

release of calcium, phosphorus and fluoride ions.[6] The

purpose of this study was to analyse the effect of black

tea extract mouth-rinse,a natural measure with reduced

side effect, in the prevention of dental caries.

MATERIALS & METHODS:

This study was carried out with the approval of

Institutional Ethical Committee and Bhabha College of

Dental Sciences, Bhopal. The study was conducted in

the Department of Oral Pathology & Microbiology on

individuals in the age range of 21-40 years and a written

informed consent for the procedure was obtained from

them.

A total of 125 individuals in the age range of

21-40 years were randomly selected as per the

inclusion and the exclusion criteria from those

reporting to the Bhabha College of Dental Sciences,

Bhopal. They were divided into three groups (a)

administered Black tea extract mouth-rinse (BTEMR)

in 50 subjects (b) administered Chlorhexidine

mouthwash (CM) in 50 subjects and (c) administered

distilled water in 25 subjects.

Inclusion Criteria-

Individuals with or without dental caries in the

age group of 21-40 years.

Exclusion Criteria-

a) Completely or partially (more than 4 teeth

missing) edentulous patients; b) patients who had

received any antibiotic therapy in the last 14 days prior

to the study; c) patients who underwent topical fluoride

application in the last 3 days or mouth wash gargles

within the last 12 hours prior to the study and d)

patients with any known systemic disease.

BTEMR made by 2 gm of dried black tea

leaves were boiled in 100 ml of tap water for 3 minutes

in a stainless-steel utensil. The solution was allowed to

cool for 10 minutes and then sieved into a disposable

glass. This BTEMR was prepared fresh before use.

The individuals were briefly explained about

the procedure prior to sample collection. They were

instructed to maintain their normal oral prophylaxis

and were instructed to avoid brushing or eating 1 hour

prior to the saliva collection. Unstimulated saliva

sample was collected from all the individuals

participating in the study prior to and after the

administration of BTEMR. Each individual of the

study population was instructed to spit around 1-2 ml

of saliva in a sterile wide mouth glass bottle. After

having collected the first saliva sample, the individuals

were instructed to gently rinse their mouth with 10-20

ml of the prepared BTEMR for 2 minutes and a second

saliva sample was collected again after 30 minutes in

another sterile glass bottle. The same procedure was

followed for the other two groups, where instead of

BTEMR, Chlorhexidine Mouthwash (CM) or distilled

water were administered.

Culture media was prepared as follows:90 gm

of Mitis Salivarius dehydrated agar was added to 1 litre

of distilled water and boiled on a Bunsen burner to

dissolve completely. To this 20 gm of sucrose per 100

ml was added. This solution was then sterilized by

autoclaving for 15 minutes at 15 lb per square inch at

1210 C. The solution was allowed to cool to

500C.Potassium tellurite (0.1 mg/ml), bacitracin (0.2

Khade R et al. Effects of Black Tea & Chlorhexidine on S.mutan

People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023

Table 1: Distribution and comparison of the salivary S.mutans colony count (CFU/ml) and the colony score in all the subjects

before and after the administration of BTEMR.

Mean

Standard Deviation

Difference of means

p-value

CFU/ml

Before 50

8.3700

5.9713

<0.0001 (HS)

(x 104)

After 50

4.2566

3.6510

4.1134

Colony

Before 50

1.3448

0.6695

0.0003 (HS)

Score

After 50

1.1875

0.7124

0.1573

HS: Highly Statistically Signiﬁcant

Graph 1: Distribution and comparison of the salivary S.mutans colony count

(CFU/ml) in all the subjects before and after the administration of BTEMR.

units /ml) and human blood (2% v/v) were added to this

solution. It was then well mixed and poured equally into

sterile petri dishes.

0.1ml of diluted 10-2saliva sample was cultured

on agar by spread plate technique. The plates were then

incubated at 37oC for 48 hours in plastic bags inflated

with expired air to enhance the carbon dioxide.

The number of colonies grown on the agar

surface were counted with a magnifying glass in front of

an illuminated source. Number of Colony Forming

Units (CFUs) per ml of saliva was calculated as follows.

CFU in 0.001 ml = 'x' (Number of colonies)

Therefore, CFU/ml = 'x' × 1000

Thus, colony forming units (CFU)/ml of saliva was

calculated and scored for each saliva sample.

Scoring of salivary S. mutans count:

0 < 10,000 CFU/ml

1 < 100,000 CFU/ml

2 = 100,000-1,000,000 CFU/ml

3 > 1,000,000 CFU/ml

CFU Colony Forming Unit.

STATISTICALANALYSIS:

Paired t test was applied to analyse the

difference in the CFU/ml. p-value of <0.05 was

considered significant for the differences in the mean of

CFU/ml and S mutans count scores before and after

rinsing with BTEMR, CM or distilled water.

RESULTS:

Graph 2: Distribution and comparison of the salivary S.mutans colony

score in all the subjects before and after the administration of BTEMR.

125 individuals in the age range of 21-40 years, divided

into three groups and were administered BTEMR (n =

50), CM (n = 50), and distilled water (n = 25) Salivary

samples were collected from each individual twice; the

first sample which was collected prior to the

administration of BTEMR, CM or distilled water, and

the second sample was collected 30 min after the

administration of BTEMR, CM or distilled water.

We compared S.mutans CFU/ml with the

Graph 3: Comparison of the mean salivary S.mutans load before and after

the administration of BTEMR.

colony score before and after administration of

BTEMR and found there was drastic reduction in

colony score from 1.3448 to 1.1875. These results were

highly significant [Table 1] [Graph 1, Graph 2 and

Graph 3]. We found in our study that the results were

The present study included a study group of almost similar when we compared the distribution and

Khade R et al. Effects of Black Tea & Chlorhexidine on S.mutan

People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023

Table 2: Distribution and comparison of the salivary S.mutans colony count (CFU/ml) and the colony score in all the subjects

before and after the administration of CM.

Mean

Standard Deviation

Difference of means p-value

CFU/ml (x 104) Before

7.1700

9.0713

After

3.9566

4.7630 3.2134 <0.0001 (HS)

Colony Score Before

1.1432

0.6787 0.0002 (HS)

After

0.8541

0.5612 0.2891

HS: Highly Statistically Signiﬁcant

Table 3 : Distribution and comparison of the salivary S.mutans colony count (CFU/ml) and the colony score in all the

subjects before and after the administration of distilled water.

NS: Non Signiﬁcant

colony score before and after the administration of CM

and BTEMR. These results were also highly significant

[Table 2]. However, when we used distilled water as the

expectorant the results were not significant [Table 3].

DISCUSSION:

Among the various health issues being faced in

day-to-day life, dental caries is one of the most common

chronic diseases of modern times. Dental caries is

associated with the frequency of fermentable

carbohydrate intake. Also, certain dietary substances

are more cariogenic than others. Simple sugars (e.g.,

Sucrose) are more cariogenic than complex sugars

(e.g., Starch).[7]Caries is initiated by S.mutans, whereas

Lactobacilli help further progression of the lesion

inside the dentin.[8]Sakeenabi and Hiremath, Ravindran

et al. and Pannu et al, concluded that S.mutans are the

major pathogens responsible for dental caries.[9-12]

The microﬂora in dental caries is highly

0.21 (NS)

by Sims. Studies of these organisms and their growth

habits suggested that in most situations Streptococci

are initial colonizers over teeth. The acid condition

created by S.mutans favors the presence of lactobacilli.

In presence of sucrose, S. mutans form extracellular

dextrans and levans which can be utilized later to

produce lactic acid.[8]Further, it is stated that S.mutans

have a short generation time and multiply faster than

lactobacilli. They are acidogenic, which results in

accumulation of acid. As the pH falls, the generation

time of S.mutans lengthens and becomes longer than

lactobacilli. But when this occurs, Lactobacilli

multiply more rapidly than Streptococci; the

environment becomes more acidic and growth of

S.mutans is inhibited. This show that caries is initiated

by S.mutans, whereas lactobacilli help further

progression of the lesion inside the dentin.[8]

Antimicrobial agents have been in the tradition

that exert a direct bactericidal effect on caries

producing bacteria. Many of these are the

complex and varies between individual lesions. Mutans chlorhexidine mouthwashes,[ 1 3 ] gels, sodium

group Streptococci, such as S. mutans and

Streptococcus sobrinus, and Lactobacilli are important

in the initiation and progression of caries. These

microorganisms are acidogenic (produce acid) by

fermenting dietary carbohydrates, which result in the

demineralization of enamel and dentin. They are also

aciduric (acid tolerant), which gives them a competitive

survival advantage.[8]

S.mutans and lactobacilli are regarded as the

two chief bacterial species, responsible for decay in

teeth. The relationship between oral cariogenic

S.mutans and Lactobacilli species has been theorized

hypochlorite solutions, etc. Their regular and long-

term use cannot be advocated due to the potential side

effects. Thus, there arises a need to introduce an agent

that is relatively safe and equally efﬁcient in targeting

the microbial etiology. BTEMR was thus studied to

know the antibacterial action that can be exerted by it

on salivary load of S.mutans.

Black tea is a fermented tea harvested from the

sp. Camellia sinensis. Major difference between black

tea and the other types of tea is that black tea contains

highest amount of catechins which are the major

components possessing the antibacterial properties.[5]

Mean

Standard Deviation

Difference of means p-value

CFU/ml (x 104) Before

8.2134

5.1533

After

7.5766

6.8756

0.6368 0.19 (NS)

Colony Score Before

1.6332

0.9867

After

1.2354

0.7586

0.3978

Khade R et al. Effects of Black Tea & Chlorhexidine on S.mutan

These catechins make up almost 30 to 40% of the

composition of dried black tea leaves [14-17]and so

brewing only 2 g of these leaves in 100 ml of water (i.e.

2% of BTEMR) was done in the present study. This

yielded a concentration of around 6000 to 8000μg/ml

of the catechin compounds. These levels asserted to be

well above the minimum inhibitory concentration of

250 to 1,000 μg/ml of the phenolic compounds of the

black tea that is required to exert an antibacterial

action.[18-20]

Black tea components are the polyphenols

which constitute the most interesting group amongst

the components of black tea leaves. The main

polyphenols in black tea are catechins (ﬂavan-3-ols).

The four main catechins are: epigallocatechin 3 gallate

(EGCG) that constitutes about 59% of total catechins,

epigallocatechin (EGC) about 19%, epicatechin 3

gallate (ECG) about 13.6% and epicatechin (EC)

about 6.4%.

The constituents of black tea synergistically

help in the inhibition of dental caries and can be

summarized into following mechanisms:

1. Remineralisation of dental hard tissues

2. Inhibition of bacterial enzymes

3. Prevention of bacterial adherence

4. Direct bactericidal action.

Lemos J ( 2005 ) suggested that the

suppression of F1Fo- ATPase and agmatine deiminase

system(AgDS) by EGCg may lead not only to energy

starvation but also to disruption of constant pH across

the cell membrane, which in turn may trigger a series

of physiological effects in the cell. As a result of

suppression of AgDS and F1Fo-ATPase cytoplasmic

acidiﬁcation and impaired acid tolerance may inhibit

the normal function of various acid intolerant

enzymes. The optimum pH range of GTFs in S.mutans

was reported to be 5.5 to 6.0. The malfunction of GTFs

at the lower pH value may lead to reduced production

of EPS and intracellular polysaccharides (IPS). The

latter of which could have been metabolized when

exogenous fermentable substrate was depleted in the

oral cavity. Therefore, the malfunction of GTFs may

disrupt both bacterial adherence to the tooth surface

and bioﬁlm integrity and may augment the starvation

stress of S.mutans cells due to the reduced preservation

The inhibition of LDH at both transcriptional and

enzymatic levels may also increase the levels of

NADH and decrease the redox potential of the cell,

leading to the NAD+ /NADH imbalance and/or

accumulation of glycolytic intermediates in the cell,

which is toxic for S.mutans. The net result would be

cytoplasmic acidiﬁcation and disrupted glycolytic

processes with diminished ATP pools, thereby

triggering a series of cascaded biological effects at

molecular levels, leading to compromised competence

to environmental stress and impaired cellular

functions, even cell death. Thus, Xu X et al

summarized that EGCg represents a natural and

alternative anticariogenic agent because (i) EGCg

inhibits growth of both S.mutans planktonic and

bioﬁlm cultures, and (ii) EGCg inhibits various

cariogenic virulence factors of S.mutans at the

transcriptional and enzymatic levels, leading to

reduced acidogenicity and compromised stress

tolerance (especially acid tolerance.

On comparison of the salivary load of

S.mutans from the culture plates of samples

obtained before and after the administration of

BTEMR, it was observed that there was a

signiﬁcant fall in the salivary S.mutans load after

rinsing with BTEMR.

The present study thus reﬂects the

antibacterial effect of BTEMR particularly on

salivary S.mutans and that regular use of BTEMR

can keep a check on the major cariogenic

microorganisms like S.mutans by exerting its

bactericidal action.

The black tea consumption orally in the

form of a drink, although being safe for most of the

people in moderate quantities, few adverse health

effects l ike gastrointestinal upset and

hepatotoxicity are possible.[22]

CONCLUSION:

This study concluded that the effect of 2%

BTEMR on salivary S.mutans load leads to signiﬁcant

reduction of the same irrespective of age or gender.

This in turn implies that a regular use of BTEMR by the

of IPS.[21] Cytoplasmic acidity, in concert with the general population can prove to be an effective natural

inhibition of enolase by EGCg, may also inhibit the

normal process of glycolysis, as described above. This

in turn will diminish the ATP pool and further suppress

the activity of the proton translocation (F1Fo-

ATPase), aggravating cytoplasmic acidiﬁcation.

anticariogenic agent with no known implicated side

effects.

Financial Support and sponsorship

Nil.

18 People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June

Khade R et al. Effects of Black Tea & Chlorhexidine on S.mutan

People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023

Conflicts of interest

There are no conflicts of interest.

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