Research Article

Comparing the antibacterial efficacy of 0.2% chlorhexidine

mouthwash and 1% stevia extract on oral microflora – An
in vivo study
Amanthi Ganapathi, Jayashri Prabakar*

ABSTRACT

Background: In recent years, the researches concerning the use of herbal products have been vastly analyzed to decrease
the disease burden caused by dental caries. It has been found that certain phytochemicals present in the plants have certain
inhibitory effect toward Streptococcus mutans which is currently the main causative organism for dental caries initiation.
Hence, in the present study, prepared herbal mouthwash has been tested for their antibacterial effect. Aim: This study aims
to compare the antibacterial action of commercially available chlorhexidine (CHX) mouthwash product and prepared stevia
mouthwash against oral microflora. Materials and Methods: A single-blind, randomized controlled trial was conducted
among 20 healthy individuals. The participants were randomly allocated into two groups: Group I (experimental group –
aqueous stevia) and Group II (controlled group – chlorhexidine). Before conducting the trial, the study design was explained
to the qualifying participant and informed consent was obtained from the voluntary patients who were willing to participate in
the study. Results: There is a significant reduction in the colony count after using CHX mouthwash when compared to the use
of stevia mouthwash. Stevioside can also serve as an efficient vehicle for topical oral medication. Furthermore, drug industries
can incorporate such extracts which can be delivered. Conclusion: This study documented that among experimental groups,
stevia was equally effective in reduction of colony count. Furthermore, this study highlights the effectiveness of stevia as
mouthwash. Further studies for investigation of the effect of stevia use on dental caries are needed.

KEY WORDS: Chlorhexidine, Mouthwash, Oral health, Stevia, Total microbial count

INTRODUCTION agent in initiation of enamel caries, both in laboratory

Dental caries is a chronic, infectious, transmissible,
and biobehavioral disease that extends throughout
the life span. The essential process of this disease
involves bacterial adherence to tooth surfaces, dental
plaque formation, and localized demineralization of
tooth enamel by acids of bacterial origin produced
from the fermentation of dietary carbohydrates.[1]
More than a century back, W. D. Miller had postulated
the chemicoparasitic theory for the etiology of dental
caries. Until today, the modern concepts of cariogram
demonstrate microorganisms as one of the major
etiological factors for dental caries. Mutans streptococci
are shown to be highly associated with caries in
humans. Considerable evidence exists implicating
Streptococcus mutans as an important etiological
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animals and humans. The presence of ≥106 mutans
streptococci/ml saliva may indicate a high caries risk
or activity.[2] These incipient lesions are managed by
restorative or by non-operative treatments.[3]
Non-operative treatment acts at various levels against
dental caries: At tooth level, microbial level, and
salivary level. One of the non-operative treatments
is the control of the cariogenic microorganisms by
antimicrobial therapy, in the form of mouthwashes,
sprays, dentifrices, gels, varnishes, chewing gums,
and lozenges. Chemical/synthetic agents, such as
chlorhexidine (CHX), triclosan, and xylitol, are
some commonly used antimicrobial agents in caries
prevention. The synthetic chemicals and natural
products, such as herbs and herbal extracts, have been
found to be biocompatible with the tissues and the
body. Herbal medicines have been tested overtime as a
solution for all oral health problems. Extracts of tulsi,

Department of Public Health Dentistry, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences,
Saveetha University, Chennai, Tamil Nadu, India

*Corresponding author: Dr.  Jayashri Prabakar, Department of Public Health Dentistry, Saveetha Dental College,
Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai  -  600  077, Tamil Nadu, India.
E-mail: shrijaya2009@gmail.com

Received on: 10-07-2019; Revised on: 14-08-2019; Accepted on: 16-09-2019

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 Amanthi Ganapathi and Jayashri Prabakar
neem, green tea, and Terminalia chebula have been
tested for their anticariogenicity, in both in vivo and in
vitro studies.[4,5] Among other strategies implemented to
eliminate and/or control the microorganisms involved
in dental caries, chemical and antimicrobial substances
have been used. Antimicrobial biomolecules of
natural origin are also currently being explored for
use in adjuvant therapy.[6] Plants have been widely
used around the world as traditional remedies in the
treatment of diseases. It is estimated that 66–75%
of the world population currently uses plant-based
medicines.[5] The main aim of research into medicinal
plants is to identify plants that possess pharmacological
activity and thus discover new substances or molecules
having antimicrobial activity that could be transformed
into medications by different chemical processes and
used to control or prevent infectious diseases.[7]
Stevia rebaudiana (bertoni leaves) is an herbaceous
perennial plant of the family Asteraceae and is
indigenous to Paraguay and Brazil. It is cultivated in parts
of Asia, Europe, and Canada. In India, the cultivation
of S. rebaudiana herbal plant is mostly seen in Gujarat
and it is commercially available in powder form. The
leaf extract of S. rebaudiana is used to sweeten foods
and is also used as a dietary supplement. The major
components are glycosides, namely, stevioside and
rebaudioside-A. These compounds exhibit characteristic
organoleptic properties and have sweetness intensities
more than 300 times that of sucrose. Leaf extract of this
plant has been used traditionally for the treatment of
diabetes.[8] In addition to being a non-caloric sweetener
known in many parts of the world, it has hypoglycemia,
antioxidant, and antihypertensive action.[9-12] Another
advantage is that no toxic or genotoxic activity has
been found in the complete extracts obtained from
S. rebaudiana bertoni leaves.[13] There are studies
reporting antimicrobial activity of extracts obtained
from S. rebaudiana on fungi and Gram-positive and
Gram-negative bacteria.[9,14,15]
CHX, a cationic bis-biguanide biocide with
low mammalian toxicity and broad-spectrum
antibacterial[16] activity, was first described in
1954.[17] The primary mechanism of action of
this biocide is membrane disruption, causing
concentration-dependent growth inhibition and cell
death.[18] Secondary interactions causing inhibition
of proteolytic and glycosidic enzymes may also
be significant.[19] With respect to dental hygiene
applications, the cationic nature of CHX enables it
to bind to tooth surfaces and oral mucosa, reducing
pellicle formation and increasing substantivity
through controlled release of the agent.[20] The efficacy
of CHX in reducing oral bacterial viability,[21-23]
strongly inhibiting plaque regrowth, and preventing
gingivitis[24] has been demonstrated in many studies.[25]
In spite of its use of stevia in the field of medicine,
2638
spotlight for the use of stevia in dentistry is minimal.
Therefore, this study was aimed to compare the
antibacterial effect of prepared stevia mouthwash in
comparison with CHX.
MATERIALS AND METHODS
Study Design
It is a single-blinded, parallel-group, randomized
controlled clinical trial.
Study Population
Final year dental students of Saveetha Dental College
and Hospitals, Chennai, were recruited for a single-
blinded, parallel-group, randomized controlled
clinical trial.
Inclusion Criteria
The following criteria were included in the study:
• Apparently healthy patients without any known
history of systemic illness above 18–25 years of age
• Participants with good to fair plaque index score
• Participants with mild-to-moderate gingivitis
• Participants with habit of toothbrushing twice daily.
Exclusion Criteria
• Participants with a positive history of the usage
of antimicrobial therapy and routine use of oral
antiseptics in the previous 3 months were excluded
from the study
• Participants with a history of allergic and
idiosyncratic reactions to product ingredients were
excluded from the study
• Participants undergoing orthodontic treatment
• Participants with the presence of systemic diseases
that could alter the production or composition of
plaque and saliva were excluded from the study
• Participants who were already on any mouthwash
regime were excluded from the study. The presence
of systemic disease was another exclusion criteria
• Those who were on any medication that would alter
the salivary flow were also not included in the study
• The presence of systemic disease was other
exclusion criteria.
A total of 20 (n = 20) such patients were randomly
allocated to both groups using simple random
sampling.
Group I: (n = 10) Participants who were given aqueous
stevia mouthwash
Group II: (n = 10) Participants who were given 0.2%
CHX mouthwash.
Preparation of Aqueous Stevia Mouthwash
For 1% aqueous suspension of S. rebaudiana, 5 g of
S. rebaudiana leaf powder was dispersed in 100 ml of
distilled water with constant stirring for 2 h, 0.5  ml
Drug Invention Today | Vol 12 • Issue 11 • 2019
 Amanthi Ganapathi and Jayashri Prabakar

of glycerin was added, and the volume of 500 ml was Step 2: Two groups were assigned each having three
made up with distilled water [Figures 1 and 2]. participants. Group 1 was given with CHX mouthwash
and Group 2 was given with stevia mouthwash [Figure 3].
Blinding
Investigator was unaware of the treatment groups. Step 3: Saliva samples of the participants before using
Therefore, the investigator was blinded in this study. the mouthwash were collected in an Eppendorf tube
and saliva sample of the participants was collected on
Armamentarium the 15th day after using the mouthwash.
The following equipment/instrument/materials were
Step 4: Using biological loop streaks where make on
used for the study:
the culture plate and were incubated for 24 h and the
• Dental chair
bacterial count was made [Figure 4].
• Dental operating tool
• Tray sheet
• Disposable head cap
RESULTS
• Disposable mouth mask Table 1 depicts the comparison of mean difference of
• Disposable gloves – small size baseline and 15th  day total microbial count between
• Disposable cups the groups. A  statistically significant difference was
• Disinfecting solution. observed between the groups using Mann–Whitney
U-test. The mean difference of Group 2 total microbial
Study Procedures count was found to be more than the Group 1. Table 2
Step 1: The aqueous mouthwash of stevia was shows the comparison of mean total microbial count
prepared in the pharmacology laboratory in Saveetha at baseline and 15th  day total microbial count with
Dental College. in the groups. A  statistically significant difference
was observed within the groups using Wilcoxon
signed-rank test. The mean microbial count of
Group  1 showed a statistically significant reduction
from 1934.10 ± 439.54 to 1628.30 ± 378.47. The
mean microbial count of Group  2 showed a highly

Figure 1:  Processed stevia sample

Figure 3:  Prepared stevia mouth wash

Figure 2:  Saliva sample collection Figure 4:  Culture plates

Drug Invention Today | Vol 12 • Issue 11 • 2019 2639

 Amanthi Ganapathi and Jayashri Prabakar
Table 1: Comparison of mean difference of baseline and 15th day total microbial count between the groups
Variable Groups n Mean
Total microbial count 1 10 305.80
2 10 1322.60
Mann–Whitney U‑test (P<0.05)
Table 2: Comparison of mean total microbial count at baseline and 15th day total microbial count with in the groups
Groups Time periods n Mean
Group 1 Baseline 10 1934.10
15th day 10 1628.30
Group 2 Baseline 10 2068.60
15th day 10 746.00
Wilcoxon signed‑rank test (P<0.05)
statistically significant reduction from 2068.60 ±
485.84 to 746.00 ± 170.56.
DISCUSSION
In this study, Table 1 depicts the comparison of mean
difference of baseline and 15th day total microbial count
between the groups. A statistically significant difference
was observed between the groups using Mann–Whitney
U-test. The mean difference of Group 2 total microbial
count was found to be more than the Group 1. Table 2
shows the comparison of mean total microbial count
at baseline and 15th day total microbial count within the
groups. A statistically significant difference was observed
within the groups using Wilcoxon signed-rank test. The
mean microbial count of Group 1 showed a statistically
significant reduction from 1934.10 ± 439.54 to 1628.30
± 378.47. The mean microbial count of Group 2 showed
a highly statistically significant reduction from 2068.60
± 485.84 to 746.00 ± 170.56.
As per the caries management by risk assessment
guidelines, antibacterial mouthwash is an important
caries preventive therapy to control effectively and
for prevention against dental caries in high-risk
individuals.[26,27] Apart from the use of well-known
antibacterial mouthwashes, such as CHX, there is
a recent flash in the use of natural products as oral
care therapies. The antibacterial property of the
phytochemicals, essential oils, and flavonoids extracted
from plant or food products, against S. mutans, has
been well established in many in vitro and in vivo
studies.[28] The hypothesis generated in this study was
based on the established antibacterial properties of the
glycosides from plant products on oral flora. The leaves
of S. rebaudiana are a natural sweetener. It has around
100 phytochemicals. Stevia has two main glycosides
that are stevioside (110–270  times sweeter than
sugar) and rebaudioside-A (180–400  times sweeter
than sugar). The leaves also contain 80–85% water.
Ethanolic and methanolic extracts of stevia leaves have
been found to be against both Gram positive and Gram
negative. CHX gluconate is a cationic bisbiguanide
having low toxicity and broad-spectrum antibacterial
2640
Standard deviation Standard error mean P‑value
133.07 42.08 <0.05*
506.66 160.22
Standard deviation Minimum Maximum P
439.54 1076.00 2412.00 <0.05*
378.47 1000.00 2067.00
485.84 1256.00 2874.00 <0.001*
170.56 547.00 1081.00
activity. When used as a mouthwash, it has a flushing
action; its effects in the oral cavity are attributed to its
lethal effects on the bacteria.
It results in membrane disruption of the bacteria,
causing a concentration-dependent growth inhibition
and cell death. Secondary interactions leading to the
inhibition of proteolytic and glycosidic enzymes are
also significant. The cationic nature of CHX helps it to
bind to the tooth structure and oral mucosa, reducing
pellicle formation and increasing substantivity through
controlled release of the agent.[29] CHX has been used
as a mouthwash for over 40 years now and has been
an antibacterial agent of choice in control of both the
quantity of bacterial colonies and formation of plaque.
It has been an agent of choice for its effectiveness as
well as low scope of resistance. CHX has been regarded
as a “gold” standard in chemical plaque control for over
45 years in dentistry for the prevention of plaque and
gingivitis.[30] In few in vitro studies, aqueous extracts
of stevia have been shown to be ineffective against
S. mutans.[31,32] Despite such an evidence, an aqueous
extract of the stevia leaves was planned to be used as
mouthwash in this study, for safety and palatability
of patient’s use. In this study, there was a statistically
significant difference in the mean difference between
pre-  and post-rinse parameters in the stevia group as
well as in the CHX group.
This means that both mouthwashes were effective in
favorably altering the oral flora. While comparing the
efficacy of stevia mouthwash over CHX mouthwash,
it was found that stevia mouthwash performed as
good as the CHX mouthwash in altering the salivary
parameters. This is in accordance to a study by
Brambilla et al. where 20 volunteers rinsed with stevia
solutions and sucrose solutions. Post-rinse, the plaque
pH was measured at 7 time points after each rinse.[33] It
was found that the pH dropped to acidic with sucrose
solutions and not with stevia solution, concluding
that stevia is non-acidogenic. The pre-rinse pH of the
plaque collected from the volunteers in the study was
normal, whereas in our study, the pre-rinse salivary
pH was acidic, as it was collected from high caries risk
Drug Invention Today | Vol 12 • Issue 11 • 2019
 Amanthi Ganapathi and Jayashri Prabakar
patients, which became normal after the rinse with
stevia extract. Thus, it can be inferred from the current
study that stevia extract is not only non-acidogenic but
also tends to lower the acidic pH of saliva.
CONCLUSION
The mean difference of Group 2 total microbial
count was found to be more than the Group 1 sample
microbial count. The reduction in total microbial
count was observed in both the groups. Hence, stevia
can be used as an effective alternative for preventing
oral diseases and enhancing oral health. Furthermore,
this study highlights the effectiveness of stevia as
mouthwash. Further studies for investigation of the
effect of stevia use on dental hygiene are needed.
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Source of support: Nil; Conflict of interest: None Declared
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