[Downloaded free from http://www.jisponline.com on Saturday, August 20, 2022, IP: 103.171.117.

162]

Original Article

Effect of 0.4% Triphala and 0.12%

Department of Public
Health Dentistry,
Sinhgad Dental College
and Hospital, Pune,
1
Department of Public
Health Dentistry, Sharad
Pawar Dental College
and Hospital, Wardha,
Maharashtra, India
The work belongs to
the Department of
Public Health Dentistry,
Sinhgad Dental College
and Hospital, Pune,
India
Access this article online
Website:
www.jisponline.com
DOI:
10.4103/jisp.jisp_338_20
Quick Response Code:
chlorhexidine mouthwash on dental
plaque, gingival inflammation, and
microbial growth in 14–15‑year‑old
schoolchildren: A randomized
controlled clinical trial
Ketaki Bhor, Vittaldas Shetty, Vikram Garcha, Kadambari Ambildhok,
Vineet Vinay, Gargi Nimbulkar1
Abstract:
Context: A  strong correlation exists between plaque and dental caries and periodontal diseases. Ayurvedic
drugs have been used since ancient times; oral rinses made from these are used in periodontal therapy.
Triphala is one of these with wide spectrum of activity. Aims: To assess and compare the effect of 0.4%
Triphala and 0.12% chlorhexidine  (CHX) mouthwash on dental plaque, gingival inflammation, and microbial
count of Streptococcus mutans, Streptococcus sanguinis, and Lactobacilli from dental plaque sample of
14–15‑year‑old schoolchildren of Pune city during 90 days supervised use. Settings and Design: A randomized,
controlled, double‑blind, parallel‑group clinical trial was conducted among 72 schoolchildren aged 14–15 years.
Subjects and Methods: Children were divided into two study groups: Group A with 0.4% Triphala
mouthwash (n  =  36) and Group B with 0.12% CHX mouthwash  (n  =  36). The plaque Index  (Loe H  [1967]),
gingival index (Loe H and Silness J [1963]), and microbial analysis were recorded at baseline, 30 days, and
90 days interval. Statistical Analysis Used: Statistical analysis was done using unpaired t‑test for group‑wise
comparison and one‑way analysis of variance test, followed by Tukey’s post hoc test for intragroup comparison.
P < 0.05 was considered statistically significant. Results: The results showed that 0.4% Triphala and 0.12%
CHX have similar inhibitory effect on plaque accumulation, gingivitis, and growth of S. mutans, S. sanguinis, and
Lactobacilli. Conclusion: Herbal mouthwash proved to be helpful in reducing plaque microbial counts, plaque,
and gingival inflammation and opens new arenas in the field of herbal dentistry and chemical plaque control.
Key words:
Chlorhexidine, dental plaque, gingival inflammation, microbial growth, Triphala
INTRODUCTION significant delay in the colonization of mutans
Streptococci.[4,5] Streptococcus mutans (S. mutans)

O ral health, now recognized as equally

important in reference to general health,
has become an integral part of the overall health
metabolizes sucrose in a peculiar way, producing
dextran, thus promoting the firm adherence of

Address for
correspondence:
Dr. Ketaki Bhor,
Department of Public
Health Dentistry, Sinhgad
Dental College and
Hospital, Pune ‑ 411 041,
Maharashtra, India.
E‑mail: ketaki.bhor@gmail.
com
Submitted: 10-May-2020
Revised: 13-Mar-2021
Accepted: 21-Mar-2021
Published: 01-Nov-2021
and well‑being of an individual. Dental caries
and periodontal diseases are the two leading
oral pathologies that remain widely prevalent
and affect almost all inhabitants throughout the
lifetime.[1] Dental plaque has been proven to be
a paramount factor in initiation and progression
of dental caries, gingivitis, and periodontal
diseases.[2]
Gram‑positive streptococcus strains form the
major group of organisms during the first
few hours of plaque formation.[3] Streptococcus
sanguinis (S. sanguinis) are the primary colonizers
in the human oral cavity, and its elevated
levels in the oral cavity were correlated to a
This is an open access journal, and articles are
distributed under the terms of the Creative Commons
Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows
others to remix, tweak, and build upon the work non‑commercially,
as long as appropriate credit is given and the new creations are
licensed under the identical terms.
For reprints contact: WKHLRPMedknow_reprints@
wolterskluwer.com
How to cite this article: Bhor K, Shetty V,
Garcha V, Ambildhok K, Vineet V, Nimbulkar G.
Effect of 0.4% Triphala and 0.12% chlorhexidine
mouthwash on dental plaque, gingival inflammation,
and microbial growth in 14–15‑year‑old
schoolchildren: A randomized controlled clinical
trial. J Indian Soc Periodontol 2021;25:518-24.

518 © 2021 Indian Society of Periodontology | Published by Wolters Kluwer - Medknow

[Downloaded free from http://www.jisponline.com on Saturday, August 20, 2022, IP: 103.171.117.162]
Bhor, et al.: Effect of Triphala and CHX: Plaque, gingivitis, and oral microbes
the organisms to the tooth surface[3] along with coaggregation
mediated by a protein on the surface of the Lactobacilli,[6]
consequently contributing to the formation of bacterial plaque
and subsequently resulting in gingival inflammation and
localized decalcification of the enamel.[3]
The National Oral Health Survey and Fluoride Mapping 2003,
India, reported that 72.5% of 12‑year‑old children and 75.4%
of 15‑year‑old children had dental caries, whereas 55.4% of
12 year or higher age groups had gingivitis. Gingivitis usually
begins in childhood and features a lifelong squeal; hence,
primary care must begin early in life before the onset of the
problem.[7]
The removal of plaque is of utmost importance to control
dental caries and gingivitis that is commonly maintained by
mechanical methods. However, in children, factors such as lack
of manual dexterity and individual motivation and monitoring
limit the effectiveness of toothbrushing, particularly at
interproximal sites, and necessitate the use of chemotherapeutic
agents such as a therapeutic mouthrinse as an adjunct to
mechanical plaque control.[8,9] Currently, chlorhexidine (CHX),
a potent antibacterial substance, is employed as a gold standard
chemical plaque control agent.[10]
However, excessive use of these antimicrobial agents may result
in the development of bacterial resistance and derangement of
the oral and intestinal flora and may cause undesirable side
effects such as vomiting, diarrhea, taste alterations, and tooth
staining. Hence, the use of herbal mouthwash in the prevention
and treatment of oral conditions has increased recently.[9]
“Triphala” is among the most commonly used formula in
traditional ayurvedic medicine as it has antibacterial, antiseptic,
and anti‑inflammatory properties. The 20th shloka of Sushruta
Samhita has stated that Triphala can be used as a mouthrinsing
agent in dental ailments.[11]
There is a need to investigate a suitable alternative, like
Triphala which is locally available, renewable, culturally
accepted, affordable, and effective against the oral pathogenic
microorganisms.[12]
Hence, this study was conducted with the aim to clinically
assess and compare the effect of Triphala and commercially
available CHX mouthwash on the dental plaque, gingival
inflammation, and microbial counts of S. mutans, S. sanguinis,
and Lactobacilli counts among 14–15‑year‑old schoolchildren
after 90 days of supervised use.
SUBJECTS AND METHODS
A randomized, controlled, double‑blind, parallel‑group clinical
trial was conducted among 14–15‑year‑old children from
private schools in Pune city, India. The reporting of the study
is in accordance to the Consolidated Standards of Reporting
Trials guidelines [Figure 1]. The study protocol was approved
by the Ethical Committee of the Institutional Review Board
(SDCH/IEC/OUT/2013‑14/77) and the trial is registered
under Clinical Trials Registry of India, CTRI/2017/10/010155.
Furthermore, the necessary permissions were obtained from
the concerned school authorities.
Journal of Indian Society of Periodontology - Volume 25, Issue 6, November-December 2021 519
Preparation of Triphala and chlorhexidine mouthwashes
The Triphala mouthwash was formulated and developed
in a private laboratory using the water‑based liquid extract
of Triphala. The prepared extract was tested for microbial
activity on three oral pathogens, namely, S. mutans ATCC
25175, S. sanguinis ATCC 10556, and Lactobacillus ATCC 4356
by agar well diffusion assay using swab technique,[13] after
which a minimum inhibitory concentration and the minimum
bactericidal concentration of 0.4% was determined by the broth
dilution test.[13‑15]
CHX gluconate mouthwash (Proprietary name: Eludril,
concentration: 0.12%) procured from the market was given to
the pharmacy manufacturing center for dilution with equal
amount of sterile water. Both mouthwashes were made of
identical colors and were dispensed in 500 ml bottles for use.
The bottles were coded by the pharmacists, and at the end of
the study, the decoding was done.
Pilot study
The training and calibration of the examiner for recording
the indices was done by a subject expert before the start of
the study. Intraexaminer agreement was determined using
the weighted kappa (k = 0.81). The method of plaque sample
collection, storage, and transportation was standardized after
discussion with a microbiologist.
Study population
Schoolchildren aged 14–15 years from four private schools in
the southwest zone of Pune city who were willing to participate
in the study after giving written informed assent and consent
obtained from their parents were selected.
Sample size determination
Sample size determination was based on the expected
minimum reduction in plaque scores in the controlled
group, as observed in a previous study.[14] Sample size of
36 participants per group was determined using the formula
n = 2[Zα(σ)/d] 2 and considering 10% sample attrition rate.
Thus, a total of 72 participants were included in the study.
Sampling methodology
First stage (selection of schools)
As per the details obtained from the education officer, there
are a total of 90 private schools in Pune city. Schools with easy
accessibility were identified and approached. Four schools who
gave the requisite permissions were selected by lottery method.
Second stage (selection of study participants)
As per the below‑mentioned inclusion and exclusion criteria,
the study participants from all the four schools were screened
and a separate list of girls and boys for each of the four schools
was prepared. Then, by systematic random sampling, 18 study
participants from each school were selected such that there were
equal number of girls (n = 9) and boys (n = 9) in each group.
Inclusion criteria
Study participants aged 14–15 years having at least 20 intact
natural teeth with similar socioeconomic status and oral
hygiene practices as well as fair plaque score (Loe H 1967)[15]
and moderate gingivitis (Loe H and Silness J 1963)[16] at baseline
were selected.
[Downloaded free from http://www.jisponline.com on Saturday, August 20, 2022, IP: 103.171.117.162]

Bhor, et al.: Effect of Triphala and CHX: Plaque, gingivitis, and oral microbes

Enrollment Assessed for eligibility

School 1 assessed School 2 assessed School 3 assessed School 4 assessed

for eligibility for eligibility for eligibility for eligibility
n = 38 n = 30 n = 24 n = 44
M = 18 M = 18 M =14 M = 24
F = 20 F = 16 F = 10 F = 20

Systematic randomization

School 1 final School 2 final School 3 final School 4 final

sample size sample size sample size sample size
n = 18 n = 18 n = 18 n = 18
M=9 M=9 M=9 M=9
F=9 F=9 F=9 F=9

4 schools were randomized into two groups by lottery method

Allocation

Group A (0.4% Triphala) Group B (0.02% chlorhexidine)

School 1 and School 4 School 2 and School 3
Allocated to intervention group (n = 36) Allocated to positive control group (n = 36)
• Received allocated intervention (n = 36) • Received allocated intervention (n = 36)
• Did not receive allocated intervention (n = 0) • Did not receive allocated intervention (n = 0)

Follow-up

Lost to follow-up (n = 0) Lost to follow-up (n = 0)

Discontinued intervention (n = 0) Discontinued intervention (n = 0)

Analysis

Analyzed (n = 36) Analyzed (n = 36)

• Excluded from analysis (n = 0) • Excluded from analysis (n = 0)

Figure 1: Schematic representation of study design as per the Consolidated Standards of Reporting Trials (CONSORT) guidelines. n – no of study participants, M – male, F – female

Exclusion criteria • Microbial analysis: Mitis salivarius bacitracin agar, blood

Study participants with any systemic disorders, having history
of hypersensitivity to any product used in the study using
other mouthrinses, and under antimicrobial therapy at least 3
months before the study were excluded.
Random allocation of the two mouthwashes
The selected four schools were randomly allocated by lottery
method by a person not involved in examination into two study
groups such that two schools per group with 36 study participants
in each group. The study participants received the products
according to the specified code. The two study groups were:
• Group A: 0.4% Triphala mouthwash (n = 36)
• Group B: 0.12% CHX mouthwash (n = 36).
Materials used for the study
• Recording indices: Mouth mirror, explorer, Williams
graduated probe, tweezers, and chip syringe
• Collection of plaque samples: Eppendorf vials, transport
media, and vaccine carrier with freezing mixture
agar, and Man, Rogosa, Sharpe (MRS) agar.
Blinding
Study participants and the examiner were blinded regarding
the medicament allotted to the two intervention groups
as the codes were given by a person not involved in the
examination (pharmacist). All the microbiological procedures
were performed by the microbiologist, who was blinded with
respect to the plaque samples of different groups during the
entire course of the study.
Study tools
The data were collected using an investigator‑administered
pro forma and by clinical examinations using plaque
index (PI) by Loe H[15] and gingival index given by Loe H and
Silness J (1963).[16] The total colony count scores of the plaque
microorganism such as S. mutans, S. sanguinis, and Lactobacilli
were also recorded.

520 Journal of Indian Society of Periodontology - Volume 25, Issue 6, November-December 2021
[Downloaded free from http://www.jisponline.com on Saturday, August 20, 2022, IP: 103.171.117.162]
Bhor, et al.: Effect of Triphala and CHX: Plaque, gingivitis, and oral microbes
Study setting
Type III clinical examination, as recommended by the American
Dental Association,[17] was conducted in a chair/school bench,
in the selected four schools.
Collection of plaque samples and microbial analysis
The study participants were made to sit on a chair and
the plaque was collected from the gingival thirds of the
buccal surfaces and from the pit and fissure areas using a
Sheppard’s hook no. 5 explorer. They were instructed not to
eat or drink anything for at least 1 h before the collection of
plaque sample. The collected plaque samples were diluted
to 1:1000 using sterile peptone for the microbial analysis.
A semiquantitative, i.e., four‑quadrant streaking method was
adopted (Sitges–Serra and Linares).[18] Using a standard loop,
the plaque sample was streaked on mitis salivarius bacitracin
agar (S. mutans), blood agar (S. sanguinis), and Lactobacillus MRS
agar (Lactobacilli). The growth in all the four quadrants was
recorded in colony‑forming units per ml (CFU/ml).
Administration of mouthwash
The study participants in both the groups were given two
bottles of the mouthwash of 500 ml every 15th day, one to be
kept in school with the teachers for using in morning after
30 min after breakfast and one to be carried home for night
use after 30 min after dinner. Before using the mouthwash,
gargling with plain water was done to remove the debris. The
study participants were instructed to use 30 ml of mouthwash
by holding it in the mouth and then perform active cheek
movement for 2 min before expectorating the mouthwash. The
teachers and parents were educated and trained in the use of
mouthwash so that supervised mouth rinsing was performed
by the children for a period of 2 min.[14] The study participants
were advised not to eat or rinse for the next 30 min. They were
given the compliance checklist to tick mark after every rinse.
The emptiness of the bottle of mouthwash was correlated with
the number of mouthrinses done.
Follow‑up
Plaque, gingivitis scores, and microbiological analysis were
recorded at baseline, 1 month, and 3 months.
Statistical analysis
Statistical analysis was done with Statistical Package for the
Social Sciences (IBM SPSS Statistic for Windows, version 21.0.
Armonk, NY: IBM Corp.). For intragroup comparison, one‑way
analysis of variance (ANOVA) test followed by Tukey’s post
hoc test was applied, whereas for intergroup comparison of
PI, gingival index, and microbial colony count at 0, 30, and
90 days, un‑paired t‑test was applied. P < 0.05 was considered
for statistical significance.
RESULTS
Table 1 reports the Intragroup and intergroup comparison of
mean plaque index scores, gingival index scores, and microbial
count among Group A (0.4% Triphala) and Group B (0.02%
chlorhexidine) at varying time periods.
Mean plaque index scores in the two groups [Figure 2]
The mean plaque scores for Group A and Group B were
1.08 ± 0.16 and 1.14 ± 0.27 at baseline, 0.69 ± 0.15 and 0.75 ± 0.22
Journal of Indian Society of Periodontology - Volume 25, Issue 6, November-December 2021 521
Figure 2: Comparative evaluation of mean plaque index scores in Group A (0.4%
Triphala mouthwash) and Group B (0.12% chlorhexidine mouthwash) at varying
time periods. *P < 0.05 significant. The mean plaque index scores in Group A (0.4%
Triphala mouthwash) and Group B (0.12% chlorhexidine mouthwash) at different
time periods were statistically significant (one‑way ANOVA, P < 0.05). Tukey’s post
hoc analysis showed that there was a significant difference in the mean plaque
index scores. P – Probability value
at 1 month, and 0.41 ± 0.06 and 0.41 ± 0.03 at 3 months of
follow‑up, respectively. The intragroup difference in Group A
and Group B was statistically highly significant  (P < 0.001)
at 1 month and 3 months of follow‑up from baseline as
well as between 1 month and 3 months, but the intergroup
difference between both the groups was not statistically
significant (P > 0.05) at the three time intervals.
Mean gingival index scores in the two groups [Figure 3]
The mean gingival scores for Group A and Group B were
1.01 ± 0.16 and 1.07 ± 0.22 at baseline, 0.67 ± 0.92 and 0.64 ± 0.22
at 1‑month follow‑up, and 0.30 ± 0.05 and 0.32 ± 0.050 at
3‑month follow‑up, respectively. The intragroup difference
in Group A was statistically significant (P < 0.05) at 1 month
and 3 months of follow‑up from baseline and was statistically
highly significant (P < 0.001) between 1 month and 3 months.
The intragroup difference in Group B was statistically highly
significant (P < 0.001) at 1 month and 3 months of follow‑up
from baseline and between 1 month and 3 months, but the
intergroup difference between both the groups was not
statistically significant (P > 0.05) at the three time intervals.
Mean Streptococcus Mutans count in the two groups,
expressed as 103 CFU/ml of plaque [Figure 4]
The mean total colony counts of S. mutans in Group A and
Group B were 24.22 ± 4.49 and 22.97 ± 3.43 at baseline,
20.83 ± 3.71 and 18.61 ± 3.35 at 1‑month follow‑up, and
14.8 ± 2.60 and 14.22 ± 2.07 at 3‑month follow‑up, respectively.
The intragroup difference in Group A and Group B was
statistically highly significant  (P < 0.001) at 1 month and 3
months of follow‑up from baseline and between 1 month and
3 months, but the intergroup difference between Group A
and Group B was statistically significant at 1 month (P < 0.05).
Mean Streptococcus Sanguinis count in the two groups,
expressed as 103 CFU/ml of plaque [Figure 5]
The mean total colony counts of S. sanguinis in Group A
and Group B were 37.38 ± 5.22 and 34.5 ± 6.46 at baseline,
34.13 ± 4.53 and 35.77 ± 6.38 at 1‑month follow‑up, and
39.91 ± 3.84 and 40.69 ± 4.77 at 3‑month follow‑up, respectively,
[Downloaded free from http://www.jisponline.com on Saturday, August 20, 2022, IP: 103.171.117.162]

Bhor, et al.: Effect of Triphala and CHX: Plaque, gingivitis, and oral microbes

showing an increase in the count from baseline to 3 months
in both the groups. The intragroup difference in Group
A was statistically significant (P < 0.05) at 1 month and
3 months of follow‑up from baseline, but it was statistically
highly significant (P < 0.001) between 1 month and 3 months.
The intragroup difference in Group B was statistically
significant  (P < 0.05) between baseline and 3 months and
statistically highly significant  (P < 0.001) between 1 month
and 3 months, but the intergroup difference between both the
groups was not statistically significant (P > 0.05) at the three
time intervals.
Mean Lactobacilli count in the two groups, expressed as 103
CFU/ml of plaque [Figure 6]
The mean total colony counts of Lactobacilli in Group A and
Group B were 12.77 ± 3.65 and 12.0 ± 3.95 at baseline, 8.97 ± 2.22
and 8.91 ± 3.09 at 1‑month follow‑up, and 7.8 ± 1.8 and
6.58 ± 1.7 at 3‑month follow‑up, respectively. The intragroup
difference in Group A and Group B was statistically highly
significant (P < 0.001) at 1 month and 3 months of follow‑up
from baseline. The intergroup difference between Group A
and Group B was statistically highly significant (P < 0.001) at
3 months of follow‑up.
DISCUSSION
A direct relationship has been demonstrated between plaque
levels and the severity of gingivitis.[2] The S. mutans, S. sanguinis,
and Lactobacilli species are found in high concentration in
dental plaque as compared to saliva; therefore, direct plaque
samples were collected and analyzed for the above‑stated
microorganisms.
Children are easily accessible in schools and are known to often
practice inadequate oral hygiene measures and experience

Figure 3: Comparative evaluation of mean gingival index scores in
Group A (0.4% Triphala mouthwash) and Group B (0.12% chlorhexidine
mouthwash) at varying time periods. *P < 0.05 significant. The mean
gingival index scores in Group A (0.4% Triphala mouthwash) and Group
B (0.12% chlorhexidine mouthwash) at different time periods were
statistically significant (one‑way ANOVA, P < 0.05). Tukey’s post hoc
analysis showed that there was a significant difference in the mean plaque
index scores. P – Probability value
Figure 4: Comparative evaluation of mean Streptococcus mutans counts in Group
A (0.4% Triphala mouthwash) and Group B (0.12% chlorhexidine mouthwash)
expressed as 103 CFU/ml of plaque at varying time periods. *P < 0.05 significant.
The mean Streptococcus mutans counts in Group A (0.4% Triphala mouthwash)
and Group B (0.12% chlorhexidine mouthwash) at different time periods were
statistically significant (one‑way ANOVA, P < 0.05). Tukey’s post hoc analysis
showed that there was a significant difference in the mean plaque index scores.
P – Probability value, CFU – Colony‑forming units, TCC – Total colony count

Table 1: Intragroup and intergroup comparison of mean plaque index scores, gingival index scores, and microbial
count among Group A (0.4% Triphala) and Group B (0.02% chlorhexidine) at varying time periods
Parameters Group Mean±SD P intragroup
Baseline 1st month 3rd month Baseline‑1 month Baseline‑3 months 1-3 months
Mean plaque index Group A (Triphala) 1.08±0.16 0.69±0.15 0.41±0.06 0.001** 0.001** 0.001**
score Group B (CHX) 1.14±0.27 0.75±0.22 0.41±0.03 0.001** 0.001** 0.001**
P 0.29 0.18 0.83 ‑ ‑ ‑
Mean gingival index Group A (Triphala) 1.01±0.16 0.67±0.92 0.30±0.05 0.02* 0.01* 0.001**
score Group B (CHX) 1.07±0.22 0.64±0.22 0.32±0.050 0.001** 0.001** 0.001**
P 0.27 0.83 0.13 ‑ ‑ ‑
Mean Streptococcus Group A (Triphala) 24.22±4.49 20.83±3.71 14.8±2.60 0.001** 0.001** 0.001**
mutans count Group B (CHX) 22.97±3.43 18.61±3.35 14.22±2.07 0.001** 0.001** 0.001**
P 0.18 0.01* 0.29 ‑ ‑ ‑
Mean Streptococcus Group A (Triphala) 37.38±5.22 34.13±4.53 39.91±3.84 0.09* 0.05* 0.001**
sanguinis count Group B (CHX) 36.5±2.46 35.77±6.38 40.69±4.77 0.63 0.02* 0.001**
P 0.06 0.21 0.45 ‑ ‑ ‑
Mean Lactobacilli Group A (Triphala) 12.77±3.65 8.97±2.22 7.8±1.8 0.001** 0.001** 0.16
count Group B (CHX) 12.0±3.95 8.91±3.09 6.58±1.7 0.001** 0.001** 0.001**
P 0.38 0.93 0.001** ‑ ‑ ‑
*P<0.0 statistically significant; **P<0.001 statistically highly significant, Tests applied: For intragroup comparison - Oneway ANOVA followed Tukey’s post
hoc analysis; for intergroup comparison - Unpaired t‑test, Values expressed as mean±SD. SD - Standard deviation; CHX - Chlorhexidine; P - Probability;
ANOVA - Analysis of variance

522 Journal of Indian Society of Periodontology - Volume 25, Issue 6, November-December 2021
[Downloaded free from http://www.jisponline.com on Saturday, August 20, 2022, IP: 103.171.117.162]
Bhor, et al.: Effect of Triphala and CHX: Plaque, gingivitis, and oral microbes
Figure 5: Comparative evaluation of mean Streptococcus Sanguinis counts in
Group A (0.4% Triphala mouthwash) and Group B (0.12% chlorhexidine mouthwash)
expressed as 103 CFU/ml of plaque at varying time periods. *P < 0.05 significant.
The mean Streptococcus sanguinis counts in Group A (0.4% Triphala mouthwash)
and Group B (0.12% chlorhexidine mouthwash) at different time periods were
statistically significant (one‑way ANOVA, P < 0.05). Tukey’s post hoc analysis
showed that there was a significant difference in the mean plaque index scores.
P – Probability value, CFU – Colony‑forming units, TCC – Total colony count;
gingivitis;[19] therefore, 14–15‑year‑old schoolchildren were
selected.
The antimicrobial efficacy of aqueous extract of Triphala was
established in the in vitro phase of the study, which showed
a minimal inhibitory concentration of 0.4% for S. mutans,
S. sanguinis, and Lactobacilli. CHX rinses are often used as a
benchmark control.[20] The two most common concentrations
commercially available are 0.2% and 0.12%. In the study, 0.12%
CHX was used with the rationale to reduce side effects when
maintaining comparable efficacy as the total amount of CHX
is approximately the same: 10 mL of 0.2% CHX contains 20 mg
and 15 mL of 0.12% CHX contains 18 mg per volume.[10]
Intragroup effect on plaque
The results of the study participants in Group A and in Group
B indicated a significant reduction in PI scores when the
means were compared at different time intervals. For Group
A, the mean PI, which was 1.08 (±0.16) at baseline, reduced to
0.69 (±0.15) at 1 month and to 0.41 (±0.06) at 3 months, with
statistically highly significant differences (P < 0.001) at 1 month
and 3 months from baseline. These results could be attributed to
tannic acid which gets adsorbed well onto the hydroxyapatite
of the tooth by binding the anionic groups on the bacterial
cell wall, leading to protein denaturation and subsequently
resulting in cell death.[21]
For Group B, the mean PI, which was 1.14 (±0.27) at baseline,
reduced to 0.75 (±0.22) at one month and to 0.41 (±0.03) at 3 months,
with statistically significant differences  (P < 0.05) at 1 month
and 3 months from baseline. This result could be attributed to
substantivity of CHX. The tooth surface‑bound CHX due to its
bacteriostatic effect interferes with the bacterial adherence on the
tooth surface, thus preventing plaque formation.[22]
Intragroup effect on gingivitis: A significant parallel reduction
of GI scores was seen among the study participants of Group
A and Group B when the means were compared at different
time intervals. For Group A, the mean gingival index scores
Journal of Indian Society of Periodontology - Volume 25, Issue 6, November-December 2021 523
Figure 6: Comparative evaluation of mean Lactobacilli counts in Group A (0.4%
Triphala mouthwash) and Group B (0.12% chlorhexidine mouthwash) expressed
as 103 CFU/ml of plaque at varying time periods. *P < 0.05 significant. The
mean Lactobacilli counts in Group A (0.4% Triphala mouthwash) and Group
B (0.12% chlorhexidine mouthwash) at different time periods were statistically
significant (one‑way ANOVA, P < 0.05). Tukey’s post hoc analysis showed that
there was a significant difference in the mean plaque index scores. P – Probability
value, CFU – Colony‑forming units, TCC – Total colony count
reduced from 1.0 (±0.16) at baseline to 0.67 (±0.92) at 1 month
and to 0.30 (±0.05) at 3 months, with statistically significant
differences (P < 0.05). The result could be attributed to the
inhibitory activity of Triphala against matrix melano‑proteins‑9,
seen in pathologically elevated collagenases associated with
gingival and periodontal disease.[11]
For Group B, the mean GI scores were 1.07 (±0.22) at baseline
to 0.64 (±0.57) at 1 month and to 0.32 (±0.05) at 3 months,
with statistically highly significant differences  (P < 0.001).
The reduction in gingivitis may be parallel to the reduction in
plaque due to the antiplaque action of CHX.
Intergroup effect on plaque and gingivitis: The intergroup
comparison done at baseline, 1 month, and 3 months showed
that the mean plaque and gingival scores in Group A and
Group B were not statistically significant (P > 0.05), suggesting
that both mouthwashes had the same inhibitory effect on
plaque and gingivitis. Similar results for plaque scores were
obtained in the studies conducted by Desai et al.[11] and Naiktari
et al.,[20] whereas contrasting results were observed in the study
conducted by Bajaj et  al.[14] Bhattacharjee et  al.[23] while for
gingivitis study conducted by Bajaj et al.,[14] Naiktari et al.[20] and
Bhattacharjee et al.[23] showed similar results.  The difference
may be due to the duration of the study period where CHX
was more effective in short‑term studies, whereas Triphala had
better efficacy over a long period of time.
Effect on microbial count: The mean S. mutans and Lactobacilli
counts in plaque in Group A and Group B study participants
showed a significant reduction at follow‑up. For Group A,
the mean S. mutans and lactobacilli counts in plaque showed
statistically highly significant differences (P < 0.001) at 1 month
and 3 months from baseline. The antimicrobial activity of
Triphala can be attributed to the presence of gallic acid, Vitamin
C, ellagic acid, chebulic acid, bellericanin, β‑sitosterol, and
flavonoids present in Triphala as they inhibit the growth of
Gram‑positive and Gram‑negative bacteria.[24]
[Downloaded free from http://www.jisponline.com on Saturday, August 20, 2022, IP: 103.171.117.162]
Bhor, et al.: Effect of Triphala and CHX: Plaque, gingivitis, and oral microbes
For Group B, the mean S. mutans and Lactobacilli counts in plaque
showed statistically highly significant differences (P < 0.001) at
1 month and 3 months from baseline.
An apparent antagonism was observed between S. sanguinis
and S. mutans count (Pearson’s correlation: -0.138), suggesting
that the colonization of S. sanguinis may influence the
subsequent colonization of S. mutans, and this, in turn, may
suggest several ecological approaches toward controlling
dental caries.[5]
The intergroup comparison done at baseline, 1 month, and
3 months showed that the mean S. mutans and S. sanguinis
counts in Group A and Group B were not statistically
significant (P > 0.05), suggesting that both mouthwashes had
the same antibacterial activity at the end of the study which
was in consensus with the study done by Bajaj et al.[14] The CHX
mouthwash was more effective than Triphala mouthwash in
reducing mean Lactobacilli count at 3 months of follow‑up,
which was in contrast to the study conducted by Bajaj et al.[14]
Limitations of the study
The participants in this clinical trial may experience Hawthorne
effect and novelty effect. The experimental period was an
extended period of 3 months; still, further long‑term studies
with a larger sample size as well as including government
schools and government‑aided schools must be performed to
evaluate the antimicrobial, antigingivitis, and antiplaque effects
of Triphala and CHX mouthwash.
CONCLUSION
Triphala (0.4%) and CHX (0.12%) mouthwash showed a similar
trend in preventing plaque formation and in anti‑inflammatory
effect on gingival health with no evident side effects after
90 days of use. Hence, the use of medicinal plants against oral
diseases can be a viable alternative to other antimicrobial agents
as they offer a cheap and effective module in controlling plaque
and bacteria responsible for oral infections.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
REFERENCES
1. Sharma A, Bansal P, Grover A, Sharma S, Sharma A. Oral health
status and treatment needs among primary school going children
in Nagrota Bagwan block of Kangra, Himachal Pradesh. J Indian
Soc Periodontol 2014;18:762‑6.
2. Bhat N, Mitra R, Reddy J, Oza S, Vinayak KM. Evaluation of
efficacy of chlorhexidine and a herbal mouthwash on dental
plaque: An in vitro comparative study. Int J Pharm Bio Sci
2013;4:625‑32.
3. Thomas B, Shetty S, Vasudeva A, Shetty V. Comparative
evaluation of antimicrobial activity of triphala and commercially
available toothpastes: An in‑vitro study. Int J Public Health Dent
2011;2:8‑12.
4. Okahashi N, Nakata M, Terao Y, Isoda R, Sakurai A, Sumitomo T,
et al. Pili of oral Streptococcus sanguinis bind to salivary amylase
524 Journal of Indian Society of Periodontology - Volume 25, Issue 6, November-December 2021
and promote the biofilm formation. Microb Pathog 2011;50:148‑54.
5. Caufield  PW, Dasanayake  AP, Li  Y, Pan  Y, Hsu  J, Hardin  JM.
Natural history of Streptococcus sanguinis in the oral cavity
of infants: Evidence for a discrete window of infectivity. Infect
Immun 2000;68:4018‑23.
6. Badet C, Thebaud NB. Ecology of lactobacilli in the oral cavity:
A review of literature. Open Microbiol J 2008;2:38‑48.
7. National Oral Health Survey and Fluoride Mapping. An
Epidemiological Study of Oral Health Problems and Estimation
of Fluoride Levels in Drinking Water. Vol. 32. New Delhi: Dental
Council of India; 2004. p. 67‑78.
8. Lakade LS, Shah P, Shirol D. Comparison of antimicrobial efficacy
of chlorhexidine and combination mouth rinse in reducing the
Mutans streptococcus count in plaque. J Indian Soc Pedod Prev
Dent 2014;32:91‑6.
9. Mishra R, Tandon S, Rathore M, Banerjee M. Antimicrobial
and plaque inhibitory potential of herbal and probiotic oral
rinses in children: A randomized clinical trial. Indian J Dent Res
2014;25:485‑92.
10. Kapoor D, Kaur N, Nanda T. Efficacy of two different
concentrations of chlorhexidine mouth‑rinse on plaque re‑growth.
Indian J Dent 2011;2:11‑5.
11. Desai A, Anil M, Debnath S. A clinical trial to evaluate the effects
of triphala as a mouthwash in comparison with chlorhexidine
in chronic generalised periodontitis patient. Indian J Dent Adv
2010;2:243‑7.
12. Srinagesh J, Krishnappa P, Somanna SN. Antibacterial efficacy of
triphala against oral streptococci: An in vivo study. Indian J Dent
Res 2012;23:696.
13. Gupta R, Chandrashekar BR, Goel P, Saxena V, Hongal S, Jain M,
et al. Antimicrobial efficacy of aqueous and ethanolic extracts of
Triphala on primary plaque colonizers: An in vitro study. J Young
Pharm 2014;6:7‑13.
14. Bajaj N, Tandon S. The effect of triphala and chlorhexidine
mouthwash on dental plaque, gingival inflammation, and
microbial growth. Int J Ayurveda Res 2011;2:29‑36.
15. Löe H. The gingival index, the plaque index and the retention
index systems. J Periodontol 1967;38:610‑6.
16. Loe H, Silness J. Periodontal disease in pregnancy. I. Prevalence
and severity. Acta Odontol Scand 1963;21:533‑51.
17. James PM, Beal JF. Dental epidemiology and survey procedures.
In: Slack JL, Burt BA, editors. Dental Public Health – An
Introduction to Community Dental Health. 2nd ed. Bristol: John
Wright and Sons Ltd.; 1981. p. 86‑118.
18. Sitges‑Serra A, Linares J. Limitations of semi‑quantitative method
for catheter culture. J Clin Micro 1988;26:1074‑7.
19. Bertness J, Holt K, editors. Promoting Oral Health in Schools:
A Resource Guide. 2nd ed. Washington, DC: National Maternal
and Child Oral Health Resource Center; 2013.
20. Naiktari RS, Gaonkar P, Gurav AN, Khiste SV. A randomized
clinical trial to evaluate and compare the efficacy of triphala
mouthwash with 0.2% chlorhexidine in hospitalized patients with
periodontal diseases. J Periodontal Implant Sci 2014;44:134‑40.
21. Jagadish L, Anand Kumar VK, Kaviyarasan V. Effect of triphala
on dental bio‑film. Indian J Sci Technol 2009;2:30‑3.
22. Pai MR, Acharya LD, Udupa N. The effect of two different dental
gels and a mouthwash on plaque and gingival scores: A six‑week
clinical study. Int Dent J 2004;54:219‑23.
23. Bhattacharjee R, Nekkanti S, Kumar NG, Kapuria K, Acharya S,
Pentapati KC. Efficacy of triphala mouth rinse (aqueous extracts)
on dental plaque and gingivitis in children. J Investig Clin Dent
2015;6:206‑10.
24. Gowda DV, Muguli G, Rangesh PR, Deshpande RD.
Phytochemical and pharmacological actions of Triphala:
Ayurvedic formulation – A review. Int J Pharm Sci Rev Res
2012;15:61‑5.