DOI: 10.1111/ipd.
12018
The effects of natural compounds-containing mouthrinses on
patients with fixed orthodontic appliance treatment: clinical
and microbiological outcomes
YONG CHEN1, RICKY W. K. WONG1, CHAMINDA JAYAMPATH SENEVIRATNE2, URBAN
HAGG1, COLMAN MCGRATH3 & LAKSHMAN P. SAMARANAYAKE2
1
Discipline of Orthodontics, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China, 2Discipline of Oral
Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China, and 3Discipline of Dental Public Health,
Faculty of Dentistry, The University of Hong Kong, Hong Kong, China
International Journal of Paediatric Dentistry 2013; 23:
452–459
Aim. To investigate the effects of two natural
compounds-containing mouthrinses (NCCMs) (a
fructus mume (FM) extract–containing mouthrin-
se and an essential oil (EO)-containing mouthrin-
se) on gingival health and microbial profiles in
young orthodontic patients.
Design. This 6-month randomized, single-blinded,
parallel-controlled clinical trial consists of 90
patients with fixed appliance treatment. The sub-
jects were allocated to (1) negative control group:
oral hygiene instruction (OHI) alone; (2) test
group 1: OHI plus EO mouthrinse; and (3) test
group 2: OHI plus FM mouthrinse. Clinical exam-
inations included plaque index (PI), bleeding
Introduction
Previous studies have shown that fixed ortho-
dontic appliances create new locations for pla-
que retention and thereby increase the risk of
gingival inflammations and dental caries1–3.
Therefore, antimicrobial mouthrinses have
been recommended to be used as an adjunct
to enhance plaque removal and maintain gin-
gival health for orthodontic patients4–6.
Although the clinical benefits of the mouth-
rinses that contain active agents such as
chlorhexidine (CHX), triclosan, and cetylpy-
ridinium chloride (CPC) have been well docu-
mented7–9; however, their side effects are of
concern both in public and practitioners.
Long-term CHX mouthrinse use results in
Correspondence to:
Dr Ricky W.K. Wong, 2/F, Orthodontics, Prince Philip
Dental Hospital, 34 Hospital Road, Hong Kong, China.
E-mail: fyoung@hkucc.hku.hk
452
index (BI) and modified gingival index (MGI).
Salivary microbial quantifications included total
aerobic and anaerobic bacteria, Streptococci and
Lactobacilli counts. Clinical and microbiological
examinations were conducted at baseline, 3rd and
6th months (T1, T2, and T3).
Results. BI was significantly reduced in both the
FM mouthrinse and EO mouthrinse groups com-
pared with the negative control group at T3
(P < 0.05). There were no significant intergroup
differences in salivary bacteria counts in all groups
(P > 0.05).
Conclusion. Both NCCMs effectively reduced gin-
gival bleeding without causing significant altera-
tions of microbial profile in young orthodontic
patients.
taste sensation alterations, tooth staining, and
desquamation or soreness of oral mucosa10,11.
Triclosan application has been suspected to
cause resistant strains of bacteria and allergic
contact dermatitis12,13, and CPC mouthrinse
has been found to cause tooth staining and
burning sensation14. These adverse reports
increase the demands to explore alternative
agents for oral health promotion15.
The application of therapeutic compounds
derived from natural products such as plant, ani-
mal,microorganismandmarineorganismsinthe
treatment of oral diseases has a long history, and
numerous natural compounds-containing
mouthrinses (NCCMs) have been proved effec-
tive against periodontal diseases16–20. Currently,
essential oil (EO)-containing mouthrinse is the
NCCM that has been widely used in the general
population as well as in orthodontic patients5,21;
however, concerns also have been raised for its
high quantity of alcohol presence22. Fructus
mume (FM) is a herbal medicine that has been
© 2012 John Wiley & Sons Ltd, BSPD and IAPD

used to relieve cough, treat ulceration and
improve digestive function for thousands of
years23. Recent in vitro studies have demon-
stratedthatFMextractnotonlyhasstrongantiox-
idant effect (FM extract contains several
antioxidant flavonoids such as naringin and ru-
tin) and anti-inflammation capacities (FM
extract inhibits prostaglandin (PG) E2 and nitric
oxide (NO) production)24,25, but also has ade-
quate antimicrobial effects on oral pathogens in
planktonic and biofilm status26,27. Thus, this
water-soluble natural extract is likely to be used
as a nonalcohol-containing NCCM in the man-
agement of gingivitis. As high-performance
liquid chromatography (HPLC) data have shown
that the main ingredients of FM extract are
organic acids that include citric acid, tartaric acid,
oxalic acid, etc.26, we developed a two-stage
mouthrinse whereby the second-stage sodium
bicarbonatesolutionwasusedtoneutralizeresid-
ualofthefirst-stageacidicFMsolution.
In addition, although the clinical effects of
EO mouthrinse on orthodontic patients have
been proved5, its microbial effect remains
unclear. Therefore, the aim of this study was
to investigate the clinical and microbiological
effects of the two NCCMs (the FM extract-
containing mouthrinse and an EO-containing
mouthrinse) in the improvement of gingival
health for the patients with fixed appliance
treatment.
Material and methods
Study design
A 6-month, randomized, single-blinded, par-
allel-controlled clinical trial involving 90 sub-
jects (mean age 17.7  3.9 years; 52% (47)
women). Subjects were recruited from the
patients undergoing fixed orthodontic appli-
ance therapy in the Faculty of Dentistry, the
University of Hong Kong (from October to
November 2010 and follow-up for 6 months).
Subjects were selected based on the following
inclusion criteria: (1) age  13 years, (2)
nonsmoker, (3) generally healthy, (4) pre-
existing gingivitis [modified gingival index
(MGI)  1] and (5) no evidence of periodon-
titis at any site (i.e., probing depth  4 mm).
The study protocol was approved by the
© 2012 John Wiley & Sons Ltd, BSPD and IAPD
Nature compounds for gingivitis 453
Institution Review Board of The University of
Hong Kong/Hospital Authority Hong Kong
West Cluster (Reference Number: UW 10-
278). Written informed consent was obtained
from all participants (and/or their guardians).
A sample size calculation of 25 per group was
derived, which was based on the hypothesis
of detecting a 0.10 intergroup difference in
MGI with 90% power (a = 0.05)28. Five addi-
tional subjects per group were recruited to
allow for potential dropout.
The 90 participants were randomly allocated
to three groups through concealed block ran-
domization in groups of six (stratified accord-
ing to baseline MGI). To blind the examiner, a
designated dental surgery assistant identified
from sealed envelopes which group individuals
were to be assigned to. The three groups com-
prised of1 a negative control group who
received standardized oral hygiene instruction
(OHI, included the methods and times of tooth
brushing and dental flossing during orthodon-
tic treatment) alone2; test group 1 who
received OHI plus EO mouthrinse (Listerine®,
tartar control; IDS manufacturing Ltd, Bang-
kok, Thailand); Usage: Rinse with 20 mL solu-
tion for 30 s, twice daily; and3 test group 2
who received OHI plus the FM mouthrinse
which is a two-stage mouthrinse: solution A
5% FM extract (five times concentrate powder
from rough extract, Nong’s company, Hong
Kong, China) and Solution B 2% sodium
bicarbonate (Wong Li international Ltd, Hong
Kong, China), the vehicle for both solutions is
distilled water. Usage procedure is as follows:
rinse with 20 mL solution A for 30 s and then
immediately following rinse with 20 mL solu-
tion B for 30 s twice daily. Compliance was
monitored by the designated assistant, and
the mouthrinses were dispensed at 1-month
intervals. All clinical examinations and saliva
microbiological assays were performed by
one trained and masked examiner (YC) at
baseline (T1), 3rd (T2) and 6th (T3) months,
respectively.
Clinical examinations
The gingival health status of participants was
assessed by the plaque index (PI)29: 0 = no
plaque; 1 = discontinuous band of plaque at
454 Y. Chen et al.
the gingival margin; 2 = up to 1 mm continu-
ous band of plaque at the gingival margin;
3 = band of plaque wider than 1 mm but less
than one-third of the surface; 4 = plaque cov-
ering one-third or more of the surface, but
less than two-thirds of the surface; and
5 = plaque covering two-thirds or more of
the surface. One measurement for each tooth
was scored for all categories; bleeding index
(BI)30: 0 = absence of bleeding after 30
seconds, 1 = bleeding observed after 30
seconds, and 2 = immediate bleeding; MGI31:
0 = absence of inflammation, 1 = mild
inflammation (either marginal or papillary
gingival unit), 2 = mild inflammation (entire
marginal and papillary gingival unit),
3 = moderate inflammation, and 4 = severe
inflammation, on the facial side of at Ramfj-
ord index teeth index (upper right first molar,
upper left central incisor, upper left first
premolar, lower left first molar, lower right
central incisor, lower right first premolar)32.
Saliva microbiological assay
Saliva sample collection (unstimulated): No
intake of food or drink and no application of
oral hygiene procedure or mouthrinses 2 h
before collection; the subjects were in a
seated position with their head tilted forward.
The procedure was accomplished in a quiet
and well-ventilated room, and samples were
collected after clinical examinations. The
examiner instructed each participant to rinse
with distilled water once and then collect
saliva produced during a 5-min period in a
sterile plastic bottle33.
Salivary microbe quantification: Saliva sam-
ples were packed on ice and plated within
2 hours. Serial 10-fold dilutions were made,
and 0.05 mL of each dilution was inoculated
onto horse blood agar (HBA) (Oxoid Colum-
bia blood base, Oxoid Limited, Basingstoke,
Hampshire, UK) for aerobic culture and
anaerobic culture, whereas Mitis Salivarius
agar (BD Difcoe; Mitis Salivarius Agar,
Sparks, MD, USA) and Rogosa agar (BD Dif-
coe; Rogosa SL Agar, Sparks, MD, USA) were
used for selective culture streptococci and lac-
tobacilli, respectively. Inoculum was spread
evenly over the agar using a spiral plater
(Autoplate® 4000; Advanced Instruments,
Inc., Norwood, MA, USA). The aerobic and
anaerobic cultures were incubated for 48 h.
Colony counts were performed on plates
yielding 30–300 bacteria colonies per plate34.
The number of viable bacteria per milliliter was
calculated by multiplying the number of colo-
nies by the reciprocal of the dilution factor.
Statistical analysis
The Statistical Package for Social Sciences
17.0 (SPSS Inc, Chicago, IL. USA) was used
for the data analysis. Kolmogorov–Smirnov
test was computed for each variable to assess
whether the variables followed a Gaussian dis-
tribution. For parametric variables (PI, MGI,
and BI), intragroup comparisons were
performed by paired t-test, and intergroup
comparisons were performed by t-test. For
nonparametric variables (log10 bacteria
counts), intergroup comparisons were per-
formed by Mann–Whitney U-test, and intra-
group comparisons were performed by
Wilcoxon test. The level of significance was
set at P < 0.05.
Results
A flow diagram of the study process is pre-
sented in Fig. 1. Seventy-nine of the 90 origi-
nal subjects (88%) completed this clinical
trial, and there was no significant difference
in the dropout rate between the three groups
(P > 0.05). No adverse reactions or side
effects were reported from any subjects, and
only time commitments were cited by partici-
pants as reasons for not attending follow-up
assessments. Intra- and intergroup compari-
sons of the clinical parameters are presented
in Table 1. There was no significant difference
in PI, BI and MGI at baseline between the
three groups. Intergroup comparison identi-
fied significant differences of BI at T3
between the negative control group and the
two mouthrinse groups (P < 0.05). There
were no significant differences in any of the
clinical parameters between the FM mouthr-
inse and the EO mouthrinse group
(P > 0.05). Intragroup comparison showed
significant changes in BI at both T2 and T3
© 2012 John Wiley & Sons Ltd, BSPD and IAPD
 Nature compounds for gingivitis 455

(aerobicandanaerobic)andMitisSalivariusagar,
482 subjects underwent primary screening while no lactobacilli was detected in Rogosa agar
culturein16ofthesamplesatT1(6inthenegative
control, 5 in the FM mouthrinse, and 5 in the EO
mouthrinse group), 14 of the samples at T2 (6, 3,
111 subjects were eligible according to
recruitment criteria and5,respectively)and13ofthesamplesatT3 (6,
3, and 4,). There were no significant intergroup
21 declined to
participate differences in total anaerobic and aerobic bacte-
90 subjects underwent clinical ria, streptococci or lactobacilli counts between
and saliva examinations three groups (P > 0.05). Intragroup comparison
showedasignificantchangeofstreptococcicount
EO FM
at T2 compared with baseline in the FM mouthr-
Control
mouthrinse mouthrinse
group (–) inse group (P < 0.01), a significant change of
group group
(n = 30) (n = 30)
(n = 30) anaerobic bacteria counts at T2 and T3 compared
with baseline in EO mouthrinse group
(P < 0.05), and a significant change of anaerobic
3 months clinical and saliva bacteria, aerobic bacteria and streptococci count
examinations (n = 84)
atT2andanaerobicbacteriacountatT3compared
with baseline in the negative control group
(P < 0.05)(Fig. 2).
6 months clinical and saliva
examinations (n = 79)

Discussion
EO FM
mouthrinse mouthrinse Control This study firstly investigated the clinical and
group (–)
group group microbiological effects of NCCMs in ortho-
(n = 25 ) (n = 26)
(n = 28) dontic patients with gingivitis. In all clinical
indices, BI was the only one which was sig-
Fig. 1. The flow chart of the study process.
nificantly changed in the whole study pro-
cess. The intragroup reductions in BI were
assessments compared with T1 in all three observed in all three groups at T2 and T3. It
groups (P < 0.01), and a significant change of is plausible that participation in a ‘trail’ in
MGI at T2 compared with T1 among the FM itself motivated and enhanced their oral
mouthrinse and EO mouthrinse groups hygiene behaviors35. However, both mouthr-
(P < 0.05). inse groups showed significant higher reduc-
Detectable levels of saliva bacteria were found tion in BI at 6 months compared with the
in all saliva samples in blood agar negative control group (OHI alone), which

Table 1. Clinical gingival assessments over time among study groups

Baseline(T1) 3rd (T2) 6th (T3)

Clinical parameters Group N Mean  SD Mean  SD Mean  SD

PI EOM 28 1.61  0.45 1.70  0.45 1.69  0.39

FMM 25 1.66  0.47 1.91  0.50† 1.66  0.40
NC 26 1.78  0.39 1.87  0.48 1.83  0.50
BI EOM 28 0.48  0.38 0.22  0.17†† 0.16  0.16†††, *
FMM 25 0.49  0.37 0.21  0.18†† 0.16  0.18†††, *
NC 26 0.54  0.32 0.26  0.21†† 0.31  0.25††
MGI EOM 28 1.68  0.42 1.54  0.26† 1.56  0.19
FMM 25 1.69  0.36 1.56  0.20† 1.59  0.17
NC 26 1.65  0.29 1.66  0.24 1.63  0.17

EOM, essential oils mouthrinse; FMM, fructus mume mouthrinse; NC, negative control.
Intergroup comparison between mouthrinse groups and the negative control group (*P < 0.05, t-test).
Intragroup comparison between 3rd and 6th month and baseline (†P < 0.05, ††P < 0.01, †††P < 0.001, paired t-test).

© 2012 John Wiley & Sons Ltd, BSPD and IAPD

456 Y. Chen et al.
Salivary bacteria counts (Log10 cfu/ml)
Fig. 2. Box plot diagram illustrating the changes of bacteria
count (total anaerobic bacteria, total aerobic bacteria,
streptococci and lactobacilli) at T1, T2 and T3. (●, outliers
and ★, extreme outlier values) (Values on the y-axis:
log10 cfu/mL).
indicated both mouthrinses exhibited to some
extent antigingivitis capacities. There was no
significant difference in BI between the FM
mouthrinse and EO mouthrinse groups at
any time point, which suggested that they
might have comparable effectiveness. How-
ever, due to the limitation of the sample size,
this study did not have a control with sodium
bicarbonate rinse; therefore, although the
sodium bicarbonate solution did not exhibit
any antimicrobial effect at the present con-
centration in vitro, this study only can verify
the clinical beneficial effect that might come
from the combination of the two rinses (FM
extract solution plus sodium bicarbonate
solution) instead of FM extract solution
alone. Intragroup differences in MGI were
evident at T2 (compared with baseline) in
both mouthrinse groups; however, there was
no significant difference in MGI in all three
groups at T3. In gingival inflammation evalu-
ation, BI was reported to have greater dis-
crimination compared with MGI (or gingival
index) as the latter reflects more the past
history rather than the current status of
inflammation36,37.
In this study, no intergroup changes in PI
were evident. This result was similar to a
previous study which showed that the PI did
not significantly change during EO mouthr-
inse application in orthodontic patients (PI
mean was 0.799 at baseline and 1.014 at
6-month in EO mouthrinse group)5. PI in
itself is a marker of oral hygiene status per se
rather than gingival health status. Some
other studies also showed that the applica-
tion of antimicrobial mouthrinses or tooth-
pastes resulted in statistically significant
reductions in gingival bleeding but no statis-
tically significant reduction in plaque
scores38,39. These clinical results suggested
that the antigingivitis mechanism of the FM
mouthrinse and EO mouthrinse is likely to
be anti-inflammatory rather than reduction
in plaque mass. In addition, their bacteria-
static effects21,26, which could slow plaque
maturation and reduce plaque pathogenicity,
might also contribute to their antigingivitis
capacity. Further studies are warranted to
support or refute this claim.
Saliva is a complex mixture containing sal-
iva gland fluid, gingival crevicular fluid
(GCF), oral bacteria, cells etc., and its diag-
nostic value has been well recognized in
recent years40. In this study, salivary aerobic
and anaerobic bacteria were used to represent
the oral bacteria load, and streptococci and
lactobacilli were examined as they commonly
exist in supragingival plaque and saliva41,42.
Investigation of bacteria count aimed to mon-
itor the possible microbial shifting caused by
the NCCMs over the 6-month period. Intra-
group differences in bacteria counts were
identified in all groups; thus, these kinds of
changes may be attributed to natural varia-
tions rather than the effects of mouthrinses
per se. No intergroup difference in the bacte-
rial load among the three groups was identi-
fied at any time points, suggesting that both
NCCMs did not cause a significant shift of
oral bacteria in this study. Although several
short-term studies indicated that antimicrobial
mouthrinses caused the reduction in strepto-
cocci count6,43, long-term studies showed that
antimicrobial mouthrinses have no effect on
streptococci or other bacteria counts in plaque
or saliva44,45.
© 2012 John Wiley & Sons Ltd, BSPD and IAPD

This study aimed to investigate the clinical
and microbiological effects of a novel NCCM
and a clinically proven NCCM in orthodontic
patients whose gingival health is challenged
by appliance-caused bacteria-induced gingival
inflammations1–3 and orthodontic periodontal
tissue remodeling-related nonplaque-induced
gingival inflammations46,47, and the present
positive results indicated both of them have
potential values for clinical application. How-
ever, due to the ethical consideration
(patient’s right to know the interventions)
and the difficulty of placebo preparation, the
present study employed a single-blinded
(examiner blinded), randomized trial; there-
fore, the test power of this study might not
be as good as a double-blinded randomized
trial. Another limitation of this study was that
most of the participants only had mild gingi-
vitis (MGI mean = 1.67), which made it diffi-
cult to have a dramatic improvement by the
mouthrinses; therefore, the potential antigin-
givitis capacity of both NCCMs might not yet
be fully revealed. In addition, as the culture
plates were only incubated for 48 h in the
present study, bacteria such as Porphyromon-
as gingivalis (Pg) may require much longer
incubation time and therefore bacterial
counts may be underestimated, and as only
limited specific bacteria species were investi-
gated, the underlying microbial effects of the
two NCCMs still warrant further study.
Conclusion
In summary, with the limitations of the pres-
ent study, it was concluded that both the FM
mouthrinse and the EO mouthrinse effec-
tively reduce gingival bleeding without caus-
ing significant alterations of microbial profile
in orthodontic patients.
Why this paper is important to paediatric dentists
• This paper provided an insight into natural com-
pounds as alternative antigingivitis agents for young
patients and inspired future studies in this area.
• Gingivitis is popular in young patients especially when
they undergoing orthodontic treatment, paediatric den-
tists should know the cost/effect of anti-gingivitis mou-
thrinses used as an adjunct to patient’s oral hygiene
routine, and make right recommendations for them.
© 2012 John Wiley & Sons Ltd, BSPD and IAPD
Nature compounds for gingivitis 457
Acknowledgements
This study was supported by the 2008 Inno-
vation in Oral Care Awards (International
Association for Dental Research/Glaxo-
SmithKline). We thank Ms Fiona Cheng for
the clinical assistance and Ms Joyce Yau for
the technical assistance. The authors reported
no conflict of interest related to this study.
Conflict of interest
The authors declare no conflict of interest.
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