Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2015; 60: 317–327

doi: 10.1111/adj.12340

Effects of curcumin on crevicular levels of IL-1b and CCL28

in experimental gingivitis
SJ Pulikkotil,* S Nath†
*Senior Lecturer, Department of Restorative Dentistry, School of Dentistry, International Medical University, Kuala Lumpur, Malaysia.
†Senior Lecturer, Department of Periodontology, Vananchal Dental College and Hospital, Garhwa, Jharkhand, India.

ABSTRACT
Background: Curcumin has anti-inflammatory properties. The aim of this study was to compare interleukin-1b (IL-1b)
and chemokine (C-C motif) ligand 28 (CCL28) levels following a topical application of curcumin (CRM), chlorhexidine
(CHX) and chlorhexidine-metronidazole (CHX-MTZ) in an experimental gingivitis human model.
Methods: Sixty systemically healthy selected subjects were randomly assigned to one of three topical antigingivitis gels.
Each gel was applied twice daily for 10 minutes as the sole method of oral hygiene for 29 days on the test quadrant
only. Modified gingival index (MGI), plaque index (PI), bleeding on probing (BOP) and probing depth (PD) were
assessed at baseline, 29 days and 60 days. Estimation of IL-1b and CCL28 levels in gingival crevicular fluid was done at
baseline and at 29 days.
Results: The increase of IL-1b in the CRM (14.52  16.6 pg/ml) and CHX-MTZ (31.63  15.96) groups was signifi-
cantly less than that of the CHX group (70.55  38.81). Similar results were also observed for CCL28 (CRM:
8.12  8.78 pg/ml; CHX-MTZ: 12.81  18.68; CHX: 41.15  22.82). All groups had a significant increase in MGI, PI
and BOP at 29 days.
Conclusions: The anti-inflammatory potential of topical curcumin was similar to CHX-MTZ but superior to CHX in
affecting IL-1b and CCL28 levels.
Keywords: CCL28, curcumin/curcuma longa, gingivitis, inflammation, interleukin-1beta.
Abbreviations and acronyms: BOP = bleeding on probing; CHX = chlorhexidine; CHX-MTZ = chlorhexidine-metronidazole; CRM =
curcumin; GCF = gingival crevicular fluid; MGI = modified gingivial index; PD = probing depth; PI = plaque index.
(Accepted for publication 17 August 2014.)

antimicrobials reduce the microbial burden and host

INTRODUCTION
modulation therapeutics modulate the host immuno-
Gingivitis is the most prevalent inflammatory periodon- inflammatory response.6–8 Although bacteria are essen-
tal disease, affecting more than 80% of the world’s tial for periodontal disease, most tissue damage is
population.1 Pathogenic biofilm around the teeth initi- caused by inflammatory mediators and free radicals.2,8
ate the disease process. The host immuno-inflammatory Current preventive and treatment approaches are only
response leads to pathogenesis and progression of partially effective as they mainly focus on biofilm man-
disease from gingivitis to periodontitis in susceptible agement rather than controlling the host inflammatory
individuals.2 Excessive production of inflammatory mediators responsible for tissue damage.9 The potential
cytokines, especially interleukins (IL) and tumour side effects seen in commonly used topical oral chemo-
necrosis factor-a (TNF-a) have been implicated in peri- therapeutics include increasing failure, long-term use
odontal destruction.2 Mechanical plaque control is the and drug resistance.10 There is a need to develop a
most effective method for controlling gingivitis.3 How- more effective and efficient preventive and treatment
ever, the perception that the general population does approach for periodontal disease which includes a com-
not undertake adequate oral hygiene has led to a search bined approach towards host modulation, inflamma-
for adjunctive agents to control the effects of biofilm.4 tion resolution, as well as direct management of
The addition of a chemical agent as an adjunct has bacteria.9 This has led to the screening of several
been suggested to improve conventional methods of medicinal plants which have equal efficacy and fewer
plaque control.4,5 Chemical agents can act in two ways: side effects.11
© 2015 Australian Dental Association 317
SJ Pulikkotil and S Nath

Turmeric has been used for the treatment of many gel, and a combination gel of chlorhexidine and met-
diseases.12 Turmeric (also known as Curcuma longa) ronidazole by assessing IL-1b and CCL28 levels from
is a member of the ginger family, Zingaberaceae.12 GCF and the clinical parameters during experimental
This yellow spice contains the polyphenol curcumin gingivitis.
in its rhizome. Extensive research on curcumin has
demonstrated a wide spectrum of therapeutic effects
MATERIALS AND METHODS
such as anti-inflammatory, antioxidant, antibacterial,
antiviral, antifungal, antitumour, antispasmodic, he- This study was a randomized, double-blinded, con-
patoprotective and wound healing properties.13–15 trolled, parallel group clinical trial. It was conducted
The anti-inflammatory properties of curcumin can be at Chhattisgarh Dental College and Research Institute,
useful in the treatment of inflammatory periodontal Rajnandgaon, India from May 2013 to September
diseases.14 This effect is mediated through its ability 2013. The clinical trial was approved by the Institu-
to inhibit cyclooxygenase-2 (COX-2), lipoxygenase tional Ethics Committee (CDEC/15/2013/01/CP) and
(LOX) and inducible nitric oxide synthase registered in the Clinical Trials Registry, India (CTRI/
(iNOS).14,15 Another mechanism is by modulating 2013/12/004223). Voluntary informed consent was
the activity of signalling pathways and transcription obtained from the subjects after providing them with
factors, especially nuclear factor-jB (NF-jB), activat- detailed information about the trial. By obtaining
ing protein-1 (AP-1) and mitogen-activated protein informed consent and in the conduct of the study, we
(MAP) kinases.15 In a recent study, curcumin effec- adhered to the principles of experimentation involving
tively inhibited activation of NF-jB in the gingival human subjects as outlined in the Declaration of
tissues.14,16 Down-regulation of these pathways leads Helsinki.
to suppression of proinflammatory cytokines such as
IL-1b, IL-6, IL-12, TNF-a IL-8, matrix metallo-
Sample size
proteinase-2 and matrix metalloproteinase-9.15,16
Curcumin can target both bacteria and host inflam- The sample size was calculated for an alpha error of
matory cells and would be highly effective in pre- 0.05 and power at 90%. As there were no similar
venting the destruction associated with periodontal studies, we selected the deviation seen in CCL28
diseases. levels as reported by Ertugrul et al.,21 which was
Gingival crevicular fluid (GCF) is an exudate whose greater than in IL-1b levels reported in other stud-
constituents can be used to assess periodontal dis- ies.18,20 On the basis of this data, the sample size was
ease.17 In our study we used two markers of inflam- calculated to 17 subjects to enable detection of a
matory status, IL-1b and a novel marker CCL28. difference of 20 ng/ml. However, considering the pos-
IL-1b is important in periodontal tissue destruction sibility of drop-out among subjects, the total number
and its level is indicative of the activeness of dis- of patients was 20 for each group.
ease.18,19 A site specific increase in IL-1b has been
observed in an experimental gingivitis model.20 The
Participants
chemokine (C-C motif) ligand 28 (CCL28) is a novel
marker for the diagnosis of gingivitis and periodonti- Sixty volunteers aged between 18 to 35 years were
tis.21 Previous studies have shown CCL28 is associ- recruited. The subjects’ inclusion was based on the
ated with increased epithelial inflammation.22–24 The following criteria: (1) exhibit good periodontal health
production of IL-1b increases following contact with with no teeth having a probing depth >3 mm; (2)
a microbial stimulus, which in turn significantly systemically healthy; (3) possessing at least 20
increases the expression of CCL28.21 natural teeth; (4) negative for hypersensitivity to
To our knowledge, there are no studies evaluating curcumin, chlorhexidine and/or metronidazole. Partic-
the effectiveness of curcumin as a topical chemother- ipants who used tobacco, who had recently used
apeutic agent for periodontal disease. Curcumin has antibiotics, aspirin or non-steroidal anti-inflammatory
a combined anti-inflammatory, antioxidant and anti- drugs (NSAIDs) during the past one month, and
bacterial effect.13 We hypothesized that curcumin pregnant and lactating mothers were excluded from
will be superior in reducing the severity of gingival this study.
inflammation compared to popular commercially
available topical antibacterials containing chlorhexi-
Test products
dine and metronidazole. We are not aware of any
studies to our knowledge that have assessed anti- Three types of commercially available chemotherapeu-
inflammatory effects along with clinical changes. The tic preparations were tested in this clinical trial: (1)
aim of this study was to investigate the anti-inflam- group I – gel containing curcumin (CRM) (Curenext
matory effects of curcuma oral gel, chlorhexidine Oral Gelâ, Abbott Health Care, Mumbai, MH,
318 © 2015 Australian Dental Association
 Anti-inflammatory effects of curcumin

India). Each gram contained curcuma longa extract margin. Its function was to induce experimental gingi-
(10 mg); (2) group II – gel containing chlorhexidine vitis on the selected treatment site and keep the gel in
(CHX) (Hexigelâ, ICPA Health Products Ltd, Mum- contact with the gingival margin of the experimental
bai, MH, India). Each gram contained chlorhexidine quadrant.
equivalent to chlorhexidine gluconate 1% w/w; (3)
group III – gel containing chlorhexidine and metroni-
Treatment phase
dazole (Metrogyl DG gelâ, JB Chemicals & Pharma-
ceuticals Ltd, Mumbai, MH, India). Each gram Two weeks after scaling, participants returned for the
contained chlorhexidine gluconate solution IP 0.25% recording of clinical data and the collection of GCF
w/w and metronidazole (10 mg). as baseline. The experimental quadrant was assessed
To ensure blinding, all tubes containing the topical for periodontal disease. Participants continued in the
agents were similar and not labelled except for their trial if they had a gingival index and plaque index
lot numbers. Neither the examiner (SN) nor the recor- score of less than 0.5 and absence of bleeding on
der (RPP) had access to the subject’s treatment code. probing. At baseline the participants were stratified
All topical agents were packaged in 20 g tubes. into three groups and distributed randomly according
to a computer generated code with 20 participants in
each group: in the first group CRM gel; in the second
Study design
group CHX gel; and in the third group CHX-MTZ
This was a randomized, double-blinded, controlled, gel was applied.
parallel clinical trial. The clinical trial was divided To induce experimental gingivitis, subjects were
into pre-screening, treatment and post-treatment instructed to wear the provided tooth shield over the
phases. Figure 1 describes the treatment study design. selected quadrant during their twice a day brushing
and flossing routine for a period of 29 days. Partici-
pants self-administered approximately 1 g of gel in the
Pre-screening phase
tooth shield before placing over the experimental quad-
The recruited subjects underwent professional scaling rant prior to their oral hygiene routine. The shield was
and tooth polishing. The importance of maintaining left on the quadrant for 10 minutes so as to permit gin-
oral hygiene was reinforced by the same operator gival contact of the experimental gel as per the manu-
(SJP). A quadrant for experimentation was randomly facturer’s instruction. The mouth was rinsed following
selected by lottery method. removal of the shield. Participants were instructed not
A tooth shield was constructed only for the experi- to consume any food or liquid at least one hour after
mental quadrant by taking an alginate impression and gel application. Thus, the tooth shield prevented any
pouring it in die stone to obtain a cast. A spacer of mechanical removal of plaque during brushing and
0.5 mm was used over this cast. The tooth shield was flossing for the purpose of inducing gingivitis in the
made of thermoplastic mouthguard material of 1 mm selected area and allowing the experimental gels to
thickness and then trimmed 2 mm beyond the gingival remain in contact during this time.

Fig. 1 Treatment study design.

© 2015 Australian Dental Association 319
SJ Pulikkotil and S Nath

During the study period participants followed their selected sites by using a UNC-15 periodontal probe
usual dietary habits and were instructed to avoid any (Hu-Friedy Inc.â, Chicago, Illinois, USA). In addition
antibacterial rinses (e.g. CHX or phenolic compounds), to this examination, the gingiva and surrounding soft
therapeutic dentifrices (e.g. triclosan or stannous fluo- tissue were visually inspected by the same examiner for
ride), irrigating devices, chewing gum and mints. All the presence of any adverse reaction.
subjects were also given verbal and written instructions Subjective evaluations of participants were done
about the correct use of oral gel and oral hygiene meth- after 29 days using a questionnaire. Subjects were
ods. They were told to report back in case of any use instructed to answer either ‘yes’ or ‘no’ for pain,
of antibiotics or anti-inflammatory drugs. Patients burning sensation, pruritis/itchiness, dryness of the
were recalled after 29 days. During this 29-day period, mouth, taste disturbance, discolouration of teeth and
each patient was given a diary to record the time of bitter taste in the mouth. Patients were asked to rate
application, duration and any adverse effects such as the severity of the side effects as mild, moderate or
pain, itching, ulceration, discomfort, discolouration severe if present.
and taste disturbance. They were instructed to report
back immediately in case of any side effects.
Examiner calibration
All measurements were performed by one trained and
Post-treatment phase
calibrated periodontist (SN). The repeatability of the
Following the day 29 visit, GCF samples and clinical examiner for MGI, PI and PD scoring was determined
recordings were made for all participants in the study prior to the commencement of the study. Ten subjects
quadrant by the same examiner (SN). During this per- were clinically examined and then measurements were
iod they reinstituted all home care procedures and reassessed after 60 minutes from the initial examina-
received professional scaling, tooth polishing and oral tion. The examiner’s repeatability of mean index
hygiene instruction to re-establish periodontal health. scores was assessed using the intraclass correlation
Subjects were recalled again at 35 and 60 days (post- coefficient. The intraclass coefficients for the mean
treatment visit) to evaluate for signs of periodontal MGI, PI and PD were 0.89, 0.87 and 0.84
health. All clinical parameters were re-recorded at
60 days.
Collection of GCF
Each participant was given four 20 g tubes of the
respective experimental gel. To check for compliance, The first (baseline) GCF samples were obtained from
participants were asked to return all used and unused/ five selected sites in the test quadrant prior to the first
partially used tubes after 29 days. This allowed us to application of the experimental gel. The second GCF
measure the amount of gel used by the patients during samples were collected after 29 days of gel application
the treatment period. from the same selected sites. The collection was
always done prior to assessment of the clinical param-
eters. Before the collection of GCF, supragingival pla-
Outcome measurements
que was carefully removed with gauze. The teeth were
The single trained and calibrated examiner (SN) con- isolated with sterile gauzes and the areas around the
ducted all clinical examinations. The examiner (SN) gingival crevice were gently dried with an air syringe.
and recorder (RPP) were blinded to the gels assigned GCF samples were obtained from each participant by
to each subject. placing colour coded, 1–5 ll calibrated microcapillary
The primary outcome was assessment of IL-1b and pipettes (Sigma Aldrich Chemicals Company Ltd,
CCL28 levels from GCF at baseline and 29 days fol- St Louis, MO, USA) extracrevicularly. The sites for
lowing treatment for all participants. The secondary GCF collection were selected as follows: labial (mid)
outcomes were Plaque Index (PI),25 Modified Gingival site of central incisor; mesiobuccal site of first premo-
Index (MGI),26 bleeding on probing (BOP) and probing lar and second molar; and mid-buccal and lingual/pal-
depth (PD) recorded at baseline, 29 and 60 days on the atal (mid) site of first molar. This ensured a fair
selected quadrant. PI and MGI were evaluated after air representation of sites from the experimental quad-
drying the teeth and a score of 0 to 3 was assigned to rant. Micropipettes were kept for not more than
the four gingival areas of teeth: distofacial, mesiofacial, 5 minutes or until a volume of 1 ll was collected.
facial and lingual surface for each tooth. The values of The fluid was immediately transferred to a plastic vial
the four sites of each tooth were averaged to determine and frozen at –70 °C. All quadrants expressed at least
the score of PI and MGI. BOP was recorded as the 3 ll of GCF. The pooled volume was calculated for
overall percentage of sites with bleeding occurring each quadrant. Pipettes in sites which did not express
within 15 seconds of probing. PD was measured from any volume of fluid after 5 minutes or those contami-
the free gingival margin to the base of the pocket at the nated with blood/saliva were discarded.
320 © 2015 Australian Dental Association
 Anti-inflammatory effects of curcumin

IL-1b and CCL28 analysis in GCF recall visits and were excluded from the analysis,
therefore a total of 56 participants completed the
IL-1b and CCL28 levels in pooled GCF samples were
study with 19 subjects in group I, 18 subjects in group
measured using a commercially available enzyme-
II and 19 subjects in group III (Fig. 2). Two sub-
linked quantitative sandwich immunosorbent assay kit
jects failed to follow-up for the final visit due to
(Raybioâ, Human Elisa kits, RayBiotech, Inc., USA),
change of residence and the other two withdrew for
specific for human IL-1b and CCL28. The results
personal reasons. The mean age was 21.95  3.34,
were reported as a concentration of IL-1b and CCL28
25.85  5.83 and 20.70  3.38 for groups I, II and
in picogram per millilitre of sample. This assay
III respectively. There were 7 males in groups I and
employs an antibody specific for human IL-1b/CCL28
III, and 11 males in group II. There was no significant
coated on a 96-well plate. Standards, controls and
difference among the treatment groups with respect to
samples were pipetted into the wells and IL-1b/
age and gender. The GCF volumes increased over the
CCL28 present in the sample were bound to the wells
29-day period with no significant difference in any
by the immobilized antibody. The wells were washed
group. Assessment of gel tube weights indicated no
and biotinylated antihuman IL-1b/CCL28 antibody
significant difference among groups for the mean
was added. After washing away unbound biotinylated
quantity of gel used. Examination of soft tissues after
antibody, HRP-conjugated streptavidin was pipetted
29 days and 60 days revealed no remarkable adverse
to the wells. The wells were again washed, a TMB
findings to any of the test products. Curcumin gel
substrate solution added and the colour developed in
application caused a mild yellowish discolouration of
proportion to the amount of IL-1b/CCL28 bound.
the teeth after 29 days, which returned to normal
The Stop Solution changed the colour from blue to
after professional scaling. No other adverse events
yellow, and the intensity of the colour was measured
were reported by any of the participants during the
at 450 nm.
study.

Statistical analysis
Clinical parameters
Data analysis was carried out using SPSS software
(Version 16.0, SPSSâ Inc., Chicago, IL, USA). The In the intragroup comparison (Table 2), there was an
values of the different parameters are expressed as increase in mean difference of MGI, PI and BI individ-
mean standard deviation. Paired t-tests were used for ually for all three groups from baseline to 29 days,
intragroup comparison. For intergroup comparisons, and the difference was significant (p < 0.001). After
one-way ANOVA followed by post hoc Tukey’s test 60 days there was a gradual decrease in all three
was carried out. The mean changes from baseline to parameters and no significant difference from baseline
29 days were calculated for all the parameters, and to 60 days in any of the groups. In the case of PD, all
changes from baseline to 60 days were calculated only values remained similar except for group II (p = 0.01)
for the clinical parameter. In each case the level of at 29 days. In the intergroup comparison (Table 3),
significance was set at p < 0.05. The power of the all groups were similar at baseline (MGI: p = 0.08;
study was calculated to be of 92%. PI: p = 0.48; PD: p = 0.49) and at 60 days (MGI:
p = 0.29; PI: p = 0.43), showing non-significant mean
differences for all clinical parameters except for PD at
RESULTS 60 days (p = 0.05). The mean difference from base-
line to 29 days was significant for MGI, PI and BI for
Characteristics of the study population
all three treatment groups (p < 0.001). CRM and
Table 1 summarizes the characteristics of the study CHX-MTZ were similar in MGI and PI scores, and
population. Four participants did not follow-up in significantly lower than the CHX group at 29 days

Table 1. Characteristics of the study participants

I II III P-value*

Number of subject 19 19 18
Age (mean  SD) 21.95  4.68 20.60  4.53 23.25  4.82 0.10
Age (range) 18–27 17–29 18–36
Mean volume of GCF at baseline (ll) 3.42  0.62 3.24  0.88 3.58  0.93 0.19
Mean volume of GCF at 29 days (ll) 3.82  0.89 3.93  0.99 3.86  0.75 0.17
Total mean gel mass administered (g) 56.46  5.97 54.89  6.75 57.53  6.93 0.13

I = curcumin group; II = chlorhexidine group; III = chlorhexidine-metronidazole group; GCF = gingival crevicular fluid; SD = standard deviation.
*ANOVA p-value was calculated.

© 2015 Australian Dental Association 321

SJ Pulikkotil and S Nath

Enrolment Assessed for eligibility (n = 75)

Excluded (n = 15)
Not meeting inclusion criteria (n = 8)
Declined to participate (n = 4)
Other reasons (n = 3)

Randomized (n = 60)

Allocation

Allocated to curcumin gel group Allocated to chlorhexidine gel group Allocated to chlorhexidine-metronidazole
intervention (n = 20) (n = 20) gel group
Received allocated intervention (n = 20) Received allocated intervention (n = 20) (n = 20)

Follow-up

Lost to follow-up (due to change of Lost to follow-up (due to personal Lost to follow-up (1 subject changed
residence) (n = 1) reason) (n = 1) residence, other withdrew due to
personal reason) (n = 2)

Analysis

Analysed (n =19) Analysed (n =19) Analysed (n =18)

Fig. 2 CONSORT study flow chart of various phases of the clinical trial.

when compared individually. BOP was significantly DISCUSSION

less in the CRM group when individually compared
The purpose of this randomized, double-blinded, con-
to the other two groups.
trolled clinical trial was to assess the effect of curcu-
min on IL-1b and CCL28 levels in GCF in a human
Cytokine levels
experimental gingivitis model. Gingivitis is inflamma-
Table 4 summarizes the intragroup and Table 5 the tion of the gingival tissues in response to the presence
intergroup comparison of cytokine levels from base- of dental plaque. Regular removal of plaque accumu-
line to 29 days. There was an increase in IL-1b levels lation can prevent as well as resolve gingivitis.27
from baseline to 29 days for all three groups and the Mechanical plaque removal is the superior treatment
intra mean difference was significant (I: 14.52  16.0, for gingival inflammation while topical application of
p = 0.002; II: 70.55  38.81 p < 0.001; III: 31.63  chemotherapeutic agents has an adjunct role in reduc-
15.96, <0.001). A similar increase in CCL28 was seen ing the severity of the disease.27 Hence in our study
with a significant difference for groups II and III we expected a reduction in the severity of gingival
only (I: 8.12  18.78, p = 0.10; II: 41.15  22.82, inflammation rather than a complete prevention or
p < 0.001; III: 12.81  18.68, p = 0.02) from base- resolution of gingivitis through the use of any topical
line. An intergroup comparison of IL-1b and CCL28 antigingivitis products. Our results also showed that
levels found all groups were similar at baseline (IL-1b: use of the three experimental gels did not prevent the
p = 0.92, CCL28: p = 0.56) but showed significant onset or development of experimental gingivitis when
changes after 29 days (p < 0.001) (Table 5). No dif- evaluated clinically. However, there was a difference
ference was seen between group I and III (IL-1b: in the cytokine levels assessed which will be discussed
p = 0.18; CCL28: p = 0.80), whereas significant further.
difference was seen when group II was compared Two popular topical antibacterials, CHX and
with group I (IL-1b: p < 0.001; CCL28: p < 0.001) CHX-MTZ combination gels, were selected for com-
and III (IL-1b: p < 0.001; CCL28: p < 0.001) at parison. CHX is a broad spectrum antiseptic having
29-day levels. both bactericidal and fungicidal action against Gram-
322 © 2015 Australian Dental Association
 Anti-inflammatory effects of curcumin

positive and Gram-negative strains and is considered

0.05* 0.70

*Statistically significant. I = curcumin group; II = chlorhexidine group; III = chlorhexidine-metronidazole group; MGI = modified gingival index; PI = plaque index; BOP = bleeding on prob-
P value
the gold standard for chemical plaque control.4–6

0.50
0.16
0.48

0.30

0.37
0.28
0.34
Baseline–60 days CHX has been recognized by the pharmaceutical
industry as the positive control for evaluating the effi-
cacy of alternative antiplaque agents.28 MTZ is effec-
tive against Gram-positive and Gram-negative
obligate anaerobe.6,10 CHX and MTZ have demon-

0.14  0.67
0.01  0.12
0.02  0.10

(-)0.06  0.25
0.16  0.74
0.12  0.27
(-)0.03  0.18
0.09  0.25
(-)0.05  0.32

0.0  0.0
0.0  0.0
0.0  0.0
strated anti-inflammatory properties although the
Difference

improvement in gingivitis is due to their antibiofilm

properties.29 The anti-inflammatory action of CHX
and MTZ is mainly due to their inhibitory action on
Table 2. Intragroup comparison and mean change from baseline of clinical parameter at different time points for all groups

oral biofilm. However, the use of topical chemothera-

peutics has certain disadvantages. Side effects of CHX
are brown discolouration of teeth, taste perturbation,
oral mucosal ulceration, paraesthesia, parotid swelling
<0.001**
<0.001*
<0.001*
<0.001*
<0.001*
<0.001*

<0.001*
<0.001*
<0.001*
P value

0.01*
0.07

0.37

and enhanced supragingival calculus formation,10

while MTZ can cause resistance against antibiotics.6
Baseline–29 days

Due to the drawbacks of the synthetic agents, the rel-

atively safe nature of herbal extracts has led to their
use in various fields as an alternative.11
Curcumin is a naturally occurring phytochemical
0.31
0.55
0.38
0.16
0.37
0.21
0.19
0.15
0.13
0.67
0.32
0.74
Difference

known to have antibacterial and anti-inflammatory














properties.13,15 It is a major constituent of the popular

0.45
1.29
0.68
0.42
0.85
0.36
0.26
0.68
0.50
0.29
0.22
0.16

Indian yellow spice turmeric derived from the rhi-

zomes of Curcuma spp. Curcumin has a number of
applications. It has been shown to prevent cancer in
the oral cavity, colon, skin, lung, liver, duodenum
and breasts.13,14 It is effective against gastric ulcers,
0.34
0.18
0.24
0.23
0.17
0.13

0.52
0.31
0.54
0.0
0.0
0.0
60 days

arthritis, inflammatory bowel disease and Crohn’s dis-















ease.13,14 Curcumin has the potential to treat inflam-

0.0
0.36
0.39
0.26
0.23
0.14
0.18

2.23
2.03
2.42
0.0
0.0

matory disease, especially periodontal disease.14,30–32

In our study we chose an experimental gingivitis
model of 29 days similar to previous studies to com-
pare the efficacy of three commercially available anti-
0.27
0.49
0.31
0.14
0.29
0.17
0.19
0.15
0.13
0.53
0.49
0.54

gingivitis topical agents.33,34 The original model

29 days

proposed by Loe et al.35 was of 21 days but we pre-














ferred the 29-day model because there is heightened

0.86
1.59
1.01
0.52
0.98
0.53
0.26
0.68
0.50
2.38
2.31
2.42

host inflammatory mediator response to plaque build-

up at this time point.33,34 Instead of the full mouth
model, we restricted it to the selected quadrant to
increase patient compliance and reliability, similar to
0.20
0.13
0.18
0.11
0.13
0.11

0.48
0.39
0.57

Paquette et al.34 and Saxton and van der Ouderaa.36 In

0.0
0.0
0.0
Baseline

this model, gingival health is first established followed














0.41
0.31
0.29
0.11
0.13
0.15

2.09
2.09
2.26
0.0
0.0
0.0

by abandonment of all oral hygiene procedures for a

period of 29 days to induce gingivitis. According to
Deinzer et al.,37 the experimental gingivitis model may
not be identical to chronic gingivitis in a natural setting
but it is preferable to study the anti-inflammatory
Group

effects of topical gel in a well-controlled environment.

III

III

III

III
II

II

II

II

ing; PD = probing depth.

I

The three groups of subjects were similar in age,

male–female ratio, average volume of GCF at baseline
and at 29 days. Researchers have shown an increase
in GCF volume after 21 days of no oral hygiene, but
Parameter

no such change was seen in our study.38 The interven-

tions might have controlled an increase. Smokers were
MGI

BOP

PD

excluded from this study due to the depressive effects

PI

© 2015 Australian Dental Association 323

SJ Pulikkotil and S Nath

Table 3. Intergroup comparison of clinical parameter at various follow-up visits from baseline (p-values only)
Time points Parameter I/II/III‡ I/II I/III II/III

Baseline MGI 0.08

PI 0.48
BOP 1
PD 0.49
29 days MGI <0.001* <0.001* 0.43 <0.001*
PI <0.001* <0.001* 0.99 <0.001*
BOP <0.001* <0.001* 0.003* <0.001*
PD 0.71 NC NC NC
60 days MGI 0.29
PI 0.43
BOP 1
PD 0.05
Difference Baseline–29 days MGI <0.001* <0.001* 0.13 <0.001*
PI <0.001* <0.001* 0.85 <0.001*
BOP <0.001* <0.001* 0.007* <0.001*
PD 0.81 NC NC NC
Baseline–60 days MGI 0.23
PI 0.15
BOP 1
PD 0.47

Only p-values are given here as the individual groups’ MGI, PI, BOP and PD values are given in Table 2.
‡To compare groups I, II and III, ANOVA p-value was calculated. Pairwise comparison and p-value was done only when comparison by
ANOVA was significant.
*Statistically significant.
I = curcumin group; II = chlorhexidine group; III = chlorhexidine-metronidazole group; MGI = modified gingival index; PI = plaque index;
BOP = bleeding on probing; PD = probing depth; NC = not calculable as all values were similar.

Table 4. Intragroup comparison of cytokines levels from baseline to 29 days

Cytokine Group Baseline 29 days Difference P-value

IL-1b I 24.71  6.84 39.27  15.71 14.52  16.0 0.002*

II 24.93  7.27 95.54  41.67 70.55  38.81 <0.001*
III 25.64  6.20 57.24  15.42 31.63  15.96 <0.001*
CCL28 I 76.48  9.65 84.59  16.94 8.12  18.78 0.10
II 81.64  15.26 122.78  19.30 41.15  22.82 <0.001*
III 78.13  16.23 90.9 4  15.90 12.81  18.68 0.02*

*Statistically significant.
I = curcumin group; II = chlorhexidine group; III = chlorhexidine-metronidazole group.

Table 5. Intergroup group comparison of cytokine levels from baseline to 29 days (p-values only)
Time points Parameter I/II/III ‡ I/II I/III II/III

Baseline IL-1b 0.92

CCL28 0.56
29 days IL-1b <0.001* <0.001* 0.17 <0.001*
CCL28 <0.001* <0.001* 0.57 <0.001*
Difference Baseline–29 IL-1b <0.001* <0.001* 0.18 <0.001*
CCL28 <0.001* <0.001* 0.80 <0.001*

Only p-values are given here as the individual groups’ cytokine levels are given in Table 4.
‡To compare groups I, II and III, ANOVA p-value was calculated. Pairwise comparison and p-value was done only when comparison by
ANOVA was significant.
*Statistically significant.
I = curcumin group; II = chlorhexidine group; III = chlorhexidine-metronidazole group.

of tobacco on gingival inflammation.34 In our study cross-over contamination of the intervention agent on
we did not use any negative or placebo controls as the contralateral side if used as a control as in split
there is evidence that experimental gingivitis would mouth design.39 An adequate amount of gel was used
lead to gingival inflammation and an increase in in all groups and usage was not dissimilar. We used a
inflammatory markers.33 There is also a possibility of tooth shield so that the experimental agent would
324 © 2015 Australian Dental Association
 Anti-inflammatory effects of curcumin

come into contact with the gingiva undisturbed for levels were higher in samples from a group of subjects
10 minutes as per the manufacturers’ instructions for with inflammation compared to a healthy control
the individual gels. We could not locate any other group.22,23 Ertugrul et al.21 found a significant
studies using a tooth shield for application of test gels increase (1.5 times) of IL-1b and CCL28 levels in
during initiation of experimental gingivitis. No GCF in naturally occurring gingivitis subjects com-
adverse effects including pain, hypersensitivity reac- pared to periodontal healthy subjects.
tions or ulceration were observed in or reported by With the sustained biofilm challenge for 29 days, it
the participants. Teeth staining was observed in the was observed that all three groups showed a signifi-
CRM group at 29 days, but this cleared after resum- cant increase in GCF IL-1b levels over their respective
ing mechanical plaque control. baseline values. The increase was reflective of the
Plaque, BOP and gingival inflammation scores induced gingival inflammation due to the accumula-
increased progressively from baseline to 29 days, simi- tion of plaque. A similar relationship between GCF
lar to other studies with or without the use of topical cytokines and clinical gingival inflammation has been
agents in experimental gingivitis.33,35,40 Refraining reported previously.38,42 The application of any topi-
from mechanical plaque control resulted in increased cal gel was not effective in reducing IL-1b levels to
plaque accumulation, leading to gingival inflammation. baseline levels in our study. At 29 days, IL-1b levels
This confirmed the superiority and effectiveness of in GCF were significantly lower in the CRM and
complete mechanical plaque removal. All three experi- CHX-MTZ groups compared to the CHX group.
mental gels could not prevent development of gingivitis Groups using CRM gel and CHX-MTZ gel showed
as well as lead to any resolution of established gingivi- reduced IL-1b levels during the development of exper-
tis. However at 29 days, the CRM group was more imental gingivitis compared to CHX gel. This again
effective in reducing the severity of clinical signs of corroborates our clinical findings. Similar results were
experimental gingivitis. Inflammatory signs of BOP observed with respect to CCL28 wherein the CRM
scored significantly less in the CRM than the other two and CHX-MTZ gel were similar in having signifi-
groups. MGI was similar in the CRM and CHX-MTZ cantly lower levels compared to CHX. In addition, we
group, while it was significantly higher for the CHX observed that curcumin prevented any significant
group. This is reflected in the high plaque scores for increase of CCL28 levels over baseline at 29 days.
CHX. Thus at 29 days, we found that CRM and Statistically better results obtained by the topical
CHX-MTZ were more efffective in controlling plaque application of curcumin can be attributed to its anti-
and gingival inflammation compared to the gold stan- inflammatory, antioxidant and antibacterial properties,
dard CHX. Pradeep et al.10 also reported the effective- which might have resolved inflammation in an earlier
ness of the CHX-MTZ combination gel in the control stage, reflected by lower cytokine levels and reduced
of gingivitis compared to the individual agents. Similar clinical inflammation when compared to CHX. Curcu-
to our result, Waghmare et al.30 and Suhag et al.31 min has been shown to be effective when evaluated as
demonstrated the superior anti-inflammatory proper- a treatment for periodontal disease.14,30–32 Curcumin
ties of curcumin mouthwash compared to CHX. CHX acts similarly to aspirin and aspirin-like anti-inflamma-
and MTZ have well-characterized antimicrobial effects tory drugs in diminishing inflammatory mediators of
and potential anti-inflammatory properties.29 The arachidonic acid metabolism, thus inhibiting prosta-
antiplaque effect of CHX-MTZ may be explained by glandins and leukotreines.15 Curcumin use reduces
CHX properties acting on the cell walls of the microor- inflammatory mediator levels of IL-6, IL-1b, TNF-a,
ganism and changing their surface structures, whereas MMP-2 and MMP-9 by modulation of signalling path-
the cytototoxic metabolites of MTZ directly interact ways and transcription factors, especially NF-jB, AP-1
with bacterial DNA, resulting in cell death.10,29 All and MAPKinase.14,15 Curcumin causes shrinkage of
clinical scores at 60 days were similar to baseline lev- tissues by reducing inflammatory oedema and vascular
els, which again reiterates the excellent results achieved engorgement of connective tissues.43 It promotes
with good mechanical plaque control. migration of fibroblasts in the wound bed and
GCF is a transudate that accumulates bacterial and enhances wound healing by an increase in fibronectin
host metabolic products. Therefore, it is an ideal diag- and transforming growth factor b transcription.43 The
nostic tool for analysing the inflammatory mediator. antioxidant activity of curcumin is comparable to vita-
To assess the anti-inflammatory effect of the test mins C and E.13 Rao et al.44 demonstrated the scav-
product, we used two markers: IL-1b and CCL28. enging effect of curcumin on superoxide radicals,
IL-b is a reliable marker associated with the presence hydroxyl radicals and lipid per oxidation. Curcumin
and severity of plaque induced gingival inflamma- suppressed the growth of Prevotella intermedia, a
tion.41,42 IL-1b is associated with bone resorption major periodontal pathogen.16 Therefore, due to the
exhibiting increased levels in diseased rather than diverse range of actions, curcumin treated sites showed
healthy sites.41,42 Studies have shown that CCL28 faster resolution of inflammatory signs.
© 2015 Australian Dental Association 325
SJ Pulikkotil and S Nath

Curcumin is a naturally occurring molecule and a (Abbott India), Mr Manoj Prabhu (Abbott India), Mr
major limiting factor is its low solubility in water (i.e. Tushar Fegade (Abbott India), Dr RM Zade and Dr
0.0004 mg/ml at pH 7.3) and low bioavailability. Ritu Prabha Patel for their support during the study.
This has been attributed to its poor absorption, high
rate of metabolism and rapid elimination from the
REFERENCES
body.45 A conventional oral formulation of curcumin
resulted in a very low availability of curcumin in the 1. Corbet EF, Zee KY, Lo EC. Periodontal diseases in Asia and
Oceania. Periodontol 2000;2002;29:122–152.
blood circulation to achieve therapeutic effects.46
2. Slots J. Periodontology: past, present, perspectives. Periodontol
Various types of nanoparticle (NPs), such as polymer 2000;2013;62:7–19.
NPs, polymeric micelles, liposome/phospholipid, 3. Axelsson P, Lindhe J, Nystrom B. On the prevention of caries
nano-/microemulsions, nanogels, solid lipid NPs, poly- and periodontal disease: results of a 15-year longitudinal study
mer conjugates, self-assemblies, are suitable for the in adults. J Clin Periodontol 1991;18:182–189.
delivery of an active form of curcumin.46 Shahani 4. van der Ouderaa FJ. Anti-plaque agents. Rationale and pros-
pects for prevention of gingivitis and periodontal disease. J Clin
et al.47 showed that curcumin formulation in the form Periodontol 1991;18:447–454.
of microparticles had a sustained release for nearly a
5. Ciancio SG. Agents for the management of plaque and gingivi-
month. Nanoformulations of curcumin vastly enhance tis. J Dent Res 1992;71:1450–1454.
aqueous solubility and thus bioavailability in animals 6. Goodson JM. Antimicrobial strategies for treatment of peri-
as well as humans, evidenced by a higher release pro- odontal diseases. Periodontol 2000;1994;5:142–168.
file of nanocurcumin over curcumin suspension.45 7. Krayer JW, Leite RS, Kirkwood KL. Non-surgical chemothera-
Toxicity of curcumin was reduced by nanoformula- peutic treatment strategies for the management of periodontal
diseases. Dent Clin North Am 2010;54:13–33.
tion while increasing therapeutic efficacy.48
8. Preshaw PM. Host response modulation in periodontics. Period-
Some limitations were identified in our study. ontol 2000;2008(48):92–110.
Even though compliance was checked, it is difficult to 9. Tonetti MS. Chapple IL; Working Group 3 of Seventh Euro-
tell if the at-home products were used correctly. pean Workshop on Periodontology. Biological approaches to
A cross-over design with a suitable control for cross- the development of novel periodontal therapies–consensus of
the Seventh European Workshop on Periodontology. J Clin
contamination would eliminate any bias of the Periodontol 2011;38:114–118.
variable host response. The absence of a negative con- 10. Pradeep AR, Kumari M, Priyanka N, Naik SB. Efficacy of
trol did not allow us to measure the magnitude of chlorhexidine, metronidazole and combination gel in the treat-
change for the individual groups. ment of gingivitis–a randomized clinical trial. J Int Acad Period-
ontol 2012;14:91–96.
11. Surathu N, Kurumathur AV. Traditional therapies in the man-
CONCLUSIONS agement of periodontal disease in India and China. Periodontol
2000;2011(56):14–24.
CRM and CHX-MTZ topical application were supe- 12. Witkin JM, Li X. Curcumin, an active constituent of the
rior to CHX in affecting the development of gingival ancient medicinal herb Curcuma longa L.: some uses and the
inflammation in human experimental gingivitis. CRM establishment and biological basis of medical efficacy. CNS
Neurol Disord Drug Targets 2013;12:487–497.
and CHX-MTZ were similar in clinical inflammation
13. Nagpal M, Sood S. Role of curcumin in systemic and oral
and cytokine levels of IL-1b and CCL28. However, a health: an overview. J Nat Sci Biol Med 2013;4:3–7.
mild superiority can be claimed for curcumin over 14. Guimar~aes MR, Coimbra LS, de Aquino SG, Spolidorio LC,
CHX-MTZ. CRM prevented any increase of CCL28 Kirkwood KL, Rossa C Jr. Potent anti-inflammatory effects of
levels as well as having the least BOP scores. The evi- systemically administered curcumin modulate periodontal dis-
ease in vivo. J Periodontal Res 2011;46:269–279.
dence from this study suggests that curcumin has
15. Kohli K, Ali J, Ansari MJ, Raheman Z. Curcumin: a natural
potential in the treatment of inflammatory periodontal antiinflammatory agent. Indian J Pharmacol 2005;37:141–147.
disease as an adjunct to good mechanical plaque con- 16. Kim SJ. Curcumin suppresses the production of interleukin-6 in
trol. Improved formulations employing recent Prevotella intermedia lipopolysaccharide-activated RAW 264.7
advances in nanotechnology would help realize the cells. J Periodontal Implant Sci 2011;41:157–163.
true antibacterial and anti-inflammatory potential of 17. Griffiths GS. Formation, collection and significance of gingival
curcumin. Topical, sustained and systemic delivery crevice fluid. Periodontol 2000;2003:32–42.
formulations of curcumin need to be explored. Fur- 18. Gonzales JR, Herrmann JM, Boedeker RH, Francz PI, Biesalski
H, Meyle J. Concentration of interleukin-1beta and neutrophil
ther studies are required to ascertain the effect of elastase activity in gingival crevicular fluid during experimental
curcumin on periodontitis patients. gingivitis. J Clin Periodontol 2001;28:544–549.
19. Reinhardt RA, Masada MP, Johnson GK, Dubois LM, Seymour
GJ, Allisson AC. IL-1 in gingival crevicular fluid following
ACKNOWLEDGEMENTS closed root planing and papillary flap debridement. J Clin Peri-
odontol 1993;20:514–519.
The materials and facilities used in the study were 20. Kinane DF, Winstanley FP, Adonogianaki E, Moughal NA. Bio-
funded by Abbott Healthcare Pvt. Ltd, Mumbai, MH, assay of interleukin 1 (IL-1) in human gingival crevicular fluid
India. The authors would like to thank Mr Manoj Naik during experimental gingivitis. Arch Oral Biol 1992;37:153–156.

326 © 2015 Australian Dental Association

 Anti-inflammatory effects of curcumin

21. Ertugrul AS, Sahin H, Dikilitas A, Alpaslan N, Bozoglan A. 36. Saxton CA, van der Ouderaa FJ. The effect of a dentifrice con-
Comparison of CCL28, interleukin-8, interleukin-1b and tumor taining zinc citrate and triclosan on developing gingivitis. J Peri-
necrosis factor-alpha in subjects with gingivitis, chronic peri- odontal Res 1989;24:75–80.
odontitis and generalized aggressive periodontitis. J Periodontal 37. Deinzer R, Weik U, Kolb-Bachofen V, Herforth A. Comparison
Res 2013;48:44–51. of experimental gingivitis with persistent gingivitis: differences
22. Luster AD. Chemokines-chemotactic cytokines that mediate in clinical and cytokine concentrations. J Periodontal Res
inflammation. N Engl J Med 1998;338:436–445. 2007;42:318–324.
23. Eksteen B, Miles A, Curbishley SM, et al. Epithelial inflamma- 38. Tsalikis L. The effect of age on the gingival crevicular fluid
tion is associated with CCL28 production and the recruitment composition during experimental gingivitis. A pilot study. Open
of regulatory T cells expressing CCR10. J Immunol 2006; Dent J 2010;4:13–26.
177:593–603. 39. Shaju JP, Nath S. Effect on interleukin 1-beta and -8 levels
24. Ezzat MH, Sallam MA, Shaheen KY. Serum mucosa-associated following use of fibrin sealant for periodontal surgery. Aust
epithelial chemokine (MEC/CCL28) in atopic dermatitis: a spe- Dent J 2014;59:156–164.
cific marker for severity. Int J Dermatol 2009;48:822–829. 40. Berglund T, Lindhe J. Gingivitis in young and adults dogs.
25. Silness J, Loe H. Periodontal disease in pregnancy. II. Correla- J Clin Periodontol 2000;20:179–185.
tion between oral hygiene and periodontal condition. Acta 41. Orozco A, Gemmell E, Bickel M, Seymour GJ. Interleukin-
Odontol Scand 1964;22:121–135. 1beta, interleukin-12 and interleukin-18 levels in gingival fluid
26. Lobene RR, Weatherford T, Ross NM, Lamm RA, Menaker L. and serum of patients with gingivitis and periodontitis. Oral
A modified gingival index for use in clinical trials. Clin Prev Microbiol Immunol 2006;21:256–260.
Dent 1986;8:3–6. 42. Faizuddin M, Bharathi SH, Rohini NV. Estimation of interleu-
27. Research, Science and Therapy Committee of the American kin-1beta levels in the gingival crevicular fluid in health and in
Academy of Periodontology. Treatment of plaque-induced inflammatory periodontal disease. J Periodontal Res
gingivitis, chronic periodontitis, and other clinical conditions. 2003;38:111–114.
J Periodontol 2001;72:1790–1800. 43. Sidhu GS, Singh AK, Thaloor D, et al. Enhancement of wound
28. Jones CG. Chlorhexidine: is it still the gold standard? Periodon- healing by curcumin in animals. Wound Repair Regen
tol 2000;1997:55–62. 1998;6:167–177.
29. Herrera D, Matesanz P, Bascones-Martınez A, Sanz M. Local 44. Rao CV, Simi B, Reddy BS. Inhibition by dietary curcumin of
and systemic antimicrobial therapy in periodontics. J Evid azoxymethane-induced ornithine decarboxylase, tyrosine protein
Based Dent Pract 2012;12:50–60. kinase, arachidonic acid metabolism and aberrant crypt foci for-
30. Waghmare PF, Chaudhari AU, Karhadkar VM, Jamkhande AS. mation in the rat colon. Carcinogenesis 1993;14:2219–2225.
Comparative evaluation of turmeric and chlorhexidine gluco- 45. Yallapu MM, Jaggi M, Chauhan SC. Curcumin nanoformula-
nate mouthwash in prevention of plaque formation and gingivi- tions: a future nanomedicine for cancer. Drug Discov Today
tis: a clinical and microbiological study. J Contemp Dent Pract 2012;17:71–80.
2011;12:221–224. 46. Lao CD, Ruffin MT 4th, Normolle D, et al. Dose escalation of
31. Suhag A, Dixit J, Dhan P. Role of curcumin as a subgingival a curcuminoid formulation. BMC Complement Altern Med
irrigant: a pilot study. Perio 2007;4:115–121. 2006;6:10.
32. Behal R, Mali MA, Gilda SS, Paradkar AR. Evaluation of local 47. Shahani K, Swaminathan SK, Freeman D, Blum A, Ma L,
drug delivery system containing 2% whole turmeric gel used as Panyam J. Injectable sustained release microparticles of curcu-
an adjunct to scaling and root planing in chronic periodontitis: min: a new concept for cancer chemoprevention. Cancer Res
a clinical and microbiological study. J Indian Soc Periodontol 2010;70:4443–4452.
2011;15:35–38. 48. Aggarwal BB, Sundaram C, Malani N, Ichikawa H. Curcumin:
33. Heasman PA, Collins JG, Offenbacher S. Changes in crevicular the Indian solid gold. Adv Exp Med Biol 2007;595:1–75.
fluid levels of interleukin-1 beta, leukotriene B4, prostaglandin
E2, thromboxane B2 and tumour necrosis factor alpha in
experimental gingivitis in humans. J Periodontal Res 1993;28: Address for correspondence:
241–247. Dr Shaju J Pulikkotil
34. Paquette DW, Simpson DM, Friden P, Braman V, Williams RC. International Medical University
Safety and clinical effects of topical histatin gels in humans School of Dentistry
with experimental gingivitis. J Clin Periodontol 2002;29:1051–
1058. Kuala Lumpur 57000
35. L€oe H, Theilade E, Jensen SB. Experimental gingivitis in man.
Malaysia
J Periodontol 1965;36:177–187. Email: Shaju_Jacob@imu.edu.my

© 2015 Australian Dental Association 327