Evaluation of calendula officinalis as an anti plaque and anti-gingivitis agent

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Original Article

Evaluation of Calendula officinalis as an
anti‑plaque and anti‑gingivitis agent
Mayur Sudhakar Khairnar, Babita Pawar, Pramod Parashram Marawar, Ameet Mani

Department of
Periodontology, Pravara
Institute of Medical
Sciences, Rural
Dental College, Loni,
Rahata, Ahmed Nagar,
Maharashtra, India

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DOI:
10.4103/0972-124X.124491
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Abstract:
Background: Calendula officinalis (C. officinalis), commonly known as pot marigold, is a medicinal herb with
excellent antimicrobial, wound healing, and anti‑inflammatory activity. Aim: To evaluate the efficacy of C. officinalis
in reducing dental plaque and gingival inflammation. Materials and Methods: Two hundred and forty patients
within the age group of 20-40 years were enrolled in this study with their informed consent. Patients having
gingivitis (probing depth (PD) ≤3 mm), with a complaint of bleeding gums were included in this study. Patients
with periodontitis PD ≥ 4 mm, desquamative gingivitis, acute necrotizing ulcerative gingivitis (ANUG), smokers
under antibiotic coverage, and any other history of systemic diseases or conditions, including pregnancy, were
excluded from the study. The subjects were randomly assigned into two groups – test group (n = 120) and
control group (n = 120). All the test group patients were advised to dilute 2 ml of tincture of calendula with 6 ml of
distilled water and rinse their mouths once in the morning and once in the evening for six months. Similarly, the
control group patients were advised to use 8 ml distilled water (placebo) as control mouthwash and rinse mouth
twice daily for six months. Clinical parameters like the plaque index (PI), gingival index (GI), sulcus bleeding
index (SBI), and oral hygiene index‑simplified (OHI‑S) were recorded at baseline (first visit), third month (second
visit), and sixth month (third visit) by the same operator, to rule out variable results. During the second visit,
after recording the clinical parameters, each patient was subjected to undergo a thorough scaling procedure.
Patients were instructed to carry out regular routine oral hygiene maintenance without any reinforcement in it.
Results: In the absence of scaling (that is, between the first and second visit), the test group showed a statistically
significant reduction in the scores of PI, GI, SBI (except OHI‑S) (P < 0.05), whereas, the control group showed
no reduction in scores when the baseline scores were compared with the third month scores. Also, when scaling
was performed during the third month (second visit), there was statistically significant reduction in the scores of all
parameters, when the third month scores were compared with the sixth month scores in both groups (P < 0.05),
but the test group showed a significantly greater reduction in the PI, GI, SBI, and OHI‑S scores compared to those
of the control group. Conclusion: Within the limits of this study, it can be concluded that calendula mouthwash
is effective in reducing dental plaque and gingivitis adjunctive to scaling.
Key words:
Anti‑gingivitis, anti‑inflammatory, anti‑plaque, Calendula officinalis

INTRODUCTION

G
Address for
correspondence:
Dr. Mayur Sudhakar
Khairnar,
Precision Dental Clinic and
Implant Center,
Shop No. 13,
New Geetanjali CHS Ltd,
Anand Nagar, Vasai West,
Thane - 401 202,
Maharashtra, India.
E‑mail: drmayurkhairnar@
gmail.com
Submission: 13‑09‑2011
Accepted: 15‑09‑2013

ingivitis is a chronic inflammatory process
limited to the gingiva, without either
attachment or alveolar bone loss. It is one of the
most frequent oral diseases, affecting more than
90% of the population, regardless of age, sex or
race. The earliest clinical sign is bleeding, which
is a sequel of the vasodilator effect caused by
an inflammatory response.[1] The prevention of
gingivitis by daily and effective supragingival
plaque control via brushing the teeth and dental
floss is necessary to arrest a possible progression
to periodontitis.[2,3]
Although mechanical plaque control methods
have the potential to maintain adequate
levels of oral hygiene, clinical experience and
population‑based studies have shown that such
methods are not being employed accurately by
a large number of people. Therefore, several
chemotherapeutic agents such as triclosan,
essential oils, and chlorhexidine have been

Journal of Indian Society of Periodontology - Vol 17, Issue 6, Nov-Dec 2013

developed to control bacterial plaque, aiming to
improve the efficacy of daily oral hygiene control
measures.[4]
The interest in plants with antibacterial and
anti‑inflammatory activity has increased as a
consequence of the current problems associated
with the wide‑scale misuse of antibiotics that
induce microbial drug resistance[5,6] and cytotoxic
effects on the host cells.
Natural products such as Azadirachta indica, Aloe
vera, Curcuma zedoaria, Punica granatum Linn.,
and other herbal products have been tested and
are found to have effective medicinal properties.
C. officinalis (family Asteraceae), mostly known
as ‘pot marigold’, is a medicinal shrub native to
the Mediterranean area, although, it is widely
spread throughout the world. It produces yellow
or orange flowers, which are used medicinally
either in the form of infusion, tinctures, liquid
extracts, creams or ointments. The plant contains
polysaccharides, flavanoids, triterpene alcohols,
741

Khairnar, et al.: Calendula officinalis as an anti‑plaque and anti‑gingivitis agent

phenol acids, tannins, glycosides, sterols, carotenoids,
saponosides, and the like.[7]
Various researchers have shown C. officinalis to have
antibacterial[8,9] and antifungal activity.[10] It also exhibits wound
healing and re‑epitheliazation,[11] anti‑inflammatory,[12,13]
antioxidant,[14] immunomodulatory[15,16] and anti‑mutagenic[17,18]
properties. It has reported no contraindications and no other
drug interactions, but individuals with a known sensitivity
to the Compositae family may be predisposed to allergic
reactions.[19] Mouth rinsing with calendula will allow its
anti‑inflammatory properties to work against the swollen,
irritated gums and its antibacterial properties deal with the
periodontopathic microorganisms.[20] Calendula is also used
for healing and soothing burns and sunburns, varicose ulcers,
relieving sore throats, mouth ulcers, gastric upset, athlete’s
foot, ear infection, and so on.

AIM
Few studies have documented the effect of C. officinalis on
gingival inflammation and gingival bleeding, but none of
the studies has used a comparative control group. Hence, the
present study was performed with the following aims:
• To evaluate the efficacy of C. officinalis in reducing plaque
and gingivitis
• To compare C. officinalis with the control formulation, with
or without adjunctive scaling to reduce gingivitis.

MATERIALS AND METHODS
Study design
This is a randomized controlled trial study with a test group
using the calendula mouthwash and a placebo control group.
The study was carried out in the Pravara Institute of Medical
Sciences University, India, between May 2009 and December
2010, under the approval of the Ethical Committee of the
University.
Subjects
Two hundred and forty patients (20 - 40 years) who visited the
Department of Periodontics at the Pravara Institute of Medical
Sciences University with chief complaints of bleeding gums
were enrolled in this study. All the subjects had at least 28 teeth
other than the third molars. All participants were informed
about the nature of the study and those who were willing to
participate, signed an informed consent form in compliance
with the guidelines of the Health Council of the University.
Inclusion criteria
• Gingivitis with bleeding on probing (PD ≤ 3 mm)
• Habits of cleaning teeth once in morning with a flat‑end,
medium bristles toothbrush, and a dentifrice
• Patients complying to continue with the prescribed
mouthwash as per the study protocol.
Exclusion criteria
• Periodontitis (PD ≥ 4 mm)
• Systemic medical disorder
• Under antibiotic coverage
• Habit of smoking and chewing smokeless tobacco
• Pregnant women.
742

Clinical design
Two hundred and forty patients were randomly assigned into
two groups: Test group n = 120 and control group n = 120. The
randomization was done randomly by a non‑operator.
Mother tinctures of C. officinalis were obtained from the Alpha
Home Pharmacy, Nasik. Each patient in the test group was
supplied with seven to eight bottles of calendula mother
tincture and a measuring cylinder. The test group patients
were advised to dilute 2 ml of calendula tincture (†Dr. Willmar
Schwabe Germany Home Pharmacy Pvt. Ltd [Manufacturer])
with 6 ml of water. This diluted (1: 3)[21] formulation was
prescribed for mouth rinsing twice daily for six months.
The control group patients were given distilled water as a
control mouthwash (placebo) and were advised to rinse their
mouth with 8 ml of it twice daily for six months. They were also
supplied a measuring cylinder. All participants were strictly
instructed not to bring any additional reinforcement into their
oral hygiene practice during the given study period. All patients
were re‑called to the clinics at the third month (second visit)
and at the sixth month (third visit). Patients with spontaneous
severe bleeding gums, requiring immediate treatment were not
considered, and thus, excluded from the study.
Clinical assessment
Clinical parameters like Turesky‑Gilmore modification
of the Quigley‑Hein plaque Index (PI), [22] gingival
index (GI),[23] sulcus bleeding index (SBI),[24] and oral hygiene
index – simplified (OHIS) were recorded at the baseline,
third month, and sixth month by the same operator, to rule
out individual variations in results. Also, after recording the
clinical parameters at the third month (during second visit),
every patient was subjected to undergo a thorough scaling
procedure by a skillful operator.
The Turesky‑Gilmore modification of the Quigley‑Hein plaque
index (PI) measures the levels of dental plaque harboring
the tooth surface in the fluid‑filled oral cavity. The gingival
index (GI) was used to assess the severity of gingivitis, the
sulcus bleeding index (SBI) was helpful in evaluating the
bleeding tendency in gingivitis patients, and lastly the oral
hygiene index‑simplified (OHI‑S) assessed the personal oral
hygiene status of an individual.
The PI score was assessed on the labial/buccal and lingual/
palatal surfaces of all the teeth other than the third molars after
applying a two‑tone disclosing agent. The GI and SBI scores
were recorded on the mesiobuccal, buccal, distobuccal, and
lingual aspects of all the teeth, except the third molars. The
gingival tissues were inspected for the presence of bleeding,
recorded 10 seconds after running the tip of a William probe
along the gingival margin (0.5‑mm penetration into the sulci).
The OHI‑S score was evaluated on six selected experimental
teeth, to determine the oral hygiene status of each participant.
The mean values of PI, GI, SBI, and OHI‑S were calculated for
the test and control groups.
Statistical analysis
The student paired ‘t’ test was used to evaluate the comparison
of mean values of the differences in all parameters from the
baseline to the third month, from baseline to the sixth month,


and from the third month to the sixth month, in the test and
control groups.

RESULTS
All the patients had a complete follow‑up of six months. The
calendula mouthwash showed good patient compliance,
with greater taste acceptance and with an experience
of freshness and willingness to continue, without any
noticeable adverse effect like abscess, ulcerations, allergic
reactions or the like.
The mean values of all clinical parameters, with standard
deviation, for the test and control groups at the baseline,
third month, and sixth month are given in Tables 1 and 2,
respectively.
After applying Student’s Paired ‘t’ test there was a highly
significant difference in the mean values of PI, GI, and SBI,
(except OHI‑S, P < 0.01) when the baseline scores were
compared with the third month scores. However, when
the baseline scores were compared with the sixth month
scores and similarly when the third month scores were
compared with the sixth month scores, the result showed a
statistically significant difference in PI, GI, SBI, and OHI‑S
in the calendula test group. (i.e., P < 0.01) [Table 3]. The
student paired ‘t’ test value showed that in the calendula
test group there is a highly significant reduction in the
mean values of PI, GI, and SBI scores from the baseline to
the third month, from the third month to the sixth month,
and from the baseline to the sixth month (P < 0.05), whereas,
the OHIS score showed no significant reduction when the
baseline score was compared with the third month score
(P > 0.05), but showed a significant reduction when the
third month score was compared with the sixth month score
and when the baseline score was compared with the sixth
month score (P < 0.05) [Figure 1 and Tables 1 and 3]. This
significant reduction in the scores of PI, GI, and SBI from
Table 1: Distribution of mean±SD values of various
parameters at the baseline, third month, and sixth month
in the calendula mouthwash test group (n=120)
Indices used as
parameters
Plaque index
Gingival index
Sulcus bleeding index
Oral hygeine indexsimplified

Mean±SD
Baseline

Third month

Sixth month

2.42±0.14
2.17±0.17
2.68±0.19
2.66±0.54

1.89±0.17
1.68±0.11
2.15±0. 19
2.52±0.45

1.25±0.10
1.15±0.15
1.44±0.12
0.85±0.18

SD – Standad deviation

Table 2: Distribution of mean±SD values of all
parameters at the baseline, third month, and sixth month
in the control group (n=120)
Indices used as
parameters
Plaque index
Gingival index

the baseline to the third month reflects the direct effect
of calendula in reducing plaque and gingival bleeding in
the absence of scaling; as also the reduction in score of all
parameters from the third month to the sixth month reflects
its effect as an adjunct to scaling. The significant reduction
in the OHIS score from the third month to the sixth month
is truly attributed to a thorough scaling done at the third
month, after recording the clinical parameters [Figure 1 and
Tables 1 and 3].
The student’s paired test value showed that in the control
group there was no significant decrease in the mean values
of PI, GI, SBI, and OHI‑S when the baseline scores were
compared with the third month scores (i.e., P > 0.05),
however, there was a significant reduction in the mean
values of all the parameters when the baseline scores were
compared with the sixth month scores in the control group.
Similarly, there was a significant reduction in the mean
values of all the parameters when the third month scores
were compared to the sixth month scores in the control
group [Table 4]. The statistical result showed that in the
control group there was no significant reduction in the mean
value of the differences in the PI, GI, SBI, and OHIS scores
from the baseline to the third month (P > 0.05), however,
the mean values of PI, GI, SBI and OHIS showed significant
reduction when the third month scores were compared with
the sixth month scores (P < 0.05) and the baseline scores were
compared with the sixth month scores (P < 0.05) [Figure 2
and Tables 2 and 4]. No significant reduction in the mean
value of the scores of all parameters from the baseline to
Table 3: Value of student’s paired ‘t’ test under comparison
of mean values of all parameters from baseline to third
month, from baseline to sixth month, and from third month
to sixth month in the calendula test group (n=120)
Indices used as
parameters

Plaque index
Gingival index

Third month

Sixth month

2.29±0.12
1.89±0.17
2.25±0.15
2.09±0.16

2.31±0.21
1.97±0.13
2.27±0.12
2.09±0.10

1.65±0.13
1.39±0.17
1.72±0.14
0.67±0.13

SD – Standard deviation


From
baseline to
sixth month

From third
month to
sixth month

*2.98
*5.32
*4.77
#1.87

*10.88
*7.65
*12.03
*9.23

*8.31
*5.55
*9.56
*15.02

*Significant, P<0.01; #Not significant, P>0.05

Table 4: Values of student’s paired ‘t’ test under
comparison of differences of all parameters from
baseline to the third month, from baseline to the sixth
month, and from the third month to the sixth month in
the placebo control group (n=120)
Indices used
as parameters

Mean±SD
Baseline

Student’s paired ‘t’ test value
From
baseline to
third month

Plaque index
Gingival index
Sulcus bleeding
index
Oral hygeine
index-simplified

Student’s paired ‘t’ test value
#From
baseline to the
third month

*From
baseline to the
sixth month

*From third
month to the
sixth month

#0.16
#0.28
#0.20

*6.87
*7.45
*7.98

*6.43
*7.45
*4.56

#0.09

*15.12

*17.87

*Significant, P<0.01; #Not significant, P>0.05
743


the third month signifies that placebo has no direct effect
on the reduction of plaque and gingival inflammation in
the absence of scaling. However, the significant reduction
in mean values of all parameters from the third month
to the sixth month was truly contributed to the thorough
scaling performed on the third month visit [Figure 2 and
Tables 2 and 4].

Also, when the percentage of reduction of scores from the
third month to the sixth month of the calendula test group
and control group were compared, the test group showed
significantly greater reduction in scores than the control group,
(Test group vs. control group from the third month to the sixth
month), PI (51.2% vs. 38.87%), GI (46.09% vs. 35.97%), SBI
(49.30% vs. 30.94%) [Tables 5 and 6].

Table 5: Values showing reduction in the percentage of
scores of all parameters from day 0 to the third month,
from the third month to the sixth month and day 0 to the
sixth month in the calendula test group (n=120)

Table 6: Values showing reduction in percentage of
scores of all parameters from baseline to the third
month, from the third month to the sixth month, from
baseline to the sixth month in the control group

Indices used
as parameters

Indices used
as parameters

Plaque index
Gingival index
Sulcus bleeding
index
Oral hygeine
index-simplified

Reduction of score in percentage
From day 0
to the third
month (%)

From third
month to the
sixth month (%)

From day 0
to the sixth
month (%)

28.04
29.17
24.65

51.2
46.09
49.30

93.60
88.69
86.11

55.55

196.47

212.94

Plaque index
Gingival index
Sulcus bleeding
index
Oral hygeine
index-simplified

Reduction of score in percentage
From baseline
From third
From baseline
to the third
month to the
to the sixth
month (%)
sixth month (%)
month (%)
−0.86
−4.06
−0.08

38.87
35.97
30.94

40
35.97
30.81

0

211.95

211.95

Figure 1: Bar diagram showing mean values of PI, GI, SBI, and OHI‑S at baseline, third month, and sixth month in test group

Figure 2: Bar diagram showing mean values of PI, GI, SBI, and OHI‑S at baseline, third month, and sixth month in control group
744



DISCUSSION
Dental plaque is the main etiological agent of most forms
of periodontal disease. To this day, mechanical methods of
dental plaque removal are widely regarded as being effective
means of helping to control progression of dental caries and
periodontal diseases, which rank as one of the most common
diseases in humans.[25]
However, for a variety of reasons, a mechanical routine does
not appear to be sufficient for a majority of patients. Several
chemotherapeutic agents are available commercially in the
form of mouth rinses, which are known to have marked
antiplaque and antigingivitis activity. Among them, the
most commonly used mouth rinses are Chlorhexidine,
Listerine, Povidone–Iodine, and Cetylpyridinium chloride.
The antibacterial activity of these agents has been extensively
studied and their efficacy proved. Unfortunately, the toxic
qualities of these agents do not seem to be reserved only for
bacteria, but also for fibroblasts,[26‑29] macrophages,[30] PMNs,[31]
epithelial cells, and erythrocytes.[32] However, Preethi et al.,[12]
has demonstrated that calendula extract has no direct adverse
effect on fibroblasts, but in fact, it can inhibit the cytotoxic
effect on L929 fibroblast cells induced by LPS‑stimulated
macrophages. Also, Saini et al.,[33] has shown that calendula
significantly inhibits human gingival fibroblast‑mediated
collagen degradation and MMP‑2 activity. Lagarto et al., have
demonstrated very low acute and subchronic oral toxicities of
C. officinalis extract in Wistar rats.[34]
The result of our trial demonstrated that use of the C. officinalis
mouthwash resulted in significantly greater reduction in plaque
and gingivitis in comparison to the control placebo mouthwash.
There is scarcity of material on the application of calendula
as an anti‑plaque and anti‑gingivitis agent; the only study
found was the one done by Yusoff et al., which reported that a
calendula‑containing mouthwash (plandula) was effective in
reducing the PI score by 23.9% and the GI score by 62.3% during
a 14‑day study period.[35] Yusoff used plandula mouthwash,
which contained Plantago, Fragaria, and Chamomilla, in addition
to Calendula. However, in the present study, during the period
of six months, pure Calendula diluted mother tincture was used
as a mouthwash. PI was found to be reduced by 93.60%, GI
by 88.69%, and SBI by 86.11%, in six months. The variation in
results between the present study and that of Yusoff et al. may
be due to the nature of the test mouthwash used, as also the
study time period.
The calendula mouthwash group showed significant reduction
in scores of PI (28.04%), GI (29.17%), and SBI (24.65%) from the
baseline to the third month, that is, in the absence of scaling,
whereas, the control group scores of PI, GI, and SBI were
the same or slightly increased from the baseline to the third
month [Tables 3 and 6]. This result reflected that calendula had
some direct effect on reducing plaque and gingivitis, even in
absence of scaling.
However, after scaling, from the third month to the sixth
month, the calendula test group and the control group, both
showed significant reduction in PI, GI, and SBI. However, the
percentages in reduction of the score of PI, GI, and SBI were
greater in the test group compared to the control group. This

again signifies that calendula had a certain effect in reducing
plaque and gingivitis when used as an adjunct to scaling.
Apart from its effect on plaque and gingivitis, the calendula
extract also had an antimicrobial effect on periodontopathic
microorganisms and an antifungal effect on many fungal
infections. Iauk et al.,[22] demonstrated that the calendula
flower extract possesses a high degree of anti‑microbial activity
against 18 different strains of anaerobic and facultative aerobic
periodontal bacteria in vitro, suggesting that it may have an
inhibitory effect on the bacteria causing pathogenesis of the
supporting structures of the tooth. Zilda Cristaine et al.’s (2008),
in vitro study, showed calendula to possess antifungal activity
comparable to nystatin against different species of Candida
including those causing oral candidiasis.[10]
Like chlorhexidine, calendula also has the property of
substantivity as demonstrated by Schmidgall et al. in ex vivo
laboratory model. Calendula has strong bio‑adhesion to
porcine buccal membrane, property is been attributed to
polysaccharides and mucilage content in the herb.[36] Such
effects suggest that it can be used for the therapeutic application
in treatment of canker sore, apthous ulcer, sore throat,
gingivitis, etc., However, comparative study of substantivity
for chlorhexidine and calendula needs to be analyzed in future.
The aqueous extract of calendula facilitates wound healing
by increasing neovascularization and rate of deposition of
hyaluronic acid (GAGs). Also, the flavonoids in calendula are
known to inhibit lysosomal hydrolase, which degrades the
hyaluronic acid.[37] Hyaluronic acid is capable of accelerating
new bone formation through mesenchymal cell differentiation
in bone wounds.[38] Randomized controlled trials have shown
reduction in radiation‑induced oropharyngeal mucositis in
patients with head and neck cancers.[39] Similar healing after
oral application of gel containing calendula extract over oral
mucositis, induced by chemical compound 5‑fluorouracil, has
been documented in Wistar rats.[40]
It has also been suggested that calendula accelerates wound
healing through re‑epitheliazation and collagen maturation.[11]
Calendula exerts anti‑inflammatory activity through reducing
the level of proinflammatory cytokines like IL‑1β, IL‑ 6, TNF‑ α,
and INF‑α in LPS‑induced animals and also inhibits the
expression of the Cox‑2 gene.[12] Flavonoids and carotenoids
are potent antioxidant components of calendula, which have
free radical‑scavenging activity against the OH‑, NO‑, DPPH+,
and ABTS + radicals in a dose‑dependent manner. Treatment
with the extract of calendula enhanced the level of endogenous
antioxidant catalase, superoxide dismutase, and glutathione
in animals.[14] The polysaccharide fraction of calendula has
an immunomodulatory effect by stimulating the phagocytic
activity of human granulocytes in vivo[10] and the phagocytic
activity in mice.[11]
These anti‑inflammatory, antioxidant, and immunomodulatory
properties of calendula will be beneficial in the treatment
of severe periodontitis by modulating the cytokines levels,
reducing oxidative stress, and stimulating the phagocytic activity
of polymorphonuclear leukocytes (PMNs). More research, with
the host modulatory effect of C. officinalis is warranted.
745


CONCLUSION
Within the limits of the study, it can be concluded that
calendula mouthwash is effective in reducing dental plaque
and gingivitis as an adjunct to oral prophylaxis. Various
researches have documented that calendula possesses
antimicrobial, wound healing, anti‑inflammatory, antioxidant,
immunomodulatory, and anti‑mutagenic activities. However,
research data on the application of calendula to the treatment
of moderate‑to‑severe periodontitis is limited. Hence, further
multi‑centered, long‑term clinical trials are needed for further
evaluation of C. officinalis – to prove its use as an anti‑plaque
and anti‑gingivitis agent and also for its use in the treatment
of severe periodontitis.

16.

Limitation of study
The effect of using only distilled water as a placebo cannot be
ruled out in reducing plaque and gingivitis.

20.

17.

18.

19.

21.

ACKNOWLEDGMENT
The authors would like to thank Hemant Pawar for his kind support
in the statistical analysis of this study.

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How to cite this article: Khairnar MS, Pawar B, Marawar PP, Mani
A. Evaluation of Calendula officinalis as an anti-plaque and antigingivitis agent. J Indian Soc Periodontol 2013;17:741-7.
Source of Support: Nil, Conflict of Interest: None declared.

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