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125]

Original Article
Assessing the effect of pomegranate fruit
seed extract mouthwash on dental plaque
and gingival inflammation
Farin Kiany, Hossein Niknahad1, Mohammad Niknahad2

Oral and Dental Disease
Research Center, School
of Dentistry, Shiraz
University of Medical
Sciences, 1Department
of Pharmacology and
Toxicology, School
of Pharmacy, Shiraz
University of Medical
Sciences, 2Students’
Research Committee,
School of Dentistry,
International Branch,
Shiraz University of
Medical Sciences, Shiraz,
Iran
Address for correspondence:
Dr. Farin Kiany,
E‑mail: farinkiany@yahoo.
com
ABSTRACT
Background: Utilizing natural products in the prevention and treatment of periodontal diseases has been
increased recently and could be of benefit to low‑socioeconomic level communities. Mouthwashes are very
useful in the reduction of microbial plaque and gingival inflammation. Pomegranate (Punica granatum) is an
essential medicinal plant with various pharmacological properties. In this study, the efficacy of a mouthwash
prepared from the extract of pomegranate fresh fruit, was evaluated in the reduction of dental plaque and
gingival bleeding. Materials and Methods: One hundred and four participants diagnosed as mild to moderate
gingivitis participated in this double‑blind clinical trial. Two weeks after thorough scaling and root planing,
the participants were randomly and equally divided into four groups: group 1 ‑ Persica, Group 2 ‑ Matrica,
Group 3 ‑ pomegranate mouthwashes, and Group 4 ‑ placebo. The participants were instructed to use the
prescribed mouthwashes, twice daily, for 1 month. Pomegranate mouthwash was prepared from seeds of
fresh pomegranate fruit. Periodontal parameters including plaque and bleeding indices were assessed at
baseline (2 weeks after Phase I of treatment) and 1 month after using mouthwashes. Results: Comparison of
the plaque index showed a significant reduction from baseline to 1 month in all groups (P < 0.05). However,
there was no significant difference in reduction of plaque when comparing four groups. Assessment of the
bleeding on probing revealed a significant decrease from baseline to 1 month in all groups (P < 0.05). The
three herbal mouthwashes reduced the bleeding index significantly more than the placebo (P < 0.05).
Conclusion: Pomegranate mouthwash was beneficial in improving gingival status, including reducing plaque
and bleeding indices. Its effect was comparable to two routinely used herbal mouthwashes.
KEY WORDS: Dental plaque, Matrica, mouthwash, Persica, pomegranate, Punica granatum

Introduction and it could be beneficial to low‑socioeconomic level urban

and rural communities.[2]

G ood oral health has a major influence on one’s general

health and quality of life. Several chronic and systemic
diseases have been attributed to poor oral health. Dental
caries and gingivitis are the most important multifactorial
diseases of the oral cavity. With the increasing incidence
of oral diseases, the global need for alternative prevention
and treatment methods and safe, effective, and economical
products has been expanded. The maintenance of oral health
can be achieved mainly by mechanical and chemical means.[1]
Recently, the use of natural products for the prevention and
treatment of oral pathologic conditions has been increased,
Periodental diseases start with plaque, which is a sticky film of
food, saliva, and bacteria. Dental plaque is usually a pale yellow
biofilm, which is naturally developed on the teeth. Like any
biofilm, dental plaque is formed by colonizing bacteria trying
to attach themselves to the tooth’s smooth surface. Researchers
have shown that many of the mouthwashes are very useful in
the reduction of microbial plaque, by the way of inhibiting the
formation of plaque polysaccharide matrix.[3] Among available
mouthwashes, chlorhexidine is usually used as a positive control
to compare the efficacy of other products in most studies

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DOI: How to cite this article: Kiany F, Niknahad H, Niknahad M. Assessing the effect
10.4103/2348-2915.200016 of pomegranate fruit seed extract mouthwash on dental plaque and gingival
inflammation. J Dent Res Rev 2016;3:117-23.

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Kiany, et al.: Effect of pomegranate seed mouthwash on gingivitis
since it is believed to be an excellent choice.[4,5] However, the
incidence of side effects such as undesirable tooth discoloration,
unpleasant taste, dryness, and burning sensation in the mouth
discourage patients to use this mouthwash.[6,7]
Recently, the use of herbal mouthwashes is increasing in the
world. Persica and Matrica are two available herbal mouthwashes
in Iran. Persica is prepared mainly from Salvadora persica
extract, commonly known as “miswak” that belongs to the
family of Salvadoracea. Persica mouthwash contains two other
medicinal plants, yarrow and mint in its formulation.[8,9] The
beneficial effects of miswak in respect to oral hygiene and dental
health are partially due to its mechanical action and partially
due to its pharmacological effects.[10] It has been claimed
that miswak sticks may have antiplaque effects and may also
affect the pathogenesis of periodontal diseases by reducing the
virulence of periodontal pathogens. In the form of mouthwash,
it has been shown to decrease gingival bleeding and cariogenic
bacteria significantly.[11] Matrica is another herbal mouthwash
that contains herbal extract of Chamomile flower (Matricaria
chamomilla). This plant is a member of Asteraceae/Compositae
family.[12] Different studies have shown that the use of these
herbal medicines or their extracts would support periodontal
health and reduce the accumulation of microbial plaque and
bleeding during brushing, thus controlling gingivitis and
periodontal diseases.[13,14]
In this research, a new mouthwash produced from pomegranate
(Punica granatum) seed extract was evaluated for its antiplaque
and anti‑inflammatory effects.
Pomegranate is mostly native to Iranian plateau and Himalayas
in Northern India, and its fruit is a common edible fruit,
especially in Iran.
Pomegranate is an important medicinal plant in Iran. Its flowers
are used as astringent, hemostatic, antibacterial, antifungal,
and antiviral agent. Powder prepared from its rind is used as
tooth powder and also as medicine in cosmetic industries.
Actually pomegranate components have been used as a strong
astringent and a popular remedy in folk medicine throughout
the world.[15,16]
Clinical studies have shown that pomegranate antioxidant
contents can attack the causes of tooth decay at the biochemical
level.[17‑22]
As it was emphasized, it has previously been demonstrated that
different parts of pomegranate (bark, peel, flower, seed, pomace,
etc.,) contain many components that have direct and indirect
antioxidant, anti‑inflammatory, and antibacterial effects.[19,23]
A number of researches have indicated that pomegranate
and its extract may serve as natural alternatives to chemical
antimicrobial products due to their potency against a wide
range of bacterial and viral pathogens. Nearly, every part of the
pomegranate plant has been tested for antimicrobial activities,
including the fruit juice, peel, arils, flowers, and bark. Many
 118 Journal of Dental Research and Review ● Volume 3 ● Issue 4 ● October-December 2016
studies have utilized pomegranate peel with success. In some
cases, the combination of the pomegranate constituents offers
the most benefit. The positive clinical results on pomegranate
and suppression of oral bacteria are intriguing and worthy of
further studies.[13]
In this clinical trial, the anti‑inflammatory and plaque‑reducing
activity of extract of pomegranate seed was compared to two
regularly used herbal mouthwashes in Iran, Persica, and Matrica.
Materials and Methods
This double‑blind parallel design clinical trial study was
conducted on 104 volunteers from both sexes (58 females
and 46 males), age ranging from 15 to 45 years, with mild or
moderate gingivitis. The volunteers were randomly selected
from dental students of the International Branch of Shiraz
Dental School and from patients who were referred for
periodontal treatment to periodontology department of this
dental school. Patients with systemic disorders, participants
with a history of antimicrobial or corticosteroid therapy for
2 months before the study, those with a history of periodontal
treatment or mouthwash usage for 6 months before the study,
smokers, pregnant or breastfeeding women, and persons with
fixed or removable orthodontic appliances or removable partial
dentures were excluded from the study. All of the volunteers had
at least twenty teeth (except wisdom teeth). Informed consents
were obtained from all of the participants. After thorough
scaling, root planing, and polishing, the participants were
randomly distributed into the following groups and instructed
to use 15 ml of prescribed mouthwashes twice daily (morning
and night, for 40 s) for 1 month:
• Group 1 (n = 26): Persica mouthwash
• Group 2 (n = 26): Matrica mouthwash
• Group 3 (n = 26): Pomegranate mouthwash
• Group 4 (n = 26): Placebo.
The participants were advised to avoid from eating, drinking,
and brushing at least 1 h after using mouthwashes.
Data were collected at baseline (2 weeks after scaling) and
1 month after utilizing the mouthwashes using the following
indices:
• Plaque index (Tureskey – Gilmore–Glickman modification
of Quigley‑Hein)[24]
• Bleeding index (Lenox).[25]
Scaling and root planing were performed to establish
standardized conditions of the periodontium for all the
participants. The first measurement of indices was performed
2 weeks after the baseline scaling and root planning, to let
reepithelialization of the gingival sulcus. During the trial, no
emphasis was placed on oral hygiene procedures and each
participant kept on his/her own oral hygiene performance.
Scaling and root planing and measurement of indices were
performed by one clinician who was blind to the groups. The
mouthwashes were prescribed and handed by a second person
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Kiany, et al.: Effect of pomegranate seed mouthwash on gingivitis

and were prepared in a uniform preparation. After 2 weeks, all
the participants were recruited and the compliance was checked
and their complaints about the taste of the mouthwashes or any
sensitivity reaction after its usage were recorded.
Preparation of pomegranate mouthwash
By comparing the mean of decrease in four groups, it was shown
that the three herbal mouthwashes reduced the bleeding index
significantly more than the placebo (P < 0.05) [Table 4].
Comparison of both indices from baseline to 1 month with
P value is reported in Table 5.

Pomegranate mouthwash was prepared from the extract of
the pomace of fresh fruit of pomegranate that were cut into
small pieces, after removing the rind of the fruit. After cold
pressing and blending of the pieces, the blend was filtered
and the seeds were separated from the extract. The obtained
extract was filtered through several layers of gauze, and then,
it was diluted by adding equal volume of distilled water
containing 0.4% methylparaben as preservative and 0.04%
sodium saccharine. The extract was then pasteurized and
prepared as ready to use solution in the form of drop dosage
form in 60 ml containers.
Placebo had the same volume of distilled water plus all the
other contents of the formulation except the fruit seed extract.
Matrica (Bareej Essence, Iran) and Persica (Poursina, Iran) were
purchased from a local pharmacy.
Statistics for analyzing data
Analyzing the questionnaire that was about the taste and
acceptability of the mouthwashes revealed that pomegranate
had the best taste and tolerability among three mouthwashes
plus placebo. 92.3% of the users of pomegranate mouthwash
declared good taste [Table 6].
Discussion
Dental plaque is the primary etiologic factor in gingival
inflammation. Hence, daily and effective supragingival
plaque control is necessary to arrest its progression to
periodonitis.[26] Although mechanical plaque control methods
have the potential to maintain adequate levels of oral hygiene,
studies have shown that such methods are not being employed
accurately.[27] Therefore, several chemotherapeutic agents have
been developed to control bacterial plaque. In an urge of looking
for better antiplaque and antigingivitis agents with limited
side effects as compared to chemical products, various herbal
mouthwashes have been tried with fruitful results.

Kruskal–Wallis H‑test (Pairwise comparisons) and
Mann–Whitney U‑test were used for statistical analysis. P value
was adjusted at <0.05.
Results
A total of 104 participants (58 females and 46 males) participated
in this study with the mean age of 25.3 ± 0.123 years.
Plaque index
Comparison of plaque index showed a significant reduction
from baseline to 1 month in all groups (P < 0.05) [Table 1].
There was no significant difference in reduction of plaque when
comparing four groups [Table 2].
It has been shown that pomegranate contains agents, especially
polyphenolic flavonoids, with pharmalogical actions that could
be considered conductive to good oral health, particularly
in relation to gingivitis development. It has been suggested
that reduction of oxidative stress, direct antioxidative activity,
anti‑inflammatory effects, antibacterial activity, and direct
removal of plaque from the teeth are the mechanisms involved
in this action.[20,28‑32] Effectiveness of the extract of pomegranate
in inhibiting prostaglandin and leukotriene formation through
inhibition of the eicosanoids enzymes, cyclooxygenase and
lipoxygenase, raises the possibility of use of pomegranate
derivatives in anti‑inflammatory preparations.[33]
This study was conducted to evaluate the clinical efficacy of
pomegranate seed extract in comparison to two routinely used
herbal mouthwashes.

Bleeding index Analysis of plaque index values of this study suggested that the
three mouthwashes were helpful in reducing the plaque to the
Analysis of bleeding on probing revealed a significant decrease same extent and there was no significant difference between
from baseline to 1 month in all groups (P < 0.05) [Table 3]. them.

Table 1: Plaque index of four mouthwashes at baseline and after 1 month

Group PI median (mean±SD) Reduction P
2 weeks 1 month
Persica 1.1950 (1.1227±0.7106) 0.4350 (0.5865±0.4030) −0.5450 (−0.5362±0.5950) <0.05
Matrica 0.9050 (1.1154±0.6055) 0.5500 (0.6419±0.3861) −0.4450 (−0.4735±0.3714) <0.05
Pomegranate 0.8450 (0.9623±0.8377) 0.2600 (0.3981±0.4352) −0.4900 (−0.5642±0.5121) <0.05
Placebo 0.9050 (0.9692±0.6012) 0.5850 (0.6085±0.4424) −0.4350 (−0.3608±0.4708) <0.05
PI: Plaque index, SD: Standard deviation

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Kiany, et al.: Effect of pomegranate seed mouthwash on gingivitis

There is no study in literature comparing the antiplaque and In this study, placebo also reduced plaque and gingival bleeding
anti‑inflammatory effect of pomegranate seed extract with to some extent while in comparison to three mouthwashes, the
Persica and Matrica. reduction in bleeding index was less significant in placebo group.

In the study of Ahuja et al., pomegranate mouthwash was compared This positive effect of placebo partly can be related to hawthorne
with chlorhexidine mouthwash. Pomegranate was effective in effect. This effect (also referred to as the observer effect) refers
reducing plaque but not to the extent that chlorhexidine was.[34] to a phenomenon whereby the participants in a study improve
or modify an aspect of their behavior in response to the fact of
In a microbiologic study, Menezes et  al. showed that after change in their environment. This effect is more noticeable in
a single 1 min mouth rinsing, more reduction in plaque studies observing hygiene procedures. According to hawthorne
was observed with pomegranate (84%) as compared with effect, the participants in the placebo group would improve
chlorhexidine (79%).[19] their hygiene procedures unintentionally because of their
awareness about participating in a study that is evaluating their
Analysis of the bleeding scores of the current study revealed that competence in hygiene performance procedures.[38] By the way,
the effect of three mouthwashes on the bleeding index was the rinsing with placebo has a flushing effect removing food debris
same and better than placebo. and material alba from the mouth and by this way can interfere
with organization of the dental plaque.
In the study of Ahuja et al., pomegranate mouthwash was more
efficient in reducing gingival score and bleeding on probing By assessing the measurements of plaque and bleeding indices
than chlorhexidine.[34] at baseline, it can be implicated that the majority of the
participants had mild gingivitis with even some healthy parts
As the positive effect of Persica and Matrica in reducing of the gingiva. This is to some extent due to involvement of
dental plaque and gingival inflammation has been shown in dental students in this study, who were young and moderately
different studies,[35‑37] the results of this study emphasized that watchful about their oral hygiene. The authors presume that
pomegranate mouthwash could be as effective as these two the statistically equal effect of placebo in reducing plaque, in
customarily used herbal mouthwashes. relation to mouthwashes, partly can be because of relatively
satisfactory level of oral hygiene performance in a number of
Table 2: Comparison of decrease in plaque index from baseline the participants.
to 1 month in four groups
There are various studies that P. granatum was used in other
Group PI 1 PI 2 Difference PI
forms, different from mouthwash, or in combination with other
Group 1 herbal medications.
Mean 1.1227 0.5865 −0.5362
Median 1.1950 0.4350 −0.5450
SD 0.7106 0.4030 0.5950 A study by Salgado et al. on 10% pomegranate gel showed that
Group 2 the gel was not efficient in preventing supragingival plaque
Mean 1.1154 0.6419 −0.4735 formation and gingivitis.[27] It may be speculated that because
Median 0.9050 0.5500 −0.4450 the gel was placed into a tooth shield in a nondiluted form, it
SD 0.6055 0.3861 0.3714
Group 3
could not be solubilized with saliva to exert its antimicrobial
Mean 0.9623 0.3981 −0.5642 action.
Median 0.8450 0.2600 −0.4900
SD 0.8377 0.4352 0.5121 Sastravaha et  al. used combination of Centella asiatica and
Group 4 pomegranate as biodegradable chips in deep pockets after
Mean 0.9692 0.6085 −0.3608
scaling and root planing and found a significant decrease in
Median 0.9050 0.5850 −0.4350
SD 0.6012 0.4424 0.4708 bleeding and plaque scores.[20]
Total
Mean 1.0424 0.5588 −0.4837 In a study by Rudney et al., it was shown that mouth rinsing with
Median 0.9000 0.4400 −0.4850 pomegranate mouthwash resulted in lowering of total protein
SD 0.6896 0.4221 0.4930 content in saliva in relation to placebo. Total saliva protein is
PI: Plaque index, SD: Standard deviation normally higher among people with gingivitis and may correlate

Table 3: Bleeding index of four mouthwashes at baseline and after 1 month

Group BI median (mean±SD) Reduction P
2 weeks 1 month
Persica 13.7000 (13.1154±9.8497) 7.4000 (8.4846±7.0574) −4.2000 (−4.6308±4.5914) <0.05
Matrica 12.8000 (13.3692±9.1546) 7.6500 (6.8346±5.5248) −5.4500 (−6.2115±5.4777) <0.05
Pomegranate 9.0500 (10.7308±8.6934) 2.9000 (3.3308±3.5301) −6.6000 (−7.2077±5.6641) <0.05
Pelacebo 14.5000 (13.9346±8.6886) 10.8500 (11.9308±7.7238) −2.0000 (−1.9808±3.5611) <0.05
BI: Bleeding index, SD: Standard deviation

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Kiany, et al.: Effect of pomegranate seed mouthwash on gingivitis

Table 4: Comparison of decrease in bleeding index from baseline in comparison to miconazole gel. It was mentioned that
to 1 month in four groups pomegranate inhibit species of streptococci and prevent them
Group BI 1 BI 2 Difference BI from producing chemicals that create favorable conditions for
Group 1 fungi and other microorganisms to thrive. These researchers
Mean 13.1154 8.4846 −4.6308 suggested that this phytotherapeutic agent might be used
Median 13.7000 7.4000 −4.2000 in the control of adherence of different microorganisms in
SD 9.84970 7.05740 4.59145 the oral cavity.[17]
Group 2
Mean 13.2769 7.4192 −5.8885
Median 12.8000 7.6500 −5.4500
Furthermore, Menezes et al. in an in vitro study showed that
SD 9.26120 5.07788 5.59516 hydroalchoholic extract of pomegranate had antibacterial
Group 3 activity against selected bacteria of dental plaque.[19]
Mean 10.0769 3.4846 −6.5923
Median 7.5500 3.3500 −5.2500 Abdollahzadeh et al. in an in vitro study showed the efficacy of
SD 8.99806 3.74526 5.74268
different concentrations of methanolic extract of pomegranate
Group 4
Mean 13.9346 11.1346 −2.8000 against common oral pathogens responsible for caries,
Median 14.5000 10.3500 −2.7000 stomatitis, and periodontal diseases.[40]
SD 8.68861 7.84530 4.17833
Total Some other researchers also reported that extracts of
Mean 12.6010 7.6308 −4.9779
pomegranate peel in different concentrations have antibacterial
Median 11.5500 5.4000 −4.2000
SD 9.19673 6.65769 5.20118 activity against some oral pathogens.[41‑43]
BI: Bleeding index, SD: Standard deviation
It has been demonstrated that antibacterial activity of
pomegranate may be related to the presence of hydrolysable
Table 5: Comparison of decrease in plaque and bleeding tannins and polyphenolics in the pomegranate extract, especially
indices from baseline to 1 month in 4 groups punicalagin and gallagic acid.[17,44]
Group Median (mean±SD)
ΔPI ΔBI It is cited that the antimicrobial effect of tannins is related to
Persica −0.545 (−0.563±0.595)^ −4.200 (−4.631±4.591)^ its molecular structure and toxicity for bacteria. Tannins may
Matrica −0.445 (−0.474±0.371)^ −5.450 (−6.212±5.478)^ act on the cell wall and cell membrane of bacteria.[17,41]
Pomegranate −0.490 (−0.564±0.512)^ −6.600 (−7.218±5.664)^
Placebo −0.435 (−0.361±0.471)^ −2.000 (−1.981±3.561)# It is noteworthy that this toxicity of juice and seeds of
P* 0.701 0.002
pomegranate would appear to be very low, owing to the use of
^
Same letter shows insignificant difference between groups. #Different letter these components as common items of dietary commerce.[45]
shows significant difference between groups, measured by Mann‑Whitney
U‑test. P value was adjusted at <0.05 and measured with Kruskal‑Wallis
H‑test. ΔPI: Plaque index 2‑plaque index 1, ΔBI: Bleeding index 2‑bleeding Conclusion
index 1, SD: Standard deviation
This study concluded that pomegranate mouthwash was
Table 6: Comparison of the taste of mouthwashes and placebo effective against dental plaque to the same degree as Persica
and Matrica were. Furthermore, its effect in reducing bleeding
Taste group Good (n=26), Not good, not bad Bad (n=26),
n (%) (n=26), n (%) n (%)
tendency of gingiva was comparable to two‑mentioned
mouthwashes. Its usage in daily oral hygiene prophylactic
Persica 4.0 (15.4) 15.0 (57.7) 7.0 (26.9)
Matrica 0 4.0 (15.4) 22.0 (84.6)
procedures and periodontal treatments can be encouraged
Pomegranate 24.0 (92.3) 2.0 (7.7) 0 because it is easy to prepare, has profound styptic action, and
Placebo 17.0 (65.4) 9.0 (34.6) 0 shows acceptable reduction in plaque and bleeding scores.
More clinical trials are required to know the effectiveness of
pomegranate and its advantages over chemical plaque control
with plaque‑forming bacterial content. Pomegranate‑treated agents, especially chlorhexidine which is still a gold standard
participants also experienced significant decreases in the for reduction of plaque scores and gingivitis.
salivary activity of the enzyme aspartate aminotransferase that
is considered a reliable indicator of cell injury and is elevated Acknowledgment
among patients with periodontitis.[39]
This paper has been extracted from Mr. Mohammad Niknahad’s
Most of the previous studies evaluated the effect of extract DDS thesis which was conducted under supervision of
of pomegranate on microorganisms of dental plaque Dr. Farin Kiany and Dr. Hossein Niknahad.
in vitro. Vasconcelos et al. in an in vitro study showed that
pomegranate gel had greater efficiency in reducing microbial The study was approved, registered with ID 8592044, and
adherence of Streptococcus sanguis, Streptococcus mitis, supported by the International Branch of Shiraz University of
Streptococcus mutans, Candida albicans to glass surface, Medical Sciences.

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Kiany, et al.: Effect of pomegranate seed mouthwash on gingivitis
Furthermore, We would like to thank Dr. Mehrdad Vossoughi
for his help with the statistical analysis.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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