EFFECTIVENESS OF SPIRULINA MOUTHWASH ON REDUCTION OF DENTAL PLAQUE AND GINGIVITIS: A CLINICAL STUDY

Abstract

Objective: The present study evaluated the effectiveness of Spirulina mouthwash on the reduction of dental plaque and gingivitis.Methods: A single-blind clinical trial was conducted among thirty patient’s aged 18-40 y visiting dental college and hospital in Bangalore city. Mouthwash was prepared using 0.5% Spirulina. Intervention protocol consisted of instructing the patients to rinse with 10 ml of mouthwash for 1 minute twice daily for 7 d. Plaque index and Gingival index were used to assess the variables at the baseline and after the intervention. The perception of the individual subjects with regard to the use of mouthwash was assessed using 10 cm long visual analog scale (VAS). Statistical analysis was carried out using Wilcoxon signed rank test for mean pre and post plaque and gingival scores respectively. Descriptive statistics was performed for VAS questionnaireResults: The results showed a highly significant difference (p<0.001) between the mean plaque scores at the baseline (2.16±0.34) and at the follow up (1.27±0.46). The mean gingival scores at the baseline (1.86±0.38) and at the follow-up (1.05±0.43) also showed a highly significant difference (p<0.001). Regarding the Visual Analog Scale, the mean values of 5 or greater than suggested the responses to be favourable as the values were reflectedConclusion: The study showed that Spirulina mouthwash resulted in significant reduction in dental plaque and gingivitis. Also, the mouthwash was convenient to use without any adverse effects. Hence, the use of herbal mouth rinses such as Spirulina should be supported.

Full text

Maniyar et al.
Int J Pharm Pharm Sci, Vol 9, Issue 7, 136-139
136
Original Article
EFFECTIVENESS OF SPIRULINA MOUTHWASH ON REDUCTION OF DENTAL PLAQUE AND
GINGIVITIS: A CLINICAL STUDY
RADHIKA MANIYAR
1
, UMASHANKAR G. K.
2
1, 2
Department of Public Health Dentistry, M R Ambedkar Dental College and Hospital, Bangalore
Email: maniyar.radhika@gmail.com
Received: 10 Mar 2017 Revised and Accepted: 27 May 2017
ABSTRACT
Objective: The present study evaluated the effectiveness of Spirulina mouthwash on the reduction of dental plaque and gingivitis.
Methods: A single-blind clinical trial was conducted among thirty patient’s aged 18-40 y visiting dental college and hospital in Bangalore city.
Mouthwash was prepared using 0.5% Spirulina. Intervention protocol consisted of instructing the patients to rinse with 10 ml of mouthwash for 1
minute twice daily for 7 d. Plaque index and Gingival index were used to assess the variables at the baseline and after the intervention. The
perception of the individual subjects with regard to the use of mouthwash was assessed using 10 cm long visual analog scale (VAS). Statistical
analysis was carried out using Wilcoxon signed rank test for mean pre and post plaque and gingival scores respectively. Descriptive statistics was
performed for VAS questionnaire
Results: The results showed a highly significant difference (p<0.001) between the mean plaque scores at the baseline (2.16±0.34) and at the follow
up (1.27±0.46). The mean gingival scores at the baseline (1.86±0.38) and at the follow-up (1.05±0.43) also showed a highly significant difference
(p<0.001). Regarding the Visual Analog Scale, the mean values of 5 or greater than suggested the responses to be favourable as the values were
reflected
Conclusion: The study showed that Spirulina mouthwash resulted in significant reduction in dental plaque and gingivitis. Also, the mouthwash was
convenient to use without any adverse effects. Hence, the use of herbal mouth rinses such as Spirulina should be supported.
Keywords: Gingivitis, Herbal mouthrinse, Phycocyanin, Spirulina
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2 017v9i7.18415
INTRODUCTION
The advances in science and technology have made treatment of
general and oral health diseases now available to human mankind,
thus increasing the longevity of life along with retention of teeth.
WHO recently published a global review of oral health which
emphasized that despite great improvements in the oral health of
populations in several countries, global problems still persist [1].
The incidence of gingival and periodontal diseases is rising steadily
affecting all the age groups. Dental plaque is a host associated
biofilm, which exists as soft deposits adhering to tooth surfaces or
other hard surfaces in the oral cavity, and consists of bacteria
embedded in a matrix of polymers of bacterial and salivary origin
[2]. There is a causal relationship between dental plaque and
gingivitis that was established decades ago [3]. Clinical control of
these diseases can most readily be achieved by reducing the oral
microbial load of the plaque biofilm [4]. Thus, plaque reduction
remains the mainstay of preventive dentistry. For this various
mechanical plaque control measures have been practised.
Despite the potential for adequate mechanical plaque control, clinical
experience and population-based studies demonstrate that such
methods are not being employed sufficiently by large numbers of the
population as it requires time, motivation and manual dexterity [5, 6].
As an adjunct to this, chemical plaque control measures have been
employed which includes the use of various anti-plaque agents and
mouthwashes. Such products influence plaque accumulation by
preventing bacterial attachment and removing bacterial biofilm [4].
Various mouthwashes are available containing triclosan,
metronidazole, chlorhexidine and many more. The use of these agents
has certain unpleasant effects such as altered taste sensation, staining
of teeth which often deters its use. Hence, the use of plants and plant
products can be an alternative solution to this.
Phytotherapy has been widely practiced in India since ages.
Spirulina is a cyanobacterium or blue-green algae (BGA) that is
associated with a wide range of nutritional and health benefits.
Spirulina is known to have an antimicrobial effect against S. aureus,
E. coli, P. aeruginosa, Klebsiella sp, Proteus sp and Embedobacter sp
[7]. At present, there is a mass of evidence in favour of the
antioxidant properties of Phycocyanin, a major pigment of Spirulina
which has been used to explain its anti-inflammatory effects.
Gingivitis is an inflammatory disease induced by bacterial biofilms that
accumulate in the gingival margin, in which a series of inflammatory
responses are initiated by pathogenic bacteria and ultimately results
in periodontal breakdown if not treated at the initial stage. So far in
dentistry, Spirulina was assessed for its oxidative properties in the
healing of oral submucous fibrosis and leukoplakia, but its effect on
plaque reduction and gingivitis remains unexplored. Thus, this is the
first study of its kind assessing the effectiveness of Spirulina
mouthwash on the reduction of dental plaque and gingivitis.
MATERIALS AND METHODS
The present study was a single-blind clinical trial conducted to
assess the effectiveness of mouthwash containing Spirulina on the
reduction of dental plaque and gingivitis measured using Plaque
index [8] and Gingival index [9].
The study was conducted among the patients visiting a dental
hospital in Bangalore.
Ethical clearance (EC-393) was obtained from the Institutional
Ethics Committee of the dental college and hospital. Informed
consent was obtained from the participants after explaining the
methodology, benefits and adverse effects of the study. The required
sample size was estimated based on the difference in the pre and
post plaque and gingival scores among the study group. It was
calculated based on the minimum difference of 0.5 expected
between the pre and post scores among the study group which was
25. Considering the loss to follow-up, the sample size was rounded
off to 30. The inclusion and exclusion criteria are as follows:
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 9, Issue 7, 2017

Maniyar et al.
Int J Pharm Pharm Sci, Vol 9, Issue 7, 136-139
137
Inclusion criteria
Individuals aged 18-40 y with a minimum of 20 teeth present, with
bleeding on probing present clinically, with at least fair plaque and
mild gingival scores respectively, and who had not received any
periodontal therapy for the past 6 mo.
Exclusion criteria
Individuals with probing depths>4 mm, undergoing orthodontic
treatment, wearing a removable prosthesis, with a history of
systemic disease, or had taken any systemic/topical antibiotics
during the past 3 mo and who were currently using any mouthwash
or have used mouthwash in past 15 d were excluded. Pregnant
women, lactating mothers, smokers, alcoholics and subjects with a
history of allergy to any chemical or herbal product were also
excluded from the study.
The investigator was trained and calibrated before the start of the
study in order to limit the intra-examiner variability. The intra-
examiner variability was calculated using kappa statistics. The
Cohen’s kappa value was 0.82 for plaque index and 0.85 for
gingival index.
The plaque index and gingival index scores were recorded at the
beginning of the intervention to obtain the baseline data, then the
subjects were instructed to use the mouthwash for one week. The
plaque scores and gingival scores were reassessed at the end of one
week to obtain the final scores. The type III clinical examination was
followed throughout the study.
The participating subjects were asked to fill a questionnaire using a
visual analog scale (VAS) designed to evaluate their perception with
regard to the mouthwash used. Subjects were asked to mark a point
on a 10-cm-long uncalibrated line with the negative extreme
response (0) at the left end and the positive extreme response (10)
at the right end [10].
Intervention
The mouthwash was prepared using 0.5% Spirulina with the help of
pharmaceutical agency in Bangalore, India. Blinding was carried out
by giving the mouthwash in an amber coloured bottle without any
labelling to the participating subjects. They were asked to swish the
oral cavity using 10 ml of the mouthwash for 1 minute, twice a day,
after breakfast and dinner. The subjects were instructed not to eat
or drink anything for at least one hour after rinsing with
mouthwash. The subjects were reminded from time to time for a
period of one week. The contact number of the investigator was
provided to the subjects to report any inconvenience or adverse
effect observed if any. No oral prophylaxis was done prior to
commencement of intervention. They were allowed to follow their
individual oral hygiene procedures.
Statistical analysis
The statistical analysis was performed using SPSS version 22.0. The
mean plaque and gingival scores between pre and post intervention
were compared using Wilcoxon signed rank test respectively.
Descriptive statistics was performed for VAS questionnaire.
RESULTS
All participants (n=30) completed the trial and there were no
missing values. There was an equitable distribution regarding the
gender among the participants (M = 15; F = 15). The age of the
participants ranged between 18-40 y with mean age being 28.5±7.3.
The mean plaque scores at the baseline were 2.16±0.34 and after
seven days, at follow-up were 1.27±0.46. (fig. 1) Statistical analysis
using Wilcoxon signed rank test showed that there was a highly
significant difference (p ≤ 0.001) between the scores at baseline and
follow-up indicating the effectiveness of Spirulina on the reduction
of dental plaque.
Fig. 1: Comparison of mean plaque scores before and after the
intervention
The mean gingival scores at baseline were 1.86±0.38 and at follow-up
were 1.05±0.43. The difference between the scores was seen to be
highly significant (p≤0.001) using Wilcoxon signed rank test.(fig. 2)
Fig. 2: Comparison of mean gingival scores before and after
intervention
The subjects filled the VAS questionnaire at the end of the
experimental period of 7 d. It comprised of four questions and the
subjects were asked to mark a point on 10 cm long scale with the
negative response at the extreme left and a positive response at the
extreme right end of the scale (table 1).
Table 1: Questionnaire responses (mean±SD) determined by visual analogue scale (VAS)
Question
Response
MEAN
±
SD
1)
How was the taste of the mouth
wash
?
Unacceptable……
. A
cceptable
5.0
±
0.5
2)
For what duration did the tast
e remain in mouth after rinsing
?
Long ……
. S
hort
6.3
±
0.7
3)
Was the ta
ste of food
and drinks affected
?
Altered ……Unaltered
7.6
±
1.4
4)
Was the use of
mouthrinse
convenient
?
Inconvenient ……
. C
onvenient
6.3
±
0.8

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The mean values of 5 or greater than 5 suggested the responses be
favourable, as the mean values were reflected towards the right end
of the Visual Analog scale. With regard to the question on the taste of
the mouthwash, the mean values were 5±0.5. When asked regarding
the duration the taste remained in mouth after rinsing, the mean
values were 6.3±0.7, suggesting the duration to be more towards
short response. With regard to the question on whether the taste of
the food was affected, the mean values were 7.6±1.4, suggesting that
the taste remained unaltered. Lastly, regarding the convenience of
the use of mouthwash, the mean values were 6.3±0.8, suggesting
that the mouthwash was convenient to use.
DISCUSSION
The traditional methods of phytotherapy are making a comeback
and an era of herbal renaissance is being revolutionised once again.
The World Health Organization estimates that 65-80% of the world’s
population use traditional medicine as the primary form of health
care [11]. Similarly, use of herbal products and dietary supplements
is an emerging trend in dentistry.
Studies have shown that mechanical plaque control measures are
inadequate, thus paving a pathway for the use of herbal products which
are free from the adverse effects as seen by the use of synthetic
chemicals. Blue-green algae, among the earliest life forms on earth and
have been a source of food or medicine for humans since centuries.
Certain BGA species include Aphanizomenon flos-aquae (AFA), Spirulina
platensis (SP), Spirulina maxima (SM), and Spirulina fusiformis (SF) [12].
Spirulina is a microscopic blue-green alga in the shape of a spiral
coil, living both in the sea and fresh water. Spirulina is the common
name for human and animal food supplements produced primarily
from two species of cyanobacteria: Arthrospira platensis, and
Arthrospira maxima. It is an edible, filamentous, alkalophilic,
photoautotrophic cyanobacterium belonging to the class
Cyanophyta. It is a rich source of many important nutrients like
proteins, complex carbohydrates, iron, vitamins A, K, B complexes,
minerals, lipids and essential fatty acids [13-15].
Spirulina is considered as “nature’s superfood”. This study showed
that the Spirulina mouthwash was effective in reducing dental
plaque scores significantly when compared to the baseline scores.
This could be attributed to the antimicrobial compounds found in
cyanobacterial exudates which include polyphenols, fatty acids,
glycolipids, terpenoids, alkaloids, carotenoids and a variety of
bacteriocins. Harder was the first to observe antimicrobial
substance secreted by alga. It has also been reported that they
produce substances that can inhibit microbial growth. Secondary
metabolites from cyanobacteria are associated with toxic, hormonal,
antineoplastic and antimicrobial effects. Secondary metabolites
influence other organisms in the vicinity and are thought to be of
phylogenetic importance [13, 16, 17].
The results of this study are in line with the in vitro study done by
Sujatha et al. who reported the antibacterial activity of green
seaweeds on oral bacteria. The biofilm inhibitory effect of Spirulina
against a broad spectrum of gram positive and gram negative
bacteria like S. aureus, S. epidermidis, S. viridians, E. coli, P.
aeruginosa, P. mirabilis, Vibrio spp was also well documented by F
Lewis Oscar et al. [18].
Antimicrobial active lipids and active fatty acids are present in a
high concentration in this alga. It was hypothesised that lipids kill
microorganisms by leading to disruption of the cellular membrane
as well as bacteria, fungi and yeasts because they can penetrate the
extensive meshwork of peptidoglycan in the cell wall without visible
changes and reach the bacterial membrane leading to its
disintegration [13].
This study showed a significant anti-gingivitis and anti-inflammatory
effect of Spirulina mouthwash, which can be attributed to the
important constituents, phycocyanin (PC) and gamma-linolenic acid
(GLA). Spirulina contains 1.3% GLA and C-PC is a natural blue pigment
accounting for 14% of Spirulina’s dry weight [12].
PC is thought to suppress inflammation by inhibiting the production
of pro-inflammatory cytokines and by inhibiting the expressions of
inducible nitric oxide synthase and cyclo-oxygeanase. GLA can be
metabolized to dihomo-GLA that undergoes oxidative metabolism by
cyclooxygenases and lipoxygenases to produce anti-inflammatory
eicosanoids [12, 19].
Bhat and Madyastha reported that phycocyanin inhibited about 95%
of peroxyl radical-induced lipid peroxidation. The in vitro evidence
also supports that C-PC has strong antioxidant properties by
scavenging radicals and inhibiting lipid peroxidation in cell
membranes [12, 20].
Lipid peroxidation mediated by Reactive Oxygen Species is believed
to be an important cause of destruction and damage to cell
membranes because a simple initiating event can result in the
conversion of hundreds of fatty acids side chain into lipid peroxides,
which alters the structural integrity and biochemical functions of
membranes. It has been revealed that lipid peroxidation levels are
increased during gingivitis and periodontitis. Phycocyanin also
attenuated PGH2-induced Thromboxane B2 formation and platelet
aggregation, implying that phycocyanin may also be a thromboxane
synthase inhibitor. It also has been shown to increase the expression
of essential enzymes and biochemical such as cytochrome p-450,
superoxide dismutase, catalase, alanine transaminase, aspartate
transaminase which further leads to the detoxification [21].
Lipopolysaccharide (LPS), an endotoxin produced by gram-negative
bacteria, stimulates the metabolism of arachidonic acid. This, in turn,
activates lipooxygenase and cyclo-oxygenase inflammatory
pathways. LPS can also affect macrophages, monocytes, fibroblasts
and, as a consequence, leads to the production of pro-inflammatory
cytokines, such as tumor necrosis factor (TNF)-α and interleukin
(IL)-1β. These are amongst the most important pro-inflammatory
cytokines and play a critical role in the destruction of periodontal
tissue, alveolar bone, and eventually tooth loss. Also, IL-1β and TNF-
α can induce the destruction of connective tissue [22]. Animal
studies done with rodents showed that phycocyanin has anti-
inflammatory activity due to prostaglandin E-2 inhibition and that it
reduces allergic inflammatory response and histamine release from
cells [23, 24].
A study done by Mahendra et al. showed that subgingivally delivered
Spirulina gel resulted in a decrease in pocket probing depths as well
as gain in clinical attachment levels in chronic periodontitis patients
[25].
According to Miranda et al., the main phenolic compounds found in
Spirulina were salicylic, trans-cinnamic, chlorogenic, quinic and
caffeic acids. These compounds are used to produce flavonoids,
which possess antioxidant activity [26].
The human clinical study showed that a hot water extract of
Spirulina rich in phycocyanin increased interferon production and
NK cytotoxicity (cancer killing cells) when taken orally [27].
The results of this study could not be compared with other studies
as an exploration of the available literature revealed that no studies
have been carried out till date to assess the same effect in vivo.
CONCLUSION
The Spirulina mouthwash was effective in reducing dental plaque
and gingivitis. Spirulina appears to be a promising agent with a wide
array of antibacterial, antioxidant, anti-inflammatory and anti-fungal
properties with low toxicity and minimal side effects. Thus, the use
of herbal mouthrinse such as Spirulina should be supported.
As this was the first attempt to evaluate the effectiveness of
Spirulina mouthwash on plaque and gingivitis, clinical trials of
longer duration with a larger sample size should be conducted.
Also in vitro studies should be carried to understand the exact
antimicrobial mechanism of Spirulina on pathogens playing an
important role in gingivitis and periodontitis.
The effects of this mouthwash should also be compared with the
benchmark control i.e. chlorhexidine. Further longitudinal studies
and clinical trials of longer duration should be carried out to
evaluate its safety.

Maniyar et al.
Int J Pharm Pharm Sci, Vol 9, Issue 7, 136-139
139
ACKNOWLEDGEMENT
We would like to thank the Cash pharmaceutical agency, Bangalore,
India for their technical support in the preparation of the Spirulina
mouthwash.
AUTHOR’S CONTRIBUTION
First author: Dr. Radhika Maniyar: conception and design,
acquisition of data, or analysis and interpretation of data and
drafting the article
Second author: Dr. Umashankar GK: supervision, proofreading and
final approval
CONFLICTS OF INTERESTS
Declared none
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How to cite this article
• Radhika Maniyar, Umashankar GK. Effectiveness of spirulina
mouthwash on the reduction of dental plaque and gingivitis: a
clinical study. Int J Pharm Pharm Sci 2017;9(7):136-139.