Anti-bacterial Effect of Marine Algae against Oral-borne Pathogens
The aim of this study is to screen and investigate marine algae that can be
used as a natural anti-bacterial agent for oral health care. The anti-bacterial
activities of marine algal extracts were evaluated by agar-well diffusion assay.
Marine algal extracts were dispensed into the wells on agar plates. After incubation,
a clear zone surrounded substances was considered as positive exhibiting anti-bacterial
properties. Among 51 marine algal extracts, Laurencia okamurae Yamada,
Dictyopteris undulata Holmes and Sargassum micracanthum (Kutzing)
Endlicher showed the potent anti-bacterial activities against tested
all oral pathogens. Sargassum micracanthum (Kutzing) Endlicher
showed the strongest anti-bacterial activity with 6.0±1.4 mm of clear
zone against Streptococcus mutans. Additionally, the growth of S.
sobrinus and S. pyogene was inhibited the most effectively by an
extract of Sargassum micracanthum (Kutzing) Endlicher. Laurencia
okamurae Yamada had the strongest susceptibility with 9.5±0.7 mm
of clear zone against S. sobrinus. Laurencia okamurae Yamada,
Dictyopteris undulata Holmes and Sargassm micracanthum (Kutzing)
Endlicher showed the superior anti-bacterial activities to chemical antibacterial
agents, such as streptomycin and CPC against oral pathogens. In addition, the
growth of S. anginosus, S. aureus and S. epidermis was
suppressed by the treatment of Laurencia okamurae Yamada, Dictyopteris
undulata, Holmes and Sargassum micracanthum (Kutzing) Endlicher.
Received: December 17, 2013;
Accepted: May 24, 2014;
Published: June 18, 2014
Oral cavity is invaded by many bacteria through the breathing and food intake
all the time. Physiological conditions of oral cavity are suitable for bacterial
growth and eventually the specific oral microbial flora exits in oral cavity.
Generally, the oral microbial flora of human includes more than 30 bacteria
and have the diverse species spectrum according to the age, health conditions
and hygiene-status of individuals. Suppression and control against oral pathogens
is important for the care of oral health.
Especially the tooth decay and halitosis are closely associated with oral pathogens.
Dental caries is a highly prevalent and a costly disease throughout the world
(Hu et al., 2011). Surgeon Generals in
United States report showed that 45% of children aged 5-17 dental caries and
the children in underserved countries are placed in severe disease risk (Anonymous,
Dental caries is a destructive disease of dental hard tissues and can progress
to inflammation and to death of vital pulp tissue eventually by spread of infection
to the periapical area of the tooth. Dental caries is an infectious disease
by several species of cariogenic bacteria and specific dietary patterns, such
as sugar intake and eating frequency (Ding et al.,
2014; Nishimura et al., 2012). Streptococcus
mutans, Streptococcus sobrinus and Lactobacillus casei are
the well-known cavity-causing bacteria. Among these oral pathogens, S. mutans
is the major contributor to tooth decay and acid production in carcinogenic
process (Banas, 2013; Kohler et
al., 1995; Nakajo et al., 2009). Most
of tooth decaying bacteria colonize on the tooth surfaces and produce acids
to dissolve tooth structures. Therefore, the anti-microbial treatments against
these bacteria may help the control and the prevention of tooth decay for oral
Several researchers have studied the anti-bacterial activity of plant extracts
against oral pathogens. Herbal medicine extracts and its constituents, such
as (-)-epigalocatechin, have anti-bacterial activities against oral pathogens:
S. mutans, Fusobacterium nucleatum, Prevotella intermedia
and Porphyromonas gingivalis (Lee et al.,
2010; Park et al., 2010; Jeon
and Han, 2004). However, as an anti-bacterial natural material against oral-borne
pathogens, the marine algae extracts and its constituents have been paid little
attention relatively, so far.
Marine algae are considered as sources of natural bioactive compounds, because
they have a great variety of secondary metabolites characterized by a broad
spectrum of biological activities. Several compounds with cytostatic, anti-viral,
anthelmintic, anti-fungal and anti-bacterial activities were studied in green,
brown and red algae (Lindequist and Schweder, 2001).
There are numerous reports concerning anti-bacterial and anti-fungal activities
of macroalgae against bacteria, fungi and yeasts related to human diseases (Wright
et al., 2011; Bhatnagar and Kim, 2010). Bromophenol,
dolabellane derivatives, phloroglycin and hydroquinone derivatives were isolated
as anti-bacterial compounds from red algae, Rhodomelaceae, Dictyota
dichotoma, Fucus versiculosus and Dictyopteris zonarioides
(Choi et al., 2000; Mayer
et al., 2010).
In current study, we focused to screen and investigate Korean marine algae
with anti-bacterial activity against tooth decaying bacteria. It is anticipated
that marine algae with anti-bacterial activity might be used as a natural anti-bacterial
alternative for oral health care.
MATERIALS AND METHODS
Sea weeds materials: All marine algae collected from the seashore of Jeju-island
Korea, were authenticated and deposited by Jeju Biodiversity Research Institute
(JBRI, Jeju, Korea) (Table 1). Marine algae were washed, dried
by freeze drier and crushed. Crushed algae were extracted with 70% methanol
(in water) at room temperature. Extracts were filtered through Whatman No.1
filter paper and were concentrated by evaporator under reduced pressure. Marine
algal extract was redissolved in DMSO to 100 mg mL-1, stored at -20°C
and used it as a stock.
Microorganisms and growth condition: Oral bacterial strains used in
this study are in the following; S. mutans ATCC (American Type Culture
Collection, Manassas, VA, USA) 25175, S. sobrinus ATCC 27607, Streptococcus
pyogenes ATCC 21059, Streptococcus anginosus KCTC (Korean Collection
for Type Culture, Daejon, Korea) 5141, Staphylococcus aureus ATCC 25923
and Staphylococcus epidermidis ATCC 12228. Streptococcus and Staphylococcus
strains were cultured at 37°C in Brain Heart Infusion medium (BHI) (BactoTM,
BD 237500, Sparks, MD, USA) and Nutrient Broth (NB) (DifcoTM, BD
234000, USA), respectively. Stock of bacteria were kept in 35% glycerol (in
phosphate buffered saline) at -70°C. All bacterial strains were cultured
by inoculating 100 μL of the thawed microbial stock suspensions into 5
mL of BHI and NB medium in a shaking incubator at 37°C for 24 h in aerobic
Agar-well diffusion assay: The anti-bacterial activities of marine algal
extracts were evaluated by agar-well diffusion assay. The assay was carried
out according to the method with some modifications (Owen
and Palombo, 2007). After the well was cut on agar plate using a sterile
cork-borer (5 mm diameter), 50 μL of each bacterial suspension (OD600
= 0.2-0.3, approximately 1x107 CFU mL-1) was spread uniformly
on the surface of each agar plate. Marine algal extracts (10 μg well-1)
were dispensed into the wells. And agar plates were left at room temperature
for 30 min to allow the liquid to diffuse into the agar before overnight incubation
All assays were carried out in triplicate. A clear zone of the inhibited microbial
growth surrounded substances was considered as positive exhibiting anti-bacterial
properties. The solvent system used for the preparation of marine algal extract
samples was used for blank well. After incubation, the inhibition zones around
wells were measured in mm using a caliper (Fazeli et
al., 2007). Cetylpridinium chloride (CPC) and streptomycin (10 μg
well-1) were used as positive control for anti-bacterial activity.
Statistical analysis: The students
t-test was used to determine the statistical significance of data value differences.
Data was expressed as Means±Standard Deviation (SD).
RESULTS AND DISCUSSION
Using fifty-one species of marine algal extracts as target natural materials,
we have screened marine algal species with anti-bacterial activity against oral-borne
pathogens. All algal extracts were provided from JBRI and anti-bacterial activity
was determined by agar-well diffusion assay. The DMSO, treated as a negative
control, did not show any clear zone around wells of all oral pathogens (Fig.
1). In addition, streptomycin and CPC were used as positive controls of
anti-bacterial activity for the comparison with marine algae extracts. Figure
1 shows the anti-bacterial activities of Laurencia okamurae Yamada
and Dictyopteris undulata Holmes against S. mutans, S. sobrinus
and S. pyogenes and the comparison with the anti-bacterial intensity
of CPC. Extracts of Laurencia okamurae Yamada and Dictyopteris undulata
Holmes had a superior anti-bacterial activity to CPC against S. mutans,
S. sobrinus and S. pyogenes.
Anti-bacterial activity of Laurencia
okamurae Yamada and Dictyopteris undulata Holmes against oral
||Agar diffusion susceptibility of oral-pathogens to Korean
domestic algal extracts
Laurencia okamurae Yamada, Dictyopteris undulata Holmes and Sargassum
micracanthum (Kutzing) Endlicher showed the potent anti-bacterial
activities against tested all 6 oral pathogens, whereas other algal extracts
had the susceptibility against only 2-5 oral pathogens (Table
1). Sargassum micracanthum (Kutzing) Endlicher showed the
strongest anti-bacterial activity with 6.0±1.4 mm of clear zone against
S. mutans, a major cariogenic bacterial species. Additionally, the growth
of S. sobrinus and S. pyogene was inhibited the most effectively
by extract of Sargassum micracanthum (Kutzing) Endlicher. Intensity
of anti-bacterial activity against oral-borne pathogens showed the following
order; Sargassum micracanthum (Kutzing) Endlicher> Dictyopteris
undulata Holmes> Laurencia okamurae Yamada. Laurencia okamurae
Yamada had the strongest susceptibility with 9.5±0.7 mm of clear zone
against only S. sobrinus. And CPC, an anti-bacterial agent in commercial
mouthwashes had the better anti-bacterial activity with 4.75±0.9, 5.75±0.9,
4.5±1.2, 4.5±0.5, 4.25±0.9 and 2.5±0.5 mm of clear
zone against S. mutans, S. sobrinus, S. pyogene, S.
anginosus, S. aureus and S. epidermidis, respectively, than
streptomycin. Extracts of Laurencia okamurae Yamada, Dictyopteris
undulata Holmes and Sargassm micracanthum (Kutzing) Endlicher
were investigated to have the superior anti-bacterial activities to streptomycin
and CPC against oral pathogens.
Interest in the personal beauty and health care has been increasing recently.
Oral health is no exception to this interest and many oral care products are
selling commercially. Simultaneously the public demands for the natural material
without bad side-effects are surging up rapidly. Cariogenic S. mutans,
S. sobrinus and S. pyogene had the great susceptibilities against
most of marine algal extracts, as compared with other oral pathogens (Table
1). Extracts of nine marine algae, including Padina arborescens Holmes,
Sargassum muticum (Yendo) Fensholt, Ishige okamurae Yendo,
Sargarssum fulvellum (Turner) C. agardh, Colpomenia sinusa
(Mertens ex Roth) Derbes et Solier in Castagne, Chondrus ocellatus Holmes,
Dictyopteris undulata Holmes, Sargassm micracanthum (Kutzing)
Endlicher and Ecklonia stolonifera Okamura, showed the potent
anti-bacterial activity against S. mutans. And the growth of S. sobrinus
was inhibited by treatment of Padina arborescens Holmes, Ishige okamurae
Yendo, Scytosiphon lomentaria (Lyngbye) Link, Grateloupia filicina
(Lamouroux) C. agardh, Laurencia okamurae Yamada, Scytosiphon
gracilis Kogame, Grateloupia lanceolata (Okamura) Kawaguchi,
Gelidium amansii (Lam.) Lamouroux, Dictyopteris undulata Holmes,
Sargassm micracanthum (Kutzing) Endlicher and Ecklonia stolonifera
Okamura. Also the extracts of Sargassum muticum (Yendo) Fensholt, Ishige
okamurae Yendo, Sargarssum fulvellum (Turner) C. agardh, Laurencia
okamurae Yamada, Colpomenia sinusa (Mertens ex Roth) Derbes et Solier
in Castagne, Dictyopteris undulata Holmes and Sargassm micracanthum
(Kutzing) Endlicher showed the anti-bacterial activities against
S. pyogene. Anti-bacterial activities against S. anginosus, S.
aureus and S. epidermis were observed by the treatment of Laurencia
okamurae Yamada, Dictyopteris undulata Holmes and Sargassm micracanthum
Among natural materials available to us, the marine algae are being recognized
as potential and promising sources of bioactive compounds (Newman
et al., 2003). Marine algae produce a great variety of secondary
metabolites with versatile activities, such as anti-viral, anthelmintic, anti-fungal
and anti-bacterial activities (Lindequist and Schweder, 2001;
Bansemir et al., 2006). Red algal genus Laurencia
(Rhodomelaceae) produces numerous specialized secondary metabolites with diverse
structural features. Its three major classes which are sesquiterpenes, diterpenes
and acetylenes have not been yet encountered in other terrestrial organisms
(Blunt et al., 2008; Suzuki
et al., 2009). Most of these metabolites are characterized by the
presence of halogen atoms in their chemical formula. Generally Laurencia
species produce bromine-containing compounds in far greater numbers than either
chlorine or iodine-containing compounds. Halogenated compounds and their metabolites
from Laurencia sp. were investigated to exhibit antimicrobial, insecticidal,
cytotoxic activities (Suzuki et al., 2009).
Brown algae Dictyotaceae (Phaeophyceae) is a class of seaweeds that
have been explored for the bioactivity potential of its metabolic products.
Dictyotaceae contains a rich and diverse source of natural products which
makes it the most studied with the great number of known metabolites. The most
bioactive products, isolated from Dictyotaceae, contains three or four
isoprenoid units in sesquiterpenes and diterpenes, respectively (Dewick,
2002). These two classes of terpenoids have many different structures and
variants by addition of halogen atoms or fragments from other biogenic pathways.
Due to the diverse compounds in Dictyopteris sp., it might be used for anti-viral
and anti-bacterial studies (De Paula et al., 2011;
Ji et al., 2009).
Dental caries and periodontal diseases are related to the oesophagus and stomach
diseases, as well as oral health problems. Therefore, there is a growing interest
in the personal hygiene products for maintaining oral healthy recently and the
ingredients in mouthwashes have been exchanged to the biosafe natural products
from chemicals, along with food and pharmaceutical materials. However, any other
alternative natural product against CPC, an antibacterial agent in mouthwashes
for prevention of dental caries and halitosis has not been utilized generally
in present. For this reason, among Korean algae which are unknown to have the
bioactivities so far, I was going to screen algae species with antibacterial
activity against oral-borne pathogens as a candidate substance for alternative
to chemical antibacterial agent. In this report, we elucidated that the anti-bacterial
activities of Laurencia okamurae Yamada and Dictyopteris undulata
Holmes against 5 oral pathogens, such as S. mutans, S. sobrinus,
S. pyogenes, S. anginosus, S. aureus and S. epidermidis,
were superior to antibiotics streptomycin and CPC. Based on this result, it
is expected that some further researches, including safety study in human, make
Laurencia okamurae Yamada and Dictyopteris undulata Holmes-extracts
to be able to use as an alternative agent for CPC. Also Laurencia okamurae
Yamada and Dictyopteris undulata Holmes are expected to be able to develop
as a potential phytotherapeutic drug for treatment and prevention of oral caries
and oral health care.
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