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Inhibition of oral pathogens and collagenase activity by seaweed extracts

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© 2012 Triveni Enterprises J. Environ. Biol.
Vikas Nagar, Lucknow, INDIA 33, 115-121 (2012)
[email protected] ISSN: 0254- 8704
Full paper available on: www.jeb.co.in CODEN: JEBIDP

Inhibition of oral pathogens and collagenase activity by seaweed extracts

Author Details
Jae-Suk Choi RIS Center, Industry-Academic Cooperation Foundation, Silla University, Busan 617-736, Korea
Yu-Mi Ha RIS Center, Industry-Academic Cooperation Foundation, Silla University, Busan 617-736, Korea
Chi-Un Joo RIS Center, Industry-Academic Cooperation Foundation, Silla University, Busan 617-736, Korea
Kwang Keun Cho Department of Animal Resources Technology, Gyeongnam National University of Science and
Technology, Chinju 660 - 758, Korea
Sung-Jo Kim Department of Periodontology, School of Dentistry, Pusan National University, Yangsan,
Gyeongnam 626-870, Korea
In Soon Choi Department of Biological Science, Silla University, Busan 617-736, Korea
(Corresponding author) e-mail: [email protected]

Abstract
Fifty-seven species of common seaweed from the Coast of Korea were screened for antimicrobial (i.e.
inhibition of Prevotella intermedia and Porphyromonas gingivalis growth) activity. As a source of bioactive
compounds, seaweeds can produce many secondary metabolites with a variety of activities. Using the agar
Publication Data diffusion method, only 17 species (29.8% ) showed inhibitory activity. Of these, methanol extracts of
Enteromorpha linza, Sargassum sagamianum, and Ulva pertusa showed strong inhibitory effects against both
Paper received: P. intermedia and P. gingivalis. The MIC values of E. linza, S. sagamianum, and U. pertusa extracts against
12 June 2010 P. intermedia were 625, 78 and 625 µg ml-1 and those against P. gingivalis were 312, 156 and 625 µg ml-1,
respectively. When these three species’ extracts were separated into five fractions according to their polarity,
Revised received: the main active agents were determined to be phenolic compounds. We then compared the antimicrobial
03 March 2011 activities of these phenolic compounds against various periodontal pathogens using a MIC test. Phenolic
compound containing extracts at concentrations of 10 to 100 µg ml-1 showed a moderate to significant
Accepted: inhibitory effect on collagenase 1, 2 and 3 activity.
21 April 2011
Key words
Antimicrobial activity, Seaweed extracts, Periodonititis, Prevotella intermedia, Porphyromonas gingivalis,
Collagenase

Introduction a gradual increase in the rate of resistance to penicillin has been

Periodontitis is a bacterial inflammatory disease, noted by several investigators over recent years (Appelbaum,
characterized by destruction of connective tissue, including alveolar 1992). At present, the antibiotics used most frequently against
bone, that can lead to tooth loss. Periodontitis is typically initiated by anaerobic bacteria include metronidazole, imipenem and
a group of Gram-negative periodontal pathogens, such as Prevotella meropenem. These are active against almost all clinically relevant
intermedia and Porphyromonas gingivalis (Socransky et al., 1999). anaerobic bacteria, although strains resistant to these agents have
been reported sporadically (Falagas and Siakavellas, 2000; Aldridge
Systemic or topical antibiotics have been used as an adjunct et al., 2001). For this reason, extensive research is now being
in the treatment of periodontal disease (Slots and Ting, 2002). carried out to find novel antimicrobial compounds.
Prevotella and Porphyromonas species, including the main
oropharyngeal pathogenic species, have traditionally been Interest in marine organisms as promising sources of
considered susceptible to penicillin (Niederau et al., 1980). However, pharmaceutical agents has increased in recent years (Mayer and

Journal of Environmental Biology
January 2012

116 Choi et al.
Hamann, 2004; Newman et al., 2003). As a source of bioactive Stock solutions were prepared by addition of 1 ml methanol or
compounds, seaweeds can produce many secondary metabolites distilled water per 100 mg of dried extract. Stock solutions were
with a variety of activities. Compounds with antiviral, antihelminthic, filtered through a 0.22 µM filter and stored at 20oC until required
antifungal and antibacterial activities have been detected in green, (Jin et al., 1997).
brown and red algae (Newman et al., 2003; Del Val et al., 2001).
There are many reports on the biological activities of macroalgae For constituent separation, seaweed powders (20 g) were
against human pathogens, fungi, and yeasts, but only a few contain extracted with 1 l of methanol-water (4:1) three times. Crude extracts
data regarding effects against P. intermedia and P. gingivalis. were evaporated under vacuum and then fractionated according to
their polarity: saccharides, lipids, phenolics, alkaloids, and nitrogen
The destruction of collagen fibers by collagenase is one of compounds (Harborne, 1998).
the unique characteristics of periodontal disease. Thus, collagenase
is an important pathogenic factor for the development of periodontal Bacterial strains and culture conditions: Standard bacterial
disease (Robertson and Simpson, 1976; Larivee et al., 1986). strains and culture conditions were used to screen for antimicrobial
Vertebrate collagenases are members of the matrix metalloproteinase activities against oral pathogens, Prevotella intermedia and
(MMP) family of proteolytic enzymes that are involved in extracellular Porphyromonas gingivalis were used. To determine the MIC values
matrix degradation and remodelling during the course of periodontal of the active fractions against several oral pathogens,
diseases. Aggregatibacter actinomycetemcomitans, Candida albicans,
Fusobacterium nucleatum subsp. vincenti, and Streptococcus
We thus assessed the P. intermedia- and P. gingivalis- mutans were used. All strains obtained from the Korean Collection
inhibitory activities of seaweed extracts and of the active fractions of for Type Cultures (KCTC; Daejeon, Korea). Anaerobic conditions
three seaweeds against several oral pathogens. Additionally, we were maintained at 5% H2, 5% CO2 and 90% N2 using the Bactron™
investigated their inhibitory effect on collagenase 1, 2 and 3 activity. Anaerobic Chamber system (SHELLAB, USA).
In this way we hope to discover therapeutic agents for periodontitis
that have few or no side effects and that are highly antimicrobial. Disk diffusion assay: In vitro antimicrobial activity against P.
intermedia and P. gingivalis was determined by a disk diffusion
Materials and Methods assay. Bacteria were incubated in GAM broth agar, supplemented
Seaweed extracts: In total, 57 species (10 green, 29 brown, 18 with 10% (v/v) sheep blood, 5 µg ml-1 hemin and 1 µg ml-1 menadione
red) of seaweed were collected from various locations in South at 37oC for 48 hr under anaerobic conditions and then adjusted to
Korea from November 2005 to April 2006. Seaweed tissues were a density of approximately 2.0 x 108 CFU ml-1. This microbial
washed with tap water to remove salt, epiphytes and sand, and suspension (1 ml) was aseptically spread on the surface of an agar
dried for 1 day at room temperature. They were then ground to a plate. Filter-paper disks (8 mm diameter) were impregnated with
powder using a coffee grinder for 5 min. For methanol and water seaweed extracts and then placed on the agar surface. Plates
extractions, 1 l of methanol was added per 20 g powder and were incubated at 37oC for 48 hr under anaerobic conditions.
incubated for 1 day. This was repeated three times and the combined Antimicrobial activity was assessed by measuring the diameter of
extracts were evaporated to dryness. Distilled water (1 l) was then the growth inhibition zone (mm) (NCCLS, 2004). Controls were
added to the remaining powder to extract water-soluble components. run simultaneously. The antimicrobial agent chloramphenicol (Sigma

Table - 1: Bacterial strains, culture conditions and NCCLS guidelines used

Bacterium /Strains Culture media /Culture conditions NCCLS guideline

Aggregatibacter actinomycetemcomitans Brucella broth + 3% horse serum M11-A6

KCTC 3698 37 oC, 72 hr, anaerobic conditions
Candida albicans RPMI 1640 medium M27-A2
KCTC 17485 37 oC, 48 hr, aerobic conditions
Fusobacterium nucleatum subsp. vincenti Schaedler broth M11-A6
KCTC 5105 37 oC, 72 hr, microaerobic conditions
Porphyromonas gingivalis GAM broth+ 10% sheep blood M11-A6
KCTC 381 + 5 µg ml-1 hemin + 1 µg ml-1 menadione
37 oC, 48 hr, anaerobic conditions
Prevotella intermedia GAM broth+ 10% sheep blood M11-A6
KCTC 25611 + 5 µg ml-1 hemin + 1 µg ml-1 menadione
37 oC, 48 hr, anaerobic conditions
Streptococcus mutans BHI broth + 3% horse serum M7-A6
KCTC 3065 37 oC, 24 hr, aerobic conditions

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Seaweed extracts with activity against oral pathogens 117

Table - 2: Antimicrobial activity against P. intermedia and P. gingivalis in seaweed methanol extracts, as determined by the disk diffusion method.
P. intermedia P. gingivalis
Seaweeds
-1 -1 -1 -1
1 mg disk 3 mg disk 5 mg disk 1 mg disk 3 mg disk-1 5 mg disk-1
Ahnfeltiopsis flabelliformis - - - - - +
Costaria costata - - - + ++ ++
Desmarestia viridis - - - + ++ ++
Enteromorpha linza - ++ +++ + ++ +++
Ishige okamurae - - - + ++ ++
Ishige sinicola - - - - + ++
Laminaria japonica - + ++ + + ++
Lomentaria catenata - - - - - +
Myagropsis myagroides - - - - ++ ++
Myelophycus simplex - - - - - +
Pachydictyon coriaceum - - - ++ +++ ++++
Petalonia fascia - - - - +
Sargassum horneri - - - + ++ ++
Sargassum sagamianum - ++ +++ ++ ++ +++
Scytsiphon lomentaria - + + + + ++
Ulva pertusa - ++ +++ ++ +++ ++++
Undaria pinnatifida - + ++ + + ++
Inhibition zone diameter was measured (+ = < 4 mm, ++ = 4 - 8 mm; +++ = 8 - 12 mm; ++++ = > 12 mm). Data are mean of triplicate determinations.

857440) was included in the assays as a positive control. All disk solution were distributed into wells of a 96 well plate. Diluted MMP
diffusion tests were performed independently in triplicate. enzymes and phenolic compounds (20 µl of each) were added,
reaction mixtures were incubated for 30 min at 37oC and diluted
Determination of MIC values: Seaweed extracts and fractions substrate (thiopeptide; 10 µl) was added. The final volume was
that showed strong activity were investigated further by a broth brought up to 100 µl immediately. Inhibition was measured by
microdilution assay, following the guidelines of the National Committee continuously reading plates at A412 for 20 min in a microplate reader
for Clinical and Laboratory Standards (NCCLS) for anaerobic (Spectra MAX 190, Molecular Devices, CA, USA). Inhibition percent
bacteria M11-A6 (NCCLS, 2004), aerobic bacteria M7-A6 (NCCLS, activity remaining were analyzed by dividing reaction velocity of
2003) and yeasts M27-A2 (NCCLS, 2002) in a 96-well U-shaped inhibitor with control and quotient multiplied with 100. All assays
microplate. Inocula were prepared from 24 hr broth cultures and were performed independently in triplicate.
suspensions were adjusted to 0.5 McFarland standard solution
turbidity. Seaweed extracts or fractions were first diluted to the highest Acute toxicity test: Acute toxicity of the moderately polar
concentration (10 µg ml-1) to be tested and then serial two-fold fractions was assessed in BALB/c mice (8-10 weeks old; 20-25
dilutions were made, resulting in concentrations ranging from 19.5 µg g body weight) (Cho et al., 2007). Animals were kept at room
to 10 µg ml-1. Microtitre plates were prepared by dispensing the temperature (24±1oC) on a 12/12 hr light/dark cycle with free
access to food and water. For the acute toxicity test, mice were
inoculum and sample (100 µl of each) into each well. The first well
fasted for 6 hr with water provided ad libitum. Phenolic fractions
of each strip contained 100 µl broth with no compound and 100 µl
were evaporated under vacuum at 35 oC using a rotary
inoculum, and represented the negative control. The second well
evaporator and then administered orally (5 g 10 ml -1 corn oil of
on each strip contained 90 µl broth, 10 µl methanol and 100 µl
5% DMSO kg b.wt. -1) to mice (n = 5). Animals were observed
inoculum, and represented the positive control. The final volume in
for any abnormal behavior for 3 hr and any mortality was
each well was 200 µl. Plates were incubated appropriately for recorded for up to 2 weeks. A group of animals treated with
each microorganism (Table 1). The MIC value was defined as the DMSO only served as a control. Animal experiments were
lowest concentration that yielded no bacterial cell growth. All MIC performed in accordance with the U.S. NIH Guidelines for the
tests were performed independently in triplicate. The antimicrobial Care and Use of Laboratory Animals.
agent chloramphenicol (Sigma 857440) was included in the assays
as a positive control. Statistics: Each independent assay was repeated at least three
times with separate cultures. Treatment means were compared to
Measurement of collagenase activity: To investigate controls using student’s t-test.
collagenase inhibition, 10 and 100 µg ml-1 concentrations of the
phenolic fraction of extracts were used. Collagenase 1 (MMP-1), 2 Results and Discussion
(MMP-8) and 3 (MMP-13) activity was measured using a MMP Screening of antimicrobial activity: Of the 57 seaweed species
Colorimetric Drug Discovery Kit: AK-404, AK-414 and AK-412 (Enzo screened, only six (10.5%) showed antimicrobial activity against P.
Life Science, Plymouth, PA, USA). Briefly, aliquots (50 µl) of buffer intermedia and 17 (29.8%) against P. gingivalis (Table 2). Among

Journal of Environmental Biology
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118 Choi et al.

the 10 species of Chlorophyta (green algae) screened, only two Fractionation of crude extracts: Each seaweed powder (20 g)
(Enteromorpha linza and Ulva pertusa; 20.0%) inhibited both was extracted in 1 l of a methanol-water (4:1) mixed three times,
microbial pathogens. Phaeophyta (brown algae) showed the highest and the crude extract was evaporated, yielding a dark greenish-
activity (37.9%) among the three classes of seaweeds screened. brown gummy residue. The fraction that was acidified to pH 2 and
Two species showed anti P. intermedia and 11 showed anti-P. extracted with chloroform yielded a moderately polar mixture of
gingivalis activity. Of the Rhodophyta (red algae), antimicrobial activity phenolic compounds (0.68, 1.30 and 0.42 g), which contained the
was present only at low levels in two species. The strongest activities majority of the antimicrobial activity. The MIC values of the phenolic
against both microbial pathogens among chlorophyta seaweed fractions of E. linza, S. sagamianum, and U. pertusa extracts against
species were exhibited by E. linza and U. pertusa; while among P. intermedia were 156, 78 and 78 µg ml-1, respectively. The MIC
Phaeophyta, the activity of S. sagamianum was highest. The values of the phenolic fractions of E. linza, S. sagamianum and U.
inhibition zones of chloramphenicol (positive control; 0.5 mg disk-1) pertusa extracts against P. gingivalis were 156, 78 and 312 µg ml-1,
were 14 and 15 mm, respectively. respectively (Table 4).

MIC values determination: The MIC values were determined MIC values of phenolics against several oral pathogens: To
using a two-fold serial dilution method. The MIC values of E. linza, determine the antimicrobial activities of phenolic fractions against
S. sagamianum and U. pertusa extracts against P. intermedia were recognized oral pathogens, MIC values were determined by the
625, 78 and 625 µg ml-1, respectively. The MIC values of E. linza, broth microdilution assay (Table 5). The MIC values of phenolic
S. sagamianum and U. pertusa extracts against P. gingivalis were fractions of E. linza, S. sagamianum, and U. pertusa extracts against
312, 156 and 625 µg ml-1, respectively. The MICs of chloramphenicol Candida albicans were 625, 78 and 312 µg ml-1, respectively. The
(positive control) against P. intermedia and P. gingivalis were 2 and MIC values against Fusobacterium nucleatum subsp. vincenti were
1 µg ml-1, respectively (Table 3). 625, 78 and 156 µg ml -1 and against Haemophilus
actinomycetemcomitans were 625, 312 and 625 µg ml-1, respectively.
Table - 3: MIC values (µg ml-1) against P. intermedia and P. gingivalis of The MIC values of S. sagamianum and U. pertusa against
methanol extracts, as determined by broth microdilution assay. Streptococcus mutans were 625 and 1,250 µg ml-1, respectively.
Seaweeds P. intermedia P. gingivalis The phenolic fraction of E. linza extract showed no inhibitory effect
against Streptococcus mutans. The three phenolics exhibited
Enteromorpha linza 625 312
Sargassum sagamianum 78 156
moderate to significant inhibition of all oral pathogens tested, except
Ulva pertusa 625 625 the phenolics of E. linza against S. mutans. The MICs of
chloramphenicol (positive control) against P. intermedia and
The MIC values were measured for 48 hr after incubation
P. gingivalis were 2 and 1 µg ml-1, respectively.

Table - 4: Comparison of antimicrobial activity against P. intermedia and P. gingivalis of seaweed extract fractions by MIC test
MIC values against P. intermedia (µg ml-1)
Seaweeds
Saccharides Lipids Phenolics Alkaloids Nitrogen compounds

Enteromorpha linza - 312 156 312 -

Sargassum sagamianum - - 78 - -
Ulva pertusa - - 78 625 -
MIC values against P. gingivalis (µg ml-1)
Enteromorpha linza - 625 156 625 -
Sargassum sagamianum - - 78 625 -
Ulva pertusa - - 312 - -
The MIC values were measured for 48 hr after incubation and “-” indicates no inhibition at 10,000 mg ml-1

Table - 5: MIC values of seaweed extract phenolic fractions against several oral pathogens.
MIC values of phenolics (µg ml-1)
Oral pathogens
E. linza S. sagamianum U. pertusa

Candida albicans 625 78 312

Fusobacterium nucleatum subsp. vincenti 625 78 156
Aggregatibacter actinomycetemcomitans 625 312 625
Streptococcus mutans - 625 1,250
The MIC values were measured for 24~72 hr after incubation and “-” indicates no inhibition at 10,000 mg ml-1

Journal of Environmental Biology
January 2012

Seaweed extracts with activity against oral pathogens 119

100 Inhibitory effect on collagenase activity: Because collagenase

(a) is important in the pathogenesis of periodontal disease, we tested
Collagenase 1 inhibition rate (%)

the effect of phenolics on collagenase activity using the collagenase

80 (MMP) assay system. In the collagenase 1 (MMP-1) assay system,
the phenolic fraction from E. linza, S. sagamianum and U. pertusa
extracts inhibited greater than 40.2, 57.1 and 48.5% of collagenase
60 activity, respectively, at 100 µg ml-1. Collagenase 2 (MMP-8) activity
levels were inhibited by 63.7, 70.1 and 64.5%, respectively and
collagenase 3 (MMP-13) activity levels were inhibited by 3.4, 71.2
40 and 14.6% at 100 µg ml-1, respectively. In the collagenase 2 assay
system, even at 10 µg ml-1, collagenase activity was inhibited by
about 10 ~ 15%, but in the collagenase 1 and 3 assay systems,
20
inhibitory effects were either absent or present at only low levels
when phenolics were applied at 10 µg ml-1 concentration (Fig. 1).
0 Acute toxicity: We evaluated the acute toxicity of the extract
E.linza S.sagamianum U.pertusa
phenolic fractions in mice. Over the 2 week observation period,
100 no death occurred in any mice administered a dose of 5 g kg-1 b.wt.
(b) Mice administered seaweed extract reacted by wandering briefly
Collagenase 2 inhibition rate (%)

and returned to normal behavior after ~10 min. According to the

80 WHO (1992), a herbal medicine is considered toxic if the LD50 is
lower than 5 g kg-1 b.wt. On this basis, these extracts would be
classified as non-toxic. Our data suggests, therefore, that these
60 fractions can be safely used by humans at moderate doses.

Periodontitis is a widespread disease that affects as many

40 as 10, 17% of the population (Brown and Löe, 1993; Choi and Kim,
2009). Bacteria commonly detected in the non-healthy gingival
pocket are the anaerobic Aggregatibacter spp., Fusobacterium
20 spp., P. gingivalis, and P. intermedia. Previous studies have shown
that P. gingivalis (Hallen et al., 2008) and P. intermedia (Kim et al.,
2007) possess a large array of virulence factors, including the
0 ability to adhere to and invade oral epithelial cells and the production
E.linza S.sagamianum U.pertusa
of proteolytic enzymes.
100
(c) Systemic or topical antibiotic therapies have been reported
Collagenase 3 inhibition rate (%)

to be useful in treating periodontal disease (Slots and Ting, 2002).

80 However, these antibiotics are known to induce side-effects
(Falagas and Siakavellas, 2000), thus, many researchers have
sought to develop new therapeutic agents for periodontitis (Park
60 et al., 2005).
Seaweeds are able to produce many secondary metabolites
with a variety of activities. Compounds with antiviral, antifungal and
40
antibacterial activities have been detected in green, brown and red
algae (Del Val et al., 2001; Newman et al., 2003). Additionally,
enzyme inhibitory activities have been detected in seaweeds (Mayer
20
et al., 1993; Chang et al., 2008; Cho et al., 2008).
Many reports have demonstrated a positive correlation
0 between the occurrence of high collagenase activity in the
E.linza S.sagamianum U.pertusa exudate from the gingival crevice and the severity of
Fig. 1: Inhibition of collagenase 1(a), 2(b) and 3(c) activities by phenolic periodontal disease (Lee et al., 1995; Larivee et al., 1986).
compounds from E. linza, S. sagamianum and U. pertusa extracts at It has been reported that collagenase is produced mainly by
10 ( ) and 100 ( ) µg ml-1. Statistically significant at p<0.01 inflammatory infiltrative cells and tissue cells, such as neutrophils,

Journal of Environmental Biology
January 2012

120 Choi et al.
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Journal of Environmental Biology
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