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Antibacterial, Antiadherence, Antiprotease, and Anti-Inflammatory Activities
of Various Tea Extracts: Potential Benefits for Periodontal Diseases
Lei Zhao,
1,2
Vu Dang La,
1
and Daniel Grenier
1
1
Oral Ecology Research Group, Faculty of Dentistry, Laval University, Quebec City, Quebec, Canada.
2
Department of Periodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China.
ABSTRACT Porphyromonas gingivalis is a key etiologic agent of chronic periodontitis. This Gram-negative anaerobic bacterium
produces several virulence factors and can induce a host inflammatory response that contributes to periodontal disease. In the present
study, we investigated green tea, white tea, oolong tea, and black tea extracts with a high polyphenol content for their effects on (i) the
growth and adherence of P. gingivalis, (ii) the activity of host and bacterial proteases, and (iii) cytokine secretion by oral epithelial cells.
All the tea extracts inhibited the growth of P. gingivalis (minimal inhibitory concentrations ranging from 200 to 500 lg/mL; minimal
bactericidal concentrations =500 lg/mL). In addition, they dose dependently reduced the adherence of P. gingivalis to oral epithelial
cells. Tea extracts also inhibited the catalytic activity of matrix metalloproteinase (MMP)-9, neutrophil elastase, and P. gingivalis
collagenase. Lastly, the tea extracts dose dependently inhibited the secretion of interleukin (IL)-6, IL-8, and chemokine (C-C motif)
ligand 5 (CCL-5) by P. gingivalis–stimulated oral epithelial cells. No marked differences in the various effects were observed among
the four tea extracts. Extracts from green tea, white tea, oolong tea, and black tea show promise for controlling periodontal disease by
their capacity to interfere with P. gingivalis growth and virulence properties, host destructive enzymes, and inflammatory mediator
secretion. Such extracts may be incorporated to oral hygiene products or locally delivered into diseased periodontal sites.
KEY WORDS: periodontal diseases Porphyromonas gingivalis tea
INTRODUCTION
Oral diseas es are the fourth most expensive diseases
to treat in most industrialized countries. More spe-
cifically, periodontal diseases are destructive inflammatory
disorders that affect the supporting tissues of the tooth and
that result in attachment loss, formation of periodontal
pockets, and resorption of the alveolar bone. If left un-
treated, these diseases may result in tooth loss. As many as
700 bacterial species can be found in subgingival plaque
samples.
1
Of these species, some, either alone or in com-
bination, have the biochemical flexibility to dominate
the oral microbiota and cause periodontitis. Period-
ontopathogens colonizing subgingival sites induce host
cellular and humoral responses, which in most cases result
in the elimination or control of the pathogens and prevent
the establishment and progression of periodontal diseases.
2
However, the continuous challenges to the host immune
system by periodontopathogens and their products initiate a
number of host-mediated destructive processes.
2,3
There is
now a consensus that Porphyromonas gingivalis,aGram-
negative anaerobic bacterium, is a key pathogen involved
in chronic periodontitis.
4
P. gingivalis produces a broad
spectrum of virulence factors, including adhesins, lipo-
polysaccharides (LPS), and proteases, that allow it and
other bacterial species in the same environment to colonize
the host and multiply, avoid destruction by host defenses,
promote inflammatory processes, and cause tissue dam-
age.
5,6
The gingival epithelium, which covers the periodontal
tissues, plays a crucial protective role as a mechanical bar-
rier that prevents the invasion of the periodontium by peri-
odontopathogens.
7
Gingival epithelial cells react to bacterial
challenges by signaling host responses and integrating in-
nate and acquired immune responses.
7
P. gingivalis has
developed different strategies to perturb the structural and
functional integrity of the gingival epithelium.
8
P. gingivalis
adheres to, penetrates, and replicates inside gingival epi-
thelial cells.
8,9
In addition, proteolytic enzymes produced
by P. gingivalis can interfere with both the cell–matrix
and cell–cell adhesion of epithelial cells, which may cause
the junctional epithelium to detach from the root surface
and regenerating tissues and promote the formation of gin-
gival pockets.
8
Interactions between P. gingivalis and epi-
thelial cells lead to the activation of several complex
signaling cascades that ultimately regulate the transcription
of target genes that encode effectors and regulators of the
immune response.
8,9
More specifically, effectors of the in-
nate immune system, including proinflammatory cytokines,
Manuscript received 14 August 2012. Revision accepted 11 January 2013.
Address correspondence to: Daniel Grenier, PhD, Oral Ecology Research Group, Faculty
of Dentistry, Laval University, 2420 rue de la Terrasse, Quebec City, QC G1V 0A6,
Canada, E-mail: daniel.grenier@greb.ulaval.ca
JOURNAL OF MEDICINAL FOOD
J Med Food 16 (5) 2013, 428–436
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2012.0207
428
chemokines, and matrix metalloproteinases (MMPs), are
upregulated and may have a direct impact on periodontal
disease progression and inflammation processes.
8,9
Tea, an aqueous aromatic infusion of cured leaves of
the plant Camellia sinensis, is the most popular beverage in
the world after water. It contains numerous components,
including catechins, caffeine, amino acids, carbohydrates,
proteins, chlorophyll, volatile compounds, fluoride, miner-
als, and other undefined compounds.
10
Teas can be classified
as nonfermented (green and white teas), semifermented
(oolong tea), and fermented (black tea). Green tea differs
from white tea by the fact that the latter is produced only from
the buds or first leaves. The chemical composition of teas
depends on how they are processed. For example, green tea
has a high catechin content; black tea has a high bisflavanol,
theaflavin, and thearubigin content;
11
white tea has a high
epigallocatechin-3-gallate (EGCG), epicatechin, and meth-
ylxanthine content.
12
Traditional Chinese medicine has con-
sidered tea as a medicine and healthful beverage since ancient
times. Several biological properties have been associated to
tea polyphenols, including antioxidant, anticarcinogenic, and
antimicrobial activities.
11
Many studies have shown that the
constituents of tea may contribute to reducing the risk of
cardiovascular disease and cancer and have a variety of other
beneficial effects on human health.
11–13
Epidemiological and
clinical studies have provided evidence that green tea con-
sumption may have potential oral health benefits.
14–17
How-
ever, few studies have compared the beneficial biological
properties of different types of tea with respect to periodontal
disease. The aim of the present study was to investigate the
effects of major types of tea on (i) the growth and adherence
of P. gingivalis, (ii) the activity of host and bacterial prote-
ases, and (iii) cytokine secretion by oral epithelial cells.
MATERIALS AND METHODS
Tea extracts
Extracts from green tea, white tea, oolong tea, and black
tea were purchased from Organic Herb, Inc. (Changsha,
China). Information provided by the company indicated that
these extracts (water/ethanol) were prepared from tea leaves
and all have a polyphenol content ‡92%. Stock solutions
were prepared by dissolving 20 mg of powder in 1 mL of
sterile distilled water and filtering the solution through a
0.45-lm-pore-size membrane filter.
Bacteria and culture conditions
P. gingivalis ATCC 33277 was grown in the Todd-Hewitt
broth (THB; BBL Microbiology Systems, Cockeysville, MD,
USA) supplemented with hemin (10 lg/mL) and vitamin K
(1 lg/mL). Bacterial cultures were incubated for 24 h at 37C
under anaerobic conditions (80% N
2,
10% H
2
,10%CO
2
).
Determination of minimal inhibitory and minimal
bactericidal concentrations
A 24-h culture of P. gingivalis was diluted in a fresh broth
medium to obtain an optical density of 0.02 at 655 nm
(OD
655
). Tea extracts (100 lL) diluted in a culture medium
(20–2000 lg/mL) were placed in the wells of flat-bottomed
96-well microplates (Sarstedt, Newton, NC) to which an
equal volume of P. gingivalis suspension was added. Wells
containing only bacteria or tea extract were used as controls.
After a 24-h incubation at 37C under anaerobic conditions,
bacterial growth was monitored by measuring the OD
655
using a microplate reader (BioTek Instruments, Winooski,
VT, USA). Minimal inhibitory concentration (MIC) values
(lg/mL) were defined as the lowest concentration of the
extract at which no growth occurred. To determine minimal
bactericidal concentration (MBC) values (lg/mL), aliquots
(10 lL) from each well with no visible growth were spread
on culture plates, which were incubated for 5 days at 37C
under anaerobic conditions. MBC values were defined as the
lowest concentration at which no colonies grew. The MIC
and MBC assays were performed in triplicate and were re-
peated three times to ensure reproducibility.
P. gingivalis adherence to human oral epithelial cells
The immortalized human oral epithelial cell line GMSM-K,
which was kindly provided by Dr. Valerie Murrah (Depart-
ment of Diagnostics Sciences and General Dentistry, the
University of North Carolina, Chapel Hill, NC, USA), was
cultured in the Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% heat-inactivated fetal bo-
vine serum (FBS) and 100 lg/mL of penicillin G/streptomycin
at 37Cina5%CO
2
atmosphere. The epithelial cells were
harvested by gentle trypsinization (0.05% trypsin-EDTA)
(Gibco-BRL, Grand Island, NY, USA), seeded (100 lL,
1.5 ·10
6
cells/mL) in sterile 96-well clear bottom black mi-
croplates (Greiner Bio One, Frickenhausen, Germany), and
incubated until they reached confluence. The wells were then
washed three times with 50 mM phosphate-buffered saline
(PBS) pH 7.2, blocked with 1% bovine serum albumin (BSA)
for 30 min to prevent nonspecific bacterial attachment, and
treated with the tea extracts diluted in the DMEM at final
concentrations ranging from 10 to 100 lg/mL for 30 min in a
5% CO
2
atmosphere at 37C. P. gingivalis cells from an
overnight culture were suspended (10
9
/mL) in a bicarbonate
buffer (0.15 M NaCl/0.1 M Na
2
CO
3
,pH9.0),incubatedfor
30 min with continuous shaking in the presence of 15 lg/mL
of fluorescein isothiocyanate isomer I (FITC; Sigma-Aldrich
Canada, Oakville, ON, Canada) in the dark, washed three
times with PBS containing 0.05% Tween 20, resuspended
in PBS in the original volume, applied at a multiplicity of
infection of 200 (200 bacteria per epithelial cell) to treated
or control epithelial cells, and incubated for 2 h at 37C
under anaerobic conditions. The incubation and washing
steps were carried out in the dark. Following the incuba-
tion, unbound P. gingivalis cellswereremovedbyaspi-
ration, and the wells were washed three times with PBS.
P. gingivalis cells that had adhered to the epithelial cell
monolayer were quantified by monitoring fluorescence
using a Synergy 2 Multi-Mode Microplate Reader (Bio-
Tek Instruments). The excitation and emission wave-
lengths were set at 488 and 522 nm, respectively. The
TEA EXTRACTS AND PERIODONTAL DISEASES 429
assays were performed in triplicate and were repeated
three times.
MMP-9, elastase, and P. gingivalis collagenase activities
Human active recombinant MMP-9 and neutrophil elas-
tase were purchased from Calbiochem (San Diego, CA,
USA). MMP-9 (1 lg/mL) diluted in the TCNB buffer
(50 mM Tris-HCl, 10mM NaCl, and 0.05% Brij 35, pH 7.5)
was incubated with tea extracts (10 to 100 lg/mL) and
gelatin DQ(150 lg/mL). A 48-h P. gingivalis culture
supernatant was incubated with tea extracts (10–100 lg/mL)
and type I collagen DQ(150 lg/mL). Elastase (50 lg/mL)
was mixed with the substrate I (Calbiochem) (4 mM)
and reaction buffer (100 mM Tris-HCl, 500 mM NaCl, pH
7.5), and then incubated with tea extracts (10–100 lg/mL).
The assay mixtures were incubated for 4 h at 37C for
MMP-9 and elastase, and for 4 h at room temperature
for P. gingivalis collagenase. Mixtures with no substrate or
enzyme were used as controls. Fluorescence was measured
using the excitation and emission wavelengths set at 490 and
525 nm, respectively. Hydrolysis of the elastase substrate
was assayed by measuring the absorbance at 415 nm. The
assay was performed in triplicate and was repeated three
times.
Preparation of P. gingivalis extract
The P. gingivalis extract was prepared using the method
described by Shenker and Slots
18
with some modifications.
Briefly, P. gingivalis was grown in THB-HK for 48 h
at 37C under anaerobic conditions. Bacterial cells from a
1-liter culture were harvested by centrifugation (10,000 g
for 20 min at 4C) and were washed with cold PBS. The
bacterial cells were sonicated for 10 min on ice using an
ultrasonic disruptor. The supernatant was collected by
centrifugation at 10,000 gfor 20 min and sterilized using a
0.45-lm filter. The protein concentration was evaluated
using a protein assay kit (DC protein assay, Bio-Rad La-
boratories, Mississauga, ON, Canada), with BSA as a
standard. The P. gingivalis sonic extract was kept at -80C
and boiled for 15 min before use.
Cytotoxicity of tea extracts and P. gingivalis sonic extract
A 3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT) assay performed according to the manufac-
turer’s protocol (Roche Diagnostics, Mannheim, Germany)
was used to determine the effect of the tea extracts (10–100
lg/mL) and the P. gingivalis extract (10 lg/mL) on the via-
bility of GMSM-K oral epithelial cells.
Cytokine secretion by P. gingivalis–stimulated
oral epithelial cells
GMSM-K human oral epithelial cells were grown and
harvested as described above. The epithelial cells were
suspended (4 ·10
5
cells/mL) in the DMEM containing 1%
heat-inactivated FBS and were seeded (1 mL) in the wells of
a 12-well plate. The plate was incubated overnight at 37C
in a 5% CO
2
atmosphere to allow cell adhesion before use.
The epithelial cells were pretreated with tea extracts
(10–100 lg/mL) at 37Cin5%CO
2
for 2 h before adding
the P. gingivalis extract (10 lg/mL). After a 24-h incuba-
tion, the supernatants were collected and stored at -20C
until used. Epithelial cells incubated with tea extract, but no
P. gingivalis extract, or with P. gingivalis extract, but no tea
extract were used as controls. Each experiment was per-
formed in triplicate. Commercial enzyme-linked immuno-
sorbent assay (ELISA) kits (R&D Systems, Minneapolis,
MN, USA) were used to quantify interleukin-6 (IL-6),
interleukin-8 (IL-8), and chemokine (C-C motif) ligand 5
(CCL5) concentrations in the cell-free supernatants ac-
cording to the manufacturer’s protocols. The absorbance at
450 nm was read using a microplate reader, with the
wavelength correction set at 550 nm. The rated sensitivities
of the kits were 9.3 pg/mL for IL-6, 31.2 pg/mL for IL-8, and
15.6 pg/mL for CCL-5.
Statistical analysis
Values are expressed as the means –standard deviations
of at least three assays. The differences between the means
were analyzed for statistical significance using the Student’s
t-test with Bonferroni corrections, and an analysis of vari-
ance was performed using the Dunnett’s post hoc multiple
comparison test. A Pvalue <.05 was considered statistically
significant.
RESULTS
The different tea extracts (green, white, oolong, and
black) exhibited a comparable dose-dependent antibacterial
activity against P. gingivalis (Fig. 1). The MICs of the tea
extracts were 200 lg/mL, except for the black tea extract,
which had an MIC of 500 lg/mL. The MBCs of the tea
extracts were 500 lg/mL. At 50 lg/mL, green tea, white tea,
oolong tea, and black tea extracts reduced P. gingivalis
growth by 21% –2%, 16% –5%, 19% –4%, and 22% –5%,
respectively (Fig. 1).
The tea extracts all exhibited a marked capacity to dose
dependently inhibit the adhesion of P. gingivalis to oral
epithelial cells (Fig. 2). The inhibitory effect of the black
tea extract was the most pronounced, while the effects of
the other tea extracts were similar. The green tea, white
tea, oolong tea, and black tea extracts (25 lg/mL) decreased
the adherence of P. gingivalis to oral epithelial cells by
63% –3%, 59% –2%, 61% –7%, and 84% –3%, respec-
tively (Fig. 2).
While the tea extracts all dose dependently inhibited
P. gingivalis collagenase, MMP-9, and neutrophil elastase
activities, the effect on the collagenase activity was more
pronounced (Fig. 3). At the lowest concentration tested (10
lg/mL), the green tea, white tea, oolong tea, and black tea
extracts significantly (P<.05) inhibited the P. gingivalis
collagenase activity by 57% –2%, 51% –1%, 51% –2%,
and 38% –1%, respectively (Fig. 3A). The tea extracts (10
lg/mL) also inhibited the MMP-9 activity by 20% –2% to
430 ZHAO ET AL.
33% –1% (Fig. 3B), and the neutrophil elastase activity by
11% –2% to 38% –3% (Fig. 3C).
No obvious cytotoxic effects were observed following a
24-h treatment with up to 100 lg/mL of tea extract (Fig. 4).
The tea extracts did, however, appear to stimulate epithelial
cell proliferation.
The ability of tea extracts to inhibit the secretion of IL-6,
IL-8, and CCL-5 by oral epithelial cells stimulated with a
P. gingivalis extract (10 lg/mL) was then tested. This
treatment of epithelial cells with P. gingivalis did not result
in a loss of cell viability (data not shown). However, treating
epithelial cells with the P. gingivalis extract significantly
(P<.05) increased IL-6, IL-8, and CCL-5 secretion by 3.1-,
1.6-, and 2.7-fold compared to control cells. The most
pronounced inhibition in cytokine secretion by tea extracts
was observed for IL-6 and CCL-5 (Fig. 5A, C). In several
cases, cytokine secretion by epithelial cells was below the
basal levels observed for nonstimulated cells. As shown in
Figure 5A, extracts from white tea and black tea were the
most efficient in decreasing the secretion of IL-6. Regarding
CCL-5 secretion, the results presented in Figure 5C show a
more pronounced inhibitory effect caused by green tea and
oolong tea extracts since the amounts secreted were below
the basal levels. Lastly, while 10 lg/mL of the tea extracts
FIG. 1. Effect of tea extracts on the growth of P. gingivalis:(A) green tea extract; (B) white tea extract; (C) oolong tea extract; (D) black tea
extract. Results are expressed as the means –standard deviations (SD) of triplicate assays for two independent experiments. A value of 100% was
assigned to growth obtained in the absence of tea extracts. *Minimal inhibitory concentrations;
{
minimal bactericidal concentrations.
FIG. 2. Effect of tea extracts on the ad-
herence of P. gingivalis to human oral epi-
thelial cells. A value of 100% was assigned to
fluorescence values obtained in the absence of
tea extracts. Results are expressed as the
means –SD of triplicate assays for two inde-
pendent experiments. *Significantly lower
than the value for the untreated control
(P<.05). GTE, green tea extract; WTE, white
tea extract; OTE, oolong tea extract; BTE,
black tea extract.
TEA EXTRACTS AND PERIODONTAL DISEASES 431
had no effect on IL-8 secretion, 25, 50, and 100 lg/mL of all
the extracts caused significant (P<.05) inhibition (Fig. 5B).
DISCUSSION
There is increasing evidence suggesting that tea has a
number of beneficial effects on health.
11,13,14,19,20
The
pharmacological benefits of tea have been associated with
various substances, including flavonoids, theaflavin, thea-
nine, alkaloids, and polysaccharides.
10
The composition of
tea is mainly determined by how the tea leaves are pro-
cessed. Unfermented teas (green and white) are rich in
methylxanthines and several kinds of catechins, including
epigallocatechin, catechin gallate, epicatechin gallate, and
EGCG,
12,21
while oolong and black teas are rich in oxidized
phenolic compounds such as gallic acid, theaflavin, and
FIG. 3. Effect of tea extracts on P. gingivalis collagenase (A), matrix metalloproteinase ( MMP)-9 (B), and neutrophil elastase (C) activities. A
value of 100% was assigned to the degradation obtained in the absence of tea extracts. Results are expressed as the means –SD of triplicate assays
for two independent experiments. *Significant inhibition of the enzyme activity compared to the untreated control (P<.05).
FIG. 4. Effect of tea extracts on oral epi-
thelial cell viability as determined using a
3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetra-
zolium bromide (MTT) assay. A value of
100% was assigned to the viability observed
in the absence of tea extracts. Results are
expressed as the means –SD of triplicate
assays for two independent experiments.
432 ZHAO ET AL.
thearubigin, but are poor in catechins.
22
In addition, the
fluoride content of black tea is reported to be five times
higher compared with green tea.
23
Anecdotal evidence
suggests that green tea has a more pronounced effect on
promoting health and preventing or treating chronic dis-
eases, including periodontitis, due to its high catechin
content.
11,13,14,19,20
It has been reported that green tea
drinkers have healthier gums and teeth,
15
and that green tea
consumption is associated with a decreased probability of
tooth loss.
16
In addition, a clinical study has shown that a
slow-release buccal delivery system containing green tea
catechins significantly reduces the periodontal pocket
depth.
17
Other compounds in tea also exhibit antibacterial, anti-
inflammatory, and antioxidant activities. For example, thea-
flavin, one of the major oxidized polyphenols in black tea,
inhibits LPS-induced ICAM-1 and VCAM-1 expression in
epithelial cells,
24
suppresses oncostatin M-induced CXCL10
production in human fibroblasts,
25
and possesses the anti-
oxidant activity comparable to that of green tea catechins.
26
Theasinensin A from oolong tea has been shown to dose
dependently inhibit the mRNA, protein, and promoter ac-
tivity of cyclooxygenase-2 in LPS-activated macrophages in
addition to inhibiting the MMP activities of human fibro-
sarcoma HT1080 cells.
27
White tea has also been reported to
have stronger antielastase, anticollagenase, and anti-
oxidative activities than green tea.
28
In the present study, we
compared four types of tea extracts (green tea, white tea,
oolong tea, and black tea) for their effects on different as-
pects involved in the initiation and progression of peri-
odontal diseases.
FIG. 5. Effect of tea extracts on the
secretion of interleukin (IL)-6 (A), IL-
8(B), and CCL-5 (C) by oral epithelial
cells stimulated with a P. gingivalis
(Pg) extract. Results are expressed as
the means –SD of triplicate assays for
two independent experiments.
{
Sig-
nificantly higher than the value for the
unstimulated control (P<.05). *Sig-
nificantly lower than the value for the
P. gingivalis-stimulated cells (P<.05).
TEA EXTRACTS AND PERIODONTAL DISEASES 433
Since P. gingivalis is widely considered as the key etio-
logic agent of periodontitis, more specifically the chronic
form,
4,6
the inhibition of this bacterium may be a potentially
valuable strategy for interfering with the initiation of peri-
odontitis. A previous study found that the MICs of green tea
polyphenols, including epigallocatechin and EGCG, against
the P. gingivalis range from 250 to 1000 lg/mL.
29
The
different types of tea used in the present study also inhibited
P. gingivalis growth, with MICs ranging from 200 to 500
lg/mL. While the exact mechanism by which tea inhibits
bacterial growth remains obscure, some studies have pro-
vided interesting clues. The tea components, theaflavins and
catechins, have been reported to irreversibly damage the
bacterial cytoplasmic membrane.
30–32
For example, EGCG
generates hydrogen peroxide in the lipid bilayer of the
bacterial cytoplasmic membrane, resulting in leakage
of intracellular materials.
31
Membrane damage may also
facilitate the diffusion of bioactive molecules into the cells.
In addition, Navarro-Martı
´nez et al.
33
provided evidence
that the antibacterial action of catechins against Steno-
trophomonas maltophila, a Gram-negative opportunistic
pathogen, is due to its ability to inhibit cytoplasmic dihy-
drofolate reductase. Dihydrofolate reductase reduces dihy-
drofolic acid to tetrahydrofolic acid, which is required by
bacteria to synthetize purine, thymidylate, and nucleic acid
precursors, which are very important for cell proliferation
and growth.
All the tea extracts significantly inhibited the adherence
of P. gingivalis to oral epithelial cells, even at the lowest
concentration tested (10 lg/mL). Gallate-type tea polyphe-
nols have already been claimed to possess an inhibitory
effect on the adherence of P. gingivalis to human oral epi-
thelial cells.
29
In addition, some catechin derivatives inhibit
Rgp and Kgp gingipains, which are involved in the adher-
ence of P. gingivalis to host cells.
34
Matsumoto et al.
35
re-
ported that high and low molecular weight oolong tea
fractions can bind to bacterial surface proteins, decreasing
cell surface hydrophobicity of Streptococcus mutans. The
inhibition of adherence observed in the present study may
thus result from the binding of tea components to P. gin-
givalis cell surface proteins.
In their natural subgingival environment, period-
ontopathogens degrade tissue proteins into low molecular
weight peptides and amino acids, which can be used as
carbon and energy sources to support bacterial growth.
36,37
Bacterial proteinases play a pivotal role in this process by
direct degradation of host proteins and the activation of la-
tent host enzymes.
36,37
Since type I collagen is the pre-
dominant protein of periodontal tissue, this constituent of
the gingival matrix may be a major source of nutrients for
P. gingivalis, which possesses a complex proteolytic system
that can degrade type I collagen into small fragments.
38
In the present study, we showed that tea extracts strongly
inhibit the collagenase activity of P. gingivalis. In addition
to reducing tissue destruction, this inhibition may affect
bacterial growth.
The MMP-9 activity has been strongly associated with
periodontitis progression.
39
Ding et al.
40
showed that
P. gingivalis can concomitantly trigger the release and ac-
tivation of MMP-9 from polymorphonuclear leukocytes. In
addition, it has been shown that infections of an engineered
human oral mucosa model with P. gingivalis result in a
significant increase in the MMP-9 protein and mRNA lev-
els.
41
Mounting evidence also points to an important role for
the elastase released from polymorphonuclear leukocytes in
periodontal destruction.
42–44
This neutrophil enzyme can
degrade several matrix proteins, including elastin, collagen,
and fibronectin, and its activity is significantly correlated
with probing depth, attachment loss, and the gingival index
of periodontal patients.
45
The present study showed that tea
extracts significantly inhibit MMP-9 and elastase activities
and may thus contribute to reducing periodontal tissue de-
struction. These results are in agreement with those reported
by Demeule et al.
46
indicating that green tea polyphenols,
especially EGCG, are potent inhibitors of MMP-9 and
MMP-12 (also known as macrophage elastase). The inhi-
bition of the MMP activity by green tea catechins has been
associated with conformational changes.
47
In a recent
study
28
comparing the anticollagenase and antielastase ac-
tivities of plant extracts, white tea was shown to inhibit
elastase and collagenase more than green tea. In the present
study, we observed no marked differences among the four
tea extracts tested.
The mechanisms underlying the destructive processes
associated with periodontitis are not only related to the di-
rect tissue damage caused by bacterial and host-derived
proteinases, but also involve indirect damage mediated by
host immune and inflammatory responses elicited by peri-
odontal pathogens. P. gingivalis cells and components can
induce a strong proinflammatory cytokine response in gin-
gival epithelial cells.
8
In the present study, an extract of
P. gingivalis upregulated the secretion of IL-6, IL-8, and CCL-
5 by oral epithelial cells. Since growing evidence
11,13,19,48–50
suggests that tea polyphenols have anti-inflammatory
properties, we evaluated the ability of green tea, white
tea, oolong tea, and black tea extracts to inhibit IL-6, IL-8,
and CCL-5 secretion by epithelial cells stimulated with
the P. gingivalis extract. Our results showed that all four tea
extracts attenuated the P. gingivalis-induced inflammatory
response to various degrees. More specifically, the expres-
sion of CCL-5, a chemokine that enhances the recruitment
and infiltration of immune cells to diseased periodontal
sites, was significantly suppressed by all four tea extracts.
Preliminary results indicated that the four tea extracts
also inhibit Aggregatibacter actinomycetemcomitans LPS-
induced inflammatory cytokines production by oral epithe-
lial cells (data not shown). Previous in vivo and in vitro
studies
24–27,49,50
have shown that tea components such as
catechins, theaflavin, and thearubigin have anti-inflammatory
activities. The molecular mechanisms may involve inter-
ference with signaling pathways such as NF-jB and AP-1. A
recent study
50
using a macrophage genome-wide DNA mi-
croassay to investigate the anti-inflammatory genes targeted
by theasinensin A showed that the activities of 63.8% of the
genes upregulated in LPS-activated macrophages are at-
tenuated by theasinensin A and that the activities of 65.7%
434 ZHAO ET AL.
of the downregulated genes are restored by theasinensin A.
The genes suppressed by theasinensin A include those
coding for tumor necrosis factor (TNF), IL-1b, and IL-6.
Interestingly, the genes coding for anti-inflammatory cyto-
kines, which were decreased in LPS-treated macrophages
were restored by theasinensin A.
To summarize, the present study showed that green
tea, white tea, oolong tea, and black tea extracts possess a
number of properties (antibacterial, antiadherence, anti-
protease, and anti-inflammatory) that may contribute to
maintaining periodontal health. None of the properties were
specifically associated with a particular type of tea, sug-
gesting that all teas may have beneficial effects. Although
the different tea extracts showed similar properties in regard
to antibacterial, antiadherence, antiprotease, and anti-
inflammatory activities, the bioactive ingredients of the
various extracts may differ considering their chemical
composition.
In conclusion, the present study compared the potential
impacts of four tea extracts on periodontal disease ther-
apeutic targets. They all exhibited comparable activities,
including the ability to inhibit (i) the growth of P. gin-
givalis and its adherence to oral epithelial cells, (ii) the
activity of host and bacterial proteases, and (iii) the se-
cretion of proinflammatory mediators by oral epithelial
cells. Bioactive molecules in tea thus hold promise as
preventive or therapeutic agents for treating periodontal
diseases.
ACKNOWLEDGMENTS
This study was supported by the Laboratoire de Contro
ˆle
Microbiologique of Universite
´Laval. We wish to thank
V. Murrah (University of North Carolina, Chapel Hill, NC,
USA) and J.M. Dirienzo (University of Pennsylvania,
Philadelphia, PA, USA) for providing the GMSM-K epi-
thelial cell line.
AUTHORS’ CONTRIBUTIONS
All authors contributed equally in data acquisition and in
writing of the manuscript. All of the authors read and ap-
proved the final version of the manuscript.
AUTHOR DISCLOSURE STATEMENT
The authors have no conflicts of interest related to this
study.
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