Antibacterial and anti-inflammatory activities of 4-Hydroxycordoin: Potential therapeutic benefits

Article abstract of DOI:10.1021/np100547b

4-Hydroxycordoin (1), a natural isopentenyloxychalcone, is a plant secondary metabolite that is relatively rare. Since there are very few reports about the biological activities of 1, its potential benefits for periodontal disease were investigated. A mar

Full text of DOI:10.1021/np100547b

2
6
J. Nat. Prod. 2011, 74, 26–31
Antibacterial and Anti-inflammatory Activities of 4-Hydroxycordoin: Potential Therapeutic
Benefits
†
†
‡
‡
§
,†
Mark Feldman, Shinichi Tanabe, Francesco Epifano, Salvatore Genovese, Massimo Curini, and Daniel Grenier*
´
Groupe de Recherche en Ecologie Buccale, Facult e´ de M e´ decine Dentaire, UniVersit e´ LaVal, 2420 Rue de la Terrasse, Qu e´ bec City, QC,
Canada, G1V 0A6, Dipartimento di Science del Farmaco, UniVersit a` G. D’Annunzio, Via Dei Vestini 31, 66013 Chieti Scalo, Chieti, Italy, and
Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, UniVersit a` degli Studi di Perugia, Via del Liceo,
0
6123 Perugia, Italy
ReceiVed August 4, 2010
4
-Hydroxycordoin (1), a natural isopentenyloxychalcone, is a plant secondary metabolite that is relatively rare. Since
there are very few reports about the biological activities of 1, its potential benefits for periodontal disease were investigated.
A marked and dose-dependent antibacterial activity of 1 was observed against the three major periodontal pathogens,
Porphyromonas gingiValis, Fusobacterium nucleatum, and PreVotella intermedia. Moreover, compound 1 showed an
antiadhesion effect, since it inhibited attachment of P. gingiValis to oral epithelial cells. Finally, using a macrophage
model, the ability of 1 to inhibit the secretion of inflammatory mediators induced by Aggregatibacter actinomycetem-
comitans lipopolysaccharide was demonstrated. The anti-inflammatory effect observed was associated with reduced
activation of the nuclear factor-κB (NF-κB) p65 and activator protein-1 (AP-1) pathways.
4
-Hydroxycordoin, (2E)-1-{2-hydroxy-4-[(3-methylbut-2-enyl)-
of the disease toward destruction or healing. The tissue homeostasis
is regulated by several host factors produced by immune and
mucosal cells in a healthy condition. However, due to the protecting
response against periodontopathogens, an accumulation of certain
inflammatory mediators, such as interleukin-1ꢀ (IL-1ꢀ), tumor
necrosis factor alpha (TNF-R), and prostaglandins, leads to chronic,
persistent inflammation and ultimately to tissue destruction mediated
oxy]phenyl}-3-(4-hydroxyphenyl)prop-2-en-1-one) (1), is a natural
isopentenyloxychalcone that is relatively rare in nature, like other
prenyloxyphenylpropanoids. Up to now, it has been isolated only
from one plant, namely, Lonchocarpus neuroscapha Benth.
1
2
(
Fabaceae). Very few reports on the biological activities of 1 are
available in the scientific literature. In 1974, de Mello and
co-workers reported that this molecule exerted a slight anticancer
activity against sarcoma 180 and Ehrlich carcinoma models in
mice. Moreover, 1 was found to exert a weak antibacterial activity
against Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa,
8
,9
by matrix metalloproteinases (MMPs). It was demonstrated that
such excessive production of inflammatory mediators is stimulated
by virulence factors, such as lipopolysaccharide (LPS) and fimbriae,
3
1
0
produced by periodontopathogens.
4
and Staphylococcus aureus.
Bacterial infections, including periodontitis, can be controlled
by several means, with the most effective being a bactericidal
approach. However, this approach has numerous drawbacks, of
which the most serious are the emergence and spread of resistant
strains. The uncontrolled use of antibiotics is reflected in the level
of resistance of various periodontopathogens in patients with
11
periodontal infections. To our knowledge, no mechanism has been
clearly described by which bacteria develop resistance against
natural plant compounds. Therefore, such substances may represent
a novel approach to control bacterial infections, as they have
potential to minimize the spread of resistant strains. Since tissue
destruction in periodontitis is also a consequence of the inflam-
matory response, the quest for developing therapeutic plant-derived
compounds for the treatment and prevention of periodontitis should
also include the investigation of their anti-inflammatory properties.
In this study, the antibacterial activity of 4-hydroxycordoin (1) was
investigated on P. gingiValis, F. nucleatum, and P. intermedia, three
bacterial species associated with periodontitis. In addition, the anti-
inflammatory effect of this natural chalcone was investigated in a
macrophage model stimulated with A. actinomycetemcomitans LPS.
Periodontitis is a complex inflammatory disease of bacterial
origin affecting the tissues that surround and support the teeth. It
involves progressive destruction of the alveolar bone around the
teeth and, if left untreated, can lead to the loss of teeth. Periodontitis
is initiated by specific bacterial species that colonize subgingival
sites along with a continuous challenge of the host immune system
by these pathogens. More than 700 microbial species have been
5
identified in subgingival plaque. However, only a limited number
of bacterial species have been associated strongly with the different
forms of periodontitis, which include Aggregatibacter actinomyce-
temcomitans, Fusobacterium nucleatum, Porphyromonas gingiValis,
PreVotella intermedia, Tannerella forsythia, and Treponema
6
,7
Results and Discussion
denticola. The accumulation and proliferation of these bacterial
species in the periodontal pockets are the initiating steps in the
onset of periodontal lesions.
Using a microplate dilution assay, 1 exhibited a dose-dependent
antibacterial activity against the three major periodontopathogens
tested (Figure 1). The most pronounced inhibition of growth and
viability was observed for P. intermedia (Figure 1C). At 1.25 µg/
mL, 1 reduced bacterial growth by 45%, while 2.5 µg/mL
represented the MIC. Moreover, it was found that 1 at 5 µg/mL
kills P. intermedia, thus representing the MBC. P. gingiValis was
less susceptible to 1 compared to P. intermedia; the MIC and MBC
values were 5 and 10 µg/mL, respectively (Figure 1A). Finally,
Although the periodontopathogens are essential for the initiation
of periodontitis, the host’s immune response modulates progression
*
Corresponding author. Tel: (418) 656-7341. Fax: (418) 656-2861.
E-mail: Daniel.Grenier@greb.ulaval.ca.
†
Universit e´ Laval.
Universit a` G. D’Annunzio.
Universit a` degli Studi di Perugia.
‡
§
1
0.1021/np100547b  2011 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/15/2010

Antibacterial Properties of 4-Hydroxycordoin
Journal of Natural Products, 2011, Vol. 74, No. 1 27
Figure 1. Antibacterial effect of 4-hydroxycordoin (1) on P. gingiValis (A), F. nucleatum (B), and P. intermedia (C). A value of 100% was
‡
given to growth obtained in the absence of 1. Values marked with indicate MBCs. The data are means ( SD of triplicate assays for two
independent experiments. *p < 0.05 compared to control with no 1.
among the three periodontopathogens tested, F. nucleatum was
found to be the most resistant to 1. The MIC was 40 µg/mL, while
the MBC was > 40 µg/mL for this bacterial species (Figure 1B).
Considering that periodontopathogens colonize the subgingival areas
where a serum exudate is found, we also carried out the above
growth studies in the presence of 1% fetal bovine serum, and similar
results were obtained (data not shown). Compound 1 has been
shown previously to exhibit a weak antibacterial activity against
4
E. coli, S. aureus, P. aeruginosa, and B. cereus. Indeed, MICs
and MBCs for the above bacterial species were in the range
4
1
000-2000 µg/mL. In contrast, very potent bacteriostatic and
bactericidal effects of 1 were observed on P. intermedia and P.
gingiValis and to a lesser extent on F. nucleatum. This suggests
that 1 may be more effective on anaerobic bacteria.
Figure 2. Effect of 4-hydroxycordoin (1) on the adherence of P.
gingiValis to human oral epithelial cells. Assays were run in
triplicate, and the means ( SD of three independent assays were
Since the adherence of bacteria to mucosal cells is a critical step
in the development of infections, the inhibition of adhesion may
represent a potentially valuable approach for preventing infectious
disease development, including periodontitis. The effect of 1 on
the adherence property of P. gingiValis to an oral epithelial cell
monolayer was investigated (Figure 2). Compound 1 inhibited the
adhesion of 14C-labeled P. gingiValis to oral epithelial cells in a
14
calculated. A value of 100% was assigned to the amount of C-
labeled bacteria bound in the absence of 1. *p < 0.05 compared to
control with no 1.
P. gingiValis and P. intermedia are obligate anaerobic Gram-
negative bacteria and important pathogens in several forms of
14
12
dose-dependent manner. The relative adherence of C-labeled P.
gingiValis to oral epithelial cells was not decreased significantly at
the lowest concentration of 1 tested (0.2 µg/mL). However, at
concentrations of 1 and 5 µg/mL, 1 inhibited 14C-labeled P.
gingiValis adherence to epithelial cells by 31% and 36%, respectively.
periodontitis. F. nucleatum is one of the most abundant anaerobic
bacteria in subgingival plaque and plays a key role in oral biofilm
formation, bridging early and late plaque colonizers by coaggre-
13
gating with a wide array of microorganisms in the oral cavity.
Previous studies showed that periodontal pathogens, including the

2
8
Journal of Natural Products, 2011, Vol. 74, No. 1
Feldman et al.
Figure 3. Effect of treating macrophages with 4-hydroxycordoin (1) on the secretion of IL-1ꢀ (A), TNF-R (B), IL-6 (C), IL-8 (D), CCL-5
E), and PGE (F) induced by A. actinomycetemcomitans ATCC 29522 LPS (A.a LPS). The data are means ( SD of triplicate assays for
†
(
2
two independent experiments. p < 0.05 compared to control with no LPS and with no 1. *p < 0.05 compared to an LPS control with
no 1.
above three bacterial species, may acquire resistance when antibiot-
2
PGE secretion. At 1 µg/mL, in the absence of 1, LPS markedly
1
1
ics are used for periodontal therapy. Our data indicate that 1, by
acting on growth and adherence property of these important
periodontopathogens, may be considered as a potential alternative
to antibiotics. However, one should not exclude the possibility that
the tested periodontal pathogens may be more resistant to 1 when
growing in a biofilm structure compared to planktonic growth
conditions. Further studies are required to determine whether
bacteria may develop resistance to 1.
increased the secretion of IL-1ꢀ (197-fold) (Figure 3A), TNF-R
(24-fold) (Figure 3B), IL-6 (62-fold) (Figure 4C), IL-8 (46-fold)
(Figure 3D), and CCL5 (17-fold) (Figure 3E), while having a lesser
2
enhancing influence on PGE production (5-fold) (Figure 3F). Our
findings indicate that 1 was able to attenuate this LPS-induced
secretion of inflammatory mediators. At 5 µg/mL, 1 significantly
(p < 0.05) reduced the secretion of all inflammatory mediators
tested. Interestingly, 1 had no effect on the inflammatory response
of unstimulated macrophages (data not shown). The most pro-
Periodontitis is strongly associated with the host inflammatory
response, which is a critical factor contributing to destruction of
tooth-supporting tissues. Therefore, therapeutic agents that in
addition to exerting antibacterial effects are able to attenuate the
inflammatory mediator production have a potential for controlling
nounced inhibition in secretion was observed for CCL5 and PGE
(Figure 3E and F, respectively) Prostaglandins, for which the levels
2
.
1
8
increase significantly during periodontitis, are potent inducers of
osteoclast generation leading to alveolar bone resorption and
14
19,20
periodontitis. There are several reports indicating that A. actino-
mycetemcomitans LPS acts as a potent inducer of various inflam-
matory mediator production by macrophages, suggesting that it may
consequently are involved in the destruction of the periodontium.
In addition, PGE
cell types including gingival fibroblasts,
2
was found to inhibit proliferation of different
21,22
thus also hampering
15,16
contribute to periodontal tissue destruction.
Macrophages, being
tissue repair. Previously, it was demonstrated that various natural
agents such as hop-derived polyphenols, cranberry-derived proan-
thocyanidins, epigallocatechin gallate, and other flavonoids inhibit
at the front line of host defenses, play important roles in the
initiation and maintenance of inflammatory processes and in the
tissue destruction observed in adult periodontitis. Moreover, these
immune cells are the constitutive targets of pathogens aiming to
2
PGE -enhanced production by various cell types and, thus, may
reduce the side effects of this inflammatory mediator on bone and
1
7
23-25
subvert immune surveillance. The ability of 1 to attenuate the A.
actinomycetemcomitans LPS-induced inflammatory response of
monocyte-derived macrophages was examined. Macrophages were
pre-exposed (2 h) to different doses of 1 prior to being stimulated
connective tissue homeostasis.
The chemokines IL-8 and CCL5 are involved in the recruitment
1
of neutrophils, eosinophils, monocytes, and TH cells to infected
2
6
sites. Due to the high levels of these chemokines in inflamed
sites, they are thought to play a crucial role in the initiation and
(
24 h) with A. actinomycetemcomitans LPS to determine its effect
27,28
on cytokine (IL-1ꢀ, TNF-R, IL-6), chemokine (IL-8, CCL5), and
progression of periodontitis.
The fact that 1 at 5 µg/mL inhibited

Antibacterial Properties of 4-Hydroxycordoin
Journal of Natural Products, 2011, Vol. 74, No. 1 29
Figure 4. Effect of treating macrophages with 4-hydroxycordoin (1) on the secretion of MMP-9 induced by A. actinomycetemcomitans
†
ATCC 29522 LPS (A.a LPS). The data are means ( SD of triplicate assays for two independent experiments. p < 0.05 compared to control
with no LPS and with no 1. *p < 0.05 compared to an LPS control with no 1.
Figure 5. Effect of treating macrophages with 4-hydroxycordoin (1) on the activation of nuclear factor-kappa B (NF-κB) p65 and activator
protein-1 (AP-1) induced by A. actinomycetemcomitans ATCC 29522 LPS (A.a LPS). The data are means ( SD of triplicate assays for
†
three independent experiments. p < 0.05 compared to control with no LPS and with no 1. *p < 0.05 compared to an LPS control with
no 1.
3
5
the secretion of IL-8 by 38% and CCL5 by 67% (Figure 3D and E,
respectively) suggests that this compound has the potential to limit
the infiltration of immune cells into periodontal sites and consequently
to modify the inflammatory processes. This finding is in agreement
with previous studies showing that natural products may exert their
anti-inflammatory properties by inhibition of chemokine secretion
periodontitis. Local inhibition of these two mediators in peri-
odontal tissues significantly reduced the inflammatory response and
36
bone loss in ligature-induced periodontitis in monkeys. Compound
1 at the highest tested concentration of 5 µg/mL had a less
pronounced inhibiting effect on the production of IL-1ꢀ and TNF-R
as compared to other inflammatory mediators tested, namely, 29%
(Figure 3A) and 23% (Figure 3B), respectively. This variability in
inflammatory response inhibition by 1 may be explained by the
different levels of secreted inflammatory factors upon LPS stimula-
tion. MMP-9, another important factor involved in periodontal tissue
destruction, is highly expressed in inflamed gingival tissues of
patients with periodontitis.
tion was also significantly inhibited by 1 at 1 and 5 µg/mL (Figure
), following a pretreatment of macrophages.
NF-κB p65 is involved in the activation of many inflammatory
23,29,30
induced by LPS from periodontopathogens.
More specifically,
it was reported that epigallocatechin-3-gallate from green tea
attenuated the severity of periapical lesions by reduction of
macrophage chemotaxis into the lesions due to inhibition of CCL5
3
1
secretion.
37,38
The LPS-induced MMP-9 produc-
IL-1ꢀ, IL-6, and TNF-R are multifunctional cytokines that play
an important role in regulating the immune response during
periodontal disease. IL-6 expression was found to be higher at sites
of periodontal inflammation and closely related to the clinical
4
3
2
39
severity of periodontitis. In addition, its levels increase in
subgingival sites of patients with periodontitis compared with
34
processes. MMP gene expression is also regulated by transcription
40
factors including NF-κB p65 and AP-1. Therefore, the effect of
1 was tested on NF-κB p65 and AP-1 activation in A. actinomyce-
temcomitans LPS-treated macrophages. The relative activity of
nuclear transcription factor NF-κB p65 and AP-1 in macrophages
stimulated with 1 µg/mL A. actinomycetemcomitans LPS increased
by 58% and 145%, respectively, compared to an untreated control
(100%) (Figure 5). This observation is in agreement with other
reports showing that A. actinomycetemcomitans LPS is a potent
3
3
periodontally healthy subjects. IL-6 promotes bone resorption
and is strongly involved in osteoclast differentiation, suggesting
that it can contribute to bone resorption associated with periodon-
titis. In this study, it was demonstrated that a 5 µg/mL treatment
of macrophages with 1 markedly decreased LPS-induced IL-6
production by 50% (Figure 3C). Thus, it may be proposed that this
chalcone may contribute to reduce the impact of host-destructive
processes mediated by IL-6 occurring during periodontal disease.
IL-1ꢀ and TNF-R are critical factors of the progression of
41
activator of NF-κB p65 and AP-1. A pretreatment of macrophages
with 5 µg/mL of 1 prior to the stimulation with LPS totally

3
0
Journal of Natural Products, 2011, Vol. 74, No. 1
Feldman et al.
neutralized the LPS-induced activation of NF-κB p65 and AP-1
Figure 5). Thus, 1 has the ability to exhibit its anti-inflammatory
cultured onto wells of 96-well plates (Sarstedt) in Dulbecco’s modified
Eagle’s medium (DMEM) to form a confluent monolayer as reported
(
4
5
elsewhere. The wells were washed three times with PBS prior to
effect, at least partly, via reduction of the activation of NF-κB p65
and AP-1. This is in agreement with previous studies reporting that
natural products with anti-inflammatory properties exert their effect
1
4
performing the adherence assay. Mixtures containing 20 µL of C-
labeled P. gingiValis (OD655 of 0.8) and 30 µL of 1 in PBS were
prepared to obtain final 1 concentrations of 0, 0.2, 1, or 5 µg/mL. The
mixtures were placed in wells coated with epithelial cells. After a 30
min incubation at 37 °C, the wells were washed three times with PBS
to remove unbound bacteria. Adhered radiolabeled bacteria were
detached by adding 100 µL of 0.5 M NaOH and incubating the plate
at room temperature for 15 min on an orbital shaker. The bacteria were
suspended in EcoLite scintillation liquid (ICN, Costa Mesa, CA), and
the radioactivity was counted using a multipurpose scintillation counter
(Beckman Coulter, Fullerton, CA).
4
1,42
by modulating the NF-κB and AP-1 pathways.
Lastly, the toxicity of 1 was evaluated toward the two cell lines
used in the present study (monocytes-derived U937 macrophages
and GMSM-K epithelial cells) using the colorimetric MTT assay.
No significant decrease was observed in viability of macrophages
and epithelial cells following a 2 h exposure to 1 at concentrations
up to 50 µg/mL.
In summary, the present study demonstrated the antibacterial and
antiadherence activities as well as anti-inflammatory properties of
LPS Preparation. A. actinomycetemcomitans ATCC 29522 was
grown in THB supplemented with 1% yeast extract and incubated at
1
. Interestingly, this natural isopentenyloxychalcone exerted these
3
7 °C under anaerobic conditions for 24 h. LPS were isolated using
properties at very low concentrations. This ability of 1 to act on
both the bacteria and the host immune response is interesting
considering that periodontitis involves these two components. It
may be proposed that 1 represents a potential therapeutic agent in
the treatment and prevention of periodontal disease. Therefore,
compound 1 may contribute to minimize the spread of antibiotic
resistance in periodontopathogens, a phenomenon associated with
the uncontrolled used of antibiotics.
46
the procedure described previously, which is based on the digestion
of a whole cell extract by proteinase K and successive solubilization
and precipitation steps. The LPS preparation was freeze-dried and stored
at -20 °C until used. Contaminating protein, which was evaluated using
a protein assay kit (DC protein assay; Bio-Rad Laboratories, Missis-
sauga, ON, Canada) with bovine serum albumin as the control, made
up less than 0.005% of the preparation.
Preparation of Monocyte-Derived Macrophages and Treatments.
U937 cells (ATCC CRL-1593.2), a human monoblastic leukemia cell
2
line, were cultivated at 37 °C in a 5% CO atmosphere in RPMI-1640
Experimental Section
medium (HyClone Laboratories, Logan, UT) supplemented with 10%
Synthesis of 4-Hydroxycordoin (1). Compound 1 was synthesized
chemically as previously reported. Briefly, commercially available
heat-inactivated fetal bovine serum (FBS) (RPMI-FBS) and 100 µg/
4
3
5
mL penicillin-streptomycin. Monocytes (2 × 10 cells/mL) were
2
3
,4-dihydroxyacetophenone was first alkylated selectively at C-4 with
,3-dimenthylallyl bromide in the presence of 1,8-diazadiazabicyclo-
incubated in RPMI-FBS containing 10 ng/mL phorbol myristic acid
(
PMA) (Sigma Chemical Co., St. Louis, MO) for 48 h to induce
[
5.4.0]undec-7-ene as the base in acetone at room temperature. This
differentiation into adherent macrophage-like cells as previously
oxyprenylated acetophenone was then made to react with 4-hydroxy-
benzaldehyde in aqueous ethanol in the presence of 60% KOH. The
desired product (1) was then recovered in 32% yield after crystallization
from n-hexane (mp ) 111-112 °C, in agreement with previously
47
reported. Following the PMA treatment, the medium was replaced
with fresh medium and differentiated cells were incubated for an
additional 24 h prior to use. Adherent macrophages were suspended in
RPMI-FBS and centrifuged at 200g for 8 min. The cells were washed,
4
3
reported data ). Its purity (>99.7%) was assessed by gas chroma-
tography-mass spectrometry.
6
suspended at a density of 1 × 10 cells/mL in RPMI supplemented
6
with 1% heat-inactivated FBS, and seeded in a six-well plate (2 × 10
Determination of Minimal Inhibitory and Minimal Bactericidal
Concentrations. P. gingiValis ATCC 33277, F. nucleatum ATCC
2
cells/well/2 mL) at 37 °C in a 5% CO atmosphere for 2 h prior to
treatments with 1 and LPS. Various concentrations of 1 (0, 0.2, 1, and
µg/mL) were added to the monocyte-derived macrophage cultures,
which were then incubated at 37 °C in a 5% CO atmosphere for 2 h
prior to adding the A. actinomycetemcomitans LPS at a final concentra-
tion of 1 µg/mL. After 24 h (37 °C in 5% CO ), the culture medium
2
5586, and P. intermedia ATCC 25611 were grown in Todd-Hewitt
5
broth (THB) (BBL Microbiology Systems, Cockeysville, MD) supple-
mented with 0.001% hemin and 0.0001% vitamin K (THB-HK).
Cultures were incubated at 37 °C under anaerobic conditions
2
2
(
N
2
-H
2
2
-CO , 80:10:10). Minimal inhibitory concentrations (MICs)
supernatants were removed and stored at -20 °C until used. Cells
incubated with no LPS and 1 as well as cells treated with LPS in the
absence of 1 were used as controls.
Cytotoxicity of LPS and 4-Hydroxycordoin (1). An MTT (3-[4,5-
diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Roche
Diagnostics, Mannheim, Germany) was used to determine the cyto-
toxicity of LPS and 1.
and minimal bactericidal concentrations (MBCs) were determined as
follows. Briefly, a 24 h culture of the above bacterial species in
THB-HK were diluted in fresh broth medium to obtain an optical
density at 655 nm (OD655) of 0.2. Equal volumes (100 µL) of bacteria
and 1 (0, 0.63, 1.25, 2.5, 5, 10, 20, and 40 µg/mL) in THB-HK were
mixed into the wells of 96-well plates (Sarstedt, Newton, NC). Control
wells with no bacteria were also prepared. After an incubation for 24 h
at 37 °C in the anaerobic chamber, bacterial growth was recorded by
measuring the OD655 using a microplate reader. MIC values (µg/mL)
of 1 for each bacterial species were determined as the lowest
concentration at which no growth occurred. To determine MBC values
Determination of Cytokine and MMP-9 Production. Commercial
enzyme-linked immunosorbent assays (ELISA) kits (R&D Systems,
Minneapolis, MN) were used to quantify IL-1ꢀ, TNF-R, IL-6, IL-8,
CCL-5, and MMP-9 in the samples, according to the manufacturer’s
protocols. The absorbance at 450 nm was read in a microplate reader
with the wavelength correction set at 550 nm. The sensitivities of the
commercial ELISA kits were 31.2 pg/mL for IL-8 and MMP-9, 15.6
pg/mL for TNF-R and CCL-5, 9.3 pg/mL for IL-6, and 3.9 pg/mL for
IL-1ꢀ.
(
µg/mL), aliquots (10 µL) of each well showing no visible growth were
spread on THB-HK agar plates, which were incubated for five days at
3
7 °C under anaerobic conditions. MBC values of 1 for each bacterial
species were determined as the lowest concentration at which no colony
formation occurred.
2
Determination of PGE Production. A competitive enzyme im-
Adherence to Human Oral Epithelial Cells. P. gingiValis cells
were radiolabeled by incubating a mid-log phase culture (OD655 of 0.5)
munoassay was performed on the above supernatant fluids according
the manufacturer’s protocol (Cayman Chemical Company, Ann Arbor,
MI). The sensitivity of the assay was 15 pg/mL.
14
with a mixture of C-labeled amino acids (Amersham Biosciences UK
Ltd., Little Chalfont, Buckinghamshire, UK) at a final concentration
of 20 µCi/mL at 37 °C for 16 h in an anaerobic chamber. Cells were
harvested by centrifugation at 10000g for 10 min, washed three times
in 10 mM phosphate-buffered saline (PBS, pH 7.2), and suspended to
an OD655 of 0.8 in PBS prereduced by overnight incubation in the
anaerobic chamber. The immortalized human oral epithelial cell line
GMSM-K (developed by Valerie Murrah, Department of Diagnostic
Sciences and General Dentistry, University of North Carolina at Chapel
Analysis of NF-KB p65 and AP-1 Activation. Macrophages were
incubated with highest tested concentration of 1 (5 µg/mL) for 1 h and
stimulated with 1 µg/mL LPS for an additional 1 h at 37 °C in a 5%
2
CO atmosphere. Macrophages without 1 served as controls. Whole-
cell extracts were prepared according to the manufacturer’s protocol
(Nuclear Extract kits, Active Motif, Carlsbad, CA), adjusted to a protein
concentration of 1 mg/mL, and stored at -80 °C until use. The extracts
were used to measure NF-κB p65 and AP-1 activation using commercial
kits (Trans-AM, Active Motif, Carlsbad, CA). Controls consisting of
4
4
Hill), which has an epithelial phenotype, was used to investigate the
effect of 1 on the adherence of P. gingiValis. GMSM-K cells were

Antibacterial Properties of 4-Hydroxycordoin
Journal of Natural Products, 2011, Vol. 74, No. 1 31
macrophages incubated in culture medium with 1 but not stimulated
with LPS were performed for anti-inflammatory assays.
Statistical Analysis. All experiments were carried out in triplicates,
and the means ( standard deviations were calculated. The statistical
analysis was performed using Student’s t test with a level of significance
of p < 0.05.
(19) Williams, R. C.; Jeffcoat, M. K.; Howell, T. H.; Hall, C. M.; Johnson,
H. G.; Wechter, W. J.; Goldhaber, P. J. Periodont. Res. 1987, 22,
4
03–407.
(
(
20) Howell, T. H.; Williams, R. C. Crit. ReV. Oral. Biol. Med. 1993, 4,
1
77–196.
21) Arai, H.; Nomura, Y.; Kinoshita, M.; Shimizu, H.; Ono, K.; Goto,
H.; Takigawa, M.; Nishimura, F.; Washio, N.; Kurihara, H. J.
Periodont. Res. 1995, 30, 303–311.
Acknowledgment. This work was supported by the Canadian
(
22) Arai, H.; Nomura, Y.; Kinoshita, M.; Nishimura, F.; Takigawa, M.;
Takahashi, K.; Washio, N.; Takashiba, S.; Murayama, Y. J. Periodont.
Res. 1998, 33, 33–39.
Institutes of Health Research (CIHR) and the Minist e` re du D e´ veloppe-
´
ment Economique de l’Innovation et de l’Exportation (MDEIE). We
thank V. Murrah (University of North Carolina at Chapel Hill) for
providing the GMSM-K epithelial cell line, and V. Dang La for
technical assistance.
(23) Inaba, H.; Tagashira, M.; Honma, D.; Kanda, T.; Kou, Y.; Ohtake,
Y.; Amano, A. Biol. Pharm. Bull. 2008, 31, 527–530.
(
24) Guti e´ rrez-Venegas, G.; Jim e´ nez-Estrada, M.; Maldonado, S. Int.
Immunopharmacol. 2007, 7, 1199–1210.
(
25) Bodet, C.; Chandad, F.; Grenier, D. Eur. J. Oral. Sci. 2007, 115, 64–
Note Added after ASAP Publication: This paper was published
on the Web on Dec 15, 2010, with an error in the structure graphic of
compound 1. The corrected version was reposted on Dec 21, 2010.
7
0.
(
(
26) Luster, A. D. N. Engl. J. Med. 1998, 338, 436–445.
27) Gamonal, J.; Acevedo, A.; Bascones, A.; Jorge, O.; Silva, A. J.
Periodont. Res. 2001, 36, 194–203.
References and Notes
(28) Gamonal, J.; Bascones, A.; Jorge, O.; Silva, A. J. Clin. Periodontol.
000, 27, 675–681.
2
(
1) Epifano, F.; Genovese, S.; Menghini, L.; Curini, M. Phytochemistry
007, 68, 939–953.
(
29) Bodet, C.; Chandad, F.; Grenier, D. J. Dent. Res. 2006, 85, 235–239.
30) Chiavaroli, A.; La, V. D.; Orlando, G.; Menghini, L.; Epifano, F.;
Grenier, D. Inflammation 2008, 31, 313–318.
2
(
(
2) De Mello, J. F.; Delle Monache, F.; Goncalves de Lima, O.; Marini-
Bettolo, G. B.; Delle Monache, G.; Lyra, F. D. A.; Fernandes de
Albuquerque, M. M.; Leoncio de Albuquerque, I. ReV. Inst. Antibiot.
(
31) Lee, Y. L.; Hong, C. Y.; Kok, S. H.; Hou, K. L.; Lin, Y. T.; Chen,
M. H.; Wang, C. C.; Lin, S. K. J. Endodon. 2009, 35, 206–211.
32) Irwin, C. R.; Myrillas, T. T. Oral. Dis. 1998, 4, 43–47.
33) Takahashi, K.; Takashiba, S.; Nagai, A.; Takigawa, M.; Myoukai, F.;
Kurihara, H.; Murayama, Y. J. Periodontol. 1994, 65, 147–153.
1
973, 13, 37–46.
(
(
(
3) De Mello, J. F.; Goncalves de Lima, O.; Fernandes de Albuquerque,
M. M.; Marini-Bettolo, G. B.; Lyra, F. D. A.; Cavalcanti de Silva, E.;
Coelho, J. S. B.; Lins de Olivera, L. ReV. Inst. Antibiot. 1974, 14,
3
9–50.
(34) Ishimi, Y.; Miyaura, C.; Jin, C. H.; Akatsu, T.; Abe, E.; Nakamura,
Y.; Yamaguchi, A.; Yoshiki, S.; Matsuda, T.; Hirano, T. J. Immunol.
1990, 145, 3297–3303.
(35) Graves, D. T.; Cochran, D. J. Periodontol. 2003, 74, 391–401.
(36) Assuma, R.; Oates, T.; Cochran, D.; Amar, S.; Graves, D. T.
J. Immunol. 1998, 160, 403–409.
(37) Smith, P. C.; Munoz, V. C.; Collados, L.; Oyarzun, A. D. J. Periodont.
Res. 2004, 39, 87–92.
(38) Ejeil, A. L.; Igondjo-Tchen, S.; Ghomrasseni, S.; Pellat, B.; Godeau,
G.; Gogly, B. J. Periodontol. 2003, 74, 188–195.
(39) Nichols, T. C.; Fischer, T. H.; Deliargyris, E. N.; Baldwin, A. S., Jr.
Ann. Periodontol. 2001, 6, 20–29.
(40) Clark, I. M.; Swingler, T. E.; Sampieri, C. L.; Edwards, D. R. Int.
J. Biochem. Cell Biol. 2008, 40, 1362–1378.
(41) La, V. D.; Bergeron, C.; Gafner, S.; Grenier, D. J. Periodontol. 2009,
80, 1875–1882.
(
4) Avila, H. P.; Smania, E. F. A.; Delle Monache, F.; Smania, A., Jr.
Bioorg. Med. Chem. 2008, 16, 9790–9794.
5) Paster, B. J.; Boches, S. K.; Galvin, J. L.; Ericson, R. E.; Lau, C. N.;
Levanos, V. A. J. Bacteriol. 2001, 183, 3770–3783.
(
(
(
(
6) Nishihara, T.; Koseki, T. Periodontology 2000 2004, 36, 14–26.
7) Feng, Z.; Weinberg, A. Periodontology 2000 2006, 40, 50–76.
8) Kinane, D. F.; Lappin, D. F. Acta Odontol. Scand. 2001, 59, 154–
1
60.
9) Madianos, P. N.; Bobetsis, Y. A.; Kinane, D. F. J. Clin. Periodontol.
005, 32, 57–71.
10) Henderson, B.; Poole, S.; Wilson, M. Microbiol. ReV. 1996, 60, 316–
41.
11) Bidault, P.; Chandad, F.; Grenier, D. Can. Dent. Assoc. 2007, 73, 721–
25.
12) Horz, H. P.; Conrads, G. Expert ReV. Anti Infect. Ther. 2007, 5, 703–
15.
(
2
(
(
(
(
3
7
7
(42) Bremner, P.; Heinrich, M. J. Pharm. Pharmacol. 2002, 54, 453–472.
(43) Genovese, S.; Epifano, F.; Curini, M.; Dudra-Jastrzebska, M.; Luszczki,
J. J. Bioorg. Med. Chem. Lett. 2009, 19, 5419–5422.
13) Kolenbrander, P. E.; Andersen, R. N.; Blehert, D. S.; Egland, P. G.;
Foster, J. S.; Palmer, R. J., Jr. Microbiol. Mol. Biol. ReV. 2002, 66,
4
86–505.
(
44) Gilchrist, E. P.; Moyer, M. P.; Shillitoe, E. J.; Clare, N.; Murrah, V. A.
(
(
14) Kornman, K. S. Clin. Infect. Dis. 1999, 28, 520–526.
Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodon. 2000, 90,
15) La, V. D.; Howell, A. B.; Grenier, D. J. Dent. Res. 2009, 88, 627–
3
40–347.
6
32.
(
45) La, V. D.; Labrecque, J.; Grenier, D. Phytother. Res. 2009, 23, 1449–
452.
(
(
(
16) Cazalis, J.; Bodet, C.; Gagnon, G.; Grenier, D. J. Periodontol. 2008,
9, 1762–1768.
17) Rosenberger, C. M.; Finlay, B. B. Nat. ReV. Mol. Cell Biol. 2003, 4,
85–396.
18) Dewhirst, F. E.; Moss, D. E.; Offenbacher, S.; Goodson, J. M. J.
Periodont. Res. 1983, 18, 156–163.
1
7
(
(
46) Darveau, R. P.; Hancock, R. E. J. Bacteriol. 1983, 155, 831–838.
47) Rovera, G.; Santoli, D.; Damsky, C. Proc. Natl. Acad. Sci. U. S. A.
3
1
979, 76, 2779–2783.
NP100547B