Synthesis and evaluation of antibacterial and anti-inflammatory properties of naturally occurring coumarins

Article abstract of DOI:10.1016/j.phytol.2015.08.008

Coumarins are a group of heterocyclic compounds naturally present in a large variety of plant families. Nevertheless, oxyprenylated coumarins have been only recently seen as valuable and promising biologically active phytochemicals. In this study, we synthesized three naturally occurring O-prenylcoumarins (1), (2), and (3), and evaluated their antibacterial and anti-inflammatory properties in view of their therapeutic potential against periodontal disease. The three O-prenylcoumarins were synthesized using well-known schemes leading to the chromen-2-one nucleus. The periodontal pathogen Porphyromonas gingivalis was found to be highly susceptible to all three O-prenylcoumarins with minimal inhibitory concentration values in the range of 12.5-25 mg/ml; the non-prenylated forms of the coumarins did not show any activity. The antibacterial activity of (1), (2), and (3) appeared to result from its ability to permeate the cell membrane. Using the U937-3xkB-LUC human monocytic cell line, compounds (2) and (3) dose-dependently inhibited lipopolysaccharide-induced NF-kB activation, while (1) did not. The non-prenylated forms of the coumarins were either inactive or much less potent. In conclusion, O-prenylcoumarins (2) and (3) by exhibiting a dual mode of action including antibacterial and anti-inflammatory activities may represent promising targeted therapeutic agents for localized treatment of periodontal diseases.

Full text of DOI:10.1016/j.phytol.2015.08.008

Phytochemistry Letters 13 (2015) 399–405
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Synthesis and evaluation of antibacterial and anti-inflammatory
properties of naturally occurring coumarins
a
b
b
b
Jabrane Azelmat , Serena Fiorito , Vito Alessandro Taddeo , Salvatore Genovese ,
b
a,
Francesco Epifano , Daniel Grenier *
Oral Ecology Research Group, Faculty of Dentistry, Université Laval, Quebec City, Quebec, Canada
a
b
Department of Pharmacy, University “G. D’Annunzio” of Chieti-Pescara, Chieti, Italy
A R T I C L E I N F O
A B S T R A C T
Article history:
Coumarins are a group of heterocyclic compounds naturally present in a large variety of plant families.
Nevertheless, oxyprenylated coumarins have been only recently seen as valuable and promising
biologically active phytochemicals. In this study, we synthesized three naturally occurring O-
prenylcoumarins (1), (2), and (3), and evaluated their antibacterial and anti-inflammatory properties
in view of their therapeutic potential against periodontal disease. The three O-prenylcoumarins were
synthesized using well-known schemes leading to the chromen-2-one nucleus. The periodontal
pathogen Porphyromonas gingivalis was found to be highly susceptible to all three O-prenylcoumarins
with minimal inhibitory concentration values in the range of 12.5–25 mg/ml; the non-prenylated forms
of the coumarins did not show any activity. The antibacterial activity of (1), (2), and (3) appeared to result
from its ability to permeate the cell membrane. Using the U937-3xkB-LUC human monocytic cell line,
compounds (2) and (3) dose-dependently inhibited lipopolysaccharide-induced NF-kB activation, while
Received 30 March 2015
Received in revised form 3 August 2015
Accepted 4 August 2015
Available online xxx
Keywords:
Antibacterial
Anti-inflammatory
Coumarin
Periodontal disease
(
1) did not. The non-prenylated forms of the coumarins were either inactive or much less potent. In
conclusion, O-prenylcoumarins (2) and (3) by exhibiting a dual mode of action including antibacterial and
anti-inflammatory activities may represent promising targeted therapeutic agents for localized
treatment of periodontal diseases.
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
therapeutic drugs with dual antibacterial and anti-inflammatory
properties represents a valuable adjunctive therapy to control this
Periodontal diseases are common chronic inflammatory dis-
orders in adults. More specifically, approximately 5–15% of the
population is affected by severe forms of the disease (Pihlstrom
et al., 2005; Burt, 2005). If left untreated, periodontal diseases may
result in tooth loss and systemic complications, such as diabetes,
cardiovascular diseases and preterm low birth weight babies
disease.
Coumarins are a group of heterocyclic compounds naturally
present in a large variety of plant families. Numerous biological
activities, including antimicrobial, anti-inflammatory, anti-cancer
and antioxidant properties, have been demonstrated in coumarins
and their derivatives (Borges et al., 2005). Since the discovery of
the first coumarin more than 200 years ago (Vogel, 1820), a huge
number of coumarins and analogues have been either isolated or
synthesized. Until the last two decades, less attention has been
dedicated to oxyprenylated coumarins. However, they have been
recently re-considered as valuable and promising biologically
active phytochemicals. In this study, we synthesized three
coumarin derivatives (1), (2), and (3) (Fig. 1) and evaluated their
antibacterial and anti-inflammatory properties in view of their
therapeutic potential against periodontal disease. 4-Isopenteny-
loxy-5-methylcoumarin (1) has been first isolated in 1973 from
Gerbera crocea Kuntze and Gerbera serrata Druce (sin. G.
asplenifolia) (Fam. Asteraceae) (Bohlmann et al., 1973), 6-isopen-
tenyloxy-7-methoxycoumarin (2) has been obtained in 1979 from
(Pizzo et al., 2010). Specific Gram-negative anaerobic bacteria,
including Porphyromonas gingivalis, that colonize the periodontal
pocket are the primary etiologic factor of periodontal diseases
(Holt and Ebersole, 2005; Berezow and Darveau, 2011). However,
the continuous and excessive host inflammatory response to these
pathogens that results in the secretion of cytokines and matrix
metalloproteinases modulates periodontal tissue destruction (Liu
et al., 2010). Given this complex etiopathogenesis, the use of
*
Corresponding author at: Oral Ecology Research Group, Faculty of Dentistry,
Universite Laval, 2420 Rue de la Terrasse, Quebec City, Quebec G1V 0A6, Canada.
Fax: +1 418 656 2861.
E-mail address: Daniel.Grenier@greb.ulaval.ca (D. Grenier).
http://dx.doi.org/10.1016/j.phytol.2015.08.008
1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

4
00
J. Azelmat et al. / Phytochemistry Letters 13 (2015) 399–405
procedure from commercially available 2-methoxyhydroquinone
(
8) and 3,3-diethoxyethyl propionate (9) that were let to react in
ꢀ
the presence of H
3
PO
4
85% for 2 h at 100 C followed by
crystallization to yield pure 6-hydroxy-7-methoxycoumarin (10).
This latter was then prenylated by the usual way (Scheme 2).
Compound (3) has been synthesized from commercially available
3
-methoxycatechol (11) that has been submitted to a Pechmann
reaction with propiolic acid (12) in the presence of catalytic
ꢀ
amounts of conc. H
2
SO
4
at 120 C. The so obtained 8-hydroxy-7-
methoxycoumarin (13) was then prenylated in the usual way as
described above (Scheme 3).
The three synthesized O-prenylcoumarins were first tested for
their antibacterial activities against two strains of P. gingivalis, a
major causative pathogen of chronic periodontitis (Holt and
Ebersole, 2005; Berezow and Darveau, 2011). This Gram negative
anaerobic bacterium was found to be highly susceptible to all three
O-prenylcoumarins with MIC and MBC values in the range of 12.5–
Fig. 1. Structure of coumarins (1), (2), and (3).
Haplophyllum pedicellatum Bunge ex Boiss (Fam. Rutaceae) (Aby-
shev and Gashimov, 1979), and 8-isopentenyloxy-7-methoxycou-
marin (3) has been extracted in ‘80s from Artemisia carvifolia Besser
25
m
g/ml (Table 1). Doxycycline used as reference molecule
g/ml and a MBC of 12.5 g/ml. Compounds
showed a MIC of 0.78
(7), (10), and (13), the non-prenylated forms of (1), (2), and (3)
respectively, did not show any antibacterial activity against P.
m
m
(
Barua et al., 1980), Artemisia laciniata Willd., Artemisia armeniaca
Lam., Artemisia tanacetifolia Georgi (Szabo et al., 1985), Melampo-
dium divaricatum DC. (Fam. Asteraceae) (Borges-del-Castillo et al.,
gingivalis at concentrations up to 200 mg/ml (data not shown). To
determine whether the antibacterial activity of compounds (1), (2),
and (3) was specific to P. gingivalis, we also tested their effect on
two additional oral bacterial species Fusobacterium nucleatum and
Streptococcus mutans. As reported in Table 1, all three O-
prenylcoumarins were poorly active with MIC and MBC values
1
984), Coleonema calycinum (Steud.) I. Williams (Gray et al., 1986),
Flindersia australis (Fam. Rutaceae) (Reisch and Podpetschnig,
987), F. Muell., and Cyperus incompletus Boeckeler (Fam.
Cyperaceae) (Dini et al., 1993).
1
>200
mg/ml in most cases.
2. Results and discussion
To investigate the antibacterial mode of action of the three O-
prenylcoumarins on P. gingivalis, we evaluated their ability to
1
The three O-prenylcoumarins (Fig. 1) have been synthesized
permeate the bacterial cell membrane using the SYTOX Green
using well-known schemes leading to the chromen-2-one nucleus.
In particular, compound (1) has been synthesized starting from
commercially available m-cresol (4) and Meldrum’s acid (5) that
dye, which penetrates damaged cell envelope and react with DNA.
As reported in Table 2, after the addition of (1), (2), (3) or ethanol
used as positive control, an increase in fluorescence was observed
following a 60 min exposure, indicating permeabilization of the
cell membrane of P. gingivalis (ATCC 33277). The effect of the three
O-prenylcoumarins was more pronounced when they were used at
a concentration corresponding to 4-fold the MIC value.
ꢀ
were made to react under solvent-free conditions for 24 h at 120 C,
yielding the monoester (6), that in turn was cyclised in the
ꢀ
presence of a catalytic amount of conc. H
2
SO
4
at 120 C for 5 h to
provide the coumarin (7). The synthesis of (1) was then finalized by
alkylation of the OH group with 3,3-dimethylallyl bromide in
Therapeutic approaches that inhibit inflammatory mediator
production by immune cells represent an interesting strategy for
controlling inflammatory diseases such as periodontal diseases
ꢀ
acetone at 80 C for 1 h in the presence of K
2
CO
3
as the base
(
Scheme 1). Compound (2) has been obtained by a two-step
(Souza et al., 2012). The transcription factor NF-kB is activated by a
ꢀ
ꢀ
ꢀ
Scheme 1. Reagents and conditions: (a) 120 C, 24 h; (b) H
2
SO
4
conc. (cat.), 120 C, 5 h; (c) 3,3-dimethylallyl bromide (1 eq.), acetone, K
2
CO
3
(2 eq.), 80 C, 1 h.

J. Azelmat et al. / Phytochemistry Letters 13 (2015) 399–405
401
ꢀ
ꢀ
Scheme 2. Reagents and conditions: (a) H
3
PO
4
, 100 C, 2 h; (b) crystallization (c) 3,3-dimethylallyl bromide (1 eq.), acetone, K
2
CO
3
(2 eq.), 80 C, 1 h.
ꢀ
ꢀ
Scheme 3. Reagents and conditions: (a) H
2
SO
4
conc., 120 C, 2 h; (b) 3,3-dimethylallyl bromide (1 eq.), acetone, K
2
CO
3
(2 eq.), 80 C, 1 h.
wide variety of stimuli including bacterial pathogens and has many
target genes comprising genes encoding cytokines, adhesion
molecules, and matrix metalloproteinases (Kumar et al., 2004).
NF-kB is thus a central player in inflammatory diseases such as
periodontitis and inhibition of its activation should be considered
as a promising therapeutic strategy (Gupta et al., 2010). In the
present study, we used the U937-3xkB-LUC cell line to evaluate the
ability of compounds (1), (2), or (3) to inhibit activation of the NF-
kB signaling pathway induced by LPS. To exclude the possibility
that cell toxicity might have been responsible for any decrease in
NF-kB activation, the cytotoxic effect of (1), (2), and (3) on U937-
cytotoxic effect was observed after a 6 h treatment of the cells with
the compounds at concentrations up to 50 g/ml (data not shown).
Fig. 2 shows that Aggregatibacter actinomycetemcomitans LPS
induced activation of NF-kB signaling pathway, while the O-
prenylcoumarins had no effect. Compounds (2) and (3) dose-
dependently inhibited LPS-induced NF-kB activation, while (1) did
m
not. More specifically, at a concentration of 12.5 mg/ml, com-
pounds (2) and (3) decreased LPS-induced NF-kB activation by
94.7% and 48.9%, respectively. Given that the transcription factor
NF-kB controls the expression of a large array of genes involved in
inflammation (Kumar et al., 2004), compounds (2) and (3) by
inhibiting NF-kB activation show potential to reduce the host
3xkB-LUC cell line was evaluated using an MTT test. No detectable

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J. Azelmat et al. / Phytochemistry Letters 13 (2015) 399–405
Table 1
3
. Experimental
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration
(
MBC) values of compounds (1), (2), and (3).
3
.1. Chemistry
Strain
(1)
(2)
(3)
Referencea
MICb MBCc MIC MBC MIC MBC MIC MBC
All starting materials were obtained from Sigma–Aldrich
Chemical Co. (Milano, Italy), and all solvents were analytical
P. gingivalis ATCC
25
25
25
25
12.5 25
25
25
0.78 12.5
1
13
3
3277
grade. H and C NMR spectra were recorded on a Bruker AC 200
1
13
P. gingivalis W83
F. nucleatum ATCC
12.5 12.5 12.5 12.5 0.78 12.5
100 0.78 50
( H NMR, 200 MHz; C NMR, 50.32 MHz). CDCl
solvent and tetramethylsilane as an internal standard. Chemical
shifts are reported in (ppm). Reactions were routinely monitored
3
was used as the
>200 >200 >200 >200 100
2
5586
d
S. mutans ATCC 35668 >200 >200 >200 >200 >200 >200 0.05 1.56
by thin layer chromatography (TLC) using Merck silica gel F254
plates. Melting points were measured on a Büchi melting point
apparatus and are uncorrected. Gas chromatography (GC) analysis
was performed on a Hewlett-Packard gas chromatograph, model
a
Doxycycline was used as reference antibiotic for P. gingivalis and F. nucleatum
while penicillin G was used for S. mutans.
b
MIC in
MBC in
m
m
g/ml.
g/ml.
c
5
890, equipped with a flame ionisation detector (FID) and coupled
to an electronic integrator. The chromatograph was fitted with a
methyl silicone column (12.5 m  0.25 mm, 0.25 mm film thick-
ness). GC analytical conditions were as follows: carrier gas, He
Table 2
Effect of compounds (1), (2), and (3) on permeabilization of P. gingivalis ATCC 33277
cells, as determined using the SYTOX Green Dye. Compounds were used at the MIC
as well as four-fold the MIC value. Ethanol was used as positive control.
1
(
purity > 99.8%); flow rate, 0.9 ml/min; and injector and detector
ꢀ
ꢀ
temperatures, 280 C and 250 C, respectively. The oven tempera-
Conditionsa
Relative fluorescence units (x103)
ꢀ
ꢀ
ture was programmed from 50 to 270 C at a rate of 10 C/min.
Quantitative data were obtained by electronic integration of FID
area data without the use of response factor correction. GC/MS
analysis was performed using a Hewlett-Packard 6890 chromato-
graph combined with HP ChemStation Software and equipped
with an HP 5973 mass selective detector. Operation conditions
were as follows: carrier gas, He (purity > 99.8%); ionisation voltage,
70 eV; scanning speed, 1 s over 40–300 amu range; and ion source
0
min
60 min
Positive control
254 Æ 3
369 Æ 3
22 Æ 3
(
(
(
(
(
(
1) MIC
1) four-fold MIC
2) (MIC)
2) four-fold MIC
3) MIC
3) four-fold MIC
0
6 Æ 4
236 Æ 13
33 Æ 5
0
0
0
0
122 Æ 7
89 Æ 17
371 Æ 7
ꢀ
temperature, 180 C. The column and conditions of use were the
a
Background values of fluorescence were substracted.
same as reported above. Elemental analyses were carried out on a
Carlo Erba 1106 elemental analyser.
inflammatory response associated with periodontal diseases.
Compounds (7) and (10), the non-prenylated forms of (1) and
3
.1.1. Compound 6 (3-(3-methylphenoxy)-3-oxopropanoic acid)
(
2) respectively, did not attenuate the LPS-induced NF-kB
activation while compound (13) was much less active than (3)
Fig. 3).
From the data reported in this study in regard to the comparison
A solvent-free mixture of Meldrum’s acid (5) (5.0 mmol) and m-
ꢀ
cresol (4) (7.0 mmol) was made to react at 120 C for 24 h. After
(
warming, the reaction mixture was added to a 10% solution of
NaHCO
phases were discarded, while the aqueous solution was then
acidified to pH 1 with 10% HCl and then extracted with CH Cl
3
(20 ml) and extracted with EtOAc (3 Â 20 ml). The organic
of the antibacterial and anti-inflammatory activities of prenylated
coumarins and parent unprenylated compounds, it appears that
the linkage of a terpenyl side chain to the benzochromene core
markedly improves the pharmacological effects. The addition of
either a geranyloxy or a 3,3-dimethylallyloxy moiety not only
increases the lipophylicity of each molecule, thus allowing it to
better permeate the cell membrane, but it may allow the
2
2
(
3 Â 20 ml). The collected organic phases were dried and evapo-
rated under vacuum. The desired product was obtained as waxy
white solid (m.p. 51–53 C) in 88% yield. All analytical data were
ꢀ
identical to those already reported for the same compound
(Matsui, 1957).
compound to trigger
a specific target in the endocellular
environment leading to the herein reported activities. Prenylation
of secondary metabolites having a phenylpropanoid, polyketide, or
alkaloid cores is a common biosynthetic reaction occurring in
nature, mainly in bacteria, fungi, and plants (Kuzuyama et al.,
3
.1.2. Compound 7 (4-hydroxy-5-methyl-2H-chromen-2-one)
The monoester (6) (3.0 mmol) was added of 3 drops of conc.
ꢀ
H
5
2
SO
4
and the resulting mixture was made to react at 120 C for
h. After warming, the latter was diluted with H
2
O (20 ml) and
2005). In most cases, the addition of an isoprenoid side chain to an
extracted with EtOAc (3 Â 20 ml). The collected organic phases
oxygen or to a nitrogen atom leads to a pharmacologically more
efficient compound than the parent non-prenylated one. One of
the clearest examples of this phenomenon was described by
Kretzschmar et al. (2010), who reported that prenylated genistein
and naringenin were able to exert a higher estrogenic effect than
their respective parent compounds. Such a phenomenon have been
also observed with coumarins and other structurally related
phenylpropanoids and polyketides (Bruyere et al., 2011; Genovese
et al., 2015).
While periodontal disease is initiated by a specific group of
Gram negative bacteria, tissue and bone destruction is mainly
modulated by an uncontrolled inflammatory response (Berezow
and Darveau, 2011; Liu et al., 2010). O-prenylcoumarins (2) and (3)
by exhibiting a dual mode of action including antibacterial and
anti-inflammatory activities, may represent promising targeted
therapeutic agents for localized treatment of periodontal diseases.
were dried and evaporated under vacuum. The desired product
ꢀ
was obtained as a white solid (m.p. 230–232 C) in 71% yield. All
analytical data were identical to those already reported for the
same compound (Padwal et al., 2011).
3
.1.3. Compound 1 (5-methyl-4-[(3-methylbut-2-enyl) oxy]-2H-
chromen-2-one)
The same general procedure for the prenylation of phenyl-
propanoid nuclei as already reported was followed (Curini et al.,
2
003). The desired product was obtained as a yellowish solid (m.p.
ꢀ
123–125 C) in 92% yield. All analytical data were identical to those
already reported for the same compound (Bohlmann et al., 1973).
3
.1.4. Compound 10 (6-hydroxy-7-methoxy-2H-chromen-2-one)
A solution of 2-methoxyhydroquinone (8) and 3,3-diethoxye-
ꢀ
thylpropionate (9) in conc. H
PO
3 4
was well stirred for 2 h at 100 C.

J. Azelmat et al. / Phytochemistry Letters 13 (2015) 399–405
403
Fig. 2. Effect of compounds (1), (2), or (3) on A. actinomycetemcomitans LPS-induced NF-kB activation using the U937-3xkB-LUC cell line model. . . . , significant increase
compared to non-stimulated cells. *, significant decrease compared to non-treated cells.
The resulting mixture was then poured into icy water and the
3.1.6. Compound 13 (8-hydroxy-7-methoxy-2H-chromen-2-one)
The same general procedure for the Pechmann reaction as
already reported was followed (Curini et al., 2003). The desired
product was obtained as a brownish solid (m.p. 169–171 C) in 38%
yield. Analytical data were identical to those obtained for a
commercial sample used for comparison.
resulting yellow solid formed filtered under vacuum. The desired
ꢀ
product was obtained (m.p. 230–232 C) in 62% yield. Analytical
ꢀ
data were identical to those obtained for a commercial sample
used for comparison.
3
.1.5. Compound 2 (6-[(3-methylbut-2-enyl)oxy]-7-methoxy-2H-
3
.1.7. Compound 3 (7-methoxy-8-[(3-methylbut-2-enyl) oxy]-2H-
chromen-2-one)
chromen-2-one)
The same general procedure for the prenylation of phenyl-
propanoid nuclei as already reported was followed (Curini et al.,
2
The same general procedure for the prenylation of phenyl-
propanoid nuclei as already reported was followed (Curini et al.,
2
003). The desired product was obtained as a brownish solid (m.p.
003). The desired product was obtained as a yellowish solid (m.p.
ꢀ
165–168 C) in 89% yield. All analytical data were identical to those
ꢀ
211–214 C) in 78% yield. All analytical data were identical to those
already reported for the same compound (Abyshev and Gashimov,
979).
already reported for the same compound (Abyshev and Gashimov,
979).
1
1

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J. Azelmat et al. / Phytochemistry Letters 13 (2015) 399–405
Fig. 3. Effect of compounds (7), (10), or (13) on A. actinomycetemcomitans LPS-induced NF-kB activation using the U937-3xkB-LUC cell line model. . . . , significant increase
compared to non-stimulated cells. *, significant decrease compared to non-treated cells.
3.2. Stock solutions of coumarins
3.4. Broth microdilution assay for determination of minimal inhibitory
concentrations and minimal bactericidal concentrations
Stock solutions of the tested compounds were prepared
ꢀ
in dimethyl sulfoxide at 5 mg/ml and kept at 4 C protected from
light.
Minimal inhibitory concentrations (MICs) and minimal bacte-
ricidal concentrations (MBCs) were determined using a broth
microdilution assay. Overnight bacterial cultures were diluted in
fresh broth medium to obtain an optical density at 660 nm (OD660
of 0.2. Equal volumes (100 l) of bacteria and two-fold serial
dilutions (from 400 g/ml) of compounds (1), (2), or (3) in culture
)
3
.3. Bacteria and growth conditions
m
m
P. gingivalis (ATCC 33277 and W83), F. nucleatum (ATCC 25586),
medium were mixed into the wells of a flat-bottomed 96-well
microplate. Control wells with no bacteria or with solvent alone
were also prepared. Doxycycline and penicillin G were used as
and S. mutans (ATCC 35668) were used in this study. Bacteria were
routinely grown in Todd Hewitt broth (THB; BD-Canada, Mis-
sissauga, ON, Canada) supplemented with 0.001% hemin and
0
ꢀ
reference compounds. After an incubation of 24 h at 37 C, bacterial
ꢀ
.0001% vitamin K, and cultures were incubated at 37 C in an
growth was recorded visually. The MIC values (mg/ml) of the tested
2 2 2
anaerobic chamber (N :H :CO /80:10:10).
compounds were determined as the lowest concentration at which

J. Azelmat et al. / Phytochemistry Letters 13 (2015) 399–405
405
no growth occurred. To determine the MBC values (
m
g/ml),
wells at room temperature. Luminescence was recorded using the
luminometer option of a Synergy 2 microplate reader (BioTek
Instruments) within 3 min after substrate addition. Moreover,
following a treatment of U937-3xkB-LUC with the tested com-
pounds for 6 h, an MTT (3-[4,5-diethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide) assay was performed according to the
manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany)
to determine the effect of the compound on the cell viability. All
assays were performed in triplicate and the means Æ standard
deviations were calculated.
aliquots (5 l) of each well showing no visible growth were spread
m
on blood-supplemented THB agar plates, which were incubated for
ꢀ
3
days at 37 C. The MBC values were determined as the lowest
concentration at which no colony formation occurred. The MIC and
MBC values were determined in three independent experiments to
ensure reproducibility.
3.5. Membrane permeabilization assay
The ability of compounds (1), (2), and (3) to permeate P.
gingivalis cells was evaluated using the SYTOX Green dye (Life
Technologies Inc., Burlington, ON, Canada), which binds to nucleic
acid of bacteria once the bacterial membrane is compromised.
Briefly, 1.25 mM of SYTOX Green dye was added to P. gingivalis cells
suspended in 10 mM HEPES (4-(2-hydroxyethyl) piperazine-1-
Acknowledgements
We are grateful to R. Blomhoff and H. Carlsen (University of
Oslo, Norway) for providing the U937-3xkB-LUC cell line. This
study was supported by the Laboratoire de Contrôle Micro-
biologique de l’Université Laval. The authors report no conflicts of
interest related to this study.
ethanesulfonic acid) pH 7.0 to an OD660 of 0.4. Aliquots of 100 ml
were added to wells of a 96-well black microplate prior to adding
compound (1), (2), or (3) at concentrations corresponding to the
MIC value and to 4-fold the MIC value. The incubation was carried
out in a microplate reader (Synergy 2; BioTek Instruments,
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