Cytotoxic phenolic compounds in leaf buds of Populus tremuloides

Article abstract of DOI:10.1139/V09-151

Phytochemical investigations of the EtOH extract of Populus tremuloides leaf buds led to the isolation of 19 phenolic compounds. Among them, (1S,2S)-1-[4-0-E-coumaroyl-β-D-glucopyranosyloxy]cyclohexanediol was reported for the first time, and its structur

Full text of DOI:10.1139/V09-151

104
Cytotoxic phenolic compounds in leaf buds of
Populus tremuloides
´
Andre Pichette, Azadeh Eftekhari, Patricia Georges, Serge Lavoie,
Vakhtang Mshvildadze, and Jean Legault
Abstract: Phytochemical investigations of the EtOH extract of Populus tremuloides leaf buds led to the isolation of 19
phenolic compounds. Among them, (1S,2S)-1-[4-O-E-coumaroyl-b-D-glucopyranosyloxy]cyclohexanediol was reported for
the first time, and its structure was determined by spectroscopic (NMR and MS) and chemical methods. Seventeen of the
isolated compounds were tested for their cytotoxicity against lung carcinoma (A549) and colorectal adenocarcinoma
(DLD-1) human cell lines. Antibacterial activity was also evaluated against Escherichia coli and Staphylococcus aureus.
Key words: quaking aspen, coumarate, flavonoids, NMR.
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Resume : Une etude phytochimique d’un extrait ethanolique des bourgeons de feuilles du Populus tremuloides a permis
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d’isoler 19 composes phenoliques, dont le (1S,2S)-1-[4-O-E-coumaroyl-b-D-glucopyranosyloxy]cyclohexanediol, un produit
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qui n’a jamais ete rapporte anterieurement et dont la structure a ete determinee par des methodes chimiques et spectrosco-
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piques (RMN et spectrometrie de masse). Les proprietes cytotoxiques de 17 de ces produits on ete evalue contre des li-
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gnees de cellules humaines de carcinome du poumon (A549) et d’adenocarcinome (DLD-1). Leur activite antibacterienne
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a aussi ete evalue contre l’Escherichia coli et le Staphylococcus aureus.
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¨
Mots-cles : peuplier faux-tremble, coumarate, flavonoıdes, RMN.
EtOH and EtOH/H2O under reflux. After evaporation of
EtOH in vacuo, the aqueous phase was successively parti-
tioned with hexane and n-BuOH. The n-BuOH soluble ex-
tract was purified on an open Diaion1 column with a
gradient of decreasing polarity, and three fractions were ob-
tained. Each fraction was investigated for in vitro cytotoxic
and antibacterial biological activities. Cytotoxic activity
evaluations were carried out on human lung cancer (A549),
human colorectal cancer (DLD-1), and normal skin fibro-
blasts (WS1) using the resazurin reduction test as previously
described in the literature.17 Antibacterial activity was eval-
uated against Escherichia coli and Staphylococcus aureus.
The results (Table 1) show that the last fraction C was found
to exert a weak cytotoxic activity against A549 (IC50, 96
7 mg mL–1) and DLD-1 (IC50, 89 6 mg mL–1), but was in-
active toward bacterial cell lines. Thus, bioassay-guided
fractionation of fraction C was undertaken with a combina-
tion of different chromatographic techniques leading to the
isolation of a new compound 1 together with 18 known
compounds: chaenomeloidin (2),18 prunin (3),19 echinaticin
(4),20 echinacin (5),21 tremulacin (6),22 salicine (7),23 tremu-
loidin (8),24 genkwanin (9),25 rhamnocitrin (10),26 sakurane-
tin (11),27 acacetin (12),28 kaempferide (13),29 aromadendrin
(14),30 phenylmethyl coumarate (15),31 phenethyl p-couma-
rate (16),32 cinnamyl coumarate (17),33 phenylmethyl caf-
feate (18),34 and trans-ferulic acid (19)35 (Fig. 1). Known
compounds were identified by comparison of their spectro-
scopic data with the values found in the literature. NMR
spectroscopic data for phenethyl p-coumarate (16), which
was also isolated from buds of P. tremuloides, were not
available. Therefore, complete 1H and 13C NMR spectro-
scopic data for 16 are also reported.
Introduction
The buds exudates of many plant species of Populus ge-
nus are known as raw material processed by bees into prop-
olis.1 The latter product has been widely used in popular
medicine as antibacterial,2–4 anti-inflammatory,5 antioxi-
dant,6,7 and cytostatic treatments.8 The biological activity of
propolis samples is mainly due to phenolic compounds like
flavonoids, aromatic acids, and diterpenic acids,9,10 which
are the principal constituents of the buds of Populus spe-
cies.11–13 Recently, studies in the northern-type propolis
showed a potential source of biologically active substances
in Populus tremuloides,14 which are widely spread across
North America.15 In spite of its use as ointment by Amerin-
dian traditional medicine to treat numerous diseases, such as
coughs, colds, and irritated nostrils,16 few studies were car-
ried out on the medicinal applications of P. tremuloides. In
the present study, the isolation and structure elucidation of a
new phenolic compound from P. tremuloides Michaux,
along with 18 known products are described.
Results and discussion
The leaf buds of Populus tremuloides were extracted with
Received 16 July 2009. Accepted 28 September 2009. Published
on the NRC Research Press Web site at canjchem.nrc.ca on
15 January 2010.
A. Pichette,1 A. Eftekhari, P. Georges, S. Lavoie,
V. Mshvildadze, and J. Legault. Chaire de recherche sur les
´
´
agents anticancereux d’origine naturelle, Departement des
´
´
`
sciences fondamentales, Universite du Quebec a Chicoutimi,
QC G7H 2B1, Canada.
The molecular formula (C21H28O9) of 1, a white amor-
phous powder, was determined from its HR-ESI-MS spec-
1Corresponding author (e-mail: andre_pichette@uqac.ca).
Can. J. Chem. 88: 104–110 (2010)
doi:10.1139/V09-151
Published by NRC Research Press

Pichette et al.
Table 1. In vitro cytotoxicity and antibiotic results of the Diaion1 column’s fractions.
105
IC50 (mg mL–1)a
MICb
Samples
A549
>100
>100
96 7
2.8 0.5
NTd
DLD-1
>100
>100
89 6
2 1
WS1
>100
>100
80 10
>50
S. aureus
>100
>100
>100
NTd
E. coli
>100
>100
>100
NTd
Fraction A
Fraction B
Fraction C
Etoposidec
Chloramphenicolc
NTd
NTd
>5
0.37 0.06
Note: Mean values ( standard deviation) for triplicate assays.
aConcentration of extract that caused 50% inhibition of cell proliferation.
bMinimum concentration of extract that resulted in inhibition of visible growth.
cPositive control.
dNot tested.
Fig. 1. Polyphenols from Populus tremuloides.
Published by NRC Research Press

106
Can. J. Chem. Vol. 88, 2010
1
Table 2. H (400 MHz) and 13C (100 MHz) NMR spectroscopic
trum (positive-ion mode) on the basis of a quasimolecular
ion peak at m/z 447.1623 [M + Na]+ (calcd. 447.1631). In-
frared absorption bands at 3328, 1602, 1160, 982, and
833 cm–1 suggested the presence of hydroxyl groups, aro-
matic system, and an ester carbonyl groups. 13C NMR spec-
trum displayed 19 carbon signals (Table 2) separated by
DEPT spectrum into five methylenes, seven aliphatic oxy-
methines, four unsaturated methines, and three quaternary
carbons (one for an ester carbonyl). Among them, six reso-
data for compound 1 in methanol-d4.
Position dC (multiplicity)a
dH (multiplicity, J in Hz)
3.87 (m)
3.85 (m)
1
2
3
79.4 (d)
71.0 (d)
31.5 (t)
1.79 (m)
1.55 (m)
4
5
6
22.4 (t)
23.1 (t)
27.6 (t)
1.65 (m)
1.34 (m)
1.71 (m)
1.31 (m)
1
nances could be assigned to a sugar moiety. H NMR spec-
trum confirmed the presence of a hexose moiety with
1
anomeric proton at dH 4.45 (1H, d, J = 7.8 Hz). H NMR
1.82 (m)
spectrum also shows the presence of two trans-olefinic pro-
tons at dH 7.66 (1H, d, J = 15.9 Hz) and dH 6.37 (1H, d, J =
15.9 Hz) and a 1,4-disubstituted aromatic ring with four pro-
tons at dH 7.47 (2H, d, J = 8.6 Hz) and 6.81 (2H, d, J =
8.6 Hz). HMBC correlations at dH 7.47 (H-2@, H-6@) and dC
147.3 (H-7@), dH 6.81 (H-3@, H-5@) and dC 161.5 (C-4@), and
dH 7.66 (H-7@) and dC 168.6 (C-9@) suggested the presence
of a coumaroyl moiety. Analysis of the COSY, HSQC, and
HMBC spectra led to the identification of a third aglycone
sytem: the cyclohexane-1,2-diol. HMBC correlation between
the methine proton at dH 4.87 (H-4’) and the carbonyl group
at dC 168.6 (C-9@) suggested the linkage between the glu-
cose and the coumaroyl moiety. Finally, the correlation be-
tween dH 4.45 (H-1’) and dC 79.4 (C-1) indicated the
linkage site of the glucose moiety to the aglycone.
1.63 (m)
1’
2’
3’
4’
5’
6’
102.2 (d)
75.1 (d)
75.6 (d)
72.5 (d)
76.1 (d)
62.4 (t)
4.45, (d, 7.8)
3.35 (dd, 9.3, 7.8)
3.65 (t, 9.3)
4.87 (m)
3.53 (m)
3.62 (dd, 15.0, 5.4)
3.54 (m)
1@
127.2 (s)
131.3 (d)
116.9 (d)
161.5 (s)
147.3 (d)
114.8 (d)
168.6 (s)
2@, 6@
3@, 5@
4@
7@
8@
7.47 (d, 8.6)
6.81 (d, 8.6)
7.66 (d, 15.9)
6.37 (d, 15.9)
9@
Acidic hydrolysis of 1 and TLC analysis of the aqueous
phase afforded identification of glucose as the sugar compo-
nent. Absolute configuration of the glucose as D was deter-
mined by optical rotations in comparison with authentic
standard. The presence of p-coumaric acid in the organic
phase was confirmed by TLC in comparison with authentic
standard. Cyclohexane-1,2-diol was also detected in the or-
ganic phase using GC–MS and NMR analysis.36 The absolute
configuration of cyclohexane-1,2-diol could be determined
directly from the organic phase, since the other aglycon part,
namely, p-coumaric acid, is optically inactive. The organic
phase showed positive value in optical activity measurement
meaning that (1S,2S)-cyclohexane-1,2-diol has been iso-
lated.37 The structure of 1 was thus confirmed as (1S,2S)-1-
[4-O-E-coumaroyl-b-D-glucopyranosyloxy]cyclohexanediol.
Compound 16 has been identified by many authors, but
surprisingly, no complete NMR assignation was given.38
aMultiplicities were deduced from DEPT experiments.
1
Table 3. H (400 MHz) and 13C (100 MHz) NMR spectroscopic
data for compound 16 in methanol-d4.
Position
1
2, 6
3, 5
4
7
8
9
1’
2’, 6’
3’, 5’
4’
7’
8’
dC (multiplicity)a
127.3 (s)
130.0 (d)
115.9 (d)
157.7 (s)
144.6 (d)
115.5 (d)
167.4 (s)
137.9 (s)
128.9 (d)
128.5 (d)
126.6 (d)
35.2 (t)
dH (multiplicity, J in Hz)
7.42 (d, 8.1)
6.85 (d, 8.1)
7.62 (d, 15.9)
6.29 (d, 15.9)
7.26 (m)
7.32 (m)
7.25 (m)
3.02 (t, 6.9)
4.42 (t, 6.9)
1
Therefore, complete H and 13C characterization was accom-
1
plished using H, 13C, and 2D spectra (Table 3). First, the
same p-coumaroyl moieties as in 1 were identified with dH
at 6.29 (1H, d, J = 15.9 Hz, H-8), 6.85 (2H, d, J = 8.1 Hz,
H-3 and H-5), 7.42 (2H, d, J = 8.1 Hz, H-2 and H-6), and
7.62 (1H, d, J = 15.9 Hz, H-7) and dC at 115.5 (C-8), 115.9
(C-3 and C-5), 127.3 (C-1), 130.0 (C-2 and C-6), 144.6
(C-7), 157.7 (C-4), and 167.4 (C-9). Additionally, five over-
65.0 (t)
aMultiplicities were deduced from DEPT experiments.
spectra (1H, 13C, DEPT, and HSQC) and comparison with
literature allowed the identification of these components as
rhamnocitrin (10)29 and kaempferide (13).26 Moreover, the
biological results were obtained using commercial pure
products. Because of the low isolated yields of compounds
3 and 19, those compounds were also tested from commer-
cial materials.
1
lapped H NMR signals between dH 7.20–7.30 along with
two methylene triplets at 3.02 (2H, t, J = 6.9 Hz, H-7’) and
4.42 (2H, t, J = 6.9 Hz, H-8’) were attributed to a phenethyl
moiety. The HMBC correlation between H-8’ and C-9 con-
firmed the link between the phenethyl and the p-coumaroyl
groups.
All isolated compounds, except compounds 2 and 4, were
evaluated using resazurin reduction test for their cytotoxicity
against human lung cancer (A549), human colorectal cancer
Compounds 10 and 13 were isolated as a mixture. Separa-
tion of each constituent was not performed due to their
small amounts. Therefore, careful examination of NMR
Published by NRC Research Press

Pichette et al.
107
(DLD-1), and normal skin fibroblasts (WS1).17 Results pre-
sented in Table 4 are expressed as the concentration of prod-
uct inhibiting cell growth by 50% (IC50). Etoposide was used
as positive control with IC50 of 2.8 and 2.0 mmol/L against
A549 and DLD-1 cell lines, respectively. The phenolic com-
pounds were regarded as active when the IC50 was smaller
than 100 mmol/L.39 The compound 9 was found to be the
most active with IC50 ranging from 5.8 to 9.2 mmol/L. More-
over, compounds 10, 12, and 15 were moderately active
against cancer cells with IC50 ranging from 19 to 37 mmol/L.
In contrast to compounds 9, 12, and 15, compound 10 was
significantly selective toward cancer cells with IC50 of
31 mmol/L for A549 and 37 mmol/L for DLD-1 in comparison
with 87 mmol/L for normal cells, WS1. Although the cytotox-
icity of compounds 9 and 12 were known on A549 cells,40,41
the activity on human colorectal adenocarcinoma DLD-1 was
never reported. Finally, compounds 13 and 18 were found
weakly cytotoxic, and all the other compounds tested were in-
active. As far as structure–activity relationships are con-
cerned, these in vitro results suggest that the addition of a
double bond in C-2 position in molecule 9, with regard to
compound 11, increases the cytotoxic activity. Similarly, the
presence of a methoxy group in C-7 position and a hydroxyl
in C-4’ position in the flavone 9 seem to have a beneficial ef-
fect on the cytotoxic activity in comparison with the acacetin
(12), where the inversion of these groups reduces the activity.
On the other hand, the presence of hydroxyl group in R2 of
compounds 10 and 13 is detrimental for the activity in com-
parison with compounds 9 and 12, respectively. In the case
of compounds 15–19, only molecules bearing a benzyl group
exhibited cytotoxicities (15 and 18). Moreover, the hydroxyl
group in R1 of compound 18 reduces significantly the cyto-
toxicity in comparison with 15. All compounds were also
evaluated for their antibacterial activities against S. aureus
and E. coli, but no significant activity was observed.
(ppm) units relative to TMS as an internal standard, and
coupling constants were given in Hz. Preparative HPLC
was performed on an Agilent 1100 liquid chromatography
system, equipped with a solvent delivery system, an auto-
sampler and a UV-MWD detector. Samples were eluted in
an Intertsil prep-ODS column C18 (20 Â 250 mm; 10 mm)
at room temperature with a flow rate of 10 mL min–1. GC–
MS analyses were performed with an instrument (Agilent
Technologies 6890N) fitted with a mass-selective detector
(Agilent Technologies 5973), a split–splitless injection port,
and an apolar capillary column DB-5MS (30 m Â
0.25 mm  0.25 mm).
Analytical thin-layer chromatography (TLC) was per-
formed with silica gel 60 F254, 0.25 mm pre-coated TLC
´
plates (Silicycle, Quebec, Canada). Flash column chroma-
tographies (CC) were performed on silica gel (40–63 mm
´
with indicator F254, Silicycle, Quebec, Canada) and on C18
reversed-phase silica gel (carbon 11%, 40–69 mm, Silicycle,
´
Quebec, Canada). Polyamide CC-6 was purchased from
Macherey-Nagel (Germany) and Diaion HP-20 from Su-
pelco. Detection of the phenolic compounds was carried out
by spraying TLC plates with polyethylene glycol (NP/PEG)
reagent followed by heating at 110 8C and detected by UV
absorption at 254 and 365 nm. TLC identification of mono-
saccharides was performed with CH2Cl2/MeOH/H2O
(50:25:5) solvent system. The compounds were visualized
by spraying an orthophosphoric acid solution of naphtoresor-
cinol 5% in EtOH, followed by heating at 110 8C.
The commercial samples used for biological tests,
namely, prunin (3), kaempferide (13), and trans-ferulic acid
(19), were purchased from Indofine Chemical Company
(USA). Rhamnocitrin (10) was purchased from Apin Chem-
icals Ltd. (UK).
Plant material
Leaf buds of P. tremuloides Michaux were collected in
the boreal forest to the south of Chicoutimi, Quebec, Can-
ada, in April 2006. Samples were identified by Patrick Na-
Concluding remarks
´
In conclusion, the structure of a new compound 1 was de-
scribed and 19 compounds were identified from P. tremu-
loides. Among them, compounds 2 and 5 were reported for
the first time in Populus genus and compounds 3, 4, 9, 10,
12, 16, and 17 for the first time in P. tremuloides. Com-
pound 9 was found to be the most cytotoxic against lung
carcinoma cell (A549) and colorectal adenocarcinoma
(DLD-1) human cell lines. Interestingly, compound 10 was
selective toward both cancer cell lines in comparison with
normal cells. Finally, all compounds tested do not possess
antibacterial activity.
´
´
deau (Departement des sciences fondamentales, Universite
´
`
du Quebec a Chicoutimi). A voucher specimen (QFA-
A540466) was deposited at the Herbarium Louis-Marie of
´
´
Universite Laval, Quebec, Canada.
Extraction and isolation
The buds of P. tremuloides (1 kg) were exhaustively ex-
tracted with EtOH (3 L, 60 8C, three times, 2 h each time)
followed by EtOH/H2O (7:2). The extracts were filtered and
pooled. After evaporation of EtOH in vacuo, the aqueous
phase was extracted successively with hexane (500 mL Â
5) and saturated n-BuOH with H2O (500 mL Â 5). The
n-BuOH phase was decanted and evaporated in vacuo. The
residue (80 g) was fractionated using an open Diaion1 col-
umn eluted with H2O/MeOH with 30%, 50%, and 80% of
MeOH. Three fractions were obtained: A (6.46 g), B
(7.24 g), and C (58.96 g).
Fraction C was purified on silica gel CC, eluted with
CHCl3/MeOH gradient (60:1 ? 5:1, v/v), and three fractions
were obtained: C1, C2, and C3.
Fraction C1 (16.73 g) was subjected to silica gel using a
gradient of CHCl3/MeOH (90:1?60:1, v/v) as eluent. Sub-
fraction C1.1 (384 mg), obtained from CHCl3/MeOH
Experimental
General
Optical rotations were measured with an automatic polar-
imeter Rudolph Research Analytical Autopol IV. FTIR spec-
tra were recorded with a PerkinElmer SpectrumOne. High
resolution electrospray ionization mass spectrum was con-
ducted in positive mode with an Applied Biosystems/MDS
Sciex QSTARXL QqTOF MS system. 1D and 2D NMR
spectra (1H–1H COSY, HSQC, and HMBC) were performed
using an Avance 400 Bruker spectrometer equipped with a
5 mm QNP-probe. Chemical shifts were expressed in d
Published by NRC Research Press

108
Table 4. In vitro cytotoxicity results of isolated compounds
Can. J. Chem. Vol. 88, 2010
C3.5 (482 mg) was separated by HPLC using a gradient of
MeOH/H2O (10:90 ? 100:0) to give 5 (21 mg) and 7 (35 mg).
(1–19).
IC50 (mmol/L SD)a
(1S,2S)-1-[4-O-E-coumaroyl-b-D-
Compounds
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
A549
>100
NTb
DLD-1
>100
NTb
WS1
>100
NTb
>100
NTb
glucopyranosyloxy]cyclohexanediol (1)
White amorphous powder. [a]2D5 –35.38 (c 1.0, MeOH). IR
(neat) nmax: 3328, 2935, 1696, 1602, 1160, 1080, 1024, 982,
>100
NTb
>100
NTb
1
and 833 cm–1. H and 13C NMR spectroscopic data: see Ta-
ble 2. HR-ESI-MS m/z: 447.16225 [M + Na]+ (calcd. for
>100
>100
>100
81 3
9 3
31 2
>100
27 3
60 10
>100
19 2
>100
>100
45.8 0.9
>100
2.8 0.5
>100
>100
>100
>100
9.2 0.9
37 3
>100
23 6
>100
>100
19.2 0.9
>100
>100
39 3
>100
2 1
42 4
>100
>100
>100
5.8 0.3
87 3
>100
20 2
42 5
>100
26 3
>100
>100
51 7
>100
>50
C21H28O9Na: 447.16310).
Acid hydrolysis of 1
Compound 1 was dissolved in HCl 10% and heated at
110 8C for 4 h. The resulting hydrolysate was extracted
with CHCl3. The organic phase was dried (MgSO4) and the
solvent evaporated under reduced pressure. The presence of
p-coumaric acid was confirmed with standard sample on
TLC (CHCl3/MeOH 10:1 as eluent and developing with
NP/PEG reagent). The presence of cyclohexane-1,2-diol in
the organic phase ([a]D + 3.4) was confirmed with a GC–
MS analysis: Injector temperature, 250 8C; ionization volt-
age, 70 eV (EI-MS); column temperature, 40 8C for the ini-
tial 2 min followed by an increase of 15 8C min–1 up to
350 8C; carrier gas, He; column flow rate, 1 mL min–1. Cy-
clohexane-1,2-diol was detected at Rt 7.55 min. The aqueous
phase was neutralized with N,N-dioctylmethylamine (10% in
CHCl3), and the solvents were evaporated under reduced
pressure. The residue contained the monosaccharide
D-glucose ([a]D + 24.8).
16
17
18
19
Etoposidec
Note: Mean values ( standard deviation) for triplicate assays.
aConcentration that caused 50% inhibition of cell proliferation.
bNot tested.
cPositive control.
Cell lines and culture conditions
(90:1), was separated on silica gel CC with CHCl3/MeOH
(80:1) as eluent, to give three fractions: C1.1A, C1.1B, and
C1.1C. Subfraction C1.1A was purified by preparative
HPLC with a gradient elution of MeOH/H2O (50:50 ?
85:15, v/v) yielding compounds 15 (249 mg), 16 (5 mg),
and 17 (41 mg). Subfraction C1.1B (2.36 g) was applied
successively on a silica gel CC and a reversed-phase CC us-
ing MeOH/H2O gradient (50:50 ? 70:30, v/v) as eluent to
give 11 (160 mg). Compound 9 (3 mg), 12 (18 mg), and a
mixture of 10 and 13 (27 mg) were isolated after a silica
gel CC eluted with CHCl3/MeOH (90:1) and a preparative
HPLC (isocratic CH3CN/H2O 40:60) of fraction C1.2. Com-
pound 18 (50 mg) was obtained from fraction C1.3
(294 mg) after repeated silica gel CC (CHCl3/MeOH, 75:1)
and polyamide flash column (MeOH/H2O, 50:50 ? 75:25).
Fraction C2 was chromatographed on silica gel CC with a
gradient elution of CHCl3/MeOH (75:1 ? 15:1, v/v) to give
eight fractions: C2.1–C2.8. Subfraction C2.7 (558 mg) was
separated by preparative HPLC using an isocratic mobile
Lung carcinoma (A549), colorectal adenocarcinoma
(DLD-1), and normal skin fibroblast (WS1) human cell lines
were obtained from the American Type Culture Collection
(ATCC). All cell lines were cultured in minimum essential
medium containing Earle’s salts and L-glutamine (Mediatech
Cellgro, VA) to which were added 10% fetal bovine serum
(Hyclone), vitamins (1X), penicillin (100 IU mL–1) and
streptomycin (100 mg mL–1), essential amino acids (1X),
and sodium pyruvate (1X) (Mediatech Cellgro, VA). Cells
were kept at 37 8C in a humidified environment containing
5% CO2.
Cytotoxicity assay
Exponentially growing cells were plated in 96-well micro-
plates (BD Falcon) at a density of 5 Â 103 cells per well in
100 mL of culture medium (DMEM with 10% SVF) and
were allowed to adhere for 24 h before treatment. Increasing
concentrations of each compound in MeOH or DMSO were
then added (100 mL per well), and the cells were incubated
for 48 h. The final concentration of MeOH or DMSO in the
culture medium was maintained at 0.25% (v/v) to avoid sol-
vent toxicity. Cytotoxicity was assessed using resazurin17 on
an automated 96-well Fluoroskan Ascent F1TM plate reader
(Labsystems) using excitation and emission wavelengths of
530 and 590 nm, respectively. Fluorescence was propor-
tional to the cellular metabolic activity in each well. Sur-
vival percentage was defined as the fluorescence in
experimental wells compared to that in control wells after
subtraction of blank values. Each experiment was carried
phase of CH3CN/H2O/HCOOH (40:60:1) to afford
2
(19 mg) and 14 (3 mg).
Fraction C3 (5.4 g) was purified on silica gel using a gra-
dient of CHCl3/MeOH (25:1 ? 7:1) for elution to give five
subfractions: C3.1–C3.5. C3.2 was separated on silica gel CC
with CHCl3/MeOH (20:1) giving 6 (289 mg). Some purifica-
tions on different silica gel CC of subfraction C3.2.1 permit-
ted to obtain 19 (2 mg). Subfraction C3.4 (418 mg) was
separated by preparative HPLC using an isocratic mobile
phase of CH3CN/H2O (30:70) to afford compounds 1
(20 mg), 3 (8 mg), 4 (14 mg), and 8 (37 mg). Subfraction
Published by NRC Research Press

Pichette et al.
109
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Antibacterial assays
Antibacterial activity was evaluated using the microdilu-
tion method42 but with some modifications: exponentially
growing bacteria were plated in 96-well flat bottom micro-
plates (BD Flacon) at a density of 5 Â 103 Gram-negative
E. coli (ATCC 25922) or 40 Â 103 Gram-positive S. aureus
(ATCC 25923) per well in 100 mL nutrient broth (Difco).
The concentration of ethanol in the culture medium was
maintained at 0.25% (v/v) to avoid solvent toxicity. Fifty
microliters of 4% resazurin was added to each well, and the
microplates were incubated for 6 h at 37 8C. Fluorescence
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Ascent FlTM plate reader (Labsystems) using excitation and
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Acknowledgement
Keinan, E. Chem. Ind. 1986, 713.
¨
¨
(21) Gu¨venalp, Z., Ozbek, H., Unsalar, T., Kazaz, C., Demirezer,
´ ´
The financial support of Fonds Quebecois de la Re-
¨
L. O. Turk. J. Chem. 2006, 30, 391
cherche sur la Nature et les Technologies (FQRNT, fonds
forestiers 02) is gratefully acknowledged. The authors wish
to thank Carole Grenon for the HPLC separations, Catherine
Dussault and Maxime Lebrun for biological assays. Finally,
the authors also thank Dr Richard Menini for his corrections
and helpful comments on this manuscript
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