Diterpenes from the leaves of Croton zambesicus.
ABSTRACT Two new trachylobane- and one isopimarane-type diterpenoids: ent-18-hydroxy-trachyloban-3-one; ent-trachyloban-3-one; isopimara-7,15-dien-3beta-ol, were isolated from the leaves of Croton zambesicus, together with trans-phytol, beta-sitosterol, alpha-amyrin and stigmasterol. The structures were determined by extensive NMR techniques and X-ray analysis. The cytotoxicity of these compounds has been evaluated on cancer and non-cancer cell-lines.
Article: Bioactive Natural Products from Two Sudanese Medicinal Plants Diospyros mespiliformis and Croton zambesicus[show abstract] [hide abstract]
ABSTRACT: Phytochemical investigations were performed in two plant species used in Sudanese traditional medicines to treat different illnesses, Diospyros mespiliformis and Croton zambesicus. The investigations revealed compounds of triterpenes (lupane series), one trihydroxyflavone and one diterpene. The compounds have been isolated and identified using various chromatographic and spectroscopic techniques. These were lupeol (1), betulinic acid (2), betulin (3) and lupenone (4) from Diospyros mespiliformis. Compounds 1, 2, 3 in addition to diterpene ent -kaurane-3β, 16β, 17-triol (5) and vitexin (6) were re-isolated from Croton zambesicus. However,compound 5 and 6 were isolated for the first time from this source.The pure isolated compounds and semi-synthesized acetates 1Ac, 2Ac and 3Ac, which were prepared from compounds 1, 2 and 3 respectively, were subjected to two bioassays: α- glucosidase enzyme inhibition assay and antioxidant activity. Compounds, 1, 1Ac, 3 and 4 showed a marked α-glucosidase inhibitory potential, while compound 6 exhibited strong antioxidant activity.Records of Natural Products. 01/2009;
Diterpenes from the leaves of Croton zambesicus
Sebastien Blocka, Chiara Baccellia, Bernard Tinantb, Luc Van Meerveltc,
Raoul Rozenbergd, Jean-Louis Habib Jiwand, Gabriel Llabre ` se,
Marie-Claire De Pauw-Gilletf, Joelle Quetin-Leclercqa,*
aLaboratoire de Pharmacognosie, Unite ´ CHAM, Universite ´ Catholique de Louvain, UCL 72.30-CHAM, Av. E. Mounier,
72, 1200 Bruxelles, Belgium
bUnite ´ CSTR, De ´partement de Chimie, UCL, Place Pasteur 1, 1348 Louvain-la-Neuve, Belgium
cBiomolecular Architecture, Chemistry Department, K.U. Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium
dLaboratoire de Spectrome ´trie de Masse, UCL, Place Pasteur 1, 1348 Louvain-la-Neuve, Belgium
eDe ´partement de Physique Expe ´rimentale, Universite ´ de Lie `ge, Alle ´e du 6 Aou ˆt, 17, 4000 Lie `ge, Belgium
fCRCE, Histologie-Cytologie Universite ´ de Lie `ge, Rue de Pitteurs, 20, 4020 Lie `ge, Belgium
Received 9 December 2003; accepted 17 February 2004
Two new trachylobane- and one isopimarane-type diterpenoids: ent-18-hydroxy-trachyloban-3-one; ent-trachyloban-3-one;
isopimara-7,15-dien-3b-ol, were isolated from the leaves of Croton zambesicus, together with trans-phytol, b-sitosterol, a-amyrin
and stigmasterol. The structures were determined by extensive NMR techniques and X-ray analysis. The cytotoxicity of these
compounds has been evaluated on cancer and non-cancer cell-lines.
# 2004 Elsevier Ltd. All rights reserved.
Keywords: Croton zambesicus; Euphorbiaceae; Diterpene; Trachylobane; Pimarane; Cytotoxicity
Croton zambezicus Muell. Arg. (Euphorbiaceae) (Syn.
C. amabilis Muell. Arg., C. gratissimus Burch.) is a
shrub or small tree reaching 10 m in height. It’s a Gui-
neo-Congolese species widespread in Tropical Africa
(Adjanohoun et al., 1989). The leaf decoction is used in
Benin as anti-hypertensive, anti-microbial (urinary
infections) and to treat fever associated with malaria
(Adjanohoun et al., 1989; Watt and Breyer-Brandwikj,
1962). The genus Croton is well known for its diterpe-
noid content and a lot of different types of diterpenes
(phorbol esters, clerodane, labdane, kaurane, trachylo-
bane, pimarane, etc.) have been isolated from this genus.
There is very little literature concerning the phyto-
chemical study of Croton zambesicus although if this plant
is widely used in African traditional medicine. Labdane,
clerodane and trachylobane diterpenes have been
identified in the stem bark of Croton zambesicus
(Ngadjui et al., 2002). Recently we have identified a new
cytotoxic trachylobane diterpene from the leaves of C.
zambesicus (Block et al., 2002). In order to continue our
investigations on the composition of the cytotoxic
dichloromethane extract of the leaveswe have isolated and
characterised two new trachylobane and one isopimarane
2. Results and discussion
HSCCC separation of the dichloromethane extract
from the leaves of C. zambesicus gave 21 fractions.
These fractions were further purified by MPLC. From
these fractionations, we isolated five diterpenes: ent-tra-
chyloban-3b-ol (Block et al., 2002), ent-18-hydroxy-tra-
chyloban-3-one (1), isopimara-7,15-dien-3b-ol (2), ent-
trachyloban-3-one (3) and trans-phytol (4) together with
a-amyrin and sterols: b-sitosterol and stigmasterol. All
these compounds were isolated for the first time from
the leaves of C. zambesicus.
0031-9422/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
Phytochemistry 65 (2004) 1165–1171
E-mail address: firstname.lastname@example.org (J. Quetin-Leclercq).
author. Tel.:+32-2-7647254;fax: +32-2-
Compound 1 was isolated as a white crystal, whose
molecular formula, C20H30O2, was established by HR-
EIMS. Infrared absorptions at 3520 and 1702 cm?1
provided evidence of respectively hydroxyl and carbonyl
groups. The presence of a cyclopropane ring was
deduced from the1H NMR spectrum that exhibits two
signals at ?H 0.62 and 0.87 ppm (H-12 and H-13
respectively) and by the13C NMR spectrum that shows
signals at ?C20.4 (C-12), 24.2 (C-13), 22.5 (C-16) ppm.
From these observations and comparison with NMR
data from closely related structures (Block et al., 2002;
Midiwo et al., 1997; Hasan et al., 1982; Arnone et al.,
1979; Leong et al., 1997) we could conclude that com-
pound 1 belongs to the trachylobane series of diterpene.
The presence of the carbonyl group was confirmed by
the signal at ?C219.1 ppm in the13C spectrum. The
primary alcohol was revealed by the two doublets at ?H
3.63 and 3.37 ppm in the1H spectrum and by the signal
at ?C66.8 ppm in the13C spectrum (Fig. 1).
Long range1H-13C correlations (HMBC) between the
three protons at ?H0.99 (Me-19) and the13C NMR
signal at ?C66.8 and X-ray analysis supported the C-18
position for the hydroxyl group. The position of the
ketone at C-3 was also deduced from HMBC corre-
lations between the protons at ?H2.22 (H-2a), 2.62 (H-
2b), 0.99 (Me-19), 3.63 (H-18a), 3.37 (H-18b) and the
ketonic carbon at ?C219.1. The full1H and13C NMR
assignments were established with HMQC correlations.
X-ray crystallographic analysis was conducted to con-
firm the structure of 1. All the naturally-occurring tra-
chylobane diterpenes isolated so far belong to the
enantio series and comparison with other ent-trachylo-
banes confirms the ent-configuration of 1 (Block et al.,
2002; Midiwo et al., 1997; Hasan et al., 1982; Arnone et
al., 1979; Leong et al., 1997). 1 is then identified as
Compound 2 was isolated as a white solid with a
molecular composition of C20H32O as inferred from
HR-EIMS. The IR spectrum showed absorption bands
for a hydroxyl group (3314 cm?1) and for a mono-sub-
stituted double bond (3099, 1639, 909 cm?1). The com-
bined analysis of the
revealed the presence of 20 carbon signals assigned to
four methyls, seven methylenes, five methine among
which one tertiary alcohol and two olefinic carbons and
four quaternary carbons. The occurrence in the
spectrum of three dd at ?H5.80 (J=10.8 and 17.6 Hz,
H-15), at ?H4.93 (J=1.6 and 17.6 Hz, H-16B) and at ?H
4.87 (J=1.6 and 10.8 Hz, H-16A) associated with the
presence of a broad doublet at ?H5.37 (J=3.5 Hz, H-7)
and four singlets corresponding to methyl groups sug-
gested a pimarane type skeleton (Lago et al., 2000). The
position of the alcohol on the skeleton of the pimarane
was determined as C-3 by the HMBC correlation
between the proton at ?H3.26 (H-3) and the13C NMR
signals ?C27.4 (C-2) and 37.8 (C-4) and by the1H–1H
13C NMR and DEPT spectra
Fig. 1. View and atom labelling of one molecule from the asymmetric
unit of 1 (Spek, 1998).
1166S. Block et al./Phytochemistry 65 (2004) 1165–1171
COSY correlation between the protons ?H3.26 (H-3)
and 1.62 (H-2). The equatorial position of the hydroxyl
group at C-3 was deduced by the observation of the
coupling constants of the dd at 3.26 (J=4.7 and 10.9
Hz, H-3a). The position of the double bound between
C-7 and C-8 was defined by the1H-1H COSY correl-
ation between the protons ?H5.37 (H-7) and ?H1.97
(H-6). The full
established with HMQC correlations. The stereo-
chemistry at C-13 was established by comparison of the
13C NMR chemical shifts of C-15, C-16 and C-17 with
those of isopimarane diterpenoids, showing an equator-
ial position for the Me-17 and an axial position for the
vinyl group (Beier, 1978; Wenkert and Buckwalter,
1972; Rasoamiaranjanahary et al., 2003; Polonsky et al.,
1970; Anjaneyulu et al., 2003; Lago et al., 2000). Com-
pound 2 was finally identified as isopimara-7,15-dien-
3b-ol. This compound has already been synthesised
from virescenol A (Polonsky et al., 1970; Ceccherelli et
13C NMR assignments were
al., 1985) and isolated from the leaves of Guarea macro-
phylla (Lago et al., 2000) but this is the first pimarane-
type diterpene isolated from C. zambesicus. Moreover,
comparison of NMR data of 2 with those reported by
Lago (Lago et al., 2000) shows very good agreement
excepted for the chemical shifts of C-2 (27.4) and C-6
(23.1) that are inverted. Our assignments were con-
firmed by the HMBC correlation between the proton at
?H3.26 (H-3) and the13C NMR signal ?C27.4 (C-2) and
by the1H–1H COSY correlations between the protons
?H3.26 (H-3) and 1.62 (H-2) and between the protons
?H5.37 (H-7) and ?H1.97 (H-6) and by comparison with
closely related structure (Ansell et al., 1993; Meragel-
man et al., 2003; Aiyar et al., 1969, 1971). Complete13C
and1H NMR assignments of 2 are presented in Table 2.
Compound 3 was isolated as a colourless oil. Its
molecular formula was determined as C20H30O by HR-
EIMS analysis. Compound 3 contained a carbonyl
group as inferred from the IR absorption band at 1706
cm?1. The trachylobane skeleton of this compound was,
as compound 1, identified by the presence of the cyclo-
propane ring signals in the1H (?H0.61 and 0.85 ppm
respectively for H-12 and H-13) and13C (?C20.5, 24.2
and 22.5 ppm respectively for C-12, C-13 and C-16)
13C and1H NMR spectroscopic data for diterpenes 1 and 3 in CDCl3.
13C NMR at 125 MHz for 1 and 100 MHz for 3.1H NMR at 500
MHz for 1 and 400 MHz for 3. Chemical shifts are given in ppm;
multiplicities and coupling constant J (in parentheses) in Hz
NMR assignments of compound 2 in CDCl3(13C at 100 MHz and1H
at 400 MHz). Chemical shifts are given in ppm; multiplicities and
coupling constant J (in parentheses) in Hz
3.26 dd (4.7, 10.9)
1.16 dd (4.9, 11.9)
5.37 bd (3.5)
5.80 dd (10.8, 17.6)
4.87 dd (1.6, 10.8)
4.93 dd (1.6, 17.6)
S. Block et al./Phytochemistry 65 (2004) 1165–11711167
NMR spectra. The presence of the carbonyl was
deduced from the signal at ?C217.5 ppm on the13C
spectrum and the position on C-3 was deduced from
HMBC spectra showing correlation between the pro-
tons at ?H2.55 (H-2b) and 1.05 (H-18) and the carbon
at ?C217.5. The stereochemistry of 3 was based on bio-
synthetic considerations (all natural trachylobanes iso-
lated up to now belong to the enantio series) and on
comparison of spectral data from 1 and closely related
compounds (Kapingu et al., 2000; Ngouela et al., 1998;
Arnone et al., 1979; Leong et al., 1997). 3 is then iden-
tified as ent-trachyloban-3-one. In order to complete the
study on the cytotoxic activity of the dichloromethane
extract of C. zambesicus, the isolated diterpenes, a-
amyrin and sterols were tested in vitro against cancer
(HeLa, HL-60) and non-cancer (WI-38) cell lines. The
results are presented in Table 3.
The biological activities of trachylobane diterpenes
are poorly known but recently we have shown that
ent-trachyloban-3b-ol possesses cytotoxic activities on
HeLa cells (IC50on HeLa cells=7.3 mg/ml). The cyto-
toxicities of compounds 1 and 3 are a little bit lower but
no clear specificity between cell lines could be observed
even if 3 is 2.5 more active on HeLa cells (cancer cell
line) than on WI-38 (non-cancer cell line).
Different biological properties have been described for
various pimarane derivatives, including antimicrobial
and spasmolytic (Vlietinck, 1987), antihypertensive
(Ohashi et al., 2000), antituberculosis (Ulubelen et al.,
1997), antifungal (Rasoamiaranjanahary et al., 2003)
and antiinflammatory described as international patent
(Suh et al., 1999). Studies have also demonstrated that
pimarane derivatives inhibited the tumor-promoting
effect of TPA (12-O-tetradecanoylphorbol 13-acetate)
and were slightly cytotoxic (Minami et al., 2002; Chang
et al., 2000) suggesting an interesting cancer chemopre-
ventive potential. The results obtained on the cytotoxi-
city of compound 2 confirm the weak cytotoxic activity
of pimarane diterpenes. In comparison to the other
diterpenes, trans-phytol (4) shows a similar range
of activity than trachylobanes. The cytotoxic activity of
phytol is due to an induction of apoptosis (Komiya et
al., 1999). Finally, in agreement with literature data
(Chaturvedula et al., 2002; Awad et al., 2000; Mogha-
dasian, 2000), b-sitosterol, a-amyrin and stigmasterol
were not cytotoxic at the tested concentrations (IC50>
30 mg/ml on every cell lines).
High Speed Counter-Current Chromatography was
performed on a HSCCC Kromaton III, SEAB. An
Omnifit glass column (OM 6427 15?750 mm) packed
with Lichroprep Si 60 (15–25 mM, Merck) was used for
MPLC. Analytical TLC was performed on precoated
silica gel 60 F254 plates (Merck) and detection was
achieved by spraying with sulfuric anisaldehyde, fol-
lowed by heating 5 min at 105?C. The IR spectra were
recorded on a Perkin Elmer FTIR 286. The optical
rotation values were obtained on a Perkin-Elmer 241
spectropolarimeter in CH2Cl2solution. UV spectra were
photometer. NMR spectra of compounds 2 and 3 were
recorded on a Bruker Avance DRX-400 spectrometer in
CDCl3at 400 MHz (1H) and 100 MHz (13C), at 25?C.
NMR spectra of compound 1 were recorded on a Bru-
ker Avance 500 at 500 MHz (1H) and 125 MHz (13C); d
in ppm rel. to Me4Si (internal standard). HR-EIMS was
recorded at 70 eV in an AutoSpec 6 F mass spectro-
meter and EIMS at 70 eV on a Finnigan TSQ7000 triple
quadrupole; m/z (rel. intensity in%).
3.2. Plant material
The aerial parts of C. zambesicus were collected in the
surroundings of Cotonou (Benin) in December 2000
and identified by botanist Prof. V. Adjakidje (Universite ´
d’Abomey-Calavi-Benin). A voucher specimen has been
deposited at the herbarium of the Belgian national
botanical garden at Meise (BR S.P. 848.108).
3.3. Extraction and isolation
percolated at room temperature with dichloromethane
to give 34 g of extract. Part of this extract (5 g) was
fractionated by HSCCC using the two phases solvent
system heptane–acetonitrile–dichloromethane (10:7:3)
(descending mode, mobile phase: lower phase, flow rate:
2 ml/min, fraction collection: 4 min/tube, rotation: 500
rpm, volume of column: 1000 ml). 21 fractions (F1–
F21) were obtained. F6 (315 mg) was separated by
MPLC on silicagel 60 (15–25 mM) eluted with Tol–
CH3CN (93:7) giving 6 fractions (F61–F66). Fraction
Cytotoxicity data for compounds 1–4a
aResults are expressed as mean of IC50values (mg/ml)?SEM of
three independent experiments.
bHeLa, human cervix carcinoma; HL-60, human promyelocytic
leukemia; WI-38, non-cancer human lung fibroblast.
1168 S. Block et al./Phytochemistry 65 (2004) 1165–1171
F65 (59.4 mg) was finally purified by MPLC on silicagel
60 (15–25 mM) eluted with Tol–EtOAc–CH3CN (91:8:1)
to give compound 1 (20 mg). Fraction F9 (350.6 mg)
was separated by MPLC on silicagel 60 (15–25 mM)
eluted with Tol–EtOAc (98:2) giving 8 fractions (F91–
F98). Fraction F94 (49.5 mg) was purified by MPLC on
silicagel 60 (15–25 mM) with Tol–EtOAc (96:4) as
mobile phase to afford compound 2 (14 mg). Fraction
F12 contained ent-trachyloban-3b-ol, previously identi-
fied in the plant (Block et al., 2002). Fraction F14 (256
mg) was applied to MPLC on silicagel 60 (15–25 mM)
eluted with Tol–EtOAc (93:8). 7 fractions (F141–F147)
were obtained. Fraction F142 was purified by MPLC on
silicagel 60 (15–25 mM), using Tol–EtOAc (90:10) as
mobile phase to give trans-phytol (4) (8 mg). Fraction
F144 was purified by MPLC on silicagel 60 (15–25 mM),
Tol–EtOAc (92:8) was used as mobile phase and 25 mg
of compound 3 were obtained. F17 gave a-amyrin and
b-sitosterol. F18 gave stigmasterol.
3.4. ent-18-Hydroxy-trachyloban-3-one (1)
White crystals [CH2Cl2]. ? ½ ?22
UV lmaxnm (log ?): 218 (2.23); IR ?NaCl
(OH), 2988, 2928, 2859, 1702 (C¼O), 1460, 1444, 1417,
1380, 1256, 1209, 1164, 1094, 1082, 1047, 1011, 975,
844, 757;1H and13C are given in Tables 1; EI-MS 70 eV
m/z (rel. int.): 302 [M]+?(30), 284 [M?H2O]+(45), 272
(48), 269 (26), 257 (16), 246 (7), 215 (6), 201(5), 187 (4),
185 (2), 159 (1), 145 (1), 107 (3), 105 (17), 93 (22), 91
(42), 81 (36), 79 (83), 55 (100). HR-EIMS m/z: 302.2249
[M]+?(calc. for C20H30O2302.2246).
D: ?77?(CH2Cl2, c 0.1);
3.5. Isopimara-7,15-dien-3?-ol (2)
Amorphous powder. ? ½ ?22
lmaxnm (log ?): 226 (2.61); IR ?NaCl
2953, 2926, 2968, 1669, 1463, 1378, 1366, 1002;1H and
13C are given in Table 2; EI-MS 70 eV m/z (rel. int.): 288
[M]+?(5), 273 [M–CH3]+(8), 270 [M?H2O]+(4), 255
(26), 245 (9), 227 (7), 213 (9), 200 (17), 185 (19), 171
(19), 145 (44), 134 (50), 132 (66), 131 (99), 129 (100), 119
(87), 105 (69), 91 (24). HR-EIMS m/z: 288.2448 [M]+?
(calc. for C20H32O 288.2453).
D: +15?(CH2Cl2, c 0.1); UV
maxcm?1: 3314 (OH),
3.6. ent-Trachyloban-3-one (3)
Colorless oil. ? ½ ?22
(log ?): 228 (2.78); IR ?NaCl
1706 (C¼O), 1458, 1384, 1367, 1261, 1202, 1112, 1082,
1010, 844;1H and13C are given in Table 1; EI-MS 70
eV m/z (rel. int.): 286 [M]+?(77), 271 [M?CH3]+(17),
253 (1), 230 (23), 215 (12), 200 (11), 173 (5), 159 (8), 145
(12), 131 (8), 119 (14), 107 (11), 105 (100), 93 (16), 91
(15), 81 (8), 79 (10), 55 (9). HR-EIMS m/z: 286.2293
[M]+?(calc. for C20H30O 286.2296).
D: ?37?(CH2Cl2, c 0.1); UV lmaxnm
cm?1: 2969, 2933, 2860,
3.7. X-Ray structure analysis of compound 1
Colourless crystals were obtained by slow evaporation
monoclinic, space group P 21, a=7.311(1), b=42.210(1),
c=10.903(1) A˚, ?=91.10(1)?, V=3363.8(1) A˚ 3, Z=8,
Dx=1.20 g cm?3, ?=0.577 mm?1, F(000)=1328,
A total of 29,725 reflections were collected using a
Bruker SMART 6000 CCD detector and CuKaradia-
(Rint=0.052). The structure was solved by direct meth-
ods with SHELXS-97 (Sheldrick, 1997) and refined by
least-squares using F2values and anisotropic thermal
parameters for non-hydrogen atoms with SHELXL-97
(Sheldrick, 1997). The H atoms of the hydroxyl groups
were localized from difference Fourier maps; all
the other H atoms were calculated and included in the
refinement with a common isotropic temperature factor.
Final R values are: R=0.041 for 6370 observed reflec-
tions, R (all data)=0.042, wR2=0.109, S=1.02, Flack
parameter=0.08(14). The data have been deposit with
the Cambridge Crystallographic Data Centre (Nr
The four independent molecules are similar except
that the conformation of ring I (C1–C2–C3–C4–C5–
C10) is clearly a less flattened chair in molecule 3
(labelled C301–C310) than in the three other ones (in
molecule 3, the endocyclic torsion angles are : ?52, 47,
?43, 48, ?54 and 54?while the mean values for mole-
cules 1 2 and 4 are ?50, 34, ?29, 40, ?54 and 58?).
O(22)=1.220(3), C(20)–O(21)=1.425(3). The four OH
groups are hydrogen-bonded to a C¼O of another
molecule making infinite one-dimensional chains.
3.8. Cytotoxicity assay
HeLa (human cervix carcinoma cells) and WI-38
(human lung fibroblast) cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco BRL) sup-
plemented with 10% fetal calf serum (Gibco BRL) and
HL-60 (human promyelocytic leukemia) cells were rou-
BRL) containing 0.33% l-glutamine, 1% non-essential
amino acids, 1% sodium pyruvate, antibiotics (100 IU
penicillin/ml, 100 mg streptomycin/ml)andsupplemented
with 10% heat-inactivated fetal calf serum (Gibco
BRL). Cells were incubated at 37?C in a humidified
atmosphere containing 5% CO2. Stock solutions of com-
pounds were prepared at 10 mg/ml in DMSO and stored
at 4?C. The cytotoxicity of the compounds on HeLa
and WI-38 cells was evaluated using the tetrazolium salt
MTT (Sigma) colorimetric method based on the
cleavage of the reagent by dehydrogenases in viable
S. Block et al./Phytochemistry 65 (2004) 1165–11711169
cells. Briefly, 5000 HeLa or WI-38 cells per well were
seeded in 100 ml of DMEM in 96-well microculture
plates for 24 h. After 24 h adaptation, 100 ml of medium
containing various drug concentrations were added to
each well, while control cells received fresh medium
containing analogous DMSO concentrations. Each
concentration was tested in at least 8 wells. After 72 h
incubation, the medium was replaced by 100 ml DMEM
(without serum) medium containing 10 ml of MTT
solution (3 mg/ml in PBS). After 45 min in the incu-
bator, the medium was removed and 100 ml of DMSO
were added to each well. The plates were shaked and
optical densities were recorded at two wavelengths (570
nm and 620 nm), against a background control as blank
(100 ml of pure DMSO). The cytotoxicity on HL-60 cells
was evaluated using another tetrazolium salt, WST-1
(Boehringer). Briefly, 50000 HL-60 cells in 100 ml of
RPMI 1640 medium were seeded in each well of a 96-well
plate. 100 ml of fresh medium containing various drug
concentrations were added to each well while control
cells received fresh medium with analogous concentra-
tions of DMSO. Each concentration was tested in at
least 8 wells. After 72 h treatment, each well was sup-
plemented with 10 ml of WST-1 and then incubated for
45 min. Afterwards the plates were shaken and the
optical density was measured at 450 and 620 nm against
a background control as blank on a microplate reader.
For the 3 cell lines, the relative optical density was
expressed as percent of the control cells considered as
100%. In each case, camptothecin (Sigma) was used as
positive control. IC50 determination was achieved via
regression analysis of the results of at least 5 different
concentrations of each drug. Results are mean?SEM of
3 independent experiments.
The authors wish to thank M.C. Fayt for its skillfull
technical assistance. We also thank Professor J. Hanuise
for the optical rotation measurement and Prof. R.
Flammang for HR-EIMS analysis. This work was sup-
ported by a grant and founds from the ‘‘fonds spe ´ cial de
recherche’’ of the Catholic University of Louvain and
from the ‘‘Re ´ gion Wallonne’’ (project : CORD/LEVE
383/conv.114713 075665 for De Pauw-Gillet).
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