Phytochemistry 64 (2003) 1125–1131
www.elsevier.com/locate/phytochem
Diterpenes from Alomia myriadenia (Asteraceae) with cytotoxic
and trypanocidal activity
Elita Scioa,b, Antônia Ribeiroa, Tânia M.A. Alvesa, Alvaro J. Romanhac,
José Dias de Souza Filhod, Geoffrey A. Cordelle, Carlos L. Zania,*
a
Laboratório de Quı´mica de Produtos Naturais, CPqRR-FIOCRUZ, Av. Augusto de Lima, 1715, Belo Horizonte, MG 30190-002, Brazil
b
Departamento de Bioquı´mica, ICB, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-030, Brazil
c
Laboratório de Parasitologia Celular e Molecular, CPqRR-FIOCRUZ, Av. Augusto de Lima, 1715, Belo Horizonte, MG 30190-002, Brazil
d
Departamento de Quı´mica, ICEx, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
e
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612-7231, USA
Received 6 January 2003; received in revised form 17 July 2003
Abstract
Further investigation of the aerial parts of Alomia myriadenia revealed an halimane diterpene identified as ent-8S,12S-epoxy7R,16-dihydroxyhalima-5(10),13-dien-15,16-olide along with the known ent-16-hydroxylabda-7,13-dien-15,16-olide, ent-12Rhydroxylabda-7,13-dien-15,16-olide, 6,7-methylenedioxycoumarin (ayapin), and kaempferol-7-methylether (rhamnocitrin). Evaluated in a panel of human cancer cell lines, the 16-hydroxylabade diterpene was the most active, showing an ED50 value of 0.3 mg/
ml against Lu1 (human lung cancer) cells. Tested in vitro against Trypanosoma cruzi in infected murine blood, this compound
caused lysis of 100% of the parasites at 250 mg/ml.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Ageratum myriadenium; Alomia myriadenia; Compositae; Halimane; Labdane; Trypanosoma cruzi; Human cancer cell line
1. Introduction
In an ongoing program to detect new bioactive natural products, several plants of the Brazilian flora were
selected for study after a survey of the NAPRALERT
database for the most promising candidates. The ethanol extract of the aerial parts of Alomia myriadenia
Sch.Bip ex Baker (syn. Ageratum myriadenium (Baker)
R.M. King & H. Rob) showed activity in a preliminary
screening with human cancer cell lines, and the labdane
diterpene ent-12R,16-dihydroxylabda-7,13-dien-15,16olide (1) was found to be the major cytotoxic compound in this extract (Zani et al., 2000). This diterpene
was previously described in Acritopappus hagei R.M.
King & H. Rob (Bohlmann et al., 1980) and Ageratum
fastigiatum (Gardner) R.M. King & H. Rob (Bohlmann et al., 1981), both from the Asteraceae family. It
was found recently that 1 also inhibits the phytohemaglutinin (PHA) stimulated proliferation of human
* Corresponding author. Tel.: +55-31-3295-3566; fax: +55-313295-3115.
E-mail address: zani@cpqrr.fiocruz.br (C.L. Zani).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00529-6
lymphocytes present in peripheral blood mononuclear
cells (PBMC) (Souza-Fagundes et al., 2002) via induction of monocyte apoptosis (Souza-Fagundes et al.,
2003).
In the present work we report the isolation, using a
bioassay-guided fractionation protocol based on
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E. Scio et al. / Phytochemistry 64 (2003) 1125–1131
cytotoxicity against KB and Col2 cells, of a new halimane diterpene (2) and four known compounds. Evaluation of the cytotoxicity of these compounds (3–6) in a
panel of human tumour cell lines and their activity
against the protozoan parasite T. cruzi, the causative
agent of Chagas’ disease (American trypanosomiasis) is
also described.
2. Results and discussion
The aerial parts of A. myriadenia were dried, ground,
and macerated with ethanol to produce a crude ethanol
extract. This extract was partitioned between immiscible
solvents to afford fractions of increasing polarity (Zani
et al., 2000). Only the medium polarity fraction
(CH2Cl2) was cytotoxic in the bioassay with KB and
Col2 cell lines showing ED50 values 9.2 and 2.9 mg/ml,
respectively. Chromatographic separation was monitored with the bioassays using these cell lines, to isolate
the active compounds. This procedure yielded compounds 2–6, whose structural elucidation was based on
the analysis of their spectral data.
Compound 2 is a mixture of epimers, as deduced by a
series of double signals in the 1H and 13C NMR spectra.
Its HRFABMS showed [M+H]+ with m/z 349.1983, in
agreement with the molecular formula C20H28O5 (calculated: 349.2015). The presence of an a,b-unsaturated
lactone was indicated by an absorption at max 1756
cm1 in the IR spectrum and confirmed by the 1H
NMR signals at 6.11 (s, H-16) and 6.06 (s, H-14) and
the corresponding 13C NMR signals (C-13 to C-16 in
Table 1). The 13C NMR (Table 1) and DEPT spectra
showed signals due to 20 carbons comprising four methyl,
five methylene, one olefinic methine, three oxymethine,
three quaternary carbons, one of them bearing an oxygen
atom, a carbonyl carbon, and three fully substituted olefinic carbons. These fragments account for a partial molecular formula C20H26O5, indicating that the two remaining
hydrogen atoms are present in hydroxyl groups.
The a,b-unsaturated g-lactone moiety is responsible
for three of the seven unsaturations calculated for the
molecule. Another unsaturation is due to a double
bond, as deduced by 13C NMR and DEPT analyses,
and the remaining three are due to three ring systems.
Furthermore, since the lactone ring and the two
hydroxyl groups known to be present sum four oxygen
atoms, the last oxygen to complete the molecular formula must be part of a ring. These inferences and a
detailed analysis of the 1H–1H COSY, HMQC, and
HMBC spectra (Table 1) and comparison with literature data suggested a halimane-type diterpene carrying
a hydroxylated a,b-unsaturated g-lactone moiety (Hara
et al., 1995; Teresa et al., 1983).
The formation of a tetrahydrofuran ring involving C8 and C-12 ( 73.0) was deduced on the basis of the
oxygenation of these carbons, their multiplicities and
the HMBC and COSY correlations with their neighbours. In the HMBC contour map, the quaternary
oxygenated carbon C-8 showed correlations to Me-20,
Me-17, and H-11. In the 1H–1H COSY contour plot the
methine H-12 showed correlations to both H-11a and b.
A HMBC correlation between C-12 and H-14 was also
observed. The decalin ring system contains a double bond
between C-5 and C-10. C-5 showed HMBC correlations
to H-3, H-6, Me-18 and Me-19, while C-10 was correlated
with H-1, H-2, H-11, and Me-20. In the HMBC contour
plot, the C-7 oxymethine showed a correlation to Me17, while the 1H–1H COSY contour map revealed
correlations between H-7 and the methylene protons at
C-6. All these data are compatible with the structure 2.
The determination of the absolute stereochemistry of
2 by NMR analysis of its Mosher esters (Latypov et al.,
1996) was precluded by the limited amount of this natural product. However, its relative stereochemistry was
deduced on the basis of the following analysis. 1H NMR
signals assignments of both 5a- (Elgamal et al., 1999)
and 5b-cardenolides (Hanna et al., 1998, 1999) shows
clearly that a b-oriented butenolide at C-17 protects the
vicinal H-16b hydrogen, that resonates around 1.9 ppm,
while its geminal H-16a gives a signal near 2.1 ppm.
Using this system as a model, if we assume that the
butenolide at C-12 in 2 is b oriented, it can be deduced
that the vicinal H-11 resonating at d 1.85 is also boriented. NOESY experiments show the spatial proximity between this hydrogen (H-11b) and the C-20
methyl, which also correlates with the vicinal C-17
methyl that, on the other side, interacts with the oxymethine proton H-7, thus indicating that they are all boriented. In this way, except for C-16, the relative stereochemistry of all chiral centers of 2 was established
and the molecule was identified as 8S,12S-epoxy-7R,16dihydroxyhalima-5(10),13-dien-15,16-olide. The absolute stereochemistry was not established but compound
2 probably belongs to the ent-series in view of its cooccurrence with ent-labdanes. Table 1 shows the proton
and carbon assignments, including the signals of each
epimer that could be distinguished.
The 13C NMR spectrum of compound 3 showed double peaks in the low field region of the 1H NMR spectrum, again suggesting a mixture of epimers at C-16. The
data listed for 3 in Table 1 are in agreement with those
published for the epimeric mixture of ent-16-hydroxylabda-7,13-dien-15,16-olides, previously isolated as their
acetates from A. fastigiatum (Bohlmann et al., 1981).
These acetates were dextrorotatory ([]20
589+7 , c=0.5,
CHCl3) and classified as ent-labdanes. As our mixture is
also dextrorotatory ([]D +40.9 ), we assumed that 3
also belonged to this enantiomeric series.
Compound 4 was isolated as white crystals, and its 1H
and 13C NMR spectra were similar to those of
compound 3, except for the absence of duplicate signals,
Table 1
1
H and 13C NMR spectral data (d/ppm) for compounds 2, 3 and 4
Position
2
3
ca
1
26.4 (2)
2
19.9 (2)
3
39.5 (2)
34.4 (0)
135.4 (0)
6
30.1 (2)
7
8
9
10
11
71.5
86.3
51.7
128.7
41.4
(1)
(0)
(0)
(2)
HMBC (H to C)
b 2.02 (m)
a 1.90 (m)
1.62 (m)
C-10
C-4, C-10
a 1.52 (m)b
b 1.45 (m)b
NOESY
Me-20
c
39.3 (2)
18.7 (2)
C-4, C-5, C-18
42.1 (2)
Me-18
32.9 (0)
50.0 (1)
b 2.33 (dd, 10.5, 5.4)b
a 2.13 (dd, 11.4, 10.5)b
3.56 (dd,10.5, 5.4)
C-5
Me-18
Me-17
Pseudo-eq (a) 2.43
(dd, 12.7, 5.5)
[2.31 (dd, 12.7, 5.4)]c
Pseudo-ax (b) 1.80
(dd, 12.7, 10.8)
[1.86 (m, 12.7, 10.6)]c
Pseudo-ax (a)4.44
(dd, 10.8, 5.5)
[4.53 (dd, 10.6, 5.4)]c
C-10 C-8
24.4 (2)
123.4
133.8
54.4
36.8
24.4
(1)
(0)
(1)
(0)
(2)
H
HMBC (H to C)
eq 1.84 (m)
ax 0.95 (m)
1.48 (m)
C-20
eq 1.45 (m)
ax 1.17 (m)
–
1.19 (m)
b 1.97 (m)
a 1.85 (m)
5.45 (brs)
–
1.69 (m)
NOESY
c
H
Me-20
39.8 (2)
H20
19.0 (2)
42.3 (2)
C-4,C-19
C-4, C-9, C-10,
C-18,
C-19, C-20
H-6b, Me-18, H-9
Me-18, H-5
Me-19, Me-20
C-9, C-10, C-12
2.55 (m)
2.37 (m)
C-13, C-14
24.2 (2)
124.2
133.6
50.8
37.2
34.4
C-7, C-8, C-9
1.74 (m)
1.70 (m)
33.3 (0)
50.4 (1)
(1)
(0)
(1)
(0)
(2)
29.6 (2)
70.6 (1)
73.0 (1)
13
14
170.1 (0)
117.9 (1)
6.06 (sl) [6.02 (s)]c
C-12, C-13/15
169.5 (0)
117.3 (1)
5.87 (m)
C-15, C-16
H-12, Me-17, H11
171.9 (0)
116.2 (1)
15
16
170.1 (0)
97.4 (1)
6.11 (s) [6.06 (sl)]c
C-15
171.2 (0)
98.6 (1)
6.01 (brd, 7.4)
C-15
H-11, H20, H-1, H17
173.9 (0)
71.2 (2)
1.29 (s)
a 1.04 (s)
b 1.00 (s)
b 1.12 (s)
C-7,
C-3,
C-3,
C-8,
1.69 (brs)
a 0.88 (s)
b 0.86 (s)
0.78 [0.77]
C-7, C-8
C-18
C-19, C-3, C-5
C-1, C-5, C-9,
C-10
H-6a
H-6b, H-5
H-1eq, H-6a, H-11,
Me-19
4.11 (d, 7.4)
C-13, C-16
OH
22.1
28.0
28.7
20.8
(3)
(3)
(3)
(3)
a in C-7 C-16
b 2.01 (m)
a 1.85 (m)
5.49 (m)
–
1.72 (m)
1.75 (m)
1.73 (m)
NOESY
Me-20
H-3axx
C-1
C-4, 119
C-4, 6, 9, 10,
11, 18, 19, 20
H-6b, Me-18
Me-19, Me-20
–
C-10
C-8, C-12, C-13
Me-20
12
17
18
19
20
eq 1.65 (m)
ax 0.84 (m)
1.55 (m)
1.44 (m)
eq 1.41 (m)
ax 1.14 (m)
–
1.20 (dd, 5, 11)
HMBC (H to C)
C-8,
C-4,
C-4,
C-9,
C-9
C-5, C-18
C-5, C-19
C-10, C-11
H-7, Me-20
H-6a
H-6b
Me-17
Me-18 H11b
22.0
21.8
33.0
13.6
(3)
(3)
(3)
(3)
at C-16
23.1
22.2
33.4
14.1
(3)
(3)
(3)
(3)
at C-12
4.76 (m)
5.97, 1H, s
C-12, C-15
H-12, Me-17,
H11
4.98 (dd, 17, 1.7)
4.90 (dd, 17, 1.4)
1.73 (m)
a 0.88 (s)
b 0.86 (s)
0.77 (s)
C-13, C-14,
C-13
C-7,C-8, C-9
C-18
C-19, C-3, C-5
C-1,C-9, C-10
H-11, H20,
H-1, H17
E. Scio et al. / Phytochemistry 64 (2003) 1125–1131
4
5
H
4
H-6a
H-6b, H-5
H-1eq, H-6a
H-11,
Me-19
2.15 (d, 4.2)
1127
a
Obtained in CDCl3 at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts are indicated in (ppm) and coupling constants (J in Hz) are in parenthesis. NOESY experiments were run in CDCl3 at 400 MHz. Carbon type,
as determined from DEPT spectra, are represented in parenthesis at the right of their respective chemical shift: 0=quaternary, 1=methine, 2=methylene, 3=methyl.
b
Proton assignments are interchangeable within the same carbon.
c
Values for the chemical shifts and J for the second epimer present in the mixture that presented distinct signal.
1128
E. Scio et al. / Phytochemistry 64 (2003) 1125–1131
indicating that the center of epimerization no longer exists
in 4. All the data (Table 1), including NOE correlations
observed in the NOESY experiment, are compatible with
the structure ent-12-hydroxylabda-7,13-dien-15,16-olide,
previously isolated from Acritopappus confertus (Bohlmann et al., 1983). As the coupling constants of H-12
are similar to those of the corresponding proton in the
previously isolated labdane 2, it is plausible to assume
that the configuration at this center is also S. On the
basis of this analysis the diterpene 4 was identified as ent12R-hydroxylabda-7,13-dien-15,16-olide. The 13C NMR
spectroscopic assignments for 3 and 4 are reported here
for the first time. No study concerning the biological
activities of these diterpenes was found in the literature.
Based on spectral analysis and comparison with
reported data, compound 5 was characterised as ayapin
(Debenedetti et al., 1998) and compound 6 as rhamnocitrin (Lin et al., 1991). The presence of a methoxyl
group at C-7 in the latter compound was confirmed by
the absence of bathochromic shift of band II after the
addition of NaOAc, and by the fragments of m/z 121
(C7H5O2) and 167 (C8H7O4) recorded in the EIMS
spectrum. Ayapin 5 is also known to occur in A. fastigiata Benth (Pozetti, 1966). This coumarin was shown
to decrease the blood coagulation time in guinea pigs
and dogs (Souza and Pozetti, 1974), and to display trypanocidal (Lopes et al., 1997), antifungal (Tal and
Robeson, 1986) and antibacterial (Kashihara et al.,
1986) activities. Rhamnocitrin 6 is reported to show
several biological activities, including strong inhibition
of platelet aggregation induced by arachidonic acid
(Okada et al., 1995), and anti-inflammatory and antispasmodic activities (Ram et al., 1989).
Table 2 summarises the results of the bioassays of the
CH2Cl2 fraction and the isolated compounds. Compound 3 was the most active in a panel of human cancer
cell lines, displaying strong activity against the Lu1 cell
line (ED50 0.3 mg/ml). All other compounds showed
ED50 values higher than 10 mg/ml in this test system.
After the bioassay-guided fractionation using the cancer
cell lines Col2 and KB was completed, in vitro assay
with the trypomastigote form of T. cruzi was next performed. Tested in this assay at 250 mg/ml and 24 h contact at 4 C, compounds 3 and 4 caused the lysis of 100
and 99% of the parasites, respectively. This potency is
comparable to that of gentian violet, used at 250 mg/ml to
treat infected-banked blood in order to clear it from the
parasites. However, as shown above, these compounds,
especially 3, are cytotoxic at much lower concentrations,
a fact that poses a serious obstacle to their potential use
to disinfect human blood before transfusion.
Table 3 summarises the results of the bioassays for the
three labdane diterpenes isolated from A. myriadenia
(2–4). The only differences between the compounds are
the number and positions of the hydroxyl groups.
Although the number of compounds considered is small,
it seems that the presence of a 16-OH group is a relevant
feature for their cytotoxic activity. In the case of anti-T.
cruzi activity, compounds 3 and 4 were active, with each
having only one hydroxyl at different positions.
This work disclosed a new halimane type diterpene as
well the strong and selective activity of 3 on Lu1 cell
line, suggesting that this class of compound is an interesting system for further development. Preliminary
investigations on its mode of action are underway.
Studies on this species aiming at further minor components, as well as new biological activities of the isolated
compounds, are in progress.
3. Experimental section
3.1. General experimental procedures
Melting points were determined using a Fisher-Johns
MP apparatus and are uncorrected. The UV spectra
Table 2
Activity of the ethanol extract and pure compounds from Alomia myriadenia against human cancer cell lines, and Trypanosoma cruzi
Sample
CH2Cl2 fraction
1
2
3
4
5
6
Ellipticine
Cell linea
T. cruzib
Lu1
Col2
KB
LN CaP
KB VI
KB VI+
BC1
% Lysis at 250 mg/ml
NT
3.9
15.4
0.3
0.02
2.9
3.7
14.8
1.2
0.3
9.2
2.4
16.9
1.7
0.04
NT
3.0
13.7
4.2
0.8
NT
9.1
NT
1.4
0.3
NT
8.6
NT
9.9
0.2
NT
4.4
NT
2.6
0.5
10
25
NT
100
99
NT
NT
NT
a
Lu1 (lung), Col2 (colon), KB (oral epidermoid), LNCaP (prostate). KB VI+ (oral epidermoid in presence of 1 mg/ml vinblastine) e KB VI (oral
epidermoid in absence of vinblastine). Results expressed as ED50 (effective dose to inhibit cell proliferation in 50%). The minus () sign means
inactive at the highest dose used (20 mg/ml). NT means not tested.
b
Trypomastigote form of T. cruzi Y strain in infected murine blood. Results are expressed as percent of lysis of parasites after 24 h contact at
4 C. Gentian violet at 7.5 mg/ml was used as positive control (50% lysis).
E. Scio et al. / Phytochemistry 64 (2003) 1125–1131
Table 3
Structure activity relationships of labdane diterpenes isolated from
A. myriadenia
Diterpene
1
3
4
a
b
12-OH
16-OH
Cytotoxic
activitya
Trypanocidal
activityb
+
+
+
+
+
+
+
+
ED50<4 mg/ml for at least one cell line.
Lysis of >90% of the parasites at 250 mg/ml.
were obtained on a Beckman DU-7 spectrophotometer.
NMR spectra were recorded with a Bruker Avance
DPX-300 spectrometer at 300 MHz with TMS as the
internal standard. NOESY spectra were obtained at 400
MHz. IR spectra were recorded on a Mattson-Galaxy
series FTIR 3000, and optical rotations were measured
on a Perkin-Elmer 241 polarimeter. EIMS (70eV) and
HRFABMS were recorded on a Finnigan MAT 90
spectrometer, ESI-MS was recorded on a Hewlett
Packard 5989B single quadrupole mass spectrometer
coupled with a 59987A electrospray interface and a
Hitachi HPLC L-7100 system. Analytical and semi-preparative HPLC were run on a Shimadzu chromatograph equipped with a LC-6AD pump and a UV
detector at 210 and 254 nm. Analytical (4.6250 mm)
and semi-prep (20250 mm) RP-18 columns (Shimpack
prep-ODS kit) were used.
3.2. Plant material
A. myriadenia was collected near Minduri, Minas
Gerais, Brazil, in June, 1997. A voucher specimen
(BHCB 42865) was deposited at the Herbarium of the
Biological Sciences Institute of the Federal University of
Minas Gerais, Belo Horizonte, Brazil.
3.3. Extraction and bioassay-guided fractionation
The aerial parts of A. myriadenia (1 kg) were powdered, macerated in EtOH, and partitioned successively
against hexane and CH2Cl2 (Zani et al., 2000). The
CH2Cl2 fraction (21.1 g) was cytotoxic to the KB
(human oral epidermoid carcinoma, ED50 9.2 mg/ml)
and Col2 (human colon cancer, ED50 2.9 mg/ml) cell
lines and was subjected to medium pressure chromatography. Sixteen fractions were obtained using a Büchi
column (25450 mm) and elution with mixtures of
CH2Cl2-EtOAc of increasing polarity. Fractions 4 (152
mg), 7 (365 mg), 8 (331 mg), and 12 (490 mg) were the
most active against the above cell lines and were selected
for further fractionation by HPLC. A Shimadzu chromatograph equipped with a LC-6AD pump and a UV
detector set at 210 and 254 nm was used. Samples were
1129
injected into Analytical (4.6250 mm) or semi-prep
(20250 mm) RP-18 columns (Shimpak prep-ODS kit)
and eluted with different proportions of CH3CN-H2O.
Fraction 4 afforded compound 5 (13 mg); fraction 7
afforded compounds 3 (19.4 mg) and 6 (7.4). Fraction 8
gave compound 4 (30.4 mg) and fraction 12 yielded
compound 2 (2 mg).
Compound 2 was isolated as a white amorphous
powder; []D 110 (CHCl3, c 0.05); UV (MeOH) lmax
(log ") 205 (4.09), 230 (3.70) sh; IR max cm1: 1756; For
1
H NMR (CDCl3; 300 MHz) and 13C NMR (75 MHz),
see Table 1; HRFABMS m/z 349.19826 [M+H]+ (calc.
for C20H29O5, 349.20150); 349.2 (6), 331.2 (12), 307.2
(15), 205.2 (20), 154.1 (100), 136.1 (76), 91 (35), 54.8
(42).
Compound 3 was obtained as white crystals from
MeOH–H2O; mp 128–129 C; []D +40.9 (MeOH, c
0.21); UV (MeOH) lmax (log ") 213 (3.83); IR max
cm1: 3395, 2957, 1752; For 1H NMR (CDCl3; 300
MHz) and 13C NMR (75 MHz), see Table 1; EIMS (70
eV) m/z 318 [M]+. C20H30O3; 318 (5), 300 (2), 285 (3),
267 (3); 205 (6); 195 (35); 177 (52). ESI-MS (negative
mode) [M–H] (m/z 317).
Compound 4 was purified as white crystals from
MeOH–H2O; mp 108–110 C; []D 5.4 (CHCl3, c
0.19); UV (MeOH) lmax (log ") 208 (4.12); IR max
cm1: 3430, 2981, 1780, 1747; For 1H NMR (CDCl3;
300 MHz) and 13C NMR (75 MHz), see Table 1; EIMS
(70 eV) m/z 318 [M]+. C20H30O3; 318 (0.4), 303 (1), 205
(4), 195 (60). ESI-MS (negative mode) [M–H] (m/z 317).
Compound 5 was obtained as pale yellow crystals
from MeOH; mp 222–223 C; UV (MeOH) lmax (log ")
205 (4.26), 233 (4.12), 345 (3.99); IR max cm1: 1704;
1
H NMR (CDCl3; 300 MHz) d 7.58 (1H, d, J=9.5 Hz,
H-4), 6.83 (1H, s, H-5 and H-8), 6.28 (1H, d, J=9.5, H3), 6.07 (2H, s, H-9); 13C NMR (75 MHz) 161.2 (C-2),
151.2 (C-7), 144.8 (C-8a), 143.4 (C-4 and C-6), 113.4 (C3), 112.6 (C-4a), 105.0 (C-5), 102.3 (C-9), 98,4 (C-8);
EIMS m/z 190 [M]+. C10H6O4, 190 (100), 162 (84), 161
(51), 104 (6).
Compound 6 was isolated as a yellow amorphous
powder, mp 225–227 C; UV (MeOH) lmax (log ") 364
(3.16), 267 (3.27), 238 (3.30); IR max cm1: 2982; 1H
NMR (DMSO; 300 MHz); d 12.46 (1H, s, OH-5), 10.12
(1H, s, OH-3), 9.51 (1H, s, OH-40 ), 8.09 (2H, d, J=8.7
H-20 and H-60 ), 6.92 (2H, d, J=8.7, H-30 and 50 ), 6.73
(1H, d, J=1.8, H-8), 6.34 (1H, d, J=1.8, H-6), 3.85
(3H, s, OCH3-7); EIMS (70 eV) m/z 300 [M]+.
C16H12O6; 300 (100), 257 (19), 167 (5), 121 (46). ESI-MS
(negative mode) [MH] (m/z 299).
3.4. Cytotoxicity activity
The cytotoxicity assays were run according to established protocols (Likhitwitayawuid et al., 1993). To
monitor the fractionation process, KB and Col2 tumor
1130
E. Scio et al. / Phytochemistry 64 (2003) 1125–1131
cell lines were used. Tests with pure compounds were
performed with BC1 (human breast cancer), Lu1
(human lung cancer), Col2 (human colon cancer),
LNCaP (human prostate cancer), KB (human oral epidermoid carcinoma), KB-VI (KB in the presence of 1
mg/ml vinblastine) and KB-VI+ (KB in the absence of
vinblastine).
3.5. Trypanocidal activity
The assays with T. cruzi were carried out using blood
from Swiss albino mice collected in the parasitaemia
peak (7th day) after infection with the Y strain of T.
cruzi. The infected blood was diluted with normal murine blood to the concentration of 2106 trypomastigotes/ml. Stock solutions (20 mg/ml) of the compounds
were prepared in dimethylsulfoxide (DMSO). A sample
(5.0 ml) of each solution was added to 195 ml of infected
blood providing a final concentration of 500 mg/ml.
Samples of 100 ml were transferred in duplicate to the
wells of a microtitre plate (96 wells). To reproduce the
blood bank conditions, plates were incubated at 4 C
for 24 h. The experiments were repeated two or three
times. The parasite concentration was evaluated
according to the procedure described by Brener
(1962) DMSO at 2.5% v/v and gentian violet at its
IC50 concentration (7.5 mg/ml) were used as negative
and positive controls, respectively. The trypanocidal
activity was expressed in percent reduction of the
parasite number (lysis) comparing the wells with the
samples to the wells with DMSO alone. DMSO at
2.5% causes no harm to the parasites, erythrocytes,
or leukocytes. The samples that caused about 100%
reduction in parasite number were tested at lower
concentrations.
Acknowledgements
The authors thank to Rosana O. Alves for technical
assistance with T. cruzi; Dr. J. M. Pezzuto and the staff
of the Bioassay Research Facility, PCRPS-UIC for the
cytotoxity assays and Ms. Mary Lou Quinn for access
to the NAPRALERT database; to CAPES, CNPq, and
Fulbright for fellowships; and to Pronex and Fiocruz
for their financial support.
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