A cytotoxic diarylheptanoid from Viscum cruciatum

Article abstract of DOI:10.1016/S0031-9422(01)00293-X

A diarylheptanoid, 1,7-di-(3′,4′-dihydroxyphenyl)-4-hepten-3-one, hirsutanone, has been isolated from the methanolic extract of the aerial parts of Viscum cruciatum (Viscaceae) and characterized by spectroscopic methods and chemical transformations. This

Full text of DOI:10.1016/S0031-9422(01)00293-X

Phytochemistry 58 (2001) 567–569
www.elsevier.com/locate/phytochem
A cytotoxic diarylheptanoid from Viscum cruciatum
Carmen Martın-Corderoa,*, Miguel Lopez-Lazaroa, Marıa Angeles Agudoa,
Eduardo Navarrob, Juan Trujilloc, Marıa Jesus Ayusoa
aLaboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain
b
´
Departamento de Farmacologıa, Universidad de la Laguna, Canarias, Spain
c
´
Instituto de Productos Naturales y Agrobiologıa de Canarias, C.S.I.C., La Laguna, Spain
Received 30 March 2001; received in revised form 18 June 2001
Abstract
A diarylheptanoid, 1,7-di-(30,40-dihydroxyphenyl)-4-hepten-3-one, hirsutanone, has been isolated from the methanolic extract of
the aerial parts of Viscum cruciatum (Viscaceae) and characterized by spectroscopic methods and chemical transformations. This
compound showed cytotoxic activity against melanoma (UACC-62), renal (TK-10) and breast (MCF-7) cancer cell lines. # 2001
Elsevier Science Ltd. All rights reserved.
Keywords: Viscum cruciatum; Viscaceae; Diarylheptanoids; 1,7-Di-(30,40-dihydroxyphenyl)-4-hepten-3-one; Hirsutanone; 1H and 13C NMR spectra
1. Introduction
Viscum cruciatum is an evergreen hemiparasitic plant,
characterized by its whorled leaves and its red berries
´
(‘‘muerdago colorado’’), usually epiphyting various dico-
tyledonous trees and shrubs: Retama sphaerocarpa
Boiss., Olea europaea L., Rhamnus alaternus L., Rhamnus
lycioides L., Prunus amygdalus Stokes. In the course of
our pharmacological and phytochemical studies on the
extracts of Viscum cruciatum, we have reported the
cytotoxic activity (Ayuso et al., 1987a,b; 1988) and we
have isolated alkaloids and flavonoids (Martın-Cordero
et al., 1989, 1993, 1997). None of them could be clearly
shown to possess the established cytotoxic activity.
As part of our continuing study of Viscum cruciatum
for novel compounds which might posess biological
activity, we have undertaken a reexamination of the
chemical constituents of the aerial parts of Viscum cru-
ciatum and we examined its cytotoxic activity. We
report here the isolation and characterization of 1,7-di-
(30,40-dihydroxyphenyl)-4-hepten-3-one, hirsutanone (1)
(Terazawa et al., 1984) and cytotoxic activity against
three tumor cell lines.
2. Results and discussion
From the methanolic extract of the aerial parts of
Viscum cruciatum a diarylheptanoid (1) was isolated on
Si gel column. The structural elucidation and the cyto-
toxic activity of compound (1) is described in the pre-
sent study. This compound was not crystallized. Its IR
spectrum showed an absorption at 3350 cmꢀ1 corre-
sponding to phenolic groups, a band at 1650 cmꢀ1 of an a-
b unsatured carbonyl group and another band at 1600
cmꢀ1 was ascribed to double bonds. Its H NMR spec-
1
trum showed multiplets at ꢀ 2.78 and ꢀ 2.43 corresponding
to 8H which were attributed to four deshielded methylene
groups. The 13C NMR exhibited four signals at 30.0,
34.2, 34.8 and 42.1 that can be ascribed to four methyl-
ene groups, tentatively assigned in experimental section.
A signal at 200.30 corresponded to a carbonyl of an a-b
unsatured ketone. The remainder of the signals were
attributable to two aromatic moieties and a double bond.
The HRMS showed a molecular ion M+ at m/z 328.1307,
corresponding to a molecular formula C19 H20O5.
* Corresponding author. Tel.: +34-5-423-37-65; fax: +34-5-423-
37-65.
E-mail address: cordero@fafar.us.es (C. Martın-Cordero).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00293-X

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C. Martın-Cordero et al. / Phytochemistry 58 (2001) 567–569
568
By acetylation or methylation, compound 1 gave a tet-
(Cadiz, Spain). The identity was kindly verified by Dr.
Santiago Silvestre (Department of Botany of the Faculty
of Pharmacy, University of Sevilla) and a voucher speci-
men is deposited, labelled SEV. 82694.
raacetate (C27H20O9) or a tetramethylether (C23H28O5).
The respective spectral data supported the structure thus
proposed. Moreover, this same compound 1 was treated
with dichloromethane/dimethylformamide and CsF to
confirm that the phenolic groups were in ortho position
(Clark et al., 1976).
3.3. Extraction and isolation
The cytotoxic assays of isolated compound 1 were car-
ried out according to the standard protocols established
by the NCI (Monks et al., 1991). 1,7-di-(30,40-dihydroxy-
phenyl)-4-hepten-3-one, hirsutanone showed activity
against cell lines tested, the GI50 values for TK-10 cells,
MCF-7 cells and UACC-62 cells were 6.8, 1.9 and 4.8mg/
ml, respectively. Etoposide was used as a positive control
(GI50 values for TK-10 cells, MCF-7 cells and UACC-62
cells were 8.1, 0.33 and 0.97mg/ml, respectively).
Air-dried, powdered aerial parts (250 g) of Viscum
cruciatum were extracted by Soxhlet successively with
CHCl3 and MeOH. The MeOH extract (3.3 g) was
chromatographed on Si gel column eluting gradient
with n-hexane–EtOAc and CHCl3–MeOH, the com-
pound (1) was isolated from the intermediate fractions
eluting with n-hexane–Et2Ac.
3.3.1. 1,7-Di-(30,40-dihydroxyphenyl)-4-hepten-3-one (1)
KBr
max
Previous reports (Chun et al., 1999; Elattar and Virji,
2000; Hadi et al., 2000; Ramsewak et al., 2000) have
pointed to the cytotoxicity of structurally related diaryl-
heptanoids such as curcumin I (1,7 - di - (40 - hydroxy -
30methoxyphenyl)-1,4,6-heptatrien-3-one-5-hydroxy),
curcumin II (monodemethoxycurcumin) and curcumin
III (bisdemethoxycurcumin) from Curcuma longa (Zin-
giberaceae). Curcumin I was found to be more active
than other two as a cytotoxic agent (Ramsewak et al.,
2000). The structural differences between curcumin I
and the isolated compound (1,7-di-(30,40-dihydrox-
yphenyl)-4-hepten-3-one, hirsutanone) are the different
substituents at the aromatic ring (C-40, C-400) and C-5
and two double bond of the aliphatic chain. In addition,
diarylheptanoids such as yakuchinone A and B have the
ability to suppress proliferation of human cancer cells via
induction of apoptosis (Surh, 1999). These findings sug-
gest that the isolation of an active diarylheptanoid from
Viscum cruciatum may contribute to its cytotoxic activity.
Open chain diarylheptanoids were isolated previously
from Aceraceae, Betulaceae, Zingiberaceae and Legu-
minosae family (Keseru and Nogradi, 1995). It is the
first report of diarylheptanoids in the Viscaceae and
Viscum species.
Syrupy solid (210 mg) IR ꢁ
cmꢀ1: 3350, 3050, 2920,
1
1650, 1510. H NMR (200 MHz, CDCl3): ꢀ 2.43–2.78
(8H, m, H-6, H-7, H-1, H-2), 6.05 (1H, d, J=16.8 Hz, H-
4), 6.45–6.69 (6H, m, H-60, H-600, H-20, H-200, H-50, H-500),
6.86 (1H, dt, J=6.5, 16.8Hz, H-5). 13C NMR (50 MHz,
CDCl3): ꢀ 30.0 (t, C-1), 34.9 (t, C-6), 34.2 (t, C-7), 42.1 (t,
C-2), 116.0 (d, C-50, C-500), 116.2 (d, C-20, C-200), 118.2 (d,
C-60, C-600), 131.1 (d, C-4), 133.5 (s, C-10, C-100) 143.8 (s, C-
40, C-400), 145.5 (s, C-30, C-300), 147.6 (d, C-5), 200.3 (s, C-3).
EIMS m/e (rel. int. %) M+ 328.1307 (calc. for C19 H20O5
328.1310) 328 (20), 206 (19), 191 (18), 149(58), 148(38), 137
(28), 136 (28), 123 (100), 107 (22), 91 (36), 77 (58).
3.3.2. Acetylation of 1
Fifty milligrams was carried out with Ac2O–Pyr at
CHCl3
max
room temperature and in the dark for 24 h. IR ꢁ
cmꢀ1: 3010, 2920, 2823, 1760, 1660, 1620, 1500, 1368,
1256, 1180, 1110, 1010, 900. 1H NMR (200 MHz,
CDCl3): ꢀ 2.27 (12H, s), 3.0–2.4 (8H, m) 6.1 (1H, d,
J=16.8 Hz), 6.8 (1H, dt, J=6.5, 16.8 Hz) 7.1–7.0 (6H,
m). EIMS m/e (rel. int.%) M+ 496.1729 (2) (calc. for
C27H28O9 496.1733) 454 (10), 412 (20), 370 (17), 352
(12), 338 (26), 206 (18), 165 (20), 123 (100), 77 (11).
3.3.3. Methylation of 1
Compound 1 (20 mg) was suspended in Me2CO (30 ml)
and K2CO3 (300 mg) and MeI (0.5 ml) was added. The
mixture was refluxed for 14 h and extracted in the usual
3. Experimental
3.1. General experimental procedure
manner. The product was purified by CC to give the tet-
cmꢀ1: 3000, 2960, 2940,
CHCl3
max
ramethylether derivate. IRꢁ
IR spectra were measured as KBr disc and CHCl3.
NMR spectra were taken in CDCl3; 13C NMR at 50
MHz and H NMR at 200 MHz, using TMS as int.
standard. MS were recorded at 70 eV. CC: silica gel.
2840, 1660, 1620, 1590, 1510, 1460, 1258, 1155, 1135,
1030, 850. 1H NMR (200 MHz, CDCl3): ꢀ 2.87–2.48 (8H,
m), 3.85 (6H, s), 3.86 (6H, s), 6.11 (1H, d, J=16 Hz),
6.91–6.68 (7H, m). EIMS m/e (rel. int.%) M+ 384.1935
(28) (calc. for C23H28O5 384.1936), 370 (4), 219 (6), 191
(4), 177 (14), 151 (100), 137 (26), 107 (33), 77 (21.).
1
3.2. Plant material
The aerial parts of Viscum cruciatum hemiparasitic of
Prunus amygdalus Stokes were collected in January,
during the fruiting period from Zahara de la Sierra
3.3.4. Methylenedioxy derivate of 1
The methylenedioxy derivate of 1 (50 mg) was
obtained by the Clark method (Clark et al., 1976) and

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C. Martın-Cordero et al. / Phytochemistry 58 (2001) 567–569
569
CHCl3
purified by CC. IR ꢁ
cmꢀ1: 2990, 2880, 2763, 1720,
Ayuso, M.J., Martın-Cordero, C., Saenz, M.T., 1987a. Activite anti-
mitotique de Viscum cruciatum Sieber: etude comparative de deux
echantillons recoltes sur Rhamnus alaternus L. et Rhamnus lycioides
L. subsp. oleoides (L.) Jahandiez & Maire. Plantes Med. et Phyt-
other 21, 177–182.
max
1660, 1620, 1498, 1480, 1438, 1090, 1042, 937. 1H NMR
(200 MHz, CDCl3): ꢀ 2.49 (2H, m), 2.69 (2H, m), 2.81
(4H, m), 5.91 (2H, s), 5.92 (2H, s), 6.08 (1H, d, J=16
Hz), 6.90-6.60 (7H, m). EIMS m/e (rel. int. %) M+
352.1307 (13) (calc. for C21H20O5 352.1310), 204 (3), 203
(3), 160 (3), 135 (100), 105 (6), 91 (6), 77 (23).
Ayuso, M.J., Saenz, M.T., De Miguel, C., 1987b. Influence of the host
plant in the cytostatic activity of Viscum cruciatum Sieber. Phyt-
other. Res. 1, 93–94.
Clark, J.H., Herbert, L., Holland, H.L., Miller, J.M., 1976. Hydrogen
bonding in organic synthesis IV: a simple, high-yield method for the
methylenation of catechols. Tetrahedron Lett. 38, 3361–3364.
Chun, K.S., Sohn, Y., Kim, H.S., Park, K.K., Lee, J.M., Moon, A.,
Lee, S.S., Surh, Y.J., 1999. Anti-tumor promoting potential of
naturally occurring diarylheptanoids structurally related to curcu-
min. Mutant. Res. 16, 49–57.
3.4. Human tumor cell lines
The following three human cancer cell lines were used
in these experiments: the human renal adenocarcinoma
(TK-10), the human breast adenocarcinoma (MCF-7)
and the human melanoma (UACC-62) cell lines. They
were kindly provided by Dr. G. Cragg, Department of
NCI, Maryland, USA. The human tumour cytotoxi-
cities were determined following protocols established
by the National Cancer Institute, National Institute of
Health (Monks et al., 1991) TK-10, MCF-7 and UACC
-62 cell lines were cultured in RPMI 1640 medium (Bio
whittaker) containing 20% fetal calf serum, 2 mM l-
glutamine, 100 U/ml penicillin and 100 mg/ml strepto-
Elattar, T.M., Virji, A.S., 2000. The inhibitory effect of curcumin,
genistein, quercetin and cisplatin on the growth of oral cancer cells
in vitro. Anticancer Res. 20, 1733–1738.
Hadi, S.M., Asad, S.F., Singh, S., Ahmad, A., 2000. Putative
mechanism for anticancer and apoptosis-inducing properties of
plant derived poliphenolic compounds. IUBMB Life 50, 167–171.
Keseru, G.M., Nogradi, M., 1995. Chemistry of natural diarylhepta-
noids. In: Atta-ur-Rahman, X. (Ed.), Studies in natural products
chemistry Vol 17. Elsevier Science, Amsterdam, p. 357.
Martın-Cordero, C., Gil, A.M., Ayuso, M.J., 1993. Transfer of bipi-
peridyl and quinolizidine alkaloids to Viscum cruciatum (Lor-
anthaceae) hemiparasitic on shrub of Retama sphaerocarpa. J.
Chem. Ecol. 19, 2389–2393.
ꢁ
mycin. All cell lines were maintained at 37 C in a 5%
CO2 atmosphere with 95% humidity. Maintenance cul-
tures were passaged weekly, and the culture medium was
changed twice a week. According to their growth profiles,
the optimal plating densities of each cell line was deter-
mined (15ꢂ103, 5ꢂ103 and 100ꢂ103 cells/well for TK-10,
MCF-7 and UACC-62, respectively) to ensure exponen-
tial growth throughout the experimental period and to
ensure a linear relationship between absorbance at 492
nm and cell number when analysed by the SRB assay.
Data for the isolated compound was analysed according
to the standard protocols established by the NCI (Monks
et al., 1991).
Martın-Cordero, C., Pedraza, M.A., Gil, A.M., Ayuso, M.J., 1997.
Bipiperidyl and quinolizidine alkaloids in fruits of Viscum cruciatum
Sieber parasitic on Retama sphaerocarpa Boissier. J. Chem. Ecol. 23,
1913–1916.
Martın-Cordero, C., Ayuso, M.J., Richomme, P., Bruneton, J., 1989.
Quinolizidine alkaloids from Viscum cruciatum, hemiparasitic shrub
of Lygos sphaerocarpa. Planta Med. 55, 196.
Monks, A., Scudeiro, D., Skehan, P., Shoemaker, R., Paull, K., Vis-
tica, D., Hose, C., Jangley, J., Cronise, P., Vaigro-Wolff, A., Gray-
Goodrich, M., Campell, H., Mayo, J., Boyd, M., 1991. Feasibility
of a high-flux anticancer drug screen using a diverse panel of cul-
tured human tumor cell lines. J. Natl. Cancer Inst. 83, 757–766.
Ramsewak, R.S., DeWitt, D.L., Nair, M.G., 2000. Cytotoxicity, anti-
oxidant and anti-inflammatory activities of curcumins I-III from
Curcuma longa. Phytomedicine 7, 303–308.
Surh, Y., 1999. Molecular mechanism of chemopreventive effects of
selected dietary and medicinal phenolic substances. Mutant. Res.
428, 305–327.
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