PHYTOCHEMICAL ANALYSIS

Phytochem. Anal. 11, 383–386 (2000)

Lignoids in Seedlings of Virola sebifera

Ana Paula Danelutte, Alberto José Cavalheiro1 and Massuo Jorge Kato*1
1
Instituto de Quı́mica, Universidade de São Paulo, 05599-970 São Paulo, SP, Brazil
2
Instituto de Quı́mica, Universidade Estadual Paulista, 14800-900, Araraquara, SP, Brazil

Quantitative analysis carried out by high performance liquid chromatography indicated the accumulation
of a major secondary compound in seedlings of Virola sebifera which was isolated and identified as the lignan
hydroxy-otobain. This lignan occurs only in trace amounts in the seeds, where cyclolignans (aryltetralones)
are by far the major components. In addition to hydroxy-otobain, only hydroxy-aryltetralones were
detected in the seedlings, indicating a selective process in the translocation of secondary compounds.
Copyright # 2000 John Wiley & Sons, Ltd.
Keywords: HPLC; cyclolignans; seedlings; Virola sebifera; Myristicaceae.

INTRODUCTION seedlings were butenolides, but these were not detected in

Within the family Myristicaceae, Virola sebifera is one of
the most widely spread species in South America. The
trees bear fruits at a younger stage and produce a larger
number of seeds than most of the Myristicaceae species
(Howe and Smallwood, 1982). Additionally, its seeds are
capable of resisting dehydration to some extent whilst
retaining a high percentage of germination (Kato, 1995).
These factors possibly represent clues as to its wide
distribution in South America forests and also in the
cerrados (Rodrigues, 1980).
V. sebifera is chemically one of the most investigated
species of the family. Analyses carried out on different
parts of the adults plants have revealed a wide variety of
secondary compounds. The bark of V. sebifera has been
found to contain hallucinogenic alkaloids (McKeena et
al., 1984; Kawanishi et al., 1985) as well as b-sitosterol
(Corothie and Nakano, 1969; Kawanishi and Hashimoto,
1987) and diepoxylignan (von Rotz et al., 1987). Di-
benzylbutyrolactone and dibenzylbutane lignans as well
as diepoxylignan were identified as the major compounds
in the leaves (Lopes et al., 1983; Martinez et al., 1999).
Representatives of the former two types of lignans have
also been observed in the pericarps (Lopes et al., 1983,
1984a) which also accumulated polyketides (Kato et al.,
1985) and cyclolignans (Lopes et al., 1982, 1984b).
Previous investigations concerning secondary metabo-
lism in seedlings of species of the Myristicaceae have
shown that in V. venosa the major lignans, cubebin and
dihydrokusunokinin, which accumulated in the seeds were
not present in the seedlings, which accumulated instead a
polyketide (Kato et al., 1992). The major compound
identified in the roots of the seedling was shown to be the
lignan sesamin, a minor compound in the seeds. In the case
of V. surinamensis, the major compounds identified in the
* Correspondence to: Dr. M. J. Kato, Instituto de Quı́mica, Universidade de
São Paulo, 05599-970 São Paulo, SP, Brazil. E-mail: majokato@iq.usp.br
Contract/grant sponsor: PADCT.
Contract/grant sponsor: CAPES.
Contract/grant sponsor: FAPESP.
Contract/grant sponsor: CNPq.
the seeds (Lopes et al., 1994).
The chemistry of seedlings is a particularly important
aspect associated with the reproduction of tropical
species, and it has been assumed that secondary
metabolites are translocated from the seeds to the tissues
of the seedlings as part of the defence strategy against
herbivores (Janzen, 1978). Since species of the Myristi-
caceae are representative of the tropical rain forest and
their seeds are a rich source of lignans and neolignans, we
have addressed in the present study the development of
an HPLC method to evaluate the composition of
secondary metabolites in seedlings of V. sebifera.
EXPERIMENTAL
Plant material. Mature seeds of Virola sebifera were
obtained from trees growing at Estação Experimental de
Araraquara (Instituto Florestal, Araraquara, Brazil): a
voucher specimen has been deposited in the herbarium of
the Instituto de Biociências (Universidade de São Paulo,
São Paulo, Brazil). Seeds were germinated on moistened
sand and the seedlings were kept under greenhouse
conditions and analysed at an age of 2 months.
Spectral determinations. 1H and 13C NMR spectra were
obtained using a Bruker (Rheinstetten, Germany) model
AC-200 spectrophotometer at 200 and 50 MHz, respec-
tively: tetramethylsilane (TMS) was used as a internal
reference and deuterochloroform as a solvent. Mass
spectra were measured on a Finnigan (Bremen, Germany)
INCOS 50B (direct injection; 70 eV) instrument, coupled
with a Varian (Palo Alto, CA, USA) model 3400 gas
chromatograph. IR spectra were recorded from potassium
bromide discs on a Nicolet (Madison, WI, USA) FT-IR
510 spectrophotometer.
Sample preparation. The various parts of the seedlings
(seeds, leaves, hypocotyls, epicotyls and roots) were
freeze dried for 24 h and milled, weighed and extracted
three times with dichloromethane; the combined extracts

Received 2 August 1999

Copyright # 2000 John Wiley & Sons, Ltd. Revised 15 January 2000
Accepted 28 January
384 A. P. DANELUTTE ET AL.
Table 1. The gradient conditions used to analyse extracts of
Virola sebifera by HPLC (for further chromato-
graphic information see the Experimental section)
Time (min) Methanol (%) Acetonitrile (%) Water (%)
0 42 18 40
10 56 24 20
17 100 0 0
22 100 0 0
27 42 18 40
were evaporated to dryness under vacuum. Part of each
extract was suspended in methanol:water (95:5), filtered
in a Sep Pak C18 cartridge (Waters, Milford, MA, USA)
and concentrated under vacuum to dryness. The resulting
fractions were suspended in methanol:tetrahydrofuran
(9:1) at a concentration of 1 mg/mL, and an aliquot
(10 mL) was analysed by HPLC. Each sample preparation
was repeated three times and the average values for each
determination are presented.
HPLC analysis. The analyses were performed using a
Hewlett-Packard (HP; Waldbronn, Germany) series 1050
liquid chromatograph coupled with a UV detector, HP
3395 integrator and a Shimadzu (Tokyo, Japan) model
SIL-9A automatic injector. A Taxsil1 (Metachem
Technologies, Torrance, CA, USA) column (250 
4.6 mm i.d.; 5 m) with a pre-column was employed. The
mobile phase consisted of a gradient mixture of HPLC-
grade methanol, acetonitrile and water (details shown in
Table 1) at a flow rate of 0.6 mL/min. The UV absorbance
was monitored at 280 nm. Cyclolignans 1, 2, 5 and 6,
hydroxy-otobain (3) and otobain (4) (see Fig. 1) were
isolated from seed and seedling leaf extracts. Solutions
containing 0.001, 0.05, 0.25, 0.5 and 1.0 mg/mL of each
of the compounds 1–6 were prepared in methanol:aceto-
nitrile:water (42:18:10) containing 100 mg/mL of methyl
ferulate as internal standard, and an aliquot (10 mL) from
each was injected onto the HPLC.
Isolation of the lignans 1–6. The dichloromethane
extracts of seedling leaves (2.5 g) were suspended in
methanol:water (4:1) and filtered over a layer of Celite.
The filtrate was partitioned with dichloromethane, dried
with anhydrous sodium sulphate, filtered and then
concentrated under vacuum (yield 720 mg). The dichloro-
methane extract was submitted to column chromatogra-
phy over silica gel 60 H (10–40 m; Merck, Darmstadt,
Germany catalogue no. 7736) eluted with a gradient of
hexane:ethyl acetate to yield fractions 1–44. Fractions 1–
6 were bulked and purified by preparative TLC
[hexane:dichloromethane:acetone (20:78:6:0.4)] fol-
lowed by HPLC using a C18 column eluted with
methanol:water (91:9) to yield otobain (4; 1.0 mg); the
bulked fractions 10–13 were purified by preparative TLC
[(hexane:dichloromethane:acetone (20:78.6:0.4)] to yield
hydroxy-otobain (3; 14.0 mg); bulked fractions 21–22
and 23–26 were separately purified by preparative TLC
[(dichloromethane:acetone (95:5)] to yield cyclolignans
1 (14.0 mg) and 2 (2.1 mg), respectively.
The dichloromethane extract of seeds of V. sebifera
(80.0 mg), prepared as described above, was submitted to
preparative TLC [hexane:ethyl acetate:isopropanol
(70:30:1)], furnishing fractions at R f values 0.35 and
Copyright # 2000 John Wiley & Sons, Ltd.
Figure 1. Lignans found in seeds and/or seedlings of Virola
sebifera.
0.20. These fractions were further purified by preparative
TLC [fraction R f 0.35 with hexane:ethyl acetate:isopro-
panol (70:30:1); fraction R f 0.20 with hexane:dichloro-
methane:acetone (20:78.6:0.4)] to yield, respectively, 6
(25.7 mg) and 5 (12.7 mg).
(ÿ)-Hydroxy-otobain (3). (8R, 7'R, 8'S)-7'-Hydroxy-
3,4:3',4'-bis(methylenedioxy)-2,7'-cyclolignan, ‰Š21
D–

15.2 (CHCl3; c 1.59). H-NMR (CDCl3), 200 MHz, d:
1
0.83 (d, J = 6.7 Hz, H-9'), 0.97 (d, J = 6.5 Hz, H-9), 1.5
(dq, J = 4.5; 13.4 Hz, H-8'), 1.78–1.84 (m, H-8), 2.2 (sl,
OH-7'), 2.47 (dd, J = 11.8; 17.0 Hz, 1H-7), 2.74 (dd,
J = 4.0; 17.0 Hz, 1H-7), 5.51, 5.68 (2d, J = 1.4 Hz,
CH2O2), 5.88 (s, CH2O2), 6.54 (d, J = 7.9 Hz, H-5),
6.64 (d, J = 8.0 Hz, H-6), 6.64 (d, J = 7.9 Hz, H-5'), 6.71
(d, J = 1.2 Hz, H-2'), 6.74 (dd, J = 1.9; 8.0 Hz). MS m/z
(relative intensity) 324 ([M]‡, 100), 268 (17), 238 (79),
209 (38), 202 (38), 187 (50), 135 (93).
RESULTS AND DISCUSSION
A comparison of the chemical composition of the
different parts of the seedlings (seeds, leaves, epicotyls,
hypocotyls and roots) and in the seeds of V. sebifera was
effected by HPLC analysis. Preliminary evaluations
carried out using a reversed-phase column (Hypersil-
ODS) and a normal phase column (data not shown) did
not allow a complete resolution between the cyclolignans
1 and 2, and 5 and 6. A total resolution was eventually
obtained using a Taxsil column which has been
previously used to analyse taxanes in Taxus species and
podophyllotoxin lignans in extracts from Podophyllum
species (Bastos et al., 1995). The complete resolution
was achieved using a ternary gradient system of
methanol:acetonitrile:water, as shown in Table 1.
Among the tissues analysed, the germinated seeds
presented the greatest diversity of cyclolignans, and
under these conditions compounds 1–6 could be
identified in the chromatogram (Fig. 2). This profile
was very similar to that observed in mature, but non-
germinated, seeds and in seeds that had been detached
after establishment of the seedlings (data not shown).
Figure 3 shows the chromatogram of the extract obtained
Phytochem. Anal. 11: 383–386 (2000)
 LIGNOIDS IN VIROLA SEBIFERA
Figure 2. HPLC chromatogram (UV detection at 280 nm) of
the extracts of seeds of Virola sebifera. Peak numbers refer to
the compounds whose structures are shown in Fig. 1: I.S. is
the internal standard methyl ferulate. (For chromatographic
protocol see the Experimental section.)
Figure 3. HPLC chromatogram (UV detection at 280 nm) of
the extracts of 2-month-old seedlings of Virola sebifera. (For
key to peak identity see legend to Fig. 2: for chromatographic
protocol see the Experimental section.)
from leaves of 2-month-old seedlings, indicating the
presence of a major compound 3 (9.76% of dry weight in
the leaves). This compound was isolated by chroma-
tography and characterised by interpretation of its
spectrometric data as the lignan hydroxy-otobain (3).
Compound 3 was also detected in hypocotyls (1.25%),
epicotyls (0.14%) and roots (0.13%). This is the first
report of hydroxy-otobain in V. sebifera, but 3 has
previously been isolated from seeds of Myristica otoba as
an antioxidant compound (Wallace et al., 1963) and also
from seeds of Diallyanthera otoba (Kohen et al., 1966)
and Osteophloeum platyspermum (Braz Filho et al.,
1984). In the case of V. sebifera, hydroxy-otobain occurs
only in trace amounts in the seeds.
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385
Figure 4. Concentrations of lignans 1±6 (percentage dry
weight) determined by quantitative HPLC in seeds (a) and
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Among the compounds occurring in the seeds, only the
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far there is no further evidence for specific translocation
of compounds 1, 2 and 3 from seeds to the seedlings or of
de novo biosynthesis of cyclolignans in the seedling
tissues.
Acknowledgements
This work was supported by financial aid provided by PADCT, CAPES
and FAPESP, and by fellowships from CNPq.
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