Germacrenes from fresh costus roots

Article abstract of DOI:10.1016/S0031-9422(01)00291-6

Four germacrenes, previously shown to be intermediates in sesquiterpene lactone biosynthesis, were isolated from fresh costus roots (Saussurea lappa). The structures of (+)-germacrene A, germacra-1(10),4,11(13)-trien-12-ol, germacra-1(10),4,11(13)-trien-12-al, and germacra-1(10),4,11(13)-trien-12-oic acid were deduced by a combination of spectral data and chemical transformations. Heating of these compounds yields (-)-β-elemene, (-)-elema-1,3,11(13)-trien-12-ol, (-)-elema-1,3,11(13)-trien-12-al, and elema-1,3,11(13)-trien12-oic acid respectively, in addition to small amounts of their diastereomers. Acid induced cyclisation of the germacrenes yields selinene, costol, costal, and costic acid respectively. It is highly probable that the elemenes reported in literature for costus root oil are artefacts.

Full text of DOI:10.1016/S0031-9422(01)00291-6

Phytochemistry 58 (2001) 481–487
www.elsevier.com/locate/phytochem
Germacrenes from fresh costus roots
Jan-Willem de Krakera,b, Maurice C.R. Franssena,*, Aede de Groota,
Toshiro Shibatac, Harro J. Bouwmeesterb
aLaboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands
bPlant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands
cHokkaido Experimental Station for Medicinal Plants, National Institute of Health Sciences, 108 Ohashi, Nayoro, Hokkaido, 096-0065, Japan
Received 20 March 2001; received in revised form 21 May 2001
Abstract
Four germacrenes, previously shown to be intermediates in sesquiterpene lactone biosynthesis, were isolated from fresh costus roots
(Saussurea lappa). The structures of (+)-germacrene A, germacra-1(10),4,11(13)-trien-12-ol, germacra-1(10),4,11(13)-trien-12-al, and
germacra-1(10),4,11(13)-trien-12-oic acid were deduced by a combination of spectral data and chemical transformations. Heating of
these compounds yields (ꢀ)-b-elemene, (ꢀ)-elema-1,3,11(13)-trien-12-ol, (ꢀ)-elema-1,3,11(13)-trien-12-al, and elema-1,3,11(13)-trien-
12-oic acid respectively, in addition to small amounts of their diastereomers. Acid induced cyclisation of the germacrenes yields seli-
nene, costol, costal, and costic acid respectively. It is highly probable that the elemenes reported in literature for costus root oil are
artefacts. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Saussurea lappa; Asteraceae; Sesquiterpenes; Sesquiterpene lactone biosynthesis; Germacrenes; Elemenes; Eudesmanes
1. Introduction
presumably formed from farnesyl diphosphate (FPP)
via (+)-germacrene A (1) (Fig. 1). Enzyme activities
that introduce a carboxylic function in the isopropenyl
side chain of (+)-germacrene A yielding germacra-
1(10),4,11(13)-trien-12-oic acid (4) have recently been
identified in chicory (Cichorium intybus L.), a vegetable
rich in sesquiterpene lactones (de Kraker et al., 1998,
2001). Currently, investigations concerning the pathway
for sesquiterpene lactones in chicory are hampered by
the lack of a source of germacrene intermediates 1–4, in
particular germacrene acid (4). These germacrene com-
pounds are themselves not present in chicory (Sannai et
al., 1982), and to our knowledge only the isolation of
(+)-germacrene A (1) from various plant species (e.g.
Wichtman and Stahl-Biskup, 1987) and the partial pur-
ification of germacra-1(10),4,11(13)-trien-12-al (3) from
Vernonia glabra (Steetz) (Bohlmann et al., 1983) have
been described. A reason for the scarcity of reports on
these germacrenes might be their instability. They are
susceptible to proton-induced cyclisations and to heat
induced (e.g. steam distillation, GC-analysis) Cope rear-
rangement yielding the eudesmane or elemene framework,
respectively (Fig. 2) (Southwell, 1970; Takeda, 1974;
Wichtman and Stahl-Biskup, 1987; Teisseire, 1994; de
Kraker et al., 1998, 2001).
Saussurea lappa Clarke, is a member of the Asteraceae
indigenous to parts of India and Pakistan where it grows
in the Himalayas at 2500–3500 m elevation. The dried 4–5
year old roots of this plant are known as costus roots and
have a reputation for their medicinal properties as well as
their fragrance. Attempts are being made to cultivate this
plant, since it has become an endangered species due to
ruthless collection from forest areas (Paul et al., 1960;
Hatakeyama et al., 1989; Ayaz, 1996).
Various studies have shown that costus roots are extre-
mely rich in sesquiterpene lactones (3% of fresh weight),
of which the most important are dehydrocostus lactone
and (+)-costunolide (5) (Paul et al., 1960; Somasekar Rao
et al., 1960). The latter is the structurally simplest sesqui-
terpene lactone and is generally accepted as the common
intermediate in the biosynthesis of most sesquiterpene
lactones (Herz, 1977; Fischer, 1990). Costunolide (5) is
* Corresponding author. Tel.: +31-317-482976; fax: +31-317-
484914.
E-mail addresses: maurice.franssen@bio.oc.wag-ur.nl
(M.C.R. Franssen), h.j.bouwmeester@plant.wag-ur.nl
(H.J. Bouwmeester).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00291-6

482
J.-W. de Kraker et al. / Phytochemistry 58 (2001) 481–487
2. Results and discussion
Et2O extracts of commercially available dried costus
roots, costus resinoid and costus root oil were screened
on GC–MSfor the presence of germacrene A ( 1). This
germacrene can be measured as a well resolved peak at a
GC-injection port temperature of 150 ꢁC. The more polar
germacrenes 2–4 cannot be measured as a real peak,
because they will rearrange during migration through the
GC-column to their faster migrating elemenes and will be
observed as a broad ‘‘hump’’ in the baseline such as
described for hedycaryol and 7-hydroxygermacrene
(Stahl, 1984; de Kraker et al., 1998, 2001). Yet, in all of
these extracts we would detect only the Cope-rearran-
gement product b-elemene (6). It was concluded that the
germacrene A originally present in these materials had
been exposed to heat and that the commercially available
materials are not suitable for the isolation of germacrenes.
Interestingly, in the resinoid and dried roots the pre-
viously unreported elema-1,3,11(13)-trien-12-oic acid (9)
was detected in addition to (ꢀ)-elema-1,3,11(13)-trien-12-
ol (7) and (ꢀ)-elema-1,3,11(13)-trien-12-al (8).
Fig. 1. Biosynthetic route from FPP to (+)-costunolide (5) via (+)-
germacrene A (1), germacra-1(10),4,11(13)-trien-12-ol (2), germacra-
1(10),4,11(13)-trien-12-al (3), and germacra-1(10),4,11(13)-trien-12-oic
acid (4). Only the last step (dotted arrow) has not yet been demonstrated.
In contrast, the Et2O extract of fresh costus roots did
contain germacrene A (1), whereas no (ꢀ)-b-elemene (6)
was observed. At the applied GC-injection port tem-
ꢁ
perature of 150 C, also the foreseen ‘‘broad humps’’ of
the oxygenated germacrenes 2–4 were visible in the GC-
chromatogram instead of sharp peaks of the oxygenated
elemenes 7–9.
In order to isolate germacrenes 1–4, 300 g of fresh cos-
tus roots was powdered in liquid N2 and extracted with
Et2O. The organic extract was shaken with aq Na2CO3 to
remove germacrene acid (4) and other acidic/polar com-
pounds; the lactones (mainly costunolide and dehy-
drocostus lactone, 1.5 g) were partially removed by
crystallisation from pentane (Somasekar Rao et al.,
1960). Germacrenes 1–3 could be selectively extracted
from pentane with aq AgNO3 (Southwell, 1970; Min-
naard, 1997), because of the complex-formation
between the Ag+-ions and the diene-ring system present
in the germacrenes. Subsesquent flash column chroma-
tography of the obtained crude germacrene-mixture in
combination with a second AgNO3 extraction yielded:
1.5 mg germacrene A (1), 1.5 mg germacrene aldehyde (3),
and a mixture of germacrene alcohol (2) with b-costol
(11), hedycaryol, costunolide (5), and dehydrocostus lac-
tone. The isolated germacrene A contained a small
impurity of 4% (determined by GC–MS) humulene that is
extracted with aq AgNO3 as well due to the presence of a
cylic diene moiety. Purification of the germacrene alco-
hol (2) was more elaborate because b-costol (10) with its
two adjacent exocyclic bond is also extracted with aq
AgNO3, whereas a- and g-costol are not. Furthermore
germacrene alcohol, in our hands, could hardly be
separated from costunolide on silica gel. Argentation
chromatography yielded 3 mg of germacrene alcohol (2)
Fig. 2. Heat induced Cope-rearrangement of 1–4 yields (ꢀ)-b-elemene
(6), (ꢀ)-elema-1,3,11(13)-trien-12-ol (7), (ꢀ)-elema-1,3,11(13)-trien-12-al
(8), and elema-1,3,11(13)-trien-12-oic acid (9); acidic conditions are
expected to give selinene (10), costol (11), costal (12), and costic acid (13).
The presence of b-elemene (6), (ꢀ)-elema-1,3,11(13)-
trien-12-ol (7), and (ꢀ)-elema-1,3,11(13)-trien-12-al (8)
in commercially available costus root oil has drawn our
attention to these roots (Klein and Thomel, 1976;
Maurer and Grieder, 1977). Since this oil is obtained by
steam distillation and elemenes are generally considered
to be artefacts, we assumed that costus roots originally
contain the corresponding germacrenes 1–3. The repor-
ted isolation of costic acid (13) from costus roots also
suggests the presence of germacrene acid (4) (Bawdekar
and Kelkar, 1965), since HCl was used in the isolation
procedure which might have initiated the cyclisation of
germacrene acid.
The aim of the present research was to investigate the
presence of the germacrenes 1–4 in fresh costus roots,
and to develop a mild isolation procedure for these
compounds so that they can be used for further studies
on sesquiterpene lactone biosynthesis.

J.-W. de Kraker et al. / Phytochemistry 58 (2001) 481–487
483
that was, however, unlike the isolated germacrenes 1, 3 and
4, contaminated with elematrien-12-ol (7) (approx. 30%).
The germacrene acid (4) containing alkaline
(Na2CO3) extracts were carefully brought to pH 6 and
extracted with Et2O. As expected from the protocol of
Bawdekar and Kelkar (1965), the sesquiterpenoid acids
did not dissolve in aq NaHCO3 and acidification to a
lower pH was not necessary, thus preventing the unde-
sired cyclisation of germacrene acid to costic acid (13).
Extraction with aq AgNO3 and column chromato-
graphy yielded 1.5 mg of germacrene acid (4).
of p-toluenesulfonic acid the germacrene aldehyde is
completely cyclised to costal (12) (Fig. 3C). This indi-
cates that the sample is free of elemene aldehyde,
because the elemene aldehyde does not cyclise to costal.
1H NMR of the germacrene aldehyde confirmed that it
did not contain any of its Cope rearrangement product,
since a double doublet at ꢀ 5.85 of C2¼C1H–C10 typical
for the elemene aldehyde was clearly missing. Unfortu-
nately, in solution many germacrenes show broadened
NMR signals or even multiple NMR signals, due to the
existence of different conformations of the ten-membered
ring (Fig. 4) (Takeda, 1974; Minnaard, 1997). This hin-
ders a complete interpretation of the NMR. Most striking
is the presence of two aldehyde proton signals of similar
height at ꢀ 9.37 and ꢀ 9.35 that cannot be due to cou-
pling with a proton at C13, accompanied by two very
small signals at ꢀ 9.47 and ꢀ 9.45 (ratio 1:1:0.01:0.01).
The existence of different germacrene conformations
also explains the formation of a small amount of diaster-
eomeric elematrien-12-al during the Cope rearrangement
of germacrene aldehyde (Fig. 3B). Cope rearrangement is
a stereospecific reaction that preferably proceeds via a
chair-like transition state (March, 1992), via either the
UU or DD conformation of the germacrene (Fig. 4).
However, the UU conformation with the isopropenal
group in the equatorial position predominates and con-
sequently the diastereomer of 8 will be formed only in
low yield (Takeda, 1974; Piet et al., 1995; Minnaard,
1997). Small amounts of diastereomeric elemenes were
also detected for the GC-injection port induced Cope
rearrangement of germacrene A, germacratrien-12-ol
GC-analyses of germacrene aldehyde (3) at a GC-
ꢁ
injection port temperature of 150 C results in a ‘‘broad
hump’’ is (Fig. 3 A) that displays the mass spectrum of
elematrien-12-al (8) over its entire range. At a GC-
ꢁ
injection port temperature of 250 C a sharp peak of
elematrien-12-al is visible instead (Fig. 3B), because the
germacrene aldehyde is immediately rearranged in the
GC-injection port and not during its migration through
the GC-column (de Kraker et al., 2001). In the presence
Fig. 3. GC–MSanalyses of the germacrene aldehyde ( 3) at an injec-
tion port temperature of 150 ꢁC (A) yields a broad hump that starts at
the position of elematrien-12-al (12) (25.17 min). Injection at an tem-
perature of 250 ꢁC (B) gives almost complete rearrangement of 3 to
elematrien-al (12) (25.19), whereas a small amount of diastereomeric
elemene aldehyde can also be detected (24.86). In the presence of p-
toluenesulfonic acid (C) the germacrene aldehyde (3) is completely
converted into costal (8): g-costal (27.18 min), b-costal (27.60 min),
and a-costal (27.67 min).
Fig. 4. Conformations of germacrenes 1–4 denoted as UU, UD, DU,
and DD. U (up) and D (down) refer to the orientation of the C-14 and
C-15 methyl groups. Both conformations UU and DD will easily
undergo Cope rearrangement, however, the UU conformation is pre-
dominant and will yield elemenes 6–9, whereas the diastereomeric ele-
menes that origin from the DD conformation will hardly be formed.

484
J.-W. de Kraker et al. / Phytochemistry 58 (2001) 481–487
and germacratrien-12-oic acid. These diastereomers
have a mass spectrum very similar to their true elemene
and typically constitute only 2–7% of the total amount
of Cope rearrangement product formed. Although we
failed to detect the b-elemene diastereomer before (de
Kraker et al., 1998), small amounts of it are not unusual
in samples containing b-elemene/germacrene A (Dr. M.
A. Posthumus, personal communication). The structure
of the elematrien-12-ol diastereomer has tentatively
been assigned by Maurer and Grieder (1977).
elemol, whereas the petroleum extract of macerated
leaves yields hedycaryol instead (Southwell, 1970). Only
in the case of germacra1(10),4,11(13)-trien-12-ol (2), it
cannot completely be ruled out that a small quantity of
its corresponding elemene is already present in fresh
costus roots. The presence of elemenes instead of ger-
macrenes was the only significant difference between an
Et2O-extract from fresh costus roots and the commer-
cial costus root oil,1 such as thoroughly described by
Maurer and Grieder (1977). It is less certain whether the
eudesmanes 10–13 have to be regarded as artefacts as
well. Although they can be formed from germacrenes
under acidic conditions, maybe during storage in the
plant—an enzyme specifically producing b-selinene
from FPP has been isolated from Citronella mitis fruits
(Belingheri et al., 1992). It cannot be ruled out that
costus root also contain such an enzyme and the eudes-
manes present are genuine enzymatic products.
When the injection port temperature of the GC was
ꢁ
varied between 150 and 250 C, germacrene alcohol (2)
and germacrene acid (4) showed chromatogram patterns
similar to that of germacrene aldehyde (3). Both ger-
macrene A (1) and germacrene acid (4) were completely
converted into their eudesmane analogues (10 and 13,
respectively) in the presence of p-toluenesulfonic acid.
However, the GC-chromatogram of the germacrene
alcohol (2) still showed a peak of elematrien-12-ol (7)
after acid treatment, indicating that isolated sample of
germacrene alcohol is actually a mixture of germacrene-
The presence of germacrenes 1–4 together with vast
amounts of costunolide (5) in costus roots supports the
pathway for costunolide as depicted in Fig. 1. The isolated
germacrene acid (4) is currently being used to investigate
the formation of costunolide (5) during sesquiterpene
lactone biosynthesis in chicory.
1
and elemene alcohol. This was confirmed by H NMR
that showed a double doublet (J=18 and 10 Hz) at
ꢀ 5.81 typical for elematrien-12-ol (Maurer and Grieder,
1977). It is not known whether the presence of elematrien-
12-ol is caused by an intrinsic higher instability of the
germacrene alcohol and/or by the more difficult and time-
consuming purification of this compound. Although less
likely, we cannot rule out that the elematrien-12-ol was
already present in the crude plant material. In this respect
it is remarkable that the germacratrien-12-ol produced in
an enzyme assay from germacrene A (de Kraker et al.,
2001) was free of any elematrien-12-ol and could be fully
cyclised into costol (11), notwithstanding the fact that
3. Experimental
3.1. General experimental procedures
1H NMR and 13C NMR: recorded at 400 MHz with
C6D6 as solvent and TMSas internal standard. GC–
MS: HP 5890 series II gas chromatograph and HP
5972A mass selective detector (70 eV), equipped with a
capillary HP5-MScolumn (30 m Â0.25 mm, film thick-
ness of 0.25 mm) at a helium flow rate of 0.969 ml
ꢁ
the germacratrien-12-ol had been exposed to 30 C, a
temperature unlikely to occur in costus roots that are
underground.
ꢁ
minꢀ1, programmed at 55 C for 4 min followed by a
The H NMR of germacrene acid (4) was free of the
ramp of 5 ꢁC minꢀ1 to 280 ꢁC, and a standard injection
1
ꢁ
double doublet at ꢀ 5.85 from elematrien-12-oic acid (9)
(de Kraker et al., 2001), but once again only few signals
could be assigned. Similar observations were made by
Zdero et al. (1991) for 3a- and 3b-acetoxygermacra-
1(10)-,4,11(13)trien-12-oic acid whose structures could
only be resolved after Cope-rearrangement to their cor-
responding elemene derivatives.
port temperature of 250 C. CC: Silica gel flash (Jans-
sen, 0.035–0.07 nm, pore 6 nm; column width 1.8 cm)
(Still et al., 1978). 5% AgNO3/Silica gel was prepared
according to Maurer and Grieder (1977). TLC: Silica
gel (Merck) 60 F254, eluent EtOAc–hexane (1:4); spray
of 5 ml p-anisaldehyde in 5 ml H2SO4 and 95 ml EtOH,
ꢁ
1–2 min heating at 120 C.
The isolation under mild conditions from fresh costus
roots of (+)-germacrene A (1), germacra-1(10),4,11(13)-
trien-12-al (3), and germacra-1(10),4,11(13)-trien-12-oic
acid (4) clearly demonstrates that their reported elemene
derivatives (6–9) are indeed heat-induced artefacts formed
during drying of the roots or the manufacturing of costus
resinoid/oil as literature on germacrenes suggests (e.g.
Bohlmann et al., 1983; Wichtman and Stahl-Biskup, 1987;
Teisseire, 1994). Very similar, has for example, been
reported for the Australian shrub Phebalium ozotham-
noides F. Muell whose main essential oil component is
3.2. Plant material
Seeds of Saussurea lappa Clarke were introduced from
Hortus Botanicus Vilr, Moscow (USSR) to the fields of
Hokkaido Experimental Station for Medicinal Plants
(Japan) in 1981 (Accession No. 9678-81) (Hatakeyama
1
Less volatile compounds like fatty acids are not taken into
account, because they are for obvious reasons not present in the costus
oil that is produced by steam-distillation.

J.-W. de Kraker et al. / Phytochemistry 58 (2001) 481–487
485
et al., 1989). Five-year old roots were harvested in
August 2000 and transported by courier to The Neth-
erlands. Upon arrival they were cut into pieces,
powdered in liquid N2 with a Waring blender and
tones (1.5 g) were partially removed by dissolving the
ꢁ
residue in pentane and cooling at ꢀ20 C, after which
the crystallised lactones were filtered off and the solvent
removed in vacuo. This procedure was repeated twice.
The obtained oil was dissolved in 15 ml pentane and
extracted with 20% aq. AgNO3 (4Â10 ml). The Ag+
ions were complexed by adding carefully 10 ml of aq.
NH3 (25%) on ice, after which the water layer was
extracted with pentane (3Â50 ml) (Southwell, 1970).
The pentane layers were washed with brine and dried
with Na2SO4 yielding 100 mg of a yellow oil. This oil
was chromatographed with 350 ml pentane–CH2Cl2
(3:7) after which the column was eluted with CH2Cl2.
Fractions of 10 ml were collected and monitored by
GC–MSand TLC; costunolide and dehydrocostus lac-
tone could easily be distinguished by their greenish-blue
(Rf 0.30) and blue (Rf 0.35) spots respectively. After
removal of aplotaxene [a major constituent of costus
root oil (Klein and Thomel, 1976; Maurer and Grie-
der, 1977)] by a second silver-ion extraction (4Â1.5 ml
20% aq. AgNO3, etc.), fractions 2+3 yielded 4 mg of
germacrene A (1) with an impurity of 4% of humu-
lene. Fractions 19–33 yielded, after a second silver-ion
extraction, 1.5 mg germacratrien-12-al (3). Fractions
40–64 contained a mixture of costunolide (5), dehy-
drocostus lactone, b-costol (11), hedycaryol and germa-
crene alcohol (2). Attempts to separate the latter from
b-costol and costunolide by column chromatography on
silica gel using pentane–CH2Cl2 (1:9) or Et2O–pentane
(1:4) failed. Column chromatography on 5% AgNO3-
silica gel (10 gram) was more successful: costol and
subsesquently costunolide were eluted with Et2O (400
ml), whereas germacratrien-12-ol (2 mg, of which ꢂ
30% elematrien-12-ol) was eluted with 150 ml MeOH–
Et2O (1:9).
ꢁ
stored at ꢀ80 C. Dried costus roots were obtained via
internet from gaines.com (El Cajon CA, USA) and
ginseng4less.com (Petaluma CA, USA).
3.3. Chemicals and reference compounds
Costus resinoid and costus oil were obtained from
Pierre Chauvet SA (Seillans, France). Costunolide (5)
and dehydrocostus lactone were isolated from costus
resinoid according to Fischer et al. (1990) making use of
the described TLC-spot colour (Asakawa et al., 1981),
and recrystallised in pentane; NMR date were identical
to those reported in literature (Kuroda et al., 1987;
Takahashi et al., 1987). Standards of (ꢀ)-elema-
1,3,11(13)-trien-12-al (8) [NMR in C6D6 did not mark-
edly differ from those recorded in CDCl3 (Maurer and
Grieder, 1977; Bohlmann et al., 1983)], elema-1,3,
11(13)-trien-12-oic acid (9) and costal (12) were isolated/
synthesised as previously described (de Kraker et al.,
2001). A mixture of a- and g-costic acid (13) was syn-
thesised from a mixture of a- and g-costal with NaClO3
(de Kraker et al., 2001). (ꢀ)-Elema-1,3,11(13)-trien-12-
ol (7) was a gift of Dr. B. Maurer (Firmenich SA, Gen-
eva, Switzerland), standards of (+)-germacrene A (1)
and (ꢀ)-b-elemene (6) were a gift of Professor W. A.
Konig (Hamburg University, Germany).
3.4. Screening of crude materials for the presence of
germacrene A (1)
Samples of dried costus roots were powdered in liquid
N2, and 0.2 g of powdered root was shaken in 3 ml Et2O
and centrifuged after 5 min. The Et2O was passed
through a Pasteur pipette with 0.4 g silica gel. A spatula
tip of costus resinoid and a droplet of costus oil were
also dissolved in 3 ml of Et2O and passed over silica gel.
The Et2O extracts were screened at an injection port
temperature of 150 ꢁC for a germacrene A (1) peak that
should be replaced by a peak of ꢁb-elemene (6) at an
injection port temperature of 250 C (de Kraker et al.,
1998).
3.6. Isolation of germacra-1(10),4,11(13)-trien-12-oic
acid (4)
The aq. NaCO3 layers (150 ml) were carefully acid-
ified on ice to pH 6 with 5 M HCl and extracted with
Et2O (4Â60 ml). The Et2O layers were washed with
brine, dried with Na2SO4, concentrated to 15 ml, and
extracted with 20% aq AgNO3 (4Â10 ml). The Ag+
ions were precipitated with 35 ml 2 M NaCl, and the
remaining solution was extracted with Et2O (4Â50 ml).
After washing with brine and drying with Na2SO4, 20
mg of a yellow solid was obtained that mainly con-
tained germacratrien-12-oic acid (4), costunolide (5)
and dehydrocostus lactone. Column chromatography
with 350 ml of EtOAc–pentane (1:4) removed the lac-
tones after which the germacrene acid was eluted with
100 ml EtOAc. The last minor impurities were removed
by column chromatography with Et2O–pentane (1:4;
400 ml), and 1.5 mg of germacratrien-12-oic acid was
obtained.
3.5. Isolation of germacrenes 1–3
Powdered fresh costus root (300 g) was extracted
twice with 500 ml of Et2O at 6 ꢁC; the Et2O was filtered
and the volume reduced to 75 ml by rotary evaporation
at room temperature. The concentrate was extracted
with 5% aq Na2CO3 (3Â50 ml). The aq Na2CO3 layers
ꢁ
were combined and stored at 4 C. The Et2O layer was
washed with brine, dried with Na2SO4, and the solvent
removed yielding 2.8 g of a yellow-brown solid. Lac-

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J.-W. de Kraker et al. / Phytochemistry 58 (2001) 481–487
3.7. Acid induced cyclisation of germacrenes
s,1H, H-1); 4.84 (br d, J=13.3 Hz) and 4.62 (br d,
J=10.3 Hz) (ratio 1:1, 1H, H-5) 2.75–2.55 (br m, 1H, H-
7). Cope rearrangement to 8, Rl 1585 EIMS m/z (rel int)
M+ 218 (1), [M–Me]+ 203 (12), 81 (100), 67 (70), 79
(68), 41 (63), 91 (56), 53 (50), 93 (46), 95 (38), 77 (38), 55
(37), 107 (37), 105 (35), 119 (27), 161 (25), 121 (24); and
diastereomer of 8, Rl 1570 EIMS m/z (rel int) M+ 218
(ND), [M–Me]+ 203 (8), 81 (100), 79 (64), 67 (60), 41
(57), 91 (48), 93 (43), 95 (41), 53 (40), 77 (36), 105 (36),
107 (35), 68 (34), 55 (34), 119 (25), 121 (22), 175 (22).
Cyclisation yields 12, Rl 1673 (g), 1692 (b) and 1695 (a)
[MS-data are comparable with those of Maurer and
Grieder (1977) and used to determine elution order].
A small amount (ꢂ 10 nmol) of germacrene (1–4) was
dissolved in 2 ml of CH2Cl2 and a crystal of p-toluenesul-
phonic acid was added (Minnaard, 1997). After 7 min at
room temperature, the solution was washed with 1 ml of
NaHCO3 (satd.) and passed through a Pasteur pipette fil-
led with 0.25 g MgSO4. The solvent was reduced to 100 ml
under a stream of N2 and the sample was analysed by
GC–MS. In case of germacratrien-12-oic acid (4)
NaHCO3 was replaced by NaH2PO4. The experiments
were repeated with elemenes 6–9 that did not cyclise.
3.8. Compound characterisation
3.8.4. Germacra-1(10),4,11(13)-trien-12-oic acid (4),
1.5 mg
White crystals, blue TLC-spot (note: 9 gives pink
spot) Rf 0.18, Rl 1771 to Æ 1845. H NMR ꢀ 6.44-6.37
3.8.1. Germacrene A (1), 4 mg
Colourless oil, purple TLC-spot Rf 0.75, Rl 1511,
EIMS m/z (rel int) M+ 204 (8), [M–Me]+ 189 (37), 93
(100), 67 (99), 68 (94), 107 (79), 81 (77), 41 (68), 79 (67),
105 (59), 91 (57), 53 (54), 147 (47), 119 (43), 121 (41), 55
(41), 161 (36), 133 (36). Cope rearrangement to 6, Rl
1396, EIMS m/z (rel int) M+204 (1), [M–Me]+ 189 (28),
93 (100), 81 (96), 67 (83), 68 (76), 41(67), 79 (64), 107
(63), 53 (51), 91 (48), 147 (45), 121 (42), 105 (42), 55
(41), 161 (31), 119 (31), 133 (29); and diastereomer of 6,
Rl 1388, EIMS m/z (rel int) M+ 204 (1), [M–Me]+ 189
(23), 93 (100), 81 (78), 67 (61), 68 (58), 41 (52), 79 (51),
107 (50), 91 (41), 121 (35), 161 (34), 55 (33), 53 (32), 105
(31), 119 (27), 133 (23), 147 (21). Cyclisation yields 10,
Rl 1479 (g), 1491 (b) and 1500 (a) [MS-data are com-
parable with those of Maurer and Grieder (1977) and
were used in combination with the Rl’s reported by
Adams (1995) to determine the elution order].
1
(m) and 6.36 (s) (ratio 1:1, 1H, H-13); 5.44 (br m) and
5.38 (s) (ratio 1:1, 1H, H0-13); 5.30–5.15 (br s, 1H, H-1);
4.85 (br m) and 4.63 (br d, J=10.6 Hz) (ratio 1:1, 1H,
H-5); 2.75-2.55 (br m, 1H, H-7). Cope rearrangement to
9, Rl 1771 EIMS m/z (rel int) M+ 234 (1), [M–Me]+ 219
(7), 81 (100), 67 (50), 79 (47), 41 (43), 68 (40), 91 (38), 53
(34), 93 (29), 105 (29), 55 (26), 77 (26), 107 (26), 121
(23), 119 (19), 69 (17), 177 (15); and diastereomer of 9,
Rl 1750 EIMS m/z (rel int) M+ 234 (ND), [M–Me]+
219 (4), 81 (100), 79 (67), 41 (58), 67 (56), 107 (55), 91
(49), 93 (49), 53 (41), 55 (40), 68 (39), 105 (38), 121 (35),
162 (33), 147 (26), 119 (25), 163 (25). Cyclisation yields
13; Rl 1854 (g), EIMS m/z (rel int) M+ 234 (26), [M–
Me]+ 219 (100), 91 (57), 41 (35), 79 (35), 81 (34), 105
(34), 147 (34), 107 (32), 93 (31), 55 (30), 77 (30), 173
(25); Rl 1876 (b), EIMS m/z (rel int) M+ 234 (25), [M–
Me]+ 219 (67), 91 (100), 79 (95), 93 (85), 41 (78), 121
(78), 77 (63), 81 (63), 55 (61), 67 (59), 105 (55), 107 (55),
53 (45), and Rl 1879 (a), EIMS m/z (rel int) M+ 234
(27), [M–Me]+ 219 (100), 91 (57), 79 (46), 41 (38), 81
(37), 107 (36), 105 (35), 93 (35), 55 (29), 205 (26) (elution
order of isomers presumed to be the same as for 10–12).
3.8.2. Germacra-1(10),4,11(13)-trien-12-ol (2), 2 mg,
mixture with 7 (ꢂ 30%)
Colourless/slightly yellow oil, pink/purple TLC-spot
Rf 0.29, Rl 1673 to Æ 1806. Cope rearrangement to 7, Rl
1673 EIMS m/z (rel int) M+ 220 (1), [M–Me]+ 205 (4),
79 (100), 81 (94), 41 (91), 91 (90), 67 (86), 93 (85), 55
(69), 105 (66), 53(60), 119 (58), 77 (55), 68 (53), 189 (42),
121 (37), 133 (35), 145 (33), 163 (25), 161 (25); and dia-
stereomer of 7, Rl 1657, EIMS m/z (rel int) M+ 220 (1),
[M–Me]+ 205 (3), 81 (100), 41 (94), 79 (92), 93 (78), 67
(78), 91 (71), 69 (70), 55 (63), 105 (55), 119 (50), 77 (46),
53 (45), 161 (28), 133 (24), 145 (18), 189 (15). Cyclisa-
tion yields 11, Rl 1755 (g), 1775 (b) and 1781 (a) [MS-
data are comparable with those of Maurer and Grieder
(1977) and used to determine elution order].
Acknowledgements
The authors are most grateful to Dr. B. Maurer for the
gift of elematrien-12-ol and to Professor Dr. W. A. Konig
for the gift of the germacrene A and b-elemene standard.
We would also like to thank Dr. M. A. Posthumus for the
GC–MSanalyses of the costus root oil and resinoid, A. van
Veldhuizen for recording NMR data, Dr. J. B. P. A. Wijn-
berg for his help in the interpretation of the NMR data,
and ing. F. W. A. Verstappen for technical assistance.
3.8.3. Germacra-1(10),4,11(13)-trien-12-al (3), 1.5 mg
Colourless/slightly yellow oil with strong mossy
odour, pink spot Rf 0.58, Rl 1585 to Æ 1715. 1H NMR ꢀ
9.35 (s) 9.37 (s), 9.45 (s) and 9.47 (s) (ratio 1:1:0.01:0.01,
1H, H-12); 5.77 (br s) and 5.72 (s) (ratio 1:1, 1H, H-13);
5.36 (br s) and 5.33 (s) (ratio 1:1, 1H, H0-13); 5.22 (br
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