Stilbenoids from Tragopogon orientalis

Article abstract of DOI:10.1016/j.phytochem.2006.06.031

A phytochemical investigation of Tragopogon orientalis L. (Asteraceae, Cichorieae) yielded the natural products 6″-O-(7,8-dihydrocaffeoyl)-α,β-dihydrorhaponticin, 3′-O-methyl-α,β-dihydrorhaponticin, and (S)-3-(4-β-glucopyranosyloxybenzyl)-7-hydroxy-5-meth

Full text of DOI:10.1016/j.phytochem.2006.06.031

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 2182–2188
www.elsevier.com/locate/phytochem
Stilbenoids from Tragopogon orientalis
a
a
b
Christian Zidorn , Sandra Grass , Ernst P. Ellmerer ,
b
a,
*
Karl-Hans Ongania , Hermann Stuppner
a
Institut f u¨ r Pharmazie der Universit a¨ t Innsbruck, Josef-Moeller-Haus, Innrain 52, A-6020 Innsbruck, Austria
b
Institut f u¨ r Organische Chemie der Universit a¨ t Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
Received 27 April 2006; received in revised form 22 June 2006
Available online 4 August 2006
Abstract
00
A phytochemical investigation of Tragopogon orientalis L. (Asteraceae, Cichorieae) yielded the natural products 6 -O-(7,8-dihydro-
0
caffeoyl)-a,b-dihydrorhaponticin, 3 -O-methyl-a,b-dihydrorhaponticin, and (S)-3-(4-b-glucopyranosyloxybenzyl)-7-hydroxy-5-meth-
oxyphtalide as well as known compounds a,b-dihydrorhaponticin, 3-(4-methoxybenzyl)-5,7-dimethoxyphthalide, p-dihydrocoumaric
acid methyl ester, and 1-hydroxypinoresinol-1-O-b-glucopyranoside. The structures were established by HR mass spectrometry, exten-
sive 1D and 2D NMR spectroscopy, and CD spectroscopy. Moreover, the radical scavenging activities of the major compounds were
measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The chemosystematic impact of the occurrence of stilbene derivatives
in T. orientalis is discussed.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Tragopogon; Asteraceae; Tribe Cichorieae; Subtribe Scorzonerinae; Chemosystematics; Stilbenoids
1
. Introduction
closely related genus Scorzonera, which like Tragopogon
is a member of the subtribe Scorzonerinae, also yielded a
number of biogenetically related dihydroisocoumarins,
benzophenone, and dihydrostilbene derivatives. These
compounds were reported from Scorzonera cretica Willd.
(Paraschos et al., 2001) and Scorzonera humilis L. (Zidorn
et al., 2000, 2002, 2003).
The present communication reports about new (2–3)
and known (1) dihydrostilbenes, a new (4) and a known
(5) benzylphtalide, a phenylpropanoid (6), and a lignan
(7) from rootstocks of T. orientalis (see Fig. 1).
Tragopogon orientalis L. (synonym: Tragopogon praten-
ˇ
sis L. subsp. orientalis Celak.) is an annual to perennial herb
of 30–70 cm height, golden yellow ligules and a cylindrical
rootstock. The taxon is native to Eastern and Central Eur-
ope and Western Siberia. T. orientalis is common in Aus-
tria; formerly the rootstocks were used as a vegetable
similar to black salsify and the leaves as a vegetable similar
to spinach (Richardson, 1976; Wagenitz, 1987; Fischer
et al., 2005; J a¨ ger and Werner, 2005). Previous phytochem-
ical studies of T. orientalis and T. pratensis yielded flavo-
noids, phenolic acids, sterols, triterpenes, triterpene
glycosides, and triterpene saponins (Krzaczek and Smolarz,
2. Results and discussion
1
988; Smolarz and Krzaczek, 1988; Miyase et al., 1992).
Recently, a number of stilbenoids and biogenetically
Compounds 1–7 were isolated from rootstocks of
related dihydroisocoumarins have been isolated from T.
porrifolius L. subsp. porrifolius (Zidorn et al., 2005a). The
T. orientalis collected in May 2004 near Innsbruck/Tyrol/
Austria. The structures were assigned using NMR, MS,
UV, optical rotation, and CD data.
*
In detail, HRMS of compound 1 in the positive mode
showed a [M + H] signal at m/z = 423.1631 (calculated
Corresponding author. Tel.: +43 512 507 5300; fax: +43 512 507 2939.
E-mail address: Hermann.Stuppner@uibk.ac.at (H. Stuppner).
+
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.06.031

C. Zidorn et al. / Phytochemistry 67 (2006) 2182–2188
2183
OCH3
OGlc
OCH3
4
'
4'
OR2
5
'
'
3'
5
'
3
'
6
'
6
2'
2'
1
2
3
: R = H, R = H
1 2
1'
1'
: R = 7,8-dihydrocaffeoyl, R = H
4
5
1
2
: R = H, R = CH
1
2
3
10
3
O
O
2
H
H
1
R O
1
1
9
O
O
2
6
4
8
HO
O
5
5
HO
7
3
O
OH
HO
OCH3
H CO
3
OCH3
OH
4
6
OCH3
O
OCH3
HO
O
6
7
GlcO
H
O
OH
OH
OCH3
Fig. 1. Structures of new (2–4) and known (1 and 5–7) natural products from Tragopogon orientalis.
for C H O , m/z = 423.1655), which indicated a molecu-
1, but showed additional signals assignable to a 7,8-dihy-
2
1
27
9
1
13
lar formula of C H O . H NMR (Table 1) and
C
drocaffeoyl moiety (Zidorn et al., 2005b). Downfield shifts
2
1
26
9
0
0
NMR (Table 2) data included signals assignable to a
of the signals assignable to the protons in position C-6 of
the glucose moiety in comparison to compound 1
(d = 4.42 and 4.18 versus d = 3.88 and 3.70) indicated
0
0
3
,5,3 ,4 -tetra-substituted bibenzyl moiety, a b-glucose
moiety, and a methoxy-group (Biondi et al., 2005). HMBC
correlations indicated that the methoxy group was situated
H
H
that the 7,8-dihydrocaffeoyl moiety was linked via this
position with the rest of the molecule. This assumption
was proven by HMBC experiments, which showed cross-
0
in position 4 and that the glucose moiety was attached to
one of the two oxygens of the other aromatic subunit (posi-
tion 3). Conclusively, 1 is a,b-dihydrorhaponticin, a natu-
ral product, which was recently isolated from Glycrrhiza
glabra L. leaves (Biondi et al., 2005). The aglycone of
a,b-dihydrorhaponticin (1a) was synthesized from 1 by
enzymatic hydrolysis. Identification was accomplished by
mass spectrometry and NMR spectroscopy and by com-
parison of the obtained data with literature data (Matsuda
et al., 2001; Hernandez-Romero et al., 2004; Biondi et al.,
0
0
peaks from the signals assignable to the protons at C-6
to the carbonyl signal of the acyl moiety. Thus, compound
00
2
was identified as 6 -O-(7,8-dihydrocaffeoyl)-a,b-
dihydrorhaponticin, which represents a new natural
product.
HRMS of compound 3 in the positive mode showed a
+
[M + H]
signal at m/z = 437.1788 (calculated for
C H O , m/z = 437.1812), which indicated a molecular
2
2
29
9
1
13
2
005).
HRMS of compound 2 in the positive mode showed a
formula of C H O . H NMR (Table 1) and C NMR
22 28 9
spectra (Table 2) were nearly identical to those of
compound 1 but showed additional signals for a second
methoxy group. HMBC experiments showed that this
+
[
M + H]
signal at m/z = 587.2160 (calculated for
C H O , m/z = 587.2129), which indicated a molecular
3
0
35 12
1
13
0
formula of C H O . H NMR (Table 1) and
C
group was linked via position C-3 with the molecule. Con-
3
0
34 12
0
NMR data (Table 2) were similar to those of compound
clusively, compound 3 was established as 3 -O-methyl-a,

2184
C. Zidorn et al. / Phytochemistry 67 (2006) 2182–2188
Table 1
Table 2
1
a
13
a
H NMR data of stilbenoids 1–3
C NMR data of stilbenoids 1–3
Position
1
2
3
Position
1
2
3
Stilbenoid moiety
1
Stilbenoid moiety
1
2
3
4
5
6
a
b
1
2
3
4
145.5
109.3
160.1
102.7
159.2
110.7
39.2
145.5
109.3
160.0
103.0
159.2
110.9
39.2
145.1
109.3
160.5
102.4
159.8
110.5
39.2
2
3
4
5
6
6.39 1H, m
6.33 1H, t (2.0)
6.35 1H, t (2.0)
6.30 1H, t (2.0)
6.40 1H, t (2.0)
6.38 1H, t (2.0)
6.29 1H, t (2.0)
b
b
6.38 1H, m
6.30 1H,
dd (2.0, 1.5)
b
b
b
a
b
1
2
2.74 2H, m
2.74 2H, m
2.72 2H, m
2.84 2H, m
38.1
38.0
37.6
b
b
b
0
0
0
0
0
0
0
0
2.72 2H, m
2.79 4H, m
136.1
116.6
147.2
147.2
112.8
120.7
136.0
116.6
147.2
147.2
112.8
120.8
136.0
113.7
150.0
148.4
112.4
121.8
56.3
0
0
c
6.65 1H, d (2.0) 6.62 1H, d (2.5)
6.71 1H,
dd (2.0)
0
0
0
0
c
3
4
5
6
5
6
6.79 1H, d (8.0) 6.75 1H, d (8.0)
6.58 1H,
6.84 1H,d (8.0)
6.69 1H,
3 –OCH
4 –OCH
3
3
6.52 1H,
56.5
56.5
56.0
dd (8.0, 2.0)
dd (8.0, 2.5)
dd (8.0, 2.0)
3.79 3H, s
3.77 3H, s
Glucose moiety
0
0
3
4
–OCH
–OCH
3
0
0
0
00
00
00
0
0
0
1
2
3
4
5
6
102.3
74.9
78.0
71.4
78.0
62.5
102.3
74.8
77.8
71.8
75.4
64.8
101.8
75.0
78.0
71.2
78.0
62.4
3
3.81 3H, s
3.80 3H, s
Glucose moiety
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
4.78 1H, d (7.5) 4.73 1H, d (7.5)
4.78 1H, d (7.0)
b
b
b
b
b
b
b
b
b
3.42 1H, m
3.44 1H, m
3.41 1H, m
3.41 1H, m
3.88 1H,
3.41 1H, m
3.43 1H, m
3.32 1H, m
3.55 1H, m
4.42 1H,
3.41 1H, m
b
3.43 1H, m
b
3.38 1H, m
b
7
1
2
3
4
5
6
7
8
9
,8-Dihydrocaffeoyl moiety
3.38 1H, m
0
0
0
0
0
0
00
00
00
00
00
00
133.6
116.5
146.1
144.6
116.4
120.6
31.3
3.88 1H,
dd (12.0, 2.0)
3.71 1H,
dd (12.0, 1.5)
.70 1H,
dd (12.0, 5.0)
dd (12.0, 2.0)
4.18 1H,
dd (12.0, 7.0)
3
dd (12.0, 5.0)
7
1
2
3
4
5
6
7
8
9
,8-Dihydrocaffeoyl moiety
0
0
0
0
0
0
0
0
0
00
00
00
00
00
00
00
00
00
000
000
000
6.62 1H, d (2.0)
37.2
174.8
a
4
Measured in methanol-d at 75 MHz (1–2) and 125 MHz (3), respec-
6.63 1H, d (8.0)
6.47 1H, dd (8.0, 2.0)
2.73 2H, m
13
tively. Spectra were referenced to solvent signals at d
NMR shift values of compound 3 were derived from HSQC and HMBC
C
= 49.0 ppm.
C
spectra.
2.57 2H, m
b
Signal assignments for these two signals were erroneously interchanged
by Biondi et al. (2005).
a
c
Measured in methanol-d
4
at 300 MHz (1–2) and 500 MHz (3),
= 3.31 ppm.
C NMR shift values of compound 3 were derived from HSQC and
Signal assignments for these two signals were erroneously interchanged
respectively. Spectra were referenced to solvent residual at d
H
by Biondi et al. (2005).
1
3
HMBC spectra.
b
Overlapping signals.
from a carbonyl moiety connected to one of the aromatic
systems to a hydroxy group in a-position to this system.
The biogenetically related dihydroisocoumarins are dihy-
drostilbenes featuring a six-ring lactone, which is formed
from a carbonyl moiety connected to one of the aromatic
systems to a hydroxy group in b-position to this system.
HMBC experiments also indicated that the glucose moiety
was attached to the phenolic hydroxy-group of the para
substituted aromatic system and that the methoxy group
was linked to the one of the two phenolic hydroxy groups
situated between the two aromatic protons of that system.
Conclusively, compound 4 was established as 3-(4-b-gluco-
pyranosyloxybenzyl)-7-hydroxy-5-methoxyphtalide. The
configuration at C-3 was deduced from CD-spectra, which
showed positive Cotton effects at 240 and 290 nm as well
as a negative peak at 255 nm. As no CD spectra of
b-dihydrorhaponticin, which represents another new natu-
ral product. The aglycon of compound 3 was recently iso-
lated as vittarin-A from Vittaria anguste-elongata Hayata
(
Wu et al., 2005).
HRMS of compound 4 in the positive mode showed a
+
[
M + Na]
signal at m/z = 471.1233 (calculated for
C H O Na, m/z = 471.1267), which indicated a molecu-
2
2
24 10
1
13
lar formula of C H O . H NMR and C NMR data
Table 3) displayed signals assignable to a para substituted
2
2
24 10
(
aromatic moiety, a tetrasubstituted aromatic moiety with
the two protons in meta position to each other, a glucose
moiety, a methine, and a methylene group. HMBC correla-
tions indicated that compound 4 was a 3-benzylphtalide,
i.e. a dihydrostilbene derivative, with a five-ring lactone

C. Zidorn et al. / Phytochemistry 67 (2006) 2182–2188
2185
Table 3
NMR data of compound 4
ester (6) (Dorrestein et al., 2003), and as 1-hydroxypinores-
inol-1-O-b-glucopyranoside (7) (Wang et al., 1993),
respectively.
1
13
Position
H NMR
C NMR
Phtalide moiety
Compounds 1, 1a, and 2 were assessed (in triplicate) for
their radical scavenging activity in comparison to the refer-
ence compound ascorbic acid. The following IC values in
lg/ml and lmol/ml, respectively, were established for the
reduction of 40 mg/l of the DPPH radical (values in brack-
ets indicate standard deviations): IC50 (lg/ml): ascorbic
acid, 2.29 (0.06); 1 8.98 (0.52); 1a 4.82 (0.31); 2 (9.04
1
3
4
5
6
7
8
9
171.6
81.8
99.9
167.5
102.4
161.0
106.0
154.8
40.3
5.59 1H, t (6.0)
6.36 1H, s
5
0
6.30 1H, s
(
(
0.22). IC50 (lmol/ml): ascorbic acid, 13.0 (0.36); 1 21.3
1.24); 1a 18.6 (1.20); 2 15.4 (0.37). The differences in
10
3.21 1H, dd (14.5, 5.5)
3
.09 1H, dd (14.5, 6.0)
IC50 values of compounds 1, 1a, and 2 given in lmol/ml
are statistically significant (Student–Newman–Keuls test;
p < 0.05). Thus, compound 2, which has three free phenolic
hydroxy groups, two of them in ortho-position, is the best
radical scavenger on a molar basis. The second best radical
scavenging activity on a molar basis is displayed by com-
pound 1a, which also features three phenolic hydroxy
groups, however none of them combined to an ortho-dihy-
droxy moiety. Compound 1, with only two free phenolic
hydroxy groups has the weakest radical scavenging activity
of the three compounds analyzed. These results are in
accord with the structure activity relationships for phenolic
radical scavenging compounds as discussed by Rice-Evans
et al. (1996).
5
–OCH
3
3.80 3H, s
56.2
Benzyl moiety
0
1
2
3
4
5
6
129.9
131.8
117.4
157.7
117.4
131.8
0
0
0
0
7.09 1H, d (8.0)
6.79 1H, d (8.0)
6.79 1H, d (8.0)
7.09 1H, d (8.0)
0
Glucose moiety
0
0
0
0
0
0
0
0
0
0
0
0
b
1
2
3
4
5
6
4.86 1H
101.8
74.3
78.0
71.2
77.4
62.4
c
3.43 1H, m
3.42 1H, m
3.38 1H, m
3.40 1H, m
3.89 1H, dd (12.0, 2.0)
c
c
c
3
.69 1H, dd(12.0, 5.0)
The discovery of further dihydrostilbenes in T. orientalis
corroborates the finding that taxa from the tribe Scorzo-
nerinae are a rich source of this class of natural compounds
a
1
13
Measured in methanol-d
4
at 500 MHz ( H NMR) and 125 MHz (
C
NMR), respectively. Spectra were referenced to solvent residual and sol-
vent signals at d = 3.31 ppm and d = 49.0 ppm, respectively. C NMR
H C
shift values were derived from HSQC and HMBC spectra.
1
3
(Paraschos et al., 2001; Zidorn et al., 2000, 2002, 2003,
b
1
H NMR signal covered by signal of residual water.
Overlapping signal.
2005a). Dihydrostilbenes are not occurring ubiquitously.
However, dihydrostilbene derivatives are occurring in
various only loosely related plant taxa, including the prim-
itive and phylogentically old liverworts (Yoshida et al.,
1996). Moreover, the distribution of dihydrostilbene
derivatives in the higher plants (Trachaeophyta) is rather
erratic. For example dihydrostilbenoids have been reported
from ferns (Wollenweber et al., 1993), monocots (Adesa-
nya et al., 1989), and Rosids (Biondi et al., 2005), including
occasional reports from the Asteraceae family (Braca et al.,
1999). Conclusively, dihydrostilbenoids are not employable
as chemosystematic markers for higher categories (family
level or above). Moreover, it is plausible to assume that
the common ancestor of the Trachaeophyta already
possessed the genetic and enzymatic requirements for the
biosynthesis of these natural products. In many taxa of
contemporary higher plants this system is either lost or
switched off. The observed frequency and high structural
diversity of dihydrostilbenes observed in the subtribe Scor-
zonerinae might be of pronounced chemosystematic inter-
est for the phenetic characterization of this taxon, which
due to recent (macro-)molecular investigations is mono-
phyletic (Mavrodiev et al., 2004). The distribution of
benzylphtalides, which are biogenetically closely related
to dihydrostilbenes, follows the same erratic pattern;
benzylphtalides have been reported from liverworts
(Asakawa et al., 1986, 1987) as well as various families of
c
3
-benzylphtalides were available for comparison, the CD
spectra of 4 were interpreted in comparison with 3-(4,7-
dimethyl-1-naphthyl)phtalide (Pirkle et al., 1998), 6-
hydroxymellein (Krohn et al., 1997), and 3-phen-
yldihydrosiocoumarins (Paraschos et al., 2001; Zidorn
et al., 2005a). Compound 4 showed a CD-spectrum similar
to (R)-3-(4,7-dimethyl-1-naphthyl)phtalide (Pirkle et al.,
1
998) and mirror-inverted to that of (R)-6-hydroxymellein
(
Krohn et al., 1997). Conclusively, (S)-configuration for
position 3 was supposed and compound 4 was elucidated
as (S)-3-(4-b-glucopyranosyloxybenzyl)-7-hydroxy-5-meth-
oxyphtalide. Compound 4 represents a new natural prod-
uct. 3-Benzylphtalides are a rare group of natural
products; compounds similar to the aglycon of 4 have been
reported from the liverwort genera Balanteopsis and Frull-
ania (Asakawa et al., 1986, 1987, 2003; Kraut et al., 1994).
To the best of our knowledge, compound 4 represents the
first naturally occurring glucoside of a benzylphtalide.
Known compounds 5–7 were identified on the basis of
their mass spectra and NMR as 3-(4-methoxybenzyl)-5,7-
dimethoxyphthalide (5), a natural product known from
Frullania falciloba Tayl. ex Lehm. (Asakawa et al., 1987,
2
003; Mali et al., 2001), as p-dihydrocoumaric acid methyl

2186
C. Zidorn et al. / Phytochemistry 67 (2006) 2182–2188
angiosperms [monocots e.g. Shode et al. (2002), Rosids e.g.
Yoshikawa et al. (1992)].
3.2.2. Semi-preparative HPLC
Semi-preparative HPLC was performed using a Dionex
P580 system with ASI-100 automated sample injector and
an UVD17OU detector; fractions were collected employing
a Gilson Abimed 206 fraction collector. For both separa-
tions a X-terra prep MS C18 7.8 · 100 mm column with
5 lm particle size was used. Compounds 3 and 7 were sep-
arated using a flow rate of 1.5 ml/min and a gradient from
20% MeCN to 40% MeCN in 20 min. Compounds 3 and 7
were collected in the intervals between 12.4 and 13.5 min
and 8.5 and 9.5 min, respectively. Compound 4 was puri-
fied using a flow rate of 2.0 ml/min and a gradient 10–
40% MeCN in 20 min. Compound 4 was collected in the
interval between 12.2 and 13.0 min.
3
. Experimental
3
.1. Plant material
T. orientalis was collected between V o¨ ls and Kematen W
of Innsbruck/Tyrol/Austria [coordinates (WGS84): N
0
00
0
00
4
7ꢁ15 40 E 11ꢁ19 01 ; alt.: 590 m a.m.s.l.] on the 24th of
May 2004. A voucher specimen (SG20040524-A1) was
deposited in the herbarium of the Institut f u¨ r Pharmazie/
Innsbruck.
3
3
.2. Extraction and isolation
3.2.3. Enzymatic preparation of compound 1a from 1
Fractions enriched in compound 1 (30.6 mg) were dis-
.2.1. Isolation of compounds 1–7
Air-dried ground rootstocks (240 g) of T. orientalis
solved in 1.3 ml of H O (adjusted to pH 5), mixed with
2
0.5 ml of H O (adjusted to pH 5) containing 30.6 mg of cel-
2
were exhaustively extracted with MeOH. The crude
extract was dried in vacuo to give 54.0 g of residue. The
residue was redissolved in a mixture of MeOH and
lulase (Sigma, St. Louis, USA). The mixture was kept for 2
days at 37 ꢁC. Compound 1a (0.6 mg) was purified by par-
titioning with CH Cl and Sephadex LH-20 CC of the
2
2
H O and successively partitioned with petrol ether and
2
CH Cl layer.
2 2
EtOAc. The EtOAc layer was dried in vacuo yielding
2
.50 g. The EtOAc layer was fractionated by silica gel
3.3. Physical data of new compounds
column chromatography (CC) employing a gradient of
CH Cl and MeOH. Fractions were combined based on
0
0
3.3.1. 6 -O-(7,8-Dihydrocaffeoyl)-a,b-dihydrorhaponticin
(2).
2
2
their TLC profiles (mobile phases: various mixtures of
CH Cl and MeOH; detection: UV/VIS, spraying with
20
Yellow crystals, m.p. 106 ꢁC; ½aꢀ ꢁ28ꢁ (c 0.69, MeOH);
2
2
D
ZnSe
max
ꢁ1
vanillin/H SO ) resulting in 21 combined fractions (CF)
FTIR v
cm : 3394 (br), 2930, 2860, 1715, 1601, 1514,
2
4
1
of increasing polarity.
1450, 1414, 1359, 1276, 1174, 1127, 1114, 1077, 1028; H
NMR: see Table 1; C NMR: see Table 2.
1
3
A mixture (3/2, 9.7 mg) of compounds 5 and 6 was iso-
lated from CF-4 [values given in brackets indicate the mass
of the respective fraction and the ratio of the solvent mix-
ture during elution] (68.3 mg; CH Cl /MeOH, 9/1, v/v) by
0
3.3.2. 3 -O-Methyl-a,b-dihydrorhaponticin (3)
Yellow amorphous solid, glass transition 100–105 ꢁC;
2
2
2
D
0
ZnSe ꢁ1
cm : 3370
max
Sephadex LH-20 CC using MeOH as the eluant. Fractions
CF-12 (57.7 mg; CH Cl /MeOH, 8/2, v/v), CF-13
½aꢀ ꢁ16.49ꢁ (c 0.10, MeOH); FTIR v
(br), 2922, 2858, 1596, 1515, 1461, 1420, 1331, 1302,
2
2
1
(
43.8 mg; CH Cl /MeOH, 7/3, v/v), and CF-14 (347 mg;
1260, 1233, 1172, 1156, 1136, 1075, 1027, 999; H NMR:
see Table 1; C NMR: see Table 2.
2
2
1
3
CH Cl /MeOH, 7/3, v/v) were repeatedly fractionated by
2
2
Sephadex LH-20 CC (eluant: MeOH). Sephadex LH-20
of CF-14 yielded 39.8 mg of compound 1. Fractions of
CF-14 enriched in compounds 2, 3, and 7 (31.5 mg) were
combined with CF-13 and fractionated by Sephadex LH-
3.3.3. (S)-3-(4-b-Glucopyranosyloxybenzyl)-7-hydroxy-5-
methoxyphtalide (4)
20
Colorless crystals, m.p. 119 ꢁC; ½aꢀ ꢁ36ꢁ (c 0.19,
D
ZnSe
max
ꢁ1
2
0 to yield fractions further enriched in 2 (28.5 mg) and 3
MeOH); FTIR v
cm : 3373 (br), 2921, 1730, 1686,
and 7 (14.1 mg). Compound 2 (13.0 mg) was finally puri-
fied by Sephadex LH-20 CC. Sephadex LH-20 of these
fractions enriched in 3 and 7 yielded 6.8 mg of a mixture
of both compounds. This was combined with subfractions
of CF-12 also enriched in 3 and 7 (5.2 mg) and subjected to
semi-preparative RP-HPL chromatography using a gradi-
1613, 1512, 1457, 1438, 1379, 1320, 1226, 1194, 1161,
1074, 1038; H NMR and C NMR: see Table 3.
1
13
3.4. Radical scavenging activity
Methanolic solutions of test compounds were mixed
with a methanolic solution of DPPH (Sigma–Aldrich,
Steinheim, Germany). The final DPPH concentration was
40 mg/l. Compounds 1, 1a, and 2 were tested in final con-
centrations of 1, 2, 5, 10, and 20 lg/ml, respectively. After
incubation in 96 well-plates, the reaction mixture (250 ll)
was kept in the dark at ambient temperature (25 ꢁC) for
30 min. Then, the optical density of the test mixtures in
ent of H O and MeCN to yield 0.9 mg of compound 3
2
and 1.3 mg of compound 7. Compound 4 was isolated from
CF-15 (105 mg; CH Cl /MeOH, 5/5, v/v) and CF-16
2
2
(
142 mg; CH Cl /MeOH, 5/5, v/v) by repeated Sephadex
2 2
LH-20 CC and successive semi-preparative RP-HPL chro-
matography using a gradient of H O and MeCN to yield
2
2
.2 mg of compound 4.

C. Zidorn et al. / Phytochemistry 67 (2006) 2182–2188
2187
comparison to DPPH and pure methanol was measured
using a Hidex Chameleon plate reader at 515 nm. IC50 val-
ues for each replicate were calculated using the following
formula: IC50 = [(50 ꢁ LP)/(HP ꢁ LP) * (HC ꢁ LC)] +
LC. LP = low percentage, i.e. highest percent inhibition
less than 50%; HP = high percentage, i.e. lowest percent
inhibition greater than 50%; HC = high concentration,
i.e. concentration of test substance at the high percentage,
LC = low concentration, i.e. concentration of test sub-
stance at the low percentage. All compounds and concen-
trations were assayed in triplicate and mean values were
calculated for each compound. Ascorbic acid and DPPH
were obtained from Merck (Darmstadt, Germany) and
Sigma–Aldrich (Steinheim, Germany), respectively.
1997. Dihydroisocoumarins from fungi: isolation, structure eluci-
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4
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Acknowledgements
The authors thank C. Kreutz and R. Micura for measur-
ing CD spectra, J. M. Rollinger for IR spectra, and R. Spi-
taler for helping collect the plant material. This work was
supported by the Fonds zur F o¨ rderung der wissenschaftli-
chen Forschung (FWF, P15594).
2
087–2091.
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