Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 982–987
www.elsevier.com/locate/phytochem
Constituents of the stem bark of Discopodium penninervium
and their LTB4 and COX-1 and -2 inhibitory activities
Abraham Abebe Wube a, Eva-Maria Wenzig a, Simon Gibbons b, Kaleab Asres c,
Rudolf Bauer a, Franz Bucar a,*
a
b
Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens University Graz, Universitaetsplatz 4/1, A-8010 Graz, Austria
The School of Pharmacy, Centre for Pharmacognosy and Phytotherapy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK
c
The School of Pharmacy, Department of Pharmaccognosy, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia
Received 21 September 2007; received in revised form 5 November 2007
Available online 20 December 2007
Abstract
The stem bark of Discopodium penninervium afforded a withanolide, 6a,7a-epoxy-1-oxo-5a,12a,17a-trihydroxywitha-2,24-dienolide
(1) and a coloratane sesquiterpene, 7a,11a-dihydroxy-4(13),8-coloratadien-12,11-olide (4) along with five known compounds, withanone
(2), 5a,17b-dihydroxy-6a,7a-epoxy-1-oxowitha-2,24-dienolide (3), 7a,11a-dihydroxy-8-drimen-12,11-olide (5), withasomnine (6), and
(E,Z)-9-hydroxyoctadeca-10,12-dienoic acid (7). The identity of the compounds was established on the basis of spectroscopic data analysis. All compounds were assessed for inhibition of leukotriene metabolism in an in vitro bioassay using activated human neutrophile
granulocytes, and for in vitro cycloxygenase-1 and -2 inhibition from sheep cotyledons and seminal vesicles, respectively. In the leukotriene biosynthesis assay all compounds tested at a concentration of 50 lM exhibited activity with percentage inhibitions ranging from
11.5 to 36.6. The withanolide, 1, displayed a 46.4% inhibition of COX-2 and a 22.9% inhibition of LTB4 formation at 50 lM concentration. Compounds 4 and 6 inhibited LTB4 biosynthesis but showed minor inhibition of COX-1 and COX-2. The remaining compounds, on the other hand, were found to be inactive on COX enzymes.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Discopodium penninervium; Solanaceae; LTB4; Cyclooxygenase; Withanolide; Coloratane sesquiterpene; 6a,7a-Epoxy-1-oxo-5a,12a,17a-trihydroxywitha-2,24-dienolide; 7a,11a-Dihydroxy-4(13),8-coloratadien-12,11-olide
1. Introduction
Discopodium penninervium Hochst (Solanaceae), a shrub
or small tree, is the only species in the genus and endemic
to Ethiopia. The leaves and barks are used for the treatment of schistosomiasis, leprosy and stomach-ache in Ethiopian folk medicine (Geyid et al., 2005).
Previous phytochemical investigation of D. penninervium
revealed the presence of 5a,17b-dihydroxy-6a,7a-epoxy-1oxowitha-2,24-dienolide, withanone and withanolide A in
the roots (Habtemariam and Gray, 1998), and 5,6-epoxy*
Corresponding author. Tel.: +43 316 3805531; fax: +43 316 3809860.
E-mail address: franz.bucar@uni-graz.at (F. Bucar).
0031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.11.001
16-oxygenated withanolides, jaborosalactone-L, and 17epiacnistin-A in the leaves (Habtemariam et al., 1993,
2000). The latter Discopodium withanolides have been
shown to exhibit cytotoxic activities to both human and
murine cancer cell lines and immunosuppressive activity in
rat spleen cells in vitro (Habtemariam et al., 2000).
In the continuation of a search for biologically active
constituents from Ethiopian medicinal plants, we report
herein the isolation and structural elucidation of a new withanolide, 1, and a new coloratane sesquiterpene, 4, together
with five known compounds from the stem bark of D. penninervium (see Fig. 1). The anti-inflammatory activity of the
compounds was assessed in vitro using leukotriene formation, and cycloxygenase-1 and -2 inhibitory assays.
983
A.A. Wube et al. / Phytochemistry 69 (2008) 982–987
28
21
H
R1
O
2
1
3
4
18
12
19
10
5
11
9
H
20
R2
17
16
15
13
14
24
23
22
O
25
O
11
12
O
H
7
2
3
1
10
9
5
4
H
O
8
7
6
14
H
OH
H
HO
15
8
6
27
26
OH
H
O
H
Ha
13
4
1: R1 = OH; R 2 = α-OH
2: R1 = H; R2 = α-OH
3: R1 = H; R 2 = β-OH
HO
Hb
N
N
H
O
O
OH
H
H
5
O
6
agreement with a previous report by Habtemariam and
Gray (1998).The presence of an epoxide between C-6 and
C-7 was inferred from the low field resonances (56.9 and
57.3 ppm) observed for these two oxygenated carbons in
the 13C NMR spectrum. A broad triplet at d 4.36 was
assigned to a methine proton attached to C-12, as it showed
a correlation with this carbon at d 76.3 in the HMQC spectrum, although a methine carbon resonance was absent in
the 13C NMR spectrum. This assignment was further substantiated by HMBC cross peaks observed between C-12
and the methyl protons at position 18 and the 1H–1H COSY
correlation between H-11 and H-12. The remaining methine
proton resonances were placed by analysis of HMQC,
HMBC and COSY spectra. The upfield carbon resonance
at d 28.3 was assigned to C-9 on the basis of HMBC cross
peaks observed for H-12/C-9 and H-19/C-9. All the resonances for methylene protons were assigned unambiguously
by HMQC, HMBC and COSY NMR experiments. The relative configuration of the hydroxyl group at position 12 was
established on the basis of the small coupling constant of H12 and by a NOESY NMR experiment (Fig. 2), showing
OH
OH
7
H
OH
H
Fig. 1. The structures of compounds 1–7.
OH
O
2. Results and discussion
H
Compound 1 was obtained as amorphous powder and its
molecular formula was deduced as C28H38O7 by the LCESI-MS data at m/z 487 [M+H]+. The HRMS showed an
ion [MH] peak at m/z 485.2553 corresponding to the
molecular formula, C28H38O7. An absorption maximum
at 223 nm in the UV spectrum was indicative of an a,bunsaturated d-lactone structural feature, which was further
confirmed by absorption band at 1694 cm1 observed in the
IR spectrum. The broad absorption band at 3493 cm1 in
the IR spectrum was attributed to hydroxyl stretching.
The 1H NMR spectrum of 1 showed resonances for five
methyl groups, with four tertiary methyls at d 0.90, 1.18,
1.79, 1.91 and one secondary methyl at d 1.14 (d,
J = 6.7 Hz). The first two methyls resonances were assigned
to CH3-18 and CH3-19, respectively, whereas the latter two
downfield resonances were assigned in turn to CH3-27 and
CH3-28, and were attached to olefinic carbons. The methyl
signal resonated as a doublet was assigned to CH3-21.
Among the ten methine protons that appeared in the 1H
NMR spectrum, the deshielded doublet at d 5.67 and the
double of doublets at d 6.62, which were coupled to one
another, were assigned to H-2 and H-3 respectively. This
further confirmed the presence of a,b-unsaturated ketone
moiety. A 1H doublet at d 3.07 and a multiplet at d 3.33
were assigned to H-6 and H-7, respectively, and the small
coupling (3.8 Hz) observed for H-6/H-7 indicated that these
hydrogens are axial and equatorial, respectively. This is in
H O
O
H
H
H
Heq
OH
Hax
O
H
1
HO
Heq
H
O
Hax
Heq
O
Hax
H
OH
H
Heq
H
Hax
Hb
Ha
4
Fig. 2. Major NOE correlations in compounds 1 and 4.
984
A.A. Wube et al. / Phytochemistry 69 (2008) 982–987
NOE enhancement between H-12 and CH3-18. The relative
stereochemistry of C-17, C-20 and C-22, on the other hand,
are the same as in compound 2. Comparison of the above
data with those in the literature indicated that the structure
of 1 is very closely related to that of withanone (2) (Habtemariam, 1997), except for the presence of an additional
hydroxyl group at position 12 in 1. Thus, the structure of
1 was established as 6a,7a-epoxy-1-oxo-5a,12a,17a-trihydroxywitha-2,24-dienolide.
Compound 4 was obtained as white needles in n-hexane/
EtOAC. Its molecular formula C15H20O4 was determined
by HRMS (m/z; measured 263.1291 [MH]; calc.
263.1289). The proton and 13C NMR data were suggestive
of a coloratane skeleton for 4 (Wube et al., 2005). The IR
spectrum showed a strong absorption band at 1762 and
1740 cm1, indicating the presence of an a,b-unsaturated
lactone (Sakio et al., 2001). This structural feature was
further supported by an absorption maximum at 210 nm
in the UV spectrum and double bond carbon resonances
at d 129.5 (C-8) and 170.0 (C-9), and a signal for a lactone
carbonyl carbon at d 170.7 (C-12) in the 13C NMR spectrum. In addition a hydroxyl stretch band at 3436 cm1
was observed in the IR spectrum. The 13C and DEPT
NMR spectra also revealed the presence of two methyls,
four methylenes, four methines, and five quaternary
carbons.
Two 3H signals, a singlet and a doublet, were observed
in the 1H NMR spectrum of 4. The singlet at d 0.98, which
showed HMBC cross peaks with C-1 at d 35.1 and C-9 at d
170.0, was assigned to CH3-15. The methyl doublet gave
HMBC correlations with C-2 at d 32.6, C-3 at d 39.1 and
C-4 at d 153.5 was assigned to CH3-14. The signals for
the H-13a and H-13b protons were clearly assigned based
on NOE correlations observed between CH3-14 and H13a and between H-6a and H-13b in the NOESY spectrum.
Four 1H signals at d 2.13, 2.53, 4.48, and 6.22 were
assigned to H-3, H-5, H-7, and H-11, respectively. The
chemical shifts for the remaining methylene protons were
assigned by a detailed analysis of HMQC and HMBC spectra. Exocyclic double bond carbon resonances at d 153.5
(C-4) and 105.1 (C-13), observed in the 13C NMR spectrum, were characteristic of coloratane sesquiterpenes.
The carbon resonances at d 97.8 and 59.7 were assigned
to a hemiketal C-11 and the hydroxyl bearing C-7, respectively. The relative configuration of the five asymmetric
centres in the coloratane skeleton was determined by analysis of the coupling constants and NOESY experiments.
The NOESY spectrum of 4 showed correlations between
the protons of H-3 and H-1a as well as the H-3 and H-5.
These correlations require that H-3 and H-5 are cis to each
other, which would enable the C-3 and C-5 protons to have
a-orientation. Similarly, H-11 exhibited a NOE correlation
(Fig. 2) with CH3-15, and this implied that H-11 and CH315 are on the same relative axial face as each other. This
would imply that hydroxyl group at C-11 be equatorial
and alpha. A small coupling constant value of H-7 (d
4.48, d, J = 3 Hz) suggested an a-orientation for the hydro-
xyl group. On the basis of these observations, compound 4
was established structurally as 7a,11a-dihydroxy-4(13),8coloratadien-12,11-olide.
The known compounds were identified by analysis of
their physical and spectral data and by comparison with
published values as withanone (2) (Habtemariam and
Gray, 1998), 5a,17b-dihydroxy-6a,7a-epoxy-1oxowitha-2,
24-dienolide (3) (Nittala and Lavie, 1981), 7a,11a-dihydroxy-8-drimen-12,11-olide (5) (Sakio et al., 2001),
withasomnine (6) (Houghton et al., 1994), and (E,Z)-9hydroxyoctadeca-10,12-dienoic acid (7) (Murakami et al.,
1992).
The drimane sesquiterpene, 7a,11a-dihydroxy-8-drimen-12,11-olide (5), was previously isolated from the animal species, Dendrodoris carbunculosa (Sakio et al., 2001)
and this is the first report of its occurrence in the plant
kingdom. Pyrazole alkaloids appear to be rare in plants
of the Solanaceae, although they have been found to occur,
for example in Newbouldia laevis (Bignoniaceae) (Aladesanmi et al., 1998), Elytraria acaulis (Acanthaceae) (Ravikanth et al., 2001) and Withania somnifera (Solanaceae)
(Schroeter et al., 1966). The occurrence of withasomnine
(6) in D. penninervium is interesting from a chemotaxonomic point of view because it was also isolated from the
Indian medicinal plant W. somnifera. Moreover, this is
the first report of the occurrence of drimane and coloratane
sesquiterpenes in the family Solanaceae.
All compounds were evaluated for inhibition of leukotriene metabolism as well as COX-1 and -2 enzymes
in vitro. The results presented in Table 3 show that all compounds tested in the leukotriene biosynthesis assay showed
some inhibitory effects on LTB4 formation. The sesquiterpene 4 displayed the highest activity against LTB4. On
the other hand, compound 5, the biogenetic precursor of
compound 4 was found to be three times less active than
compound 4. Thus, the presence of an exocyclic methylene
group at position 4 might enhance the LTB4 inhibitory
activity of compound 4. This is the first report on LTB4
inhibitory activity of withanolides. Recently, we have
reported the leukotriene biosynthesis inhibitory effect of
drimane and coloratane sesquiterpenes obtained from the
stem bark of Warburgia ugandensis (Wube et al., 2006)
and results of our study revealed that compounds having
a dialdehyde structural feature displayed superior inhibitory effects on the stable metabolite of 5-LOX, LTB4.
The in vitro COX-1 and -2 test results revealed that at a
concentration of 50 lM, compounds 4 and 6 showed dual
COX-1 and -2 inhibition, whereas compounds 2, 3, 5,
and 7 produced no inhibitory effects. The new withanolide,
compound 1, displayed a selective inhibition of the COX-2
enzyme with an IC50 value close to 50 lM. Unfortunately,
due to lack of compound higher amounts could not be
tested. This is the second report on the cycloxygenase
inhibitory activity of withanolides and the selective COX2 inhibitory potency of compound 1 is much higher than
the withanolide obtained from W. somnifera (Jayaprakasam and Nair, 2003). As compared to compounds 2 and
A.A. Wube et al. / Phytochemistry 69 (2008) 982–987
3, the COX-2 inhibitory potency of compound 1 might be
enhanced by the hydroxyl group at position 12.
Concerning anti-inflammatory drugs, selective COX-2
inhibitors are considered to have advantages over nonselective NSAIDs by a lower risk of gastrointestinal side
effects (Smith et al., 2000). Furthermore, it has been evident
that COX-2 and 5-LOX pathways are both involved in cell
proliferation and angioneogenesis. Therefore the withanolide 1 represents interesting features of a dual inhibitor of
COX-2 and leukotriene formation and may serve for the
development of anti-inflammatory and cancer chemo preventive agents. However, further structural variations
should be evaluated in order to improve the inhibitory
potency of 1 in the COX-2 and LTB4 assays.
3. Experimental
3.1. General experimental procedures
Melting points were determined with Kofler microscope
and are uncorrected. Optical rotations were measured on a
Perkin–Elmer 241 MC polarimeter. UV spectra were
recorded on Shimadzu UV-160A spectrophotometer. Perkin–Elmer 881 infrared spectrophotometer was used in
recording the IR spectra. NMR spectra were recorded at
500 MHz for 1H and 125 MHz for 13C on a Bruker
AVANCE 500 spectrometer. All spectra were measured
in CDCl3, except for compounds 1 and 4, which were dissolved in (CD3)2CO. HRMS were determined with Micromass QTOF Ultima using the internal standard TCA,
which had [MH] = m/z 514.2839. Mass spectra were
also obtained by LC-ESI-MS analysis on a Thermo-Finnigan LCQ Deca XP Plus mass spectrometer connected to
a Surveyor LC-system (Thermo-Finnigan). The absorbance for LTB4 quantification was conducted using a
Tecan RAIN BOW photometric ELISA plate reader.
Chromatographic separation were performed by analytical TLC on Si gel 60 F254 (0.2 mm thick), column chromatography on Si gel 60 (70–240 mesh), size exclusion
chromatography on Sephadex LH-20, solid phase separation on Isolute C18 (10 g) columns, and semipreparative
HPLC with LiChrospherÒ RP-18 (10 lm, 250 times 10 mm
i.d.) column.
Türks solution, Na2EDTA, CaCl2 2H2O p.a., anhydrous D-glucose, MgCl2 6H2O KCl, Tris p.a., formic
acid and ethanol p.a. were purchased from Merck. Trypan
blue solution and eicosatetraenoic acid were obtained from
Sigma Chemicals. Ca ionophor A 23187 and epinephrinehydrogentartrate were bought from Fluka. A LTB4 EIA
kit, purified PGHS-1 and -2, indomethacin, NS-398 and
arachidonic acid were obtained from Cayman Chemicals,
Ann Arbor, MI, USA. A PGE2-EIA kit was obtained from
R&D systems, Minneapolis, MN, USA. TRIS/HCl buffer
(pH 8.0) was bought from Roth. Hematin was obtained
from porcine, ICN, Aurora, OH, USA. Zileuton was purchased from Sequoia Research Products Ltd., Oxford, UK.
985
3.2. Plant material
The stem bark of D. penninervium was collected in April
2001 from Dinsho towards Addis Ababa and Dodola in
Bale zone, Ethiopia and identified by Mr. Melaku Wondafrash, the National Herbarium, Department of Biology,
Addis Ababa University. A voucher specimen (collection
number 1487) has been deposited in the National Herbarium, Department of Biology, Addis Ababa University,
Addis Ababa, Ethiopia.
3.3. Extraction and isolation
The air-dried powdered stem barks of D. penninervium
(600 g) were extracted successively with petroleum ether
and dichloromethane in a Soxhlet apparatus. The dichloromethane extract was concentrated at reduced pressure to
give 5 g of a greenish residue. After removal of chlorophyll
with Sephadex LH-20 eluting with CH2Cl2–MeOH (1:1),
the residue (1.2 g) was subjected to solid-phase separation
using a H2O/MeOH (100:0 ? 0:100) gradient elution and
ten fractions of 100 ml each were collected. Fraction 4,
eluted with H2O-MeOH (7:3), was purified by semi-prep.
HPLC using MeCN/H2O (35:65 ? 55:45) gradient elution
for 25 min to afford 5 (2 mg). Fr. 8, eluted with H2O/MeOH
(3:7), was subjected to semi-prep. HPLC using MeCN/H2O
(4:6 ? 1:1) gradient elution for 40 min to yield 1 (3 mg), 3
(6.5 mg) and 2 (9 mg) at 24, 32 and 34.7 min, respectively.
Fr. 9 and 10 were combined and applied to a Sephadex-20
column using CH2Cl2 as eluent to give 56 subfractions of
20 ml each. Subfr. 19–27 gave 4 (11 mg) after purification
by semi-prep. HPLC using a MeCN/H2O (35:65 ? 65:35)
gradient system as eluents. Subfr. 31–46 were further chromatographed on semi-prep. HPLC using a MeCN/H2O
(45:55 ? 8:2) gradient elution for 45 min to afford 6
(2 mg) and 7 (14 mg) at 9 and 37 min, respectively.
3.4. In vitro leukotriene metabolism, cycloxygenase-1, and -2
inhibitory assays
The leukotriene metabolism inhibitory assay was conducted as described previously (Adams et al., 2004) using
human neutrophile granulocytes with some modification.
After isolation of human neutrophile granulocytes from
human blood, cell vitality test, cell concentration determination, and incubation with test sample solution the samples were diluted 40-fold and the free LTB4 concentration
was measured using a competitive LTB4 EIA kit. LTB4 biosynthesis inhibition was quantified by measuring the
absorption at 405 nm after addition of 50 ll aqueous
Na3PO4 as stop solution. Inhibition was expressed in percent in relation to a control using abs. EtOH. Inhibition values are means of three experiments and each sample was
tested in duplicate. Zileuton was used as a positive control.
The cycloxygenase-1 and -2 assays were done with purified PGHS-1 from ram seminal vesicle for COX-1 and purified PGHS-2 from sheep placental cotyledons for COX-2
986
A.A. Wube et al. / Phytochemistry 69 (2008) 982–987
as reported previously (Fiebich et al., 2005; Rollinger et al.,
2005). The percent inhibitions at 50 lM concentration of
test compounds were determined for both enzymes. Inhibition values were means of three experiments and each sample was tested in duplicate. Indometacin and NS-398 were
used as positive control for COX-1 and -2, respectively.
3.5. 6a,7a-Epoxy-1-oxo-5a,12a,17a-trihydroxywitha-2,24dienolide (1)
Colourless optically inactive amorphous powder, UV
kMeOH
nm (log e) 223 (3.86), IR mMeOH
cm1: 3493, 1694,
max
max
1
1385, 1130, 1094, H NMR (500 MHz (CD3)2CO) and
13
C NMR (125.8 MHz (CD3)2CO) data, see Table 1, ESIMS (70 eV) m/z (rel. Int.): 487 [M+H]+ (34), 469
[MOH]+ (100), 452 (62), 171 (14); HRMS found
485.2553; C28H38O7, calc. 485.2545.
3.6. 7a,11a-Dihydroxy-4(13),8-coloratadien-12,11-olide (4)
White needles from n-hexane-EtOAC mixture, m.p.
28
143–145 °C, ½aD +90.0 (MeOH; c0.90), UV kMeOH
nm
max
Table 1
1
H, 13C NMR and HMBC correlations data for compound 1 in acetoned6a
Position
1
2
3
4a
4b
5
6
7
8
9
10
11a
11b
12
13
14
15a
15b
16a
16b
17
18
19
20
21
22
23a
23b
24
25
26
27
28
dH (J in Hz)
5.67
6.62
2.56
2.77
(dd) (10.1, 2.2)
(ddd) (10.1, 5.2, 2.2)
(dd) (14.5, 3.5)
(m)
3.07
3.33
1.98
2.01
(d) (3.8)
(m)
(m)
(m)
2.73 (m)
1.60 (m)
4.36 (t) (2.2)
1.84
2.07
1.36
2.37
1.45
(m)
(m)
(m)
(m)
(m)
0.90
1.18
2.13
1.14
4.55
2.21
2.45
(s)
(s)
(m)
(d) (6.7)
(dt) (12.5, 3.5)
(m)
(dd) (18.5, 9.5)
1.79 (s)
1.91 (s)
Table 2
1
H, 13C NMR and HMBC correlations data for compound 4 in acetoned6a
Position dH (J in Hz)
1a
1b
2a
2b
3
4
5
6a
6b
7
8
9
10
11
12
13a
13b
14
15
1.78
1.99
1.35
1.80
2.13
2.53
1.85
1.89
4.48
6.22
4.86
4.67
1.09
0.98
dC, multiplicity HMBC
(dt) (12.6, 4.0) 35.1, CH2
(m)
(m)
32.6, CH2
(m)
(m)
39.1, CH
153.5, C
(d) (12.0)
44.5, CH
(m)
32.1, CH2
(m)
(d) (3.0)
59.7, CH
129.5, C
170.0, C
39.4, C
(s)
97.8, CH
170.7, C
(bs)
105.1, CH2
(bs)
(d) (6.5)
18.5, CH3
(s)
17.2, CH3
H-15
H-14
H-13a, H-14
H-3, H-6a, H-14
H-6b, H-7, H-13b, H-15
H-6a
H-6 a, H-7, H-11
H-15
H-6a, H-15
H-11
H-3
a
Chemical shifts are in ppm relative to TMS; 500 MHz for 1H and
125 MHz for 13C.
dC, multiplicity
HMBC
Table 3
Leukotriene metabolism, COX-1 and -2 inhibitory activities of compounds isolated from D. penninervium at 50 lM test concentration and
IC50 values of the positive controls zileuton, indomethacin and NS-398
203.6, C
129.3, CH
141.3, CH
37.8, CH2
H-19
Compound
74.1,
56.9,
57.3,
37.2,
28.3,
51.6,
31.4,
C
CH
CH
CH
CH
C
CH2
H-19
76.3,
50.2,
40.8,
23.1,
CH
C
CH
CH2
H-2
H-12, H-14
H-19
H-18
H-18
H-18
37.7, CH2
87.1, C
16.4, CH3
15.1, CH3
43.7, CH
9.2, CH3
79.7, CH
33.4, CH2
151.4, C
121.7, C
166.9, C
12.6, CH3
20.5, CH3
H-18, H-21
H-21
% Inhibition ± SD at 50 lM test concentration
LTB4
COX-1
COX-2
1
2
3
4
5
6
7
22.9 ± 1.98
28.7 ± 1.54
18.9 ± 6.63
36.6 ± 1.38
11.5 ± 3.98
25.6 ± 3.24
22.5 ± 6.92
NA
NA
NA
2.1 ± 9.15
NA
6.8 ± 4.59
NA
46.4 ± 2.23
NA
NA
11.83 ± 6.86
NA
6.4 ± 2.31
NA
Zileuton
Indomethacin
NS-398
10.0 lM (IC50)
ND
ND
ND
1.25 lM (IC50)
ND
ND
ND
5.0 lM (IC50)
NA, not active and ND, not determined.
(log e): 210 (3.96), IR mMeOH
cm1: 3436, 1762, 1740, 1177,
max
1
1036, 940, 904, H NMR (500 MHz (CD3)2CO) and 13C
NMR (125.8 MHz (CD3)2CO) data, see Table 2, HRMS
found 263.1291; C15H20O4, calc. 263.1289.
Acknowledgements
H-21
H-27, H-28
H-27, H-28
H-27
a
Chemical shifts are in ppm relative to TMS; 500 MHz for 1H and
125 MHz for 13C.
We thank Mrs. Elke Prettner, Department of
Pharmaceutical Chemistry, Institute of Pharmaceutical
Sciences, University of Graz for recording the IR and UV
spectroscopic and optical rotation measurements and
Mr. M. Wondafrash, the National Herbarium, Department
of Biology, Addis Ababa University for plant identification.
Financial support by the Austrian Exchange Service OEAD
is gratefully acknowledged.
A.A. Wube et al. / Phytochemistry 69 (2008) 982–987
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