Two new isoflavones from Ceiba pentandra and their effect on cyclooxygenase-catalyzed prostaglandin biosynthesis

Article abstract of DOI:10.1021/np970198+

The new isoflavone glucoside vavain 3'-O-β-D-glucoside (1) and its aglycon, vavain (2), were isolated from the bark of Ceiba pentandra, together with the known flavan-3-ol, (+)-catechin. These novel structures were elucidated by one- and two-dimensional N

Full text of DOI:10.1021/np970198+

8
J . Nat. Prod. 1998, 61, 8-12
Tw o New Isofla von es fr om Ceiba pen ta n d r a a n d Th eir Effect on
Cyclooxygen a se-Ca ta lyzed P r osta gla n d in Biosyn th esis
Ylva Noreen, Hesham El-Seedi, Premila Perera, and Lars Bohlin*
Division of Pharmacognosy, Department of Pharmacy, Biomedical Centre, Uppsala University, Box 579,
S-751 23 Uppsala, Sweden
Received April 8, 1997X
The new isoflavone glucoside vavain 3′-O-â-D-glucoside (1) and its aglycon, vavain (2), were
isolated from the bark of Ceiba pentandra, together with the known flavan-3-ol, (+)-catechin.
These novel structures were elucidated by one- and two-dimensional NMR experiments and
by MS, IR, and UV spectroscopy as 5-hydroxy-7,4′,5′-trimethoxyisoflavone 3′-O-â-D-glucoside
(1) and 5,3′-dihydroxy-7,4′,5′-trimethoxyisoflavone (2), respectively. The compounds were
isolated following bioactivity-directed fractionation, using a cyclooxygenase-1-catalyzed pros-
taglandin biosynthesis assay in vitro, in which compounds 1 and 2 and (+)-catechin exhibited
IC50 values of 381, 97, and 80 µM, respectively (standard: indomethacin, IC50 1.1 µM). When
further tested for their inhibitory effects on cyclooxygenase-2-catalyzed prostaglandin biosyn-
thesis, 1 and 2 were found to be inactive (IC50 > 1200 and >900 µM, respectively).
Ceiba pentandra (L.) Gaertner (Bombacaceae) is a
tropical tree of economic importance due to the elastic
and water resistant floss of the capsules commonly
known as kapok, used in, e.g., cushions and lifejack-
ets.1,2 This species is also widely used in traditional
medicine. In Asia, Oceania, Africa, and Central America
it is used for a variety of disorders including diarrhea,
fever, gonorrhea, parasitic infections, and wounds and
as a diuretic and an emollient.3 In the Samoan Islands,
a cold water infusion of the bark of C. pentandra
(vernacular name vavae) is used for asthma.4 Two
studies have shown antiinflammatory properties of the
bark extract, both in vitro and in vivo,3,5 which can be
related to several reports of traditional use in the
treatment of ailments of an inflammatory nature such
as asthma and cough.3,6-8 In these studies, extracts
showed suppression of carrageenan-induced rat paw
edema as well as EPP-induced rat ear edema,3 two in
vivo experimental models of inflammation. Further, an
extract was found to inhibit cyclooxygenase-1 (COX-1)-
catalyzed prostaglandin biosynthesis in vitro.3
These results prompted us to further investigate the
species phytochemically by bioactivity-directed fraction-
ation using a prostaglandin biosynthesis assay in vitro.
Previously reported constituents of this species include
polysaccharides and xylans,9,10 the naphthoquinone,
7-hydroxycadelene, sesquiterpene lactones,11 and fatty
acids.12-14 This paper deals with the isolation and
characterization of three flavonoids from the bark and
the assessment of their inhibitory properties on COX-1
and cyclooxygenase-2 (COX-2)-derived prostaglandin
biosynthesis.
aqueous ethanol extract of the bark of C. pentandra was
fractionated mainly by combining medium-pressure
liquid chromatography (MPLC), high-speed counter-
current chromatography (HSCCC), and Sephadex LH-
20 chromatographic techniques. The fractionation pro-
cedure resulted in the isolation of two new isoflavones,
vavain 3′-O-â-D-glucoside (1) and vavain (2), along with
the known flavan-3-ol, (+)-catechin. To our knowledge,
isoflavonoids have so far not been reported before from
the genus Ceiba.
The FABMS of 1 showed a peak at m/z 507 [M + H]+,
suggesting the molecular formula C24H26O12. The 1H
NMR spectrum exhibited a flavonoid pattern and showed
a signal at δ 8.15 (1H, s), typical of the proton at C-2 of
an isoflavonoid skeleton.15,16 A singlet at δ 12.83, which
disappeared on deuterium exchange, indicated a C-5
hydroxyl.17 This functionality was further confirmed by
a bathochromic shift in the UV spectrum in the presence
of AlCl3.16,18,19 Two doublets at δ 6.96 and 6.93,
integrating for one proton each, suggested a trioxygen-
ated unsymmetrical B-ring. Two meta-coupled doublets
at δ 6.54 and 6.38 (1H, J ) 2.0 Hz each) represented
H-8 and H-6, respectively. 1H NMR resonances at δ 3.8
to 3.4 and signals in the 13C NMR spectrum just below
δ 70 indicated the presence of a sugar moiety. A loss of
162 mass units from the molecular ion in the FABMS
and a signal at δ 61.71, shown by DEPT to represent a
CH2 group, suggested glucose or galactose. The carbo-
hydrate moiety was confirmed as glucose by acid hy-
drolysis and subsequent TLC sugar analysis. The
glucose 13C NMR spectral data were in agreement with
Resu lts a n d Discu ssion
Guided by an in vitro assay testing for inhibition of
COX-1-catalyzed prostaglandin biosynthesis, a 70%
* To whom correspondence should be addressed. Tel.: + 46 18
4714492. Fax: + 46 18 509101. E-mail: lars.bohlin@pharmacog.uu.se.
X Abstract published in Advance ACS Abstracts, December 1, 1997.
S0163-3864(97)00198-5 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/23/1998

Inhibitory Isoflavones from Ceiba
J ournal of Natural Products, 1998, Vol. 61, No. 1
9
Ta ble 1. 1H-NMR Spectral Data of Compounds 1 and 2
compound
1
2
(AcCN-d3 + D2O)
(AcCN-d3 + D2O)
20 °C
(AcCN-d3) 60 °C
20 °C
(AcCN-d3) 60 °C
1H δ (integral,
mult, J (Hz))
1H δ (integral,
mult, J (Hz))
long-range
COSY
1H δ (integral,
mult, J (Hz))
1H δ (integral,
long-range
COSY
proton
H-2
mult, J (Hz))
8.15 (1H, s)
8.12 (1H, s)
8.10 (1H, s)
8.07 (1H, s)
H-6
H-8
H2′
H-6′
H-1′′
6.38 (1H, d, 2.0)
6.54 (1H, d, 2.0)
6.96 (1H, d)
6.93 (1H, d)
4.99 (1H, d, 7.3)
6.40 (1H, d, 2.3)
6.54 (1H, d, 2.3)
7.07 (1H, d, 2.0)
6.97 (1H, d, 2.0)
4.94 (1H, d, 7.3)
3.36-3.55 (4H, m)
H-8, 7-OMe
H-6, 7-OMe
H-6′, H-1′′
H-2′, 5′-OMe
H-2′, H-2′′-H-5′′
H-1′′, H-2′′-H-5′′
6.39 (1H, d, 2.3)
6.55 (1H, d, 2.3)
6.71 (1H, d, 2.0)
6.73 (1H, d, 2.0)
6.39 (1H, d, 2.2)
6.53 (1H, d, 2.3)
6.75 (1H, d, 1.9)
6.76 (1H, d, 2.0)
H-8, 7-OMe
H-6, 7-OMe
5′-OMe
H-2′′-H-5′′ 3.46-3.57 (4H, m)
H-6A′′
H-6B′′
OMe
OMe
OMe
3.80 (1H, dd)
3.81 (1H, dd, 2.5, 2.7, 11.9) H-2′′-H-5′′
3.67 (1H, dd, 2.6, 3.6, 12.7) 3.66 (1H, dd, 5.3, 5.5, 11.8) H-2′′-H-5′′
3.86 (6H, s); 3.83 (3H, s)
pos. 7: 3.90 (3H, s)
pos. 4′: 3.86 (3H, s)
pos. 5′: 3.88 (3H, s)
H-6, H-8
3.88 (3H, s);
3.85 (3H, s);
3.82 (3H, s)
pos. 7: 3.90 (3H, s) H-6, H-8
pos. 4′: 3.87 (3H, s)
pos. 5′: 3.88 (3H, s) H-6′
H-6′
Ta ble 2. 13C-NMR Spectral Data of Compounds 1 and 2
compound
1
2
(AcCN-d3 + D2O) 20 °C
(AcCN-d3) 60 °C
(AcCN-d3) 60 °C
carbon
13C
DEPT
C-H COSY (δ 1H)
13C
HMBC
HMQC
13C
HMBC
H-2′
H-2, H-2′, H-6′
H-2
H-6
H-8
H-6, H-8
H-6
H-2, H-8
H-6, H-8
H-2
H-6′
H-2′
H-2′, H-6′
H-6′
H-2′
HMQC
2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
155.90
123.75
181.60
162.62
99.30
CH
C
C
C
CH
C
CH
C
C
C
CH
C
8.15
155.88
124.45
182.16
164.06
99.67
155.98
125.02
182.56
164.41
99.89
H-2
H-2, H-2′, H-6′
H-2
H-6
H-8
6.38
6.54
H-6
H-8
H-6
H-8
166.74
93.58
167.47
93.89
167.71
94.11
158.92
106.74
127.89
111.01
151.26
139.12
154.01
109.00
101.81
74.05
159.52
107.50
127.91
113.10
152.02
141.19
154.94
110.21
103.15
75.22
H-2, H-8
H-6, H-8
159.84
107.82
128.53
111.29
151.52
138.30
154.66
107.58
6.96
H-6′
H-2′
H-2′
H-6′
C
C
H-2′, H-6′
H-2′
5′
6′
CH
CH
CH
CH
CH
CH
CH2
CH3
CH3
CH3
6.93
4.99
H-6′
H-1′′
H-2′′, H-3′′,
H-4′′, H-5′′
1′′
2′′
3′′
4′′
5′′
6′′
OMe
OMe
OMe
3.4-3.6
3.4-3.6
3.4-3.6
3.4-3.6
3.8
76.85a
70.30
78.19a
71.98
77.28a
61.71
78.31a
63.31
H-6′′
56.93
61.87
56.93
3.85
57.58
61.89
57.27
MeO-7
MeO-4′
MeO-5′
57.55
61.93
57.63
MeO-7
MeO-4′
MeO-5′
3.85
a
Assignments with the same superscript letter may be interchanged.
the literature.20-22 The anomeric glucose proton ap-
peared at δ 4.99 as a doublet with a coupling constant
of J ) 7.3 Hz, indicating a â-linkage of the glucose unit
to the aglycon.16 Signals attributed to three methoxy
groups were observed at δ 3.83 (3H, s) and δ 3.86 (6H,
s). The above data in conjunction with the 13C NMR
spectrum (DEPT sequence) were found to be consistent
with an isoflavone glucoside structure. Assignments of
the protonated carbons were made using C-H COSY
(HETCOR). The results are shown in Tables 1 and 2.
In the 13C NMR spectrum of 1 one methoxyl group
was found at δ 61.87, and there were another two at δ
56.93. The shift of the former below δ 60 indicated its
placement between two oxygenated carbons23 and sug-
gested the following configuration of the B-ring: the
glucose unit occurred at position C-3′ and two methoxyl
groups were affixed to positions C-4′ and C-5′.17 To
confirm this, HMBC and long-range COSY experiments
were performed (Figure 1). Because of viscosity prob-
F igu r e 1. HMBC connectivities of 1.
lems (the compound had tendencies to form a gel in
solution), these spectra were recorded at an elevated
temperature (60 °C). 1H and 13C NMR spectra were
rerun at this temperature. From long-range COSY
connectivities (Figure 3) the proposed configuration of
the B-ring was confirmed, since cross-peaks from H-2′
to H-1′′ were found, as well as from H-6′ to OMe.
Furthermore, connectivities from both H-6 and H-8 to
OMe were observed, which placed the third methoxyl
group at position C-7. The structure of 1 was thus

10 J ournal of Natural Products, 1998, Vol. 61, No. 1
Noreen et al.
many antiinflammatory drugs, although the effects
observed in vitro may not have an in vivo coun-
terpart.24-26 With the COX-1-catalyzed prostaglandin
biosynthesis assay in vitro, as employed in the bioac-
tivity-directed fractionation, vavain 3′-O-â-D-glucoside
(1), vavain (2), and (+)-catechin exhibited IC50 values
of 381, 97, and 80 µM, respectively. The values can be
compared with indomethacin (IC50 of 1.1 µM), one of the
strongest inhibitors of prostaglandin biosynthesis27 and
acetylsalicylic acid (aspirin), a much weaker inhibitor
in this test system, exhibiting an IC50 value of 1 mM.
Compounds 1, 2, and (+)-catechin were further tested
for their inhibitory activities on COX-2-catalyzed pros-
taglandin biosynthesis, with 1 and 2 found to be
essentially inactive. The IC50 value for 1 was over 1.2
mM and that for 2 over 900 µM (maximum dissoluble
concentration of 2 in solvents available to the assay).
The isoflavones 1 and 2 thus exhibited a certain COX-1
selectivity. Further studies of the inhibitory activities
on the COX-1- and COX-2-catalyzed prostaglandin
biosynthesis by (+)-catechin are described in the ac-
companying paper.28
F igu r e 2. HMBC connectivities of 2.
F igu r e 3. Important long-range COSY connectivities observed
for 1.
It cannot be ruled out that the aglycon, vavain (2), is
an artifact of the glucoside, 1, and thus formed by
hydrolysis during extraction. Simultaneous occurrence
of isoflavonoid aglycons and their O-glycosides have,
however, been reported previously from several plant
species.29-31 Vavain 3′-O-â-D-glucoside (1) did not de-
compose under any of the laboratory conditions applied.
Exp er im en ta l Section
F igu r e 4. Long-range COSY connectivities for 2.
Gen er a l Exp er im en ta l P r oced u r es. EIMS, HR-
FABMS, and positive-ion FABMS (with glycerol as
matrix) spectra were recorded with a J EOL J MS SX/
SX102A instrument.
established as 5-hydroxy-7,4′,5′-trimethoxyisoflavone 3′-
O-â-D-glucoside (vavain 3′-O-â-D-glucoside).
1
The H NMR and 13C NMR data of 2 were similar to
1H and 13C NMR, DEPT, and C-H COSY (HETCOR)
and further spectra were obtained in acetonitrile-d3 +
D2O (5-10%) at 20 °C with TMS as internal standard
using a 270 MHz J EOL J NM Ex-270/4000 NMR instru-
ment. 1H and 13C NMR, inverse heteronuclear correla-
tions HMBC and HMQC, as well as long-range COSY
spectra were recorded at 60 °C in acetonitrile-d3 on a
400 MHz Varian VXR-400 NMR instrument, IR spectra
were recorded with a Nicolet MX-S spectrophotometer,
and UV spectra were recorded with a Perkin-Elmer
Lambda 2 UV/vis spectrophotometer. Melting points
are uncorrected and were determined using a Digital
melting point apparatus (Model IA 8103, Electrothermal
Engineering Ltd, Southend-on-Sea, Essex, U.K.). The
optical rotation was determined in Me2CO or EtOH at
ambient temperature using a Perkin-Elmer Model 241
polarimeter.
TLC was performed on precoated aluminum sheets
[silica 60 F254, 0.25 mm (Merck, Darmstadt, Germany)]
and preparative TLC on precoated glass sheets [silica
60 F254, 0.5 mm (Merck)], with detection provided by
UV light (254 and 366 nm) and by spraying with
vanillin-sulfuric acid reagent followed by heating (120
°C).
1 apart from the absence of sugar resonances and the
fact that three separate methoxy signals could be
1
observed in the H NMR spectrum when run at 20 °C
1
[δ 3.81 (3H, s), 3.85 (3H, s), 3.88 (3H, s)]. The H NMR
spectrum exhibited the same indication of a C-5 hy-
droxyl, which was confirmed by UV data with AlCl3 shift
reagent, as performed for 1. The EIMS and FABMS of
2 showed peaks at m/z 344 and 345 [M + H]+, respec-
tively, suggesting the molecular formula C18H16O7. The
presence of a [M - 15]+ ion in the MS indicated the loss
of a methyl group. Ions at m/z 315 and 301 indicated
two other methyl groups. An ion at m/z 167 was
assigned to a retro-Diels-Alder fragmentation product.
Ring A was thus substituted with one methoxyl and one
hydroxy group. The long-range COSY spectrum (Figure
4) in conjunction with the HMQC spectrum allowed the
assignment of the individual methoxyl units to positions
C-4′, C-5′, and C-7, respectively (Tables 1 and 2). The
HMBC spectrum (Figure 2) confirmed the structure.
Thus, 2 was assigned as 5,3′-dihydroxy-7,4′,5′-trimeth-
oxyisoflavone, which has been given the trivial name
vavain.
The antiinflammatory effect of the isolated com-
pounds was assessed employing an in vitro assay testing
for inhibition of COX-catalyzed prostaglandin biosyn-
thesis. Prostaglandins are important mediators in the
inflammatory process, and the inhibition of COX is
considered to be involved in the mechanism of action of
A three-phase liquid extraction procedure has been
described by Nyiredy et al.32 and was carried out in this
investigation using a separatory funnel, partitioning the
plant extract, and retrieving each phase separately.

Inhibitory Isoflavones from Ceiba
J ournal of Natural Products, 1998, Vol. 61, No. 1 11
MPLC was performed with SEPARO MPLC equipment
(Bæckstro¨m Separo AB, Lidingo¨, Sweden).33 SEPARO
variable-length glass columns with an inner diameter
of 1.5 or 2.5 cm, packed with silica gel 60, 40-63 µm
(Merck), were used. An FMI Lab pump, Model QD
(Fluid Metering, Inc., Oyster Bay, NY) was used at a
flow of 20-30 mL/min. Fractions of 9 mL were collected
with a Gilson Model 201 fraction collector. Columns
were eluted with continuous gradients running from
hexane, over CH2Cl2 to MeOH, and H2O afforded by a
SEPARO constant-volume mixing chamber combined
with an open reservoir. Initially, the mixing chamber
contained 50 mL of nonpolar solvent and the reservoir
the first of 15-20 premixed binary (less polar/more
polar solvent) gradient mixtures, of 20-40 mL each,
which were successively fed to the reservoir during
separation. High-speed counter-current chromatogra-
phy (HSCCC) was carried out using an Ito Multi-Layer
Coil CCC instrument (PC Inc., Potomac, MD). Coil #10
with a volume of 370 mL was used at 800 rpm. The
flow was 3 mL/min provided by an LKB 2150 HPLC
pump (LKB, Bromma, Sweden). Fractions collected
ranged from 9 to 15 mL. Column chromatography was
performed over Sephadex LH-20 (Pharmacia, Uppsala,
Sweden).
A portion of the EtOAc extract (1.5 g) was adsorbed
onto silica gel and chromatographed on a SEPARO
column eluted with a continuous gradient from hexane
over CH2Cl2 to MeOH of increasing polarity. Eluted
fractions were evaluated by TLC and combined upon
similar appearance, yielding 13 fractions. Fraction 8
(170 mg) was combined with several fractions (ca. 70
mg) exhibiting similar TLC profiles from two earlier
HSCCC separations of portions of the EtOAc extract;
the solvent system used in both cases was n-hexane-
EtOAc-MeOH-H2O (1:8:4:10) [first separation, EtOAc
extract (500 mg), aqueous phase was used as mobile
phase, retention of stationary phase was 32%; second
separation, EtOAc extract (400 mg), organic phase as
mobile phase, 62% retention of stationary phase]. The
combined fractions (240 mg) were subjected to a third
HSCCC separation [CHCl3-MeOH-1-BuOH-H2O (10:
10:1:6), organic phase as mobile phase, 70% retention
of stationary phase] from which 10 fractions were
obtained. Fraction 2 contained (+)-catechin, which was
further purified over Sephadex LH-20, eluted by MeOH-
1-PrOH (1:1), yielding 19 mg. Fraction 10 (the contents
remaining in the stationary phase after completion of
the separation) was purified over Sephadex LH-20 (1-
PrOH), which afforded vavain 3′-O-â-D-glucoside (1, 36
mg).
During bioactivity-directed fractionation, extracts and
crude and semi-purified fractions, dissolved in 10%
EtOH or 0.5-20% DMSO to a final concentration of 100
µg/mL, were tested for inhibitory effects in a COX-1-
catalyzed prostaglandin biosynthesis assay in vitro.34
The AcCN/CH2Cl2 extract was separated by MPLC,
with a continuous gradient from n-hexane to CH2Cl2
over MeOH to H2O, which afforded 20 fractions. Frac-
tion 11 (180 mg) was chromatographed over Sephadex
LH-20 eluted with 1-PrOH and further separated by
preparative TLC (CH2Cl2-MeOH, 9:1), which yielded
4 mg of vavain (2). Fraction 12 (300 mg) was rechro-
matographed on a SEPARO column with the same
continuous gradient as employed previously, followed
by chromatography over Sephadex LH-20 with 1-PrOH
as eluent, which yielded 3.7 mg of 2.
For IC50 value determinations compound 1 and (+)-
catechin were dissolved in 10% aqueous EtOH, while 2
was dissolved in 20% DMSO (yielding final concentra-
tions of 2% and 4% of the respective solvent under the
assay conditions). Materials for COX-1 and COX-2
prostaglandin biosynthesis assay were as follows: [14C]-
arachidonic acid (Amersham), l-epinephrine (Apoteks-
bolaget, Stockholm, Sweden), arachidonic acid, aspirin
(acetylsalicylic acid), indomethacin, prostaglandins E2
and F2R, and reduced glutathione (Sigma). For chro-
matographic separation, silica gel 60, 63-200 µm
(Merck) was used. Chemicals and solvents were of
analytical grade.
Acid Hyd r olysis of 1. Compound 1 (24 mg) was
refluxed for 75 min with 4 N HCl in MeOH (35 mL).
The acid hydrolysate was concentrated, extracted with
EtOAc, and examined by TLC on silica gel in CHCl3 and
1H NMR for the liberated aglycon, which was identified
by direct comparison (1H NMR, TLC) with the isolated
naturally occurring 2. The acidic mother liquor was
neutralized with Na2CO3, filtered, and evaporated to
dryness for examination of the sugar moiety, which
proved to be glucose by detection on TLC [EtOAc-
isopropanol-H2O (65:23:12)], and sprayed with freshly
prepared anisaldehyde-H2SO4 reagent followed by
heating.35
P la n t Ma t er ia l. C. pentandra (L.) Gaertner bark
was collected at the island of Savai’i of Western Samoa
in November 1991 and authenticated by Prof. Paul A.
Cox, Department of Botany and Range Science, Brigham
Young University, Provo, UT. A voucher specimen (SA
8) is deposited at the Division of Pharmacognosy,
Department of Pharmacy, Uppsala University, Sweden.
5-Hyd r oxy-7,4′,5′-tr im eth oxyisofla von e 3′-O-â-D-
glu cosid e (va va in 3′-O-â-D-glu cosid e) (1) was crys-
tallized from EtOH as light yellow crystals: mp 140-
141 °C; [R]20D -8.0° (c 0.1, EtOH); UV (MeOH) λmax (log
ꢀ) 261 (+AlCl3) 270 nm; IR (KBr) vmax 3390 (OH), 2931-
2852 (CH, aliphatic), 1660 (CdO), 1618, 1585, 1571,
1512, 1447, 1349, 1305, 1249, 1212, 1200, 1170, 1155,
E xt r a ct ion a n d Isola t ion . Powdered bark of C.
pentandra (1.25 kg) was extracted three times in aque-
ous 70% EtOH (1 kg of material dry weight to 10 L of
aqueous EtOH first extraction, 1:5 second and third
extractions) at room temperature overnight with oc-
casional stirring. The dried crude extract (68.8 g) was
subjected to a three-phase liquid extraction procedure
with a solvent system consisting of n-hexane-CH2Cl2-
AcCN-H2O (26:13:44:13) (910 mL/455 mL/1540 mL/455
mL × 2) yielding three extracts: hexane (2.3 g), AcCN/
CH2Cl2 (3.9 g), and H2O. The H2O extract was further
extracted with EtOAc (120 × 3 mL), thus yielding an
EtOAc extract (4.5 g) and an H2O extract (46.9 g).
1
1104, 1077, 1054, 993, 831; H and 13C NMR data, see
Tables 1 and 2, respectively; HR-positive FABMS m/z
[M + Na]+ 529.1529 (C24H26O12Na requires 529.1322);
FABMS m/z 507 [M + H]+, 491, 437, 373, 345 ([M + H]
- 162)+, 315, 285, 257, 193, 167, 151.
5,3′-D ih y d r o x y -7,4′,5′-t r im e t h o x y is o fla v o n e
(va va in ) (2) was crystallized from AcCN-CH2Cl2 as a

12 J ournal of Natural Products, 1998, Vol. 61, No. 1
Noreen et al.
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Nuttige Planten van Nederlandsch-Indie¨, 2nd ed.; 1927. Djakar-
ta); Veeman und Zonen: Wageningen, The Netherlands, 1950;
Vols. I and II, pp 1050-1055.
(7) Quisumbing, E. Technical Bulletin (Republic of Philippines
Department of Agriculture and Natural Resources) 1951, No. 16,
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6, 174.
white amorphous solid: mp 169-170 °C; UV (MeOH)
λmax (log ꢀ) 260 (+AlCl3) 270 nm; IR (KBr) vmax 3423
(OH), 2966-2854 (CH, aliphatic), 1665 (CdO), 1624,
1588, 1511, 1447, 1377, 1357, 1293, 1202, 1167, 1061,
998, 829 cm-1; 1H and 13C NMR data, see Tables 1 and
2, respectively; HR-positive FABMS m/z [M + H]+
345.0979 (C18H17O7 requires 345.0974); EIMS (70 eV)
m/z 344 [M+] (100), 329 (46), 315 (4), 301 (24), 297 (7),
269 (5), 257 (4), 230 (13), 192 (2), 167 (17), 142 (8), 135
(7), 115 (5); FABMS m/z 345 [M + H]+, 331, 299, 275,
245, 215, 207, 183, 167, 115.
(+)-Ca tech in was crystallized from AcCN-CH2Cl2
1
(2:1) (19 mg) and identified by comparison of its H and
13C NMR (MeOH-d4) spectra with literature data,36
(9) Raju, T. S.; Gowda, D. C.; Anjaneyalu, Y. V. Carbohydr. Res.
1989, 191, 321-332.
[R]20 +5.9° (c 0.1, Me2CO).
D
COX-1 a n d COX-2-Ca ta lyzed P r osta gla n d in Bio-
syn th esis Assa y. The assay followed the method
developed by White and Glassman.34 Bovine cyclooxy-
genase-1 (prostaglandin endoperoxide H synthase-1)
was derived as described by Takeguchi et al.37 COX-2
(prostaglandin endoperoxide H synthase-2) enzyme
purified from sheep placental cotyledons was procured
from Cayman Chemical Company, Ann Arbor, MI.
Bovine seminal vesicle microsomes (10 µL, 20-30 µg
protein) or purified sheep placental COX-2 enzyme (10
µL, 5 units, 0.8 µg) were preincubated with 50 µL of
cofactor solution (reduced glutathione and l-epinephrine,
0.3 mg/mL each in Tris buffer, pH 8.2) in an ice-water
bath for 15 min. Vehicle or test solution (20 µL) and
20 µL of [14C]arachidonic acid (16 Ci/mol, 30 µM) were
added and incubated at 37 °C for 10 min. A blank was
kept in the ice-water bath. After incubation, the
reaction was terminated by adding 10 µL of 2 N HCl
followed by 5 µL of a carrier solution (PGE2 and PGF2R
0.2 mg/mL of each in EtOH). The unmetabolized
arachidonic acid was separated from the prostaglandin
products by column chromatography and eluted with
n-hexane-dioxane-glacial acetic acid (70:30:1). The
prostaglandin products were then eluted with EtAOc-
MeOH (85:15), and the samples were counted in a
Packard scintillation spectrometer. Indomethacin (2 µg/
mL) was used as a positive control, yielding 70-90%
inhibition. IC50 values were determined by regression
analysis.
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1989, 191, 333-341.
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Prod. 1993, 56, 2041-2045.
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9, 2225-2229.
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11, 1617-1622.
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C. Phytochemistry 1980, 19, 1831-1832.
(16) Mabry, T. J .; Markham, K. R.; Thomas, M. B. The Systematic
Identification of Flavonoids; Springer: Berlin, 1970.
(17) Ali, A. A.; El-Emary, N. A.; El-Moghazi, M. A.; Darwish, F. M.;
Frahm, A. W. Phytochemistry 1983, 22, 2061-2063.
(18) Voirin, B. Phytochemistry 1983, 22, 2107-2145.
(19) Tahara, S.; Moriyama, M.; Ingham, J .; Mizutani, J . Phytochem-
istry 1991, 30, 2769-2775.
(20) Merfort, I.; Wendisch, D. Planta Med. 1987, 53, 434-437.
(21) Agarwal, P. K. Phytochemistry 1992, 31, 3307-3330.
(22) Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983,
41, 27-66.
(23) Sree Rama Murthy, M.; Venkata Rao, E.; Ward, R. S. Magn.
Reson. Chem. 1986, 24, 225-230.
(24) Gryglewski, R. J . In Advances in Lipid Research; Paoletti, R.,
Kritchevsky, D., Eds.; Academic Press: Inc.: New York, 1978;
Vol. 16, pp 327-343.
(25) Wagner, H. Planta Med. 1989, 55, 235-241.
(26) Mukherjee, A.; Hale, V. G.; Borga, O.; Stein, R. Inflammation
Res. 1996, 45, 531-540.
(27) Redl, K.; Breu, W.; Davis, B.; Bauer, R. Planta Med. 1994, 60,
58-62.
(28) Noreen, Y.; Ringbom, T.; Perera, P.; Bohlin, L. J . Nat. Prod.
1997, 60, 2-7.
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(30) Ahnut, S.; Dietmar, H. Z.; Mues, R.; Barz, W.; Mackenbrock,
K.; Ko¨ster, J .; Markham, K. Phytochemistry 1984, 23, 1073-
1075.
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Adsorption Column Chromatography Using Simple Computa-
tional Models. Licentiate Thesis, Department of Chemistry,
Organic Chemistry, Royal Institute of Technology, Stockholm,
1996.
(34) White, H. L.; Glassman, A. T. Prostaglandins 1974, 7, 123-129.
(35) Stahl, E. Du¨nnschicht-Chromatographie. Ein Laboratoriums-
handbuch; Springer-Verlag: Berlin, 1962; p 534.
(36) Balde´, A. M.; Pieters, L. A.; Geryely, A.; Kolodziej, H.; Claeys,
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2372-2376.
Ack n ow led gm en t. We are grateful to Rolf Ander-
sson for recording NMR spectra, Suresh Gohil for MS
measurements, and J an Straube and Kerstin Ekehov-
Karlsson for skillful technical assistance. We also thank
Prof. Paul A. Cox for plant collection and authentication.
The Swedish Council for Forestry and Agricultural
Science is gratefully acknowledged for financial support
and the Swedish Institute for providing a scholarship
for H.E.
Refer en ces a n d Notes
(1) Baker, H. G. In Ecology and Economic Development in Tropical
Africa; Brokensha, D., Ed.; Institute of International Studies,
Research Series; University of California: Berkeley, CA, 1965;
pp 185-216.
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