Phenolic Compounds from Nymphaea odorata
Zhizhen Zhang,†Hala N. ElSohly,*,†Xing-Cong Li,†Shabana I. Khan,†Sheldon E. Broedel, J r.,‡
Robert E. Raulli,‡Ronald L. Cihlar,§Charles Burandt,†and Larry A. Walker*,†,⊥
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, and Department of
Pharmacology, School of Pharmacy, University of Mississippi, University, Mississippi 38677, Dorlin Pharmaceuticals,
Baltimore, Maryland 21227, and Department of Microbiology and Immunology, Georgetown University,
Washington, D.C. 20057
Received September 11, 2002
Assay-guided fractionation of the ethanol extract of Nymphaea odorata resulted in the identification of
two lignans, one new (1) and one known (2), together with six known flavonol glycosides (3-8). The
structures of 1-8 were established by spectroscopic analysis as nymphaeoside A (1), icariside E4(2),
kaempferol 3-O-R-L-rhamnopyranoside (afzelin, 3), quercetin 3-O-R-L-rhamnopyranoside (4), myricetin
3-O-R-L-rhamnopyranoside(myricitrin, 5), quercetin 3-O-(6′′-O-acetyl)-?-D-galactopyranoside(6), myricetin
3-O-?-D-galactopyranoside (7), and myricetin 3-O-(6′′-O-acetyl)-?-D-galactopyranoside (8). Compounds 3,
4, and 7 showed marginal inhibitory effect against fatty acid synthase with IC50values of 45, 50, and 25
Fatty acid synthase (FAS) is an enzyme essential tothe
infective process in Candida albicans.1,2Early work on the
structure-activity relationship among a series of structur-
ally related FAS inhibitors showed that the fungal and
human enzymes could be differentially inhibited.3This
suggested that with appropriate inhibitors FAS could be
used as a therapeutic target. In the course of our continued
efforts of searching for fatty acid synthase (FAS) inhibitors
from natural sources,4we investigated an active ethanolic
extract of Nymphaea odorata.
Nymphaea odorata Ait. (Nymphaeaceae), or American
white water lily, is a herbaceous hydrophyte native to the
southeastern United States. Previous work on this plant
resulted in the isolation of five compounds with plant
growth-inhibitory properties.5Bioactivity-guided fraction-
ation of the leaves of N. odorata resulted in the isolation
of eight constituents (1-8). This work describes the isola-
tion, structure elucidation, and biological activity of these
Compound 1 was obtained as a colorless amorphous
solid. Its molecular formula of C27H38O12was determined
by HRESIMS and indicated nine degrees of unsaturation.
The13C NMR spectrum of 1 displayed 27 signals, of which
21 were assigned to the aglycone moiety including two
aromatic rings, three methoxy groups, one hydroxypropyl
group, and one 1,3-propanediol group. The remaining six
signals corresponded to a deoxyhexose. The
spectrum of 1 displayed signals for a 1,3,4-trisubstituted
aromatic ring [δ 7.11 (1H, br s, H-2), δ 7.08 (1H, d, J ) 8.0
Hz, H-5), δ 6.93 (1H, d, J ) 8.0 Hz, H-6), δ 3.85 (3H, s,
OCH3-3)], a 1′,3′,4′,5′-tetrasubstituted aromatic ring [δ 6.57
(2H, s, H-2′,6′), δ 3.83 (6H, s, OCH3-3′,5′)], a 1,2,3-
trioxygenated propyl function [δ 4.99 (1H, d, J ) 5.2 Hz,
H-7), δ 4.22 (1H, m, H-8), δ 3.95 (1H, m, H-9b), δ 3.57 (1H,
m, H-9a)], and a hydroxypropyl group [δ 2.65 (2H, t, J )
8.0 Hz, H-7′), δ 1.82 (2H, m, H-8′), δ 3.57 (2H, m, H-9′)].
These data suggested that the aglycone of 1 is a lignan,
identified as 1-[4-hydroxy-3-methoxyphenyl)-2-[4-(3-hy-
these inferences were confirmed by HMQC and HMBC
correlations (Figure 1). Acid hydrolysis of 1 afforded
rhamnose, which was identified by co-TLC with an au-
thentic sample. The characteristic NMR signals of δH5.38
(1H, br s, H-1′′) and δH1.26 (3H, d, J ) 5.9 Hz, H-6′′), as
well as their protonated carbon signals at δC101.4 (C-1′′)
* Towhom correspondence should be addressed. (H.N.E.) Tel: 662-915-
7610. Fax: 662-915-7989. E-mail: firstname.lastname@example.org. (L.A.W.) Tel: 662-
915-1005. Fax: 662-915-1006. E-mail:email@example.com.
†National Center for Natural Products Research, University of Missis-
⊥Department of Pharmacology, University of Mississippi.
548 J . Nat. Prod. 2003, 66, 548-550
10.1021/np020442j CCC: $25.00© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/19/2003
and δC18.8 (C-6′′) also indicated the presence of a rham-
nose moiety. A HMBC correlation between H-1′′ (δ 5.38)
of rhamnose and C-4 (δ 146.0) of aglycone established that
the rhamnose was attached toC-4 of aglycone. The R-pyra-
nose form of rhamnose in 1 was confirmed8,9by a set of
carbon signals at δ 101.4, 72.2, 72.0, 73.8, 70.7 and 18.8,
which correlated with δ 5.38 (br s), 4.12 (br s), 3.95 (m),
3.48 (t, J ) 9.5 Hz), 3.91 (m), and 1.26 (d, J ) 5.9 Hz),
respectively, in the HMQC spectrum. The stereochemistry
for C-7 and C-8 was shown tobeerythro6from thechemical
shift value at δ 6.00 (1H, d) and the J coupling constant
value (4.7 Hz) of H-7 in 1a, the corresponding acetate
derivative of 1. HMQC and HMBC analyses allowed for
the complete assignments of the1H and13C signals of 1
new natural product named nymphaeoside A.
The known compounds were identified by comparison of
their spectral data with reported values as icarisideE4(2),10
kaempferol 3-O-R-L-rhamnopyranoside (afzelin, 3),11,quer-
cetin 3-O-R-L-rhamnopyranoside (4),12myricetin 3-O-R-L-
rhamnopyranoside (myricitrin, 5),13quercetin 3-O-(6′′-O-
acetyl)-?-D-galactopyranoside (6),14myricetin 3-O-?-D-
galactopyranoside (7),14and myricetin 3-O-(6′′-O-acetyl)-
?-D-galactopyranoside (8).14This is the first report of the
isolation of 2-8 from this plant.
Compounds 1-8 were evaluated in the fatty acid syn-
thase (FAS) inhibition assay.4The results showed com-
pounds 3, 4, and 7 had weak inhibitory effects against FAS
with IC50 values of 45, 50, and 25 µg/mL, respectively.
Cerulenin15was used as a positive control (IC50of 0.19 µg/
E xperimental Section
General E xperimental Procedures. Optical rotations
weredetermined on a J ASCO DIP-370 digital polarimeter. UV
spectra were recorded on a Hewlett-Packard 8435 spectrom-
eter. IR spectra were obtained on an ATI Mattson Genesis
Series FTIR spectrometer. The NMR spectra were recorded
on a Bruker Avance DRX-400 spectrometer operating at 400
measured on a Bruker Avance DRX-500 operating at 500 MHz
using standard pulse programs and acquisition parameters.
HRESIMS weremeasured on a Bruker-Magnex BioAPEX 30es
ion cyclotron high-resolution HPLC-FT spectrometer by direct
injection into an electrospray interface. Si gel (40 µm, J . T.
Baker) and RP Si gel (RP-18, 40 µm, J . T. Baker) were used
for low-pressure chromatography. HPLC was performed using
an ODS column (Phenomenex, Prodigy ODS prep, 21.2 mm
i.d. × 250 mm, 10 µm). TLC was performed on Si gel 60 F254
(EM Science) using CHCl3/MeOH (4:1, solvent A), toluene/
EtOAc/MeOH (4:1:1, solvent B), and CHCl3/EtOAc (6:1, solvent
C) or reversed-phaseKC18F Si gel 60 (Whatman) using MeOH/
H2O (70:30, solvent D). The detailed procedures for the
bioassays are described in a previous paper.4
1H and 100 MHz for
13C. 2D NMR spectra were
Plant Material. The plant material (leaves) was collected
in Florida in J uly 1996 and identified by Dr. Charles Burandt.
A voucher specimen is on deposit at the National Center for
Natural Products Research, The University of Mississippi
(voucher # BUR 190796 1A).
E xtraction and Isolation. The dried and powdered (60
mesh) plant material (400 g) was percolated with 95% EtOH
(3000 mL × 3). The ethanolic extract was evaporated to
dryness (50.1 g, IC5025 µg/mL). Part of the EtOH extract (33.0
g) was chromatographed over a silica gel column (300 g) eluting
with CHCl3/MeOH (9:1, 4:1, 1:1, 1:4, and 0:1, each 1000 mL).
On the basis of TLC analysis, nine combined fractions were
obtained: A (5.19 g), B (0.70 g), C (0.70 g), D (2.17 g), E (8.09
g), F (3.68 g), G (5.28 g), H (4.10 g), and I (2.25 g). Part of
fraction B (0.66 g) was rechromatographed over a silica gel
column (120 g) using CHCl3/MeOH (7:1 and 4:1, each 1500
mL) to afford five fractions: B1(235.2 mg), B2(250 mg), B3(9
mg), B4 (50.0 mg), and B5 (60 mg). Part of B2 (100 mg) was
further applied ontoa ODS column (10 g) washing with MeOH/
H2O (60:40) and then MeOH. The aqueous MeOH fraction (20
mg) was purified by HPLC (MeOH/H2O, 60:40, 3 mL/min, UV
276 nm) to yield 1 (10.6 mg, tR 22.5 min). Part of B4(40 mg)
was separated using an ODS column (10 g) and washing with
MeOH/H2O (0:100, 50:50, and 80:20, each 200 mL). The 50%
MeOH fraction (22 mg) was refractionated using HPLC
(MeOH/H2O, 50:50) to afford 2 (9.5 mg, tR 21 min), 3 (2.1 mg,
tR 29.4 min), and 6 (2.6 mg, tR 26 min).
Part of the FAS inhibitory fraction E (6.0 g) was chromato-
graphed over a ODS column (200 g) eluting with MeOH/H2O
mixtures (100:0, 50:50, and 0:100, each 1000 mL) to give
fractions E1(2.50 g), E2(1.48 g), E3(1.51 g), and E4(0.50 g).
Fraction E3was further purified using HPLC (MeOH/H2O, 50:
50, 3 mL/min, UV 276 nm) to afford 4 (2.6 mg, tR 54.1 min), 5
(8.0 mg, tR36.5 min), 7 (3.0 mg, tR33.0 min), and 8 (6.0 mg, tR
40.5 min). Fractions F-I, E1, and E2, rich in very polar
compounds (possibly tannins), were not further investigated.
Nymphaeoside (1): colorless amorphous powder; [R]22D
-23.1° (c 0.5, MeOH); UV (MeOH) λmax(log ?) 214 (4.05), 230
(sh), 276 (3.17) nm; IR (KBr) νmax3401, 1587, 1504, 1456, 1417,
1220, 1119, 1057, 1019, 973 cm-1;1H NMR (CD3OD) δ 1.26
(3H, d, J ) 5.9 Hz, H-6′′), 1.82 (2H, m, H-8′), 2.65 (2H, t, J )
8.0 Hz, H-7′), 3.48 (1H, t, J ) 9.5 Hz, H-4′′), 3.57 (4H, m, H-9,
9′), 3.83 (6H, s, OCH3-3′, 5′), 3.85 (3H, s, OCH3-3), 3.91 (1H,
m, H-5′′), 3.95 (1H, m, H-3′′), 4.12 (1H, br s, H-2′′), 4.22 (1H,
m, H-8), 4.99 (1H, d, J ) 5.2 Hz, H-7), 5.38 (1H, br s, H-1′′),
6.57 (2H, br s, H-2′,6′), 6.93 (1H, d, J ) 8.0 Hz, H-6), 7.08 (1H,
d, J ) 8.0 Hz, H-5), 7.11 (1H, br s, H-2);13C NMR (CD3OD) δ
154.3 (C-3′,5′), 151.6 (C-3), 146.0 (C-4), 139.9 (C-1), 138.0 (C-
1′), 134.6 (C-4′), 120.4 (C-6), 119.2 (C-5), 112.4 (C-2), 106.8 (C-
2′,6′), 101.4 (C-1′′), 87.2 (C-8), 73.8 (C-4′′), 73.7 (C-7), 72.2 (C-
2′′), 72.0 (C-3′′), 70.7 (C-5′′), 62.1 (C-9′), 61.4 (C-9), 56.6 (OCH3-
3′,5′), 56.4 (OCH3-3), 35.4 (C-8′), 33.4 (C-7′), 18.8 (C-6′′);
HRESIMS m/z 572.2698 [M + NH4]+, 577.2258 [M + Na]+
(calcd for C27H38O12, 572.2701 [M + NH4]+, 577.2255 [M +
Acetylation of 1. Compound 1 (6 mg) was dissolved in
Ac2O/pyridine (1:2, 3 mL), and the mixture was allowed to
stand at room temperature for 24 h. After addition of 10 mL
of H2O, the resulting mixture was applied ontoa low-pressure
ODS column (5 g) washing with H2O (50 mL) and then MeOH
(30 mL). The MeOH fraction was evaporated togive1a (5 mg).
1a:colorless amorphous powder; [R]22D -35.4° (c 0.5,
MeOH); UV (MeOH) λmax(log ?) 214 (4.18), 230 (sh), 276 (3.14)
nm; IR (KBr) νmax 2940, 1744, 1589, 1508, 1460, 1369, 1220,
1124, 1039 cm-1;1H NMR (CDCl3) δ 1.16 (3H, d, J ) 5.9 Hz,
H-6′′), 1.88 (2H, m, H-8′), 1.95 (3H, s, -COCH3), 1.99 (3H, s,
-COCH3), 2.04 (6H, s, -COCH3× 2), 2.11 (3H, s, -COCH3),
2.19 (3H, s, -COCH3), 2.55 (2H, t, J ) 7.1 Hz, H-7′), 3.70 (6H,
s, OCH3-3′,5′), 3.80 (3H, s, OCH3-3), 4.05 (2H, m, H-9′), 4.12
(1H, m, H-5′′), 4.20 (1H, m, H-9a), 4.39 (1H, m, H-9b), 4.55
(1H, br t, H-8), 5.07 (1H, t, J ) 9.8 Hz, H-4′′), 5.31 (1H, br s,
H-1′′), 5.49 (1H, br s, H-2′′), 5.53 (1H, m, H-3′′), 6.00 (1H, d,
J ) 4.7 Hz, H-7), 6.32 (2H, s, H-2′,6′), 6.82 (1H, d, J ) 8.0 Hz,
H-6), 6.92 (1H, br s, H-2), 6.98 (1H, d, J ) 8.0 Hz, H-5);13C
NMR (CDCl3) δ 171.5 (CdO), 171.3 (CdO × 2), 170.4 (CdO ×
F igure 1. Key HMBC (H to C) correlations of 1.
Notes J ournal of Natural Products, 2003, Vol. 66, No. 4 549
2), 170.1 (CdO), 153.4 (C-3′,5′), 150.7 (C-3), 145.1 (C-4), 137.8 Download full-text
(C-1′), 133.6 (C-1)a, 133.5 (C-4′)a, 119.8 (C-6), 118.7 (C-5), 112.0
(C-2), 105.6 (C-2′, 6′), 97.7 (C-1′′), 81.1 (C-8), 74.5 (C-7), 71.4
(C-4′′), 70.1 (C-2′′)b, 69.3 (C-3′′)b, 67.7 (C-5′′), 64.1 (C-9′), 63.1
(C-9), 56.3 (OCH3-3, 3′, 5′), 32.9 (C-7′), 30.5 (C-8′), 21.4
(-COCH3), 21.3 (-COCH3), 21.2 (-COCH3× 2), 21.1 (-CO-
CH3), 20.7 (-COCH3), 17.7 (C-6′′); assignments were based on
COSY, HMQC, and HMBC spectra (a,bThe signals may be
interchangeable); HRESIMS m/z 829.2871 [M + Na]+(calcd
for C39H50O18, 829.2889 [M + Na]+).
Acid Hydrolysis of 1. Compound 1 and authentic rham-
nose were spotted on a silica gel TLC plate and hydrolyzed in
situ by exposure to HCl vapor at 70 °C for 25 min. The TLC
plate was then developed with CHCl3/MeOH/AcOH/H2O (14:
6:2:1) and sprayed with 10% H2SO4for detection. Rhamnose
was detected with an Rfvalue of 0.2.
Acknowledgment. The authors thank Dr. Chuck Dunbar
for HRESIMS analyses, and Mr. Frank Wiggers for running
gradient HMQC and HMBC NMR (500 MHz) experiments.
This work was supported in part by the United States
Department of Agriculture, Agricultural Research Service
Specific Cooperative Agreement No. 58-6408-2-0009, and by
NCDDG Grant #5 RO1 CA 88456-02.
Supporting Information Available:
2-8. This material is available free of charge via the Internet at http://
1H and13C NMR data of
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