Iridoid glycosides and grayanane diterpenoids from the roots of Craibiodendron henryi

Article abstract of DOI:10.1021/np050261s

Four new iridoid glycosides, 10-O-trans-p-coumaroylscandoside (1), 10-O-cis-p-coumaroylscandoside (2), 10-O-trans-p-coumaroyldesacetyl asperulosidic acid (3), and 10-O-cis-p-coumaroyldesacetyl asperulosidic acid (4), and two new grayanane diterpenoids, 14β-O-(2S,3S-nilyl)-2α, 3β,5β,6β,16α-pentahydroxygrayanane (5) and 14β-O-(2S,3S-nilyl)-2α,3β,5β,6β,16α- pentahydroxygrayan-10(20)-ene (6), have been isolated from Craibiodendron henryi. The structures of these compounds were determined by chemical and spectroscopic methods including ¹H-¹H COSY, HMQC, HMBC, and NOESY experiments. Antioxidant activities and vasodilator effects of these compounds were assessed.

Full text of DOI:10.1021/np050261s

1646
J. Nat. Prod. 2005, 68, 1646-1650
Iridoid Glycosides and Grayanane Diterpenoids from the Roots of
Craibiodendron henryi
Xiang-zhong Huang, Ying-hong Wang, Shi-shan Yu,* Guang-miao Fu, You-cai Hu, Yue Liu, and Li-hua Fan
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education &
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
No. 1 Xiannongtan Street, Beijing 100050, People’s Republic of China
Received July 21, 2005
Four new iridoid glycosides, 10-O-trans-p-coumaroylscandoside (1), 10-O-cis-p-coumaroylscandoside (2),
10-O-trans-p-coumaroyldesacetyl asperulosidic acid (3), and 10-O-cis-p-coumaroyldesacetyl asperulosidic
acid (4), and two new grayanane diterpenoids, 14â-O-(2S,3S-nilyl)-2R,3â,5â,6â,16R-pentahydroxygray-
anane (5) and 14â-O-(2S,3S-nilyl)-2R,3â,5â,6â,16R-pentahydroxygrayan-10(20)-ene (6), have been isolated
from Craibiodendron henryi. The structures of these compounds were determined by chemical and
1
spectroscopic methods including H-1H COSY, HMQC, HMBC, and NOESY experiments. Antioxidant
activities and vasodilator effects of these compounds were assessed.
Craibiodendron henryi W. W. Smith (Ericaceae) is a well-
known toxic plant, distributed mainly in hilly and valley
regions of south, central, and northwest Yunnan Province
of China. Up to the present, chemical and biological work
on this plant has been limited to a report of two grayanane
diterpenoids, rhodojaponins II and III.1 The n-BuOH-
soluble fraction of an ethanolic extract was found to show
Figure 1. Selected HMBC (H f C) correlations of 1.
antioxidant activity in a microsomal lipid peroxidation
induced by ferrous-cysteine model and moderate vasodi-
lator activity in a phenylepherine-induced vasoconstriction
assay using rat aortic rings. Chemical investigation of the
plant has led to the isolation of four new iridoid glycosides
(1-4) and two new grayanane diterpenoids (5, 6), along
with three known iridoid glycosides and two grayanane
diterpenoids. In this paper we describe the isolation and
structural elucidation of the new compounds and the
unambiguous assignment of spectroscopic data by a com-
bination of NMR techniques, including 1H-1H COSY,
Figure 2. Selected NOESY correlations of 1 and 3.
HMQC, HMBC, and NOESY.
related to those reported for scandoside (1a).9 The only
Results and Discussion
evident difference was that 1 has resonances due to an
additional p-coumaroyl group, which was assigned an
E-configuration from the large coupling constant between
The n-BuOH fraction of the ethanolic extract was
subjected to column chromatography on polyamide, silica
gel, RP-18, Sephadex LH-20, and HPLC to afford four new
iridoid glycosides (1-4) and two new grayanane diterpe-
noids (5, 6), along with five known compounds. The known
compounds were characterized as vaccinoside,2 androme-
doside,3 splendoside,4 rhodojaponin III,1 and rhodomollein
I.5
Compound 1 was isolated as an amorphous powder. The
molecular formula was determined to be C25H28O13 by
HRFABMS. Its UV spectrum showed absorption bands
characteristic for a conjugated enol-ether system and an
aromatic system at 242, 303, and 314 nm. The IR spectrum
of 1 indicated hydroxyl (3407 cm-1), conjugated carbonyl
(1682 cm-1), and aromatic (1604 and 1516 cm-1) groups.
The 1H and13C NMR spectroscopic data of 1 displayed
signals characteristic of an iridoid glycoside.6-9 The 13C
NMR spectrum contained 25 carbon signals including six
for the glucopyranosyl unit, nine for a p-coumaroyl group,
a carboxyl group, and the remaining nine for the iridoid
skeleton. The 13C NMR chemical shifts of 1 were closely
1
H-2′′ and H-3′′ (J ) 16.0 Hz) in the H NMR spectrum.
The C-7 and C-10 downfield shifts (δ 133.7 from δ 129.9; δ
62.3 from δ 61.1) and the C-8 upfield shift (δ 140.6 from δ
147.3) established the attachment of the (E)-p-coumaroyl
group to the C-10 carbon. The assumption was confirmed
by the correlations between two proton signals of C-10 at
δ 5.33 and 5.06 and the ester carbonyl (δ 167.0) of an (E)-
p-coumaroyl group in the HMBC experiment (Figure 1).
The C-6 hydroxyl in 1 was assigned as a â-substituent on
the basis of the coupling constant between H-5 and H-6 (J
) 6.5 Hz) in the 1H NMR spectrum. The correlation of H-5
to H-9 and the absence of the correlation of H-5 to H-6 in
a NOESY experiment (Figure 2) supported the above
conclusion. On the basis of the reported procedure,10 the
absolute configuration of the glucose moiety was deter-
mined as D-glucose. Therefore the structure of 1 was
determined to be 10-O-trans-p-coumaroylscandoside.
Compound 2, an amorphous powder, was found to have
the same molecular formula, C25H28O13, as 1 (HRFABMS).
1
The similarity of the H and 13C NMR spectroscopic data
of 2 to those of compound 1 indicated that they had the
same skeleton structure except for the NMR data assign-
* To whom correspondence should be addressed. Tel: +8610-63165324.
Fax: +8610-63017757. E-mail: yushishan@imm.ac.cn.
10.1021/np050261s CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/28/2005

Iridoid Glycosides from Craibiodendron henryi
Journal of Natural Products, 2005, Vol. 68, No. 11 1647
Chart 1
able to the p-coumaroyl group. Specifically, a small coupling
constant (13.0 Hz) observed for two vicinal olefinic protons
suggested the presence of a (Z)-p-coumaroyl ester unit in
2, which was further confirmed by the NOESY correlation
between two olefinic protons. The HMBC correlations
between protons (δ 5.01 and 5.23) of C-10 and the ester
carbonyl function (δ 166.2) indicated that this (Z)-p-
coumaroyl group was located at the C-10 position. From
these data compound 2 was clearly assigned as 10-O-cis-
p-coumaroylscandoside.
those of 3, except for the chemical shifts and J values due
to H-2′′ and H-3′′ of the p-coumaroyl unit. A small J value
(13.0 Hz) between H-2′′ and H-3′′ of the p-coumaroyl group
was observed, indicating the Z isomer. Additionally, the
p-coumaroyl group was determined to be linked at C-10
on the basis of the HMBC correlations between two protons
(δ 5.16 and 5.42) of C-10 and the ester carbonyl function
(δ 166.3). Thus, compound 4 was assigned as the Z isomer
of compound 3.
Compound 5 was isolated as an amorphous powder. The
molecular formula was determined to be C25H42O9 by
HRFABMS. Its IR spectrum showed the presence of
hydroxyl groups (3390 cm-1) and an ester carbonyl group
Compound 3, obtained as an amorphous powder, had a
molecular formula of C25H28O13 as determined by HR-
FABMS. The UV spectrum of 3 exhibited absorption
maxima at 236, 301, and 314 nm, indicating the presence
of a conjugated enol-ether system and a coumaroyl moiety.
Its IR spectrum displayed absorptions due to hydroxyl
groups at 3410 cm-1, a conjugated carbonyl group at 1682
cm-1, and an aromatic moiety at 1604 and 1516 cm-1. The
1H and 13C NMR spectroscopic data of 3 revealed expected
signals for a â-glucopyranosyl unit, a p-coumaroyl group
with an E double bond (J ) 16.0 Hz), and an iridoid
skeleton. Compound 3 showed the same substituted hy-
droxyl group patterns as that in 1. However, differences
1
(1724 cm-1). The H NMR spectrum contained signals for
four methyl singlets (δ 1.49, 1.60, 1.64, 1.88), two methyl
doublets (δ 1.20, 1.26), and five oxygenated methines (δ
4.12, 4.17, 4.42, 5.16, 6.31). The 13C NMR (DEPT) spectrum
revealed 25 carbon signals, including six methyls, four
methylenes, nine methines (five oxygenated), and six
quaternary carbons (one ester carbonyl and three oxygen-
ated). The 1H-1H COSY spectrum indicated the existence
of the following fragments: CH-CH(OH)-CH(OH), CH-
(OH)-CH2, and CH-CH2-CH2-CH. These structural
features suggested that 5 was a grayanane-type diterpe-
noid with seven sites of oxygenation. In addition to the
signals for a grayanane-type diterpenoid skeleton, there
were the resonances due to an extra nilyl group. The
assignment of the nilyl group was determined on the basis
of the fragment of successive connectivities, CH3-CH-CH-
1
in chemical shifts were observed in the H and13C NMR
spectroscopic data, indicating the existence of a different
relative configuration. The coupling constant between H-5
and H-6 (J ) 5.5 Hz) together with the downfield shifts of
C-1 and C-8 and the upfield shifts of C-4, C-5, C-6, C-7,
and C-11 indicated that the orientation of the hydroxyl
group at C-6 was R. Moreover, H-6 of 3 correlated with H-5
and H-9 in a NOESY experiment (Figure 2), which con-
firmed that the relative configuration of the hydroxyl group
at C-6 was R. On comparing the NMR spectra of 3 with
those of asperulosidic acid (3a),9 a close resemblance was
observed except for the presence of an (E)-p-coumaroyl
group in 3 instead of an acetyl group in asperulosidic acid.
These spectroscopic data also implied that the (E)-p-
coumaroyl group of 3 was attached at the C-10, the same
position as the acetyl group of asperulosidic acid, which
was further supported by the HMBC correlations of two
protons (δ 5.01 and 5.60) of C-10 to the ester carbonyl (δ
167.1) of the (E)-p-coumaroyl group. Thus, compound 3 was
identified as 10-O-trans-p-coumaroyldesacetylasperulosidic
acid.
1
(OH)-CH3, in the H-1H COSY spectrum. This assump-
tion was confirmed by protons of C-5′ correlated with C-1′,
C-2′, and C-3′, but protons of C-4′ only correlated with C-2′
and C-3′ in the HMBC spectrum of 5. Alkaline hydrolysis
of 5 yielded the aglycon 5a. The 1H and 13C NMR and
optical rotation data of 5a were in agreement with those
of rhodojaponin VI.11,12 Thus, the structure of 5a was
established as 2R,3â,5â,6â,14â,16R-hexahydroxygrayanane.
The downfield shifts of H-14 (δ 5.08 in the aglycon (5a) to
δ 6.31 in 5) and C-14 (δ 80.1 in the aglycon (5a) to δ 83.0
in 5) indicated that the nilyl group of 5 was located at C-14.
This deduction was confirmed by the HMBC correlations
among H-14/C-15 and H-14/C-16 and the ester carbonyl
carbon. The absolute stereochemistry of the nilate moiety
was assigned by comparison of the specific rotations of
methyl nilate (8) with reported values for the stereoisomers
of this compound in the literature.13-16 The specific rotation
values ([R]23D (c 5.00, CHCl3)) of methyl nilate obtained by
Compound 4, an amorphous powder, gave the molecular
1
formula C25H28O13 as determined by HRFABMS. The H
and 13C NMR spectroscopic data for 4 were identical with

1648 Journal of Natural Products, 2005, Vol. 68, No. 11
Huang et al.
mass Autospec-Ultima ETOF spectrometer. Column chroma-
tography was performed with polyamide (30-60 mesh), silica
gel (200-300 mesh), RP-18 (40-70 µm), and Sephadex LH-
20. HPLC was carried out on a Shimadzu LC-6AD, and the
detector was a SPD-10A. A reversed-phase C18 column (YMC-
Pack ODS-A φ 20 × 250 mm, 10 µm) was employed. TLC was
carried out with glass precoated silica gel GF254 plates. Spots
were visualized under UV light or by spraying with 7% H2-
SO4 in 95% EtOH followed by heating.
Plant Material. The roots of Craibiodendron henryi (8.0
kg) were collected from Gejiu City of Yunnan Province in
China, in August of 2003. The plant was identified by associate
Prof. Lin Ma of the Department of Natural Products Chem-
istry, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College. A voucher speci-
men (No. 211) is deposited in the herbarium of Institute of
Materia Medica, Chinese Academy of Medical Sciences &
Peking Union Medical College.
methylation of nilic acid (7) derived from alkaline hydroly-
sis of 5 were as follows: (λ (nm)) 589, +34.6; 578, +35.2;
546, +37.6; 436, +47.0. These results indicated the nilate
moiety present in 5 had the (+)-erythro, i.e., (2S, 3S),
configuration. Therefore the structure of 5 was determined
as 14â-O-(2S,3S-nilyl)-2R,3â,5â,6â,16R-pentahydroxygray-
anane.
Compound 6, colorless needles, was assigned a molecular
formula of C25H40O8, as deduced from its positive HR-
FABMS. The IR spectrum showed characteristic absorp-
tions for hydroxyl (3433 cm-1), ester carbonyl (1711 cm-1),
1
and double-bond (1637 cm-1) groups. The H NMR spec-
trum contained three methyl singlets (δ 1.52, 1.56, 1.66),
two methyl doublets (δ 1.26, 1.38), five oxygenated me-
thines (δ 4.12, 4.18, 4.30, 4.95, 5.71), and two olefinic
protons (δ 5.26, 5.45). The 13C NMR (DEPT) spectrum
revealed 25 carbon signals, including five methyls, five
methylenes (one olefinic), nine methines (five oxygenated),
and six quaternary carbons (one ester carbonyl, one olefinic,
and two oxygenated). The 1H-1H COSY spectrum of 6
indicated the existence of the following fragments: CH-
CH(OH)-CH(OH), CH(OH)-CH2, and CH-CH2-CH2-
CH, the same as those in 5. These structural features
suggested that 6 was a grayanane-type diterpenoid with
six sites of oxygenation. The 1H and13C NMR data of 6 were
very similar to those of 5. By comparison of the 13C NMR
data of 6 and 5, the major difference was that 6 had an
exomethylene instead of a methyl and an oxygenated
quaternary carbon in 5. This indicated that 6 was a C-10/
20 dehydrated derivative of 5. The exomethylene at C-10/
20 was confirmed by the HMBC correlations between two
proton signals of C-20 and C-1 and C-9. The NOESY
spectrum and the J values for each proton in 6 were
comparable with those of 5, thereby indicating that the
relative stereochemistry of 6 was the same as 5. In
addition, 6 was hydrolyzed with KOH to afford the aglycon
Extraction and Isolation. The roots of C. henryi (8.0 kg)
were air-dried, ground, and extracted with EtOH under reflux.
The ethanolic extract was evaporated to almost dryness in
vacuo, and the resulting mixture was partitioned successively
between H2O and EtOAc and between H2O and n-BuOH. The
n-BuOH phase was concentrated to give a black mass (171 g),
which was applied to polyamide column chromatography
eluting with H2O, 20% EtOH, 60% EtOH, and 95% EtOH to
give Fractions A1 (5.8 g), A2 (6.1 g), A3 (107.5 g), and A4 (3.8
g), respectively. Fraction A2 was subjected to a silica gel column
eluting with a gradient of increasing MeOH in CHCl3 to yield
Fractions B1-B7. Fraction B1 (91 mg) was chromatographed
on a silica gel column with CHCl3-MeOH (20:1) and an RP-
18 column with MeOH-H2O (47:53) to yield 8 (51 mg).
Fraction B2 (146 mg) was subjected to an RP-18 silica gel
column using MeOH-H2O (45:55) as a solvent to provide 9
(59 mg). From fraciton B3 (789 mg), repeated silica gel and
Sephadex LH-20 column chromatography led to the isolations
of 10 (14 mg) and 11 (17 mg). Compound 7 (129 mg) was
obtained by applying fraction B5 (707 mg) to a silica gel column
eluting with CHCl3-MeOH (15:1). Fraction B6 (1.36 g) was
purified by Sephadex LH-20 column chromatography eluted
with CHCl3-MeOH (2:1) and then was separated into fractions
C1-C3 (C1, 817 mg; C2, 88 mg; C3, 107 mg) by RP-18 column
chromatography eluted with MeOH-H2O (29:71). Fraction C1
was purified by reversed-phase HPLC using 42% MeOH in
H2O as the mobile phase to afford 5 (94 mg) and 6 (25 mg).
Fraction C3 was similarly purified by reversed-phase HPLC
using MeOH-H2O (39:61) to obtain 1 (18 mg), 2 (8 mg), 3 (31
mg), and 4 (7 mg), respectively.
1
6a. The H and 13C NMR data of 6a were in accordance
with those of rhodomollein I,5 which was established as
2R,3â,5â,6â,14â,16R-hexahydroxygrayan-10(20)-ene. The
absolute configuration of the nilate moiety in 6 was
assumed to be the same as that of 5. Thus, the structure
of 6 was determined to be 14â-O-(2S,3S-nilyl)-2R,3â,5â,6â,-
16R-pentahydroxygrayan-10(20)-ene.
All seven iridoid glycosides showed low-level antioxidant
activity in the microsomal lipid peroxidation induced by
ferrous-cysteine testing. The inhibition rate of MDA for the
seven iridoid glycosides, at a concentration of 1.0 × 10-5
M, was found to be 24.3, 23.9, 17.7, 14.1, 17.9, 17.5, and
27.9%, respectively, compared to 49.7% for vitamin E, a
well-known antioxidant.
Vasodilator effects of four grayanane diterpenoids (1.0
× 10-5 M) on phenylepherine-induced vasoconstriction of
rat aortic rings in the presence of indomethacin (Indo) and
Nω-L-nitroarginine (L-NA) were (33 ( 1.1), (27 ( 2.1), (33
( 1.8), and (35 ( 2.4)%, respectively, compared to (105 (
4.7)% for sodium nitroprusside as a positive control. All
four compounds exhibited weak vasodilator activities.
Compound 1: amorphous powder; mp 160-162 °C; [R]23
D
-65.4 (c 0.54, MeOH); UV (MeOH) λmax (log ꢀ) 242 (3.87), 303
(3.56), 314 (4.01) nm; IR (KBr) νmax 3407, 2924, 1682, 1632,
1
1604, 1516, 1205, 1142, 1076, 839 cm-1; H NMR (pyridine-
d5, 500 MHz) δ 7.91 (1H, d, J ) 16.0 Hz, H-3′′), 7.89 (1H, s,
H-3), 7.58 (2H, d, J ) 8.5 Hz, H-5′′, 9′′), 7.17 (2H, d, J ) 8.5
Hz, H-6′′, 8′′), 6.57 (1H, d, J ) 16.0 Hz, H-2′′), 6.04 (1H, s,
H-7), 5.40 (1H, d, J ) 7.5 Hz, H-1′), 5.37 (1H, d, J ) 8.0 Hz,
H-1), 5.33 (1H, d, J ) 14.5 Hz, H-10), 5.06 (1H, d, J ) 14.5
Hz, H-10), 4.92 (1H, br s, H-6), 4.48 (1H, dd, J ) 12.0, 2.0 Hz,
H-6′), 4.33 (1H, dd, J ) 12.0, 5.5 Hz, H-6′), 4.28 (1H, dd, J )
9.0, 8.5 Hz, H-3′), 4.26 (1H, m, H-4′), 4.12 (1H, dd, J ) 9.0, 7.5
Hz, H-2′), 3.97 (1H, m, H-5′), 3.39 (1H, dd, J ) 7.0, 6.5 Hz,
H-5), 3.12 (1H, dd, J ) 8.0, 7.0 Hz, H-9); 13C NMR (pyridine-
d5, 125 MHz) δ 173.0 (C, C-11), 167.0 (C, C-1′′), 161.5 (C, C-7′′),
152.5 (CH, C-3), 145.6 (CH, C-3′′), 140.6 (C, C-8), 133.7 (CH,
C-7), 130.8 (2CH, C-5′′, 9′′), 125.9 (C, C-4′′), 116.8 (2CH, C-6′′,
8′′), 114.7 (CH, C-2′′), 111.7 (C, C-4), 101.0 (CH, C-1′), 98.9
(CH, C-1), 82.1 (CH, C-6), 78.8 (CH, C-5′), 78.3 (CH, C-3′), 74.8
(CH, C-2′), 71.3 (CH, C-4′), 62.6 (CH2, C-6′), 62.3 (CH2, C-10),
47.2 (CH, C-9), 46.3 (CH, C-5); ESIMS (positive-ion mode) m/z
559 [M + Na]+; HRFABMS m/z 559.1460 [M + Na]+ (calcd for
C25H28O13Na, 559.1428).
Experimental Section
General Experimental Procedures. Melting points were
determined on an XT-4 micro melting point apparatus and are
uncorrected. IR spectra were recorded as KBr disks on a
Nicolet Impact 400 FT-IR spectrophotometer. 1D- and 2D-
1
NMR spectra were obtained at 500 and 125 MHz for H and
13C, respectively, on an Inova 500 FT-NMR spectrometer in
pyridine-d5 with solvent peaks as references. ESIMS were
measured on an Agilent 1100 Series LC/MSD trap mass
spectrometer. HRFABMS data were measured with a Micro-
Compound 2: amorphous powder; mp 137-139 °C; [R]23
D
-27.6 (c 0.22, MeOH); UV (MeOH) λmax (log ꢀ) 242 (3.99), 302
(3.67), 315 (4.13) nm; IR (KBr) νmax 3407, 2924, 1682, 1633,

Iridoid Glycosides from Craibiodendron henryi
Journal of Natural Products, 2005, Vol. 68, No. 11 1649
1
1604, 1516, 1205, 1142, 1076, 839 cm-1; H NMR (pyridine-
14.5 Hz, H-15R), 2.31 (1H, d, J ) 14.5 Hz, H-15â), 2.20 (1H,
d, J ) 7.5 Hz, H-9), 2.16 (1H, m, H-11R), 1.88 (3H, s, H-20),
1.76 (1H, m, H-12â), 1.64 (1H, m, H-11â), 1.64 (3H, s, H-18),
1.60 (3H, s, H-19), 1.49 (3H, s, H-17), 1.26 (3H, d, J ) 6.0 Hz,
H-4′), 1.20 (3H, d, J ) 7.0 Hz, H-5′); 13C NMR (pyridine-d5,
125 MHz) δ 175.3 (C, C-1′), 86.7 (CH, C-3), 83.0 (CH, C-14),
82.8 (C, C-5), 80.9 (CH, C-2), 78.8 (C, C-16), 78.2 (C, C-10),
71.8 (CH, C-6), 69.7 (CH, C-3′), 61.8 (CH2, C-15), 56.8 (CH,
C-1), 55.3 (CH, C-9), 54.6 (CH, C-13), 50.4 (C, C-8), 48.9 (C,
C-4), 48.8 (CH, C-2′), 44.5 (CH2, C-7), 29.4 (CH3, C-20), 27.2
(CH2, C-12), 26.8 (CH3, C-18), 24.1 (CH3, C-17), 21.8 (CH2,
C-11), 21.7 (CH3, C-4′), 20.2 (CH3, C-19), 14.2 (CH3, C-5′);
ESIMS (positive-ion mode) m/z 509 [M + Na]+; HRFABMS m/z
509.2748 [M + Na]+ (calcd for C25H42O9Na, 509.2727).
d5, 500 MHz) δ 8.03 (2H, d, J ) 8.5 Hz, H-5′′, 9′′), 7.87 (1H, s,
H-3), 7.16 (2H, d, J ) 8.5 Hz, H-6′′, 8′′), 6.92 (1H, d, J ) 13.0
Hz, H-3′′), 6.04 (1H, s, H-7), 5.93 (1H, d, J ) 13.0 Hz, H-2′′),
5.39 (1H, d, J ) 7.5 Hz, H-1′), 5.35 (1H, d, J ) 8.0 Hz, H-1),
5.23 (1H, d, J ) 14.5 Hz, H-10), 5.01 (1H, d, J ) 14.5 Hz, H-10),
4.89 (1H, br s, H-6), 4.48 (1H, dd, J ) 12.0, 2.5 Hz, H-6′), 4.31
(1H, dd, J ) 12.0, 5.5 Hz, H-6′), 4.27 (1H, t, J ) 9.0 Hz, H-3′),
4.21 (1H, t, J ) 9.0 Hz, H-4′), 4.10 (1H, dd, J ) 9.0, 7.5 Hz,
H-2′), 3.97 (1H, m, H-5′), 3.35 (1H, dd, J ) 7.0, 6.5 Hz, H-5),
3.08 (1H, dd, J ) 8.0, 7.0 Hz, H-9); 13C NMR (pyridine-d5, 125
MHz) δ 173.0 (C, C-11), 166.2 (C, C-1′′), 160.7 (C, C-7′′), 152.6
(CH, C-3), 144.7 (CH, C-3′′), 140.3 (C, C-8), 133.8 (2CH, C-5′′,
9′′), 132.9 (CH, C-7), 126.4 (C, C-4′′), 116.0 (2CH, C-6′′, 8′′),
115.6 (CH, C-2′′), 111.8 (C, C-4), 100.9 (CH, C-1′), 98.8 (CH,
C-1), 82.1 (CH, C-6), 78.9 (CH, C-5′), 78.3 (CH, C-3′), 74.9 (CH,
C-2′), 71.5 (CH, C-4′), 62.7 (CH2, C-10), 62.3 (CH2, C-6′), 47.3
(CH, C-9), 46.4 (CH, C-5); ESIMS (positive-ion mode) m/z 559
[M + Na]+; HRFABMS m/z 559.1449 [M + Na]+ (calcd for
C25H28O13Na, 559.1428).
Compound 5a: amorphous powder; mp 257-259 °C; [R]23
D
-27.5 (c 0.38, EtOH); IR (KBr) νmax 3464, 2925, 1385, 1105,
795 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.15 (1H, dd, J )
7.5, 4.5 Hz, H-2), 5.08 (1H, s, H-14), 4.73 (1H, dd, J ) 9.5, 3.5
Hz, H-6), 4.05 (1H, d, J ) 4.5 Hz, H-3), 2.97 (1H, d, J ) 7.5
Hz, H-1), 2.90 (1H, dd, J ) 14.5, 3.5 Hz, H-7R), 2.62 (1H, dd,
J ) 14.5, 9.5 Hz, H-7â), 2.62 (1H, m, H-12R), 2.53 (1H, m,
H-13), 2.35 (1H, d, J ) 14.5 Hz, H-15R), 2.31 (1H, d, J ) 14.5
Hz, H-15â), 2.23 (1H, d, J ) 7.5 Hz, H-9), 2.18 (1H, m, H-11R),
1.90 (3H, s, H-20), 1.72 (1H, m, H-12â), 1.66 (1H, m, H-11â),
1.63 (3H, s, H-18), 1.57 (3H, s, H-19), 1.53 (3H, s, H-17); 13C
NMR (pyridine-d5, 125 MHz) δ 86.9 (CH, C-3), 83.4 (C, C-5),
80.8 (CH, C-2), 80.1 (CH, C-14), 79.8 (C, C-16), 78.2 (C, C-10),
72.7 (CH, C-6), 60.8 (CH2, C-15), 58.0 (CH, C-1), 56.2 (CH, C-9),
54.9 (CH, C-13), 52.3 (C, C-8), 49.2 (C, C-4), 44.9 (CH2, C-7),
29.5 (CH3, C-20), 27.1 (CH2, C-12), 26.2 (CH3, C-18), 24.0 (CH3,
C-17), 21.9 (CH2, C-11), 20.4 (CH3, C-19); ESIMS (positive-
ion mode) m/z 409 [M + Na]+.
Compound 3: amorphous powder; mp 173-175 °C; [R]23
D
-19.3 (c 0.13, MeOH); UV (MeOH) λmax (log ꢀ) 236 (3.70), 301
(3.67), 314 (3.88) nm; IR (KBr) νmax 3410, 2929, 1682, 1633,
1604, 1516, 1203, 1076, 839 cm-1; H NMR (pyridine-d5, 500
1
MHz) δ 8.15 (1H, d, J ) 1.0 Hz, H-3), 7.90 (1H, d, J ) 16.0
Hz, H-3′′), 7.56 (2H, d, J ) 9.0 Hz, H-5′′, 9′′), 7.16 (2H, d, J )
9.0 Hz, H-6′′, 8′′), 6.60 (1H, d, J ) 16.0 Hz, H-2′′), 6.34 (1H, s,
H-7), 5.85 (1H, d, J ) 8.5 Hz, H-1), 5.60 (1H, d, J ) 15.0 Hz,
H-10), 5.47 (1H, dd, J ) 5.5, 2.0 Hz, H-6), 5.34 (1H, d, J ) 7.5
Hz, H-1′), 5.20 (1H, d, J ) 15.0 Hz, H-10), 4.36 (1H, m, H-6′),
4.31 (1H, m, H-4′), 4.30 (1H, m, H-6′), 4.25 (1H, dd, J ) 9.0,
8.5 Hz, H-3′), 4.16 (1H, dd, J ) 8.5, 7.5 Hz, H-2′), 3.82 (1H, m,
H-5′), 3.47 (1H, dd, J ) 8.0, 5.5 Hz, H-5), 2.88 (1H, dd, J )
8.5, 8.0 Hz, H-9); 13C NMR (pyridine-d5, 125 MHz) δ 169.8 (C,
C-11), 167.1 (C, C-1′′), 161.6 (C, C-7′′), 153.8 (CH, C-3), 145.6
(CH, C-3′′), 144.5 (C, C-8), 132.3 (CH, C-7), 130.8 (2CH, C-5′′,
9′′), 126.0 (C, C-4′′), 116.8 (2CH, C-6′′, 8′′), 114.8 (CH, C-2′′),
109.2 (C, C-4), 101.7 (CH, C-1), 101.6 (CH, C-1′), 78.3 (CH,
C-3′), 78.3 (CH, C-5′), 74.9 (CH, C-2′), 74.5 (CH, C-6), 71.2 (CH,
C-4′), 63.0 (CH2, C-10), 62.5 (CH2, C-6′), 46.1 (CH, C-9), 42.7
(CH, C-5); ESIMS (positive-ion mode) m/z 559 [M + Na]+;
HRFABMS m/z 559.1443 [M + Na]+ (calcd for C25H28O13Na,
559.1428).
Compound 6: colorless needles; mp 210-212 °C; [R]23
D
-23.6 (c 0.19, EtOH); UV (MeOH) λmax (log ꢀ) 213 (1.54) nm;
IR (KBr) νmax 3433, 2968, 1711, 1637, 1452, 1383, 1284, 1039,
906 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.71 (1H, s, H-14),
5.45 (1H, s, H-20R), 5.26 (1H, s, H-20â), 4.95 (1H, dd, J ) 8.0,
5.0 Hz, H-2), 4.30 (1H, dd, J ) 8.5, 3.5 Hz, H-6), 4.18 (1H, d,
J ) 5.0 Hz, H-3), 4.12 (1H, m, H-3′), 3.23 (1H, d, J ) 8.0 Hz,
H-1), 2.95 (1H, br s, H-13), 2.73 (1H, m, H-2′), 2.66 (1H, dd, J
) 13.0, 8.5 Hz, H-7R), 2.56 (1H, dd, J ) 13.0, 3.5 Hz, H-7â),
2.38 (1H, d, J ) 13.5 Hz, H-15R), 2.20 (1H, d, J ) 7.0 Hz,
H-9), 2.14 (1H, d, J ) 13.5 Hz, H-15â), 2.03 (1H, m, H-12R),
1.92 (1H, m, H-11R), 1.81 (1H, m, H-12â), 1.66 (3H, s, H-19),
1.64 (1H, m, H-11â), 1.56 (3H, s, H-18), 1.52 (3H, s, H-17),
1.38 (3H, d, J ) 7.0 Hz, H-5′), 1.26 (3H, d, J ) 6.5 Hz, H-4′);
13C NMR (pyridine-d5, 125 MHz) δ 174.7 (C, C-1′), 149.0 (C,
C-10), 113.5 (CH2, C-20), 88.2 (CH, C-3), 82.9 (CH, C-14), 82.5
(C, C-5), 82.0 (CH, C-2), 79.1 (C, C-16), 70.9 (CH, C-6), 69.1
(CH, C-3′), 62.9 (CH2, C-15), 55.8 (CH, C-13), 52.0 (CH, C-9),
51.8 (CH, C-1), 51.0 (CH, C-2′), 48.0 (C, C-8), 47.9 (C, C-4),
40.4 (CH2, C-7), 27.5 (CH3, C-17), 26.7 (CH3, C-18), 24.1 (CH2,
C-12), 23.4 (CH2, C-11), 21.1 (CH3, C-4′), 20.2 (CH3, C-19), 15.2
(CH3, C-5′); ESIMS (positive-ion mode) m/z 491 [M + Na]+;
HRFABMS m/z 491.2650 [M + Na]+ (calcd for C25H40O8Na,
491.2621).
Compound 4: amorphous powder; mp 146-148 °C; [R]23
D
-55.6 (c 0.53, MeOH); UV (MeOH) λmax (log ꢀ) 237 (4.00), 301
(3.87), 314 (4.07) nm; IR (KBr) νmax 3406, 2927, 1685, 1633,
1604, 1516, 1203, 1140, 839 cm-1; H NMR (pyridine-d5, 500
1
MHz) δ 8.13 (1H, s, H-3), 8.04 (2H, d, J ) 8.5 Hz, H-5′′, 9′′),
7.15 (2H, d, J ) 8.5 Hz, H-6′′, 8′′), 6.92 (1H, d, J ) 13.0 Hz,
H-3′′), 6.29 (1H, s, H-7), 5.94 (1H, d, J ) 13.0 Hz, H-2′′), 5.82
(1H, d, J ) 8.5 Hz, H-1), 5.45 (1H, dd, J ) 5.5, 2.0 Hz, H-6),
5.42 (1H, d, J ) 14.0 Hz, H-10), 5.31 (1H, d, J ) 7.5 Hz, H-1′),
5.16 (1H, d, J ) 14.0 Hz, H-10), 4.35 (1H, m, H-6′), 4.29 (1H,
m, H-4′), 4.26 (1H, dd, J ) 9.0, 8.5 Hz, H-3′), 4.25 (1H, m,
H-6′), 4.13 (1H, dd, J ) 8.5, 7.5 Hz, H-2′), 3.80 (1H, m, H-5′),
3.41 (1H, dd, J ) 8.0, 5.5 Hz, H-5), 2.83 (1H, dd, J ) 8.5, 8.0
Hz, H-9); 13C NMR (pyridine-d5, 125 MHz) δ 169.7 (C, C-11),
166.3 (C, C-1′′), 160.7 (C, C-7′′), 153.7 (CH, C-3), 144.6 (CH,
C-3′′), 144.2 (C, C-8), 133.8 (2CH, C-5′′, 9′′), 132.8 (CH, C-7),
126.4 (C, C-4′′), 116.0 (2CH, C-6′′, 8′′), 115.7 (CH, C-2′′), 109.3
(C, C-4), 101.5 (CH, C-1), 101.4 (CH, C-1′), 78.3 (CH, C-5′),
78.2 (CH, C-3′), 74.9 (CH, C-2′), 74.5 (CH, C-6), 71.4 (CH, C-4′),
62.9 (CH2, C-10), 62.5 (CH2, C-6′), 46.1 (CH, C-9), 42.6 (CH,
C-5); ESIMS (positive-ion mode) m/z 559 [M + Na]+; HR-
FABMS m/z 559.1402 [M + Na]+ (calcd for C25H28O13Na,
559.1428).
Compound 6a: amorphous powder; mp 225-227 °C; [R]23
D
-34.8 (c 0.39, EtOH); IR (KBr) νmax 3448, 2925, 1637, 1385,
1
1078, 804 cm-1; H NMR (pyridine-d5, 500 MHz) δ 5.67 (1H,
s, H-20R), 5.26 (1H, s, H-20â), 4.94 (1H, dd, J ) 8.0, 5.0 Hz,
H-2), 4.69 (1H, dd, J ) 8.5, 3.5 Hz, H-6), 4.66 (1H, s, H-14),
4.13 (1H, d, J ) 5.0 Hz, H-3), 3.32 (1H, d, J ) 8.0 Hz, H-1),
3.10 (1H, br s, H-13), 2.61 (1H, dd, J ) 13.0, 8.5 Hz, H-7R),
2.55 (1H, dd, J ) 13.0, 3.5 Hz, H-7â), 2.52 (1H, d, J ) 13.5
Hz, H-15R), 2.34 (1H, d, J ) 13.5 Hz, H-15â), 2.26 (1H, d, J )
7.0 Hz, H-9), 2.00 (1H, m, H-12R), 1.91 (1H, m, H-11R), 1.84
(1H, m, H-12â), 1.66 (1H, m, H-11â), 1.56 (3H, s, H-19), 1.53
(3H, s, H-18), 1.40 (3H, s, H-17); 13C NMR (pyridine-d5, 125
MHz) δ 150.9 (C, C-10), 112.4 (CH2, C-20), 88.8 (CH, C-3), 84.2
(C, C-5), 83.9 (CH, C-2), 81.3 (CH, C-14), 80.6 (C, C-16), 69.4
(CH, C-6), 61.5 (CH2, C-15), 54.0 (CH, C-13), 53.7 (CH, C-9),
52.5 (CH, C-1), 50.3 (C, C-8), 48.1 (C, C-4), 42.0 (CH2, C-7),
25.3 (CH3, C-17), 25.1 (CH3, C-18), 24.5 (CH2, C-12), 23.9 (CH2,
C-11), 19.2 (CH3, C-19); ESIMS (positive-ion mode) m/z 391
[M + Na]+.
Compound 5: amorphous powder; mp 103-105 °C; [R]23
D
-12.3 (c 0.13, EtOH); UV (MeOH) λmax (log ꢀ) 274 (0.86) nm;
IR (KBr) νmax 3390, 2964, 1724, 1458, 1377, 1265, 1038, 997,
796 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 6.31 (1H, s, H-14),
5.16 (1H, dd, J ) 7.5, 4.5 Hz, H-2), 4.42 (1H, dd, J ) 9.5, 3.5
Hz, H-6), 4.17 (1H, m, H-3′), 4.12 (1H, d, J ) 4.5 Hz, H-3),
3.02 (1H, d, J ) 7.5 Hz, H-1), 2.70 (1H, dd, J ) 14.5, 3.5 Hz,
H-7R), 2.69 (1H, m, H-12R), 2.66 (1H, dd, J ) 14.5, 9.5 Hz,
H-7â), 2.66 (1H, m, H-2′), 2.52 (1H, m, H-13), 2.37 (1H, d, J )

1650 Journal of Natural Products, 2005, Vol. 68, No. 11
Huang et al.
Alkaline Hydrolysis of 5 and 6. Compounds 5 (47 mg)
and 6 (50 mg) were dissolved in 5% KOH in EtOH-H2O (1:1),
and the solution was refluxed at 80 °C for 3 h, respectively.
Then the solution was removed, acidified, saturated with NaCl,
and extracted with EtOAc to yield 5a (26 mg) and 6a (30 mg).
The aqueous phase was acidified and extracted with EtOAc
to give nilic acid (7) (7 and 9 mg), respectively.
was replaced every 15 min. Cumulative concentrations of Phe
(1 × 10-8 to 3 × 10-7 M) were added to make the aorta
precontract. After the contracted value responded to 80% of
the peak value (1 × 10-5 M Phe value), cumulative concentra-
tions of Ach were added to the above organ bath. The
relaxations caused by Ach were recorded as the control group.
Then, the ring was washed with PSS solution and balanced
for 30 min, during which the solution was replaced every 5
min. Indomethacin (Indo 10-5 M, COX inhibitor) and Nω-L-
nitroarginine (L-NA 3 × 10-7 M, NOS inhibitor) were added
to the organ bath and thermoregulated at 37 °C for 15 min,
after which the same processes as above were performed to
obtain the L-NA + Indo group relaxations. The ring was
washed again, and then the above processes were carried out
after the isolated compound solution in DMSO (10-5 M) was
added 8 min before the L-NA and Indo to record the L-NA +
Indo + compound group relaxations.
Methylation of 7. Compound 7 (7 mg) was methylated
using CH2N2 in Et2O at 0 °C. The reaction mixture was kept
in ice for 30 min and then allowed to stand overnight at room
temperature. This procedure resulted in 8 (6 mg), which was
identified as methyl (2S,3S)-nilate.
Methyl (2S,3S)-nilate (8): colorless oil; [R]23D +34.6 (c 5.00,
CHCl3); 1H NMR (500 MHz, CDCl3) δ 3.88 (1H, m, H-3′), 3.71
(3H, s, OCH3), 2.45 (1H, m, H-2′), 1.18 (3H, d, J ) 6.0 Hz,
H-4′), 1.15 (3H, d, J ) 7.0 Hz, H-5′); 13C NMR (125 MHz,
CDCl3) δ 176.2 (C, C-1′), 69.3 (CH, C-3′), 51.6 (CH3, OCH3),
46.9 (CH, C-2′), 20.6 (CH3, C-4′), 13.9 (CH3, C-5′); ESIMS m/z
155 [M + Na]+.
Acknowledgment. Financial support was from the key
project of the National Natural Science Foundations of China
(20432030), National Key Basic R&D (973) project No.
2004CB518906. The authors are grateful to the Department
of Instrumental Analysis, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical
College for measuring the IR, UV, NMR, and MS spectra. We
also thank the Department of Pharmacology of the Institute
of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, for bioactivity tests. We also
wish to acknowledge D.-Q. Yu of the institute for constructive
advice.
Absolute Configuration of Glucose Moieties in 1-4.
Compounds 1-4 (5 mg) were each refluxed with 4% HCl (5
mL) at 95 °C for 1 h. The reaction mixture was extracted with
EtOAc. The H2O layer was neutralized with 1 N NaOH, then
concentrated in vacuo and dried to give a glucose residue.
According to the reported procedure,10 the absolute configu-
ration of glucose was confirmed by comparison of the retention
times of glucose derivatives obtained from the above residues
with those of standard samples using HPLC (tR (min): D-
glucose (40.7), L-glucose (38.5)). HPLC was performed with an
Inertsil sil-100A column (250 × 4.6 mm, 5 µm, Dikma) eluting
with n-hexane-ethanol (95:5); flow rate 1.2 mL/min; detection
at 230 nm.
References and Notes
Antioxidant Activity. The antioxidant activity of com-
pounds was determined by measuring the content of MDA, a
compound that was produced during microsomal lipid peroxi-
dation induced by ferrous-cysteine.17 MDA was detected by
using the thiobarbituric acid (TAB) method. Briefly, 1 mg/mL
microsomal protein, different concentrations of compound or
vehicle, and 0.2 mM cysteine in 0.1 M PBS were incubated
for 15 min at 37 °C. After the addition of 0.5 mM ferrous
sulfate, the mixture was incubated for 15 min at 37 °C again.
An equal volume of 20% TCA was added to terminate the
reaction, and the mixture was centrifuged for 10 min at 3000
rpm. The supernatant was reacted with 0.67% TBA for 10 min
at 100 °C. After cooling, the MDA was quantified by determin-
ing the absorbance at 532 nm and then calculated in the
inhibition rate.
Preliminary Vasodilator Assays. According to ref 18
Wistar rats (6-7 weeks old, 200-250 g) were killed by a blow
on the heads. The aortas were cut in rings 3-5 mm in length
and balanced in PSS solution. An MP100WSW system on a
PC-586 was used to record the relaxations and contractions.
The presence of functional endothelium was assessed in all
preparations by determining the ability of acetylcholine (Ach,
10-5 M) to induce more than 80% relaxation of rings precon-
tracted with phenylepherine (Phe, 10-7 M). The prepared
endothelium-intact aortic rings were balanced in an organ bath
in 15 mL of PSS solution for 30 min, during which the solution
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NP050261S