3-hydroxy-3-methylglutaryl flavonol glycosides from Oxytropis falcata

Article abstract of DOI:10.1021/np300292f

Five new 3-hydroxy-3-methylglutaryl (HMG) flavonol 3-O-glycosides, named oxytroflavosides A-E (1-5), and two new rhamnocitrin 3-O-glycosides, oxytroflavosides F and G (6 and 7) were isolated from the n-BuOH-soluble fraction of an EtOH extract of Oxytropis falcata together with seven known kaempferol glycosides (8-14), of which six were isolated from the genus Oxytropis for the first time. The structures of these compounds were elucidated by spectroscopic techniques and chemical methods. The absolute configuration of HMG in compounds 1-5 was determined to be S through spectroscopic analysis of the mevalonamide obtained by amidation and reduction of the HMG moiety. Compounds 1-10 were evaluated for anti-inflammatory activities using lipopolysaccharide-induced RAW 264.7 cells, but none of them showed inhibitory effects on NO production.

Full text of DOI:10.1021/np300292f

Article
pubs.acs.org/jnp
3-Hydroxy-3-Methylglutaryl Flavonol Glycosides from Oxytropis
falcata
Shan-Shan Wang,† Xiao-Jing Zhang,† Sheng Que,‡ Guang-Zhong Tu,§ Dan Wan,† Wei Cheng,†
Hong Liang,† Jia Ye,† and Qing-Ying Zhang*,†
†State Key Laboratory of Natural and Biomimetic Drugs and Department of Natural Medicines, School of Pharmaceutical Sciences,
Peking University Health Science Center, Beijing 100191, People’s Republic of China
‡Department of Chemistry, Minorities Teachers College, Qinghai Teachers University, Xining 810008, People’s Republic of China
§Beijing Institute of Microchemistry, Beijing 100091, People’s Republic of China
S
* Supporting Information
ABSTRACT: Five new 3-hydroxy-3-methylglutaryl (HMG) flavonol 3-O-
glycosides, named oxytroflavosides A−E (1−5), and two new rhamnocitrin
3-O-glycosides, oxytroflavosides F and G (6 and 7) were isolated from the n-
BuOH-soluble fraction of an EtOH extract of Oxytropis falcata together with
seven known kaempferol glycosides (8−14), of which six were isolated from
the genus Oxytropis for the first time. The structures of these compounds
were elucidated by spectroscopic techniques and chemical methods. The
absolute configuration of HMG in compounds 1−5 was determined to be S
through spectroscopic analysis of the mevalonamide obtained by amidation
and reduction of the HMG moiety. Compounds 1−10 were evaluated for
anti-inflammatory activities using lipopolysaccharide-induced RAW 264.7
cells, but none of them showed inhibitory effects on NO production.
Oxytropis falcata Bunge (Leguminosae), known as “King of
Herbs” in Tibetan medicines, is mainly distributed at lake and
river beaches, in ravines, on slopes, in steppe meadows, and in
shrubberies throughout the Qinghai-Tibet Plateau in China. As
a commonly used folk medicine, it is mainly used to treat
arthritis, influenza, leprosy, and constipation. Modern pharma-
cological studies showed that its total flavonoids had good anti-
inflammatory and analgesic activities in vivo,1 and 2′,4′-
dihydroxychalcone, a main constituent of this plant, exhibited
significant inhibitory activity against NO production in
lipopolysaccharide (LPS)-stimulated RAW 264.7 cells2 and
antiproliferative activity against several cancer cells in vitro.3
Chemical investigations of this plant were initiated in the past
decade,4 and flavonoids were shown to be major constituents.
Isoflavans,5 24-hydroxyoleanane-type triterpenoids,6 and N-
benzoylindole analogues7 have been reported from O. falcata.
However, few details of chemical constituents of higher polarity
have been published. In this study we report the isolation and
identification of seven new rhamnocitrin 3-O-glycosides (1−7),
five of which are 3-hydroxy-3-methylglutaryl (HMG)-moiety-
conjugated compounds (1−5), from the n-BuOH-soluble
fraction of an EtOH extract of the whole plant of O. falcata.
Seven known kaempferol glycosides (8−14) were also isolated.
Their anti-inflammatory activities against NO production in
lipopolysaccharide (LPS)-induced RAW 264.7 cells were also
described.
RESULTS AND DISCUSSION
■
Compound 1 was isolated as an amorphous, yellow powder,
and its molecular formula was determined to be C34H40O19 by
HRESIMS at m/z 753.2238 [M + H]+ (calcd 753.2236). The
IR spectrum showed typical absorption bands for OH (3408
cm−1), carbonyl (1723 cm−1), conjugated carbonyl (1656
cm−1), and benzyl groups (1597 and 1499 cm−1). The UV
spectrum revealed absorption bands at 267 and 348 nm,
suggesting a flavonol skeleton. The H and 13C NMR data
1
exhibited signals characteristic of 5,7,4′-trisubstituted flavonol
glycosides, with an OCH3 [13C: δ 56.1; 1H: δ 3.85 (s)] and two
sugar units evidenced by signals of two anomeric carbons [δ
98.6 (C-1″), 100.6 (C-1″′)] and their corresponding anomeric
protons [δ 5.58 (H-1″, d, J = 8.0 Hz), 5.06 (H-1″′, s)]. The
HMBC correlation between δ 3.85 (OCH3) and δ 165.1 (C-7)
placed the OCH3 at C-7 of the aglycone. Acid hydrolysis of 1
yielded rhamnocitrin, i.e., 5,4′-dihydroxy-7-methoxyflavonol, by
comparison of its NMR data with literature,8 and two sugars.
The sugars were identified as D-galactose and L-rhamnose by
GC analysis, in which the retention times of derivatives of the
sugar residues and standard sugars were compared, as described
in the Experimental Section. The glycosidic linkage was
established by the HMBC correlations between δ 5.58 (H-1″)
and δ 132.9 (C-3) and between δ 5.06 (H-1″′) and δ 74.8 (C-
2″), indicating that the galactose unit was located at C-3 of
Received: April 24, 2012
Published: July 9, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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a
Scheme 1. Determination of the Absolute Configuration of HMG Group in Compound 1
a
Key: (a) (S)-1-phenylethylamine, DMF, Et3N, PyBOP, HOBt; (b) LiBH4, THF; (c) Ac2O, pyridine.
aglycone and the rhamnose unit was attached to C-2 of
1
galactose. The remaining signals in the H and 13C NMR
spectra of 1 were identified as an HMG moiety by HMBC
correlations between δ 2.37 (H-2a″″), 2.21 (H-2b″″) and δ
170.1 (C-1″″), between δ 2.23 (H-4″″) and δ 172.6 (C-5″″), and
between δ 0.99 (H-6″″) and δ 68.7 (C-3″″), 45.3 (C-2″″), 45.2
(C-4″″). Characteristic HMG fragment ions appeared in the
negative ESIMS spectrum.9 The HMG substituent was placed
at C-6 of galactose by HMBC correlations between δ 3.91, 4.00
(H-6 of galactose) and δ 170.1 (C-1″″). The absolute
configuration of the chiral carbon in the HMG group was
determined as follows (Scheme 1).10 Amidation with (S)-1-
phenylethylamine afforded compound S1. Reduction of S1 with
LiBH4, followed by acetylation with Ac2O, yielded 5-O-acetyl-1-
1
[(S)-phenylethyl]mevalonamide (S2). Its H NMR data were
identical to those of (3R)-5-O-acetyl-1-[(S)-phenylethyl]-
mevalonamide rather than the (3S) isomer.11 Thus, 1 was
characterized unambiguously as rhamnocitrin-3-O-[(S)-3-hy-
galactose, which was further supported by an HMBC
correlation between δ 5.08 (H-4″) and δ 170.3 (C-1″″). The
3S-configuration of the HMG moiety was determined as
described above. Thus, the structure of 3 was identified as
rhamnocitrin-3-O-[(S)-3-hydroxy-3-methylglutaryl-(1→4)]-[α-
L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside, and it was
named oxytroflavoside C.
droxy-3-methylglutaryl-(1→6)]-[α-L-rhamnopyranosyl-(1→
2)]-β-D-galactopyranoside, and it was named oxytroflavoside A.
Compound 2 had the same molecular formula as 1,
C34H40O19, on the basis of HRESIMS. The UV, IR, 1H
NMR, and 13C NMR data were similar to those of compound
1. Acid hydrolysis also produced D-Gal, L-Rha, and rhamnoci-
trin. The 1D and 2D NMR spectra allowed the assignment of
all signals, and the only difference between the two compounds
was the location of the HMG moiety; it was placed at C-3, not
C-6, of galactose in compound 2 on the basis of an HMBC
correlation between δ 4.88 (H-3″) and δ 170.1 (C-1″″). The
absolute configuration of the HMG group was also determined
to be S using the method mentioned above. Consequently,
compound 2 was characterized as rhamnocitrin-3-O-[(S)-3-
hydroxy-3-methylglutaryl-(1→3)]-[α-L-rhamnopyranosyl-(1→
2)]-β-D-galactopyranoside, and it was named oxytroflavoside B.
Compound 3 had a molecular formula of C34H40O19, the
Compound 4 had a molecular formula of C28H30O15, as
1
deduced from HRESIMS. The H and 13C NMR data differed
from those of 1 by the absence of a set of signals corresponding
to one rhamnose, and this was consistent with the results of
acid hydrolysis, which yielded only D-Gal and rhamnocitrin.
Further analysis of the 1D and 2D NMR spectra and
determination of the absolute configuration of the HMG
group confirmed the structure of 4 to be rhamnocitrin-3-O-
[(S)-3-hydroxy-3-methylglutaryl-(1→6)]-β-D-galactopyrano-
side, and it was named oxytroflavoside D.
Compound 5 had a molecular formula of C34H40O20 by
HRESIMS. The 1H and 13C NMR data were similar to those of
4, but differed in the occurrence of only one sugar unit in 4, and
two sugar residues were observed in 5 on the basis of the
signals of two anomeric carbons [δ 101.6 (C-1″), 100.1 (C-1″′)]
and their corresponding anomeric protons [δ 5.35 (H-1″), 5.02
(H-1″′)]. Acid hydrolysis yielded D-glucose and D-galactose,
determined by preparation of trimethylsilylated derivatives and
comparison with standards in GC analysis. The β-anomeric
configuration of each sugar was assigned from the large
1
same as compounds 1 and 2, by HRESIMS. The H and 13C
NMR data were again similar to those of compound 1,
indicative of a rhamnocitrin 3-O-diglycoside with an HMG
substituent. Acid hydrolysis and coupling patterns of anomeric
protons confirmed the presence of β-D-galactose and α-L-
rhamnose, and their positons as determined by HMBC were
the same as in compound 1. A significant downfield shift of H-4
of galactose (δ 5.08) compared with that of compound 1 (δ
3.62−3.65) implied that the HMG substituent was at C-4 of
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coupling constant (J = 7.5 Hz) of each anomeric proton. In
addition, a blue shift of 25 nm for absorption band I in the UV
spectrum compared with that of 4 implied the glycosylation of
the C-4′ OH group,12 which was supported by an HMBC
correlation between δ 5.02 (glc H-1) and δ 159.3 (C-4′).
Consequently, compound 5 was characterized as rhamnocitrin-
3-O-[(S)-3-hydroxy-3-methylglutaryl-(1→6)]-β-D-galactopyra-
nosyl-4′-O-β-D-glucopyranoside, and it was named oxytroflavo-
side E.
than that of 2 and 3, probably due to the lesser steric hindrance
of C-6 compared with that of C-3 and C-4 of galactose. By
detailed comparison of the 13C NMR data of compounds 1−3
with that of 6, we can conclude that HMG esterification of the
OH group generally leads to a downfield shift of 2−3 ppm for
the carbon that HMG directly links to, as well as an upfield shift
of 2−3 ppm for the adjacent carbons.
The determination of the absolute configuration of the HMG
group was a key step in the process of structural elucidation.
Generally, the methods designed to address this problem were
all composed of three steps: reduction, amidation with (S)-1-
phenylethylamine, and acylation with Ac2O. The original
method was to reduce the HMG ester with borane in the
first step, unfortunately giving an unreasonable result of 3R-
configuration for the HMG group.11c This was contradictory to
the biosynthetic mevalonic acid pathway, in which naturally
occurring HMG esters are formed via acylation of an OH group
by (3S)-HMG-CoA. The unreasonable results were later
revised to be 3S-configuration by replacing borane with
LiBH4 or LiEt3BH.11a,b Although the latter method was reliable,
its yield was low (10−30%) and, thus, was not applicable to
natural compounds with only small amounts available.
Recently, a refined method succeeded in enhancing the
reaction yield to 70% simply by reversing the steps of reduction
and amidation.10 Considering the limited amounts of some
compounds, the refined method was adopted in this study, and
the S-configuration of the HMG groups in compounds 1−5
was determined unambiguously.
Compound 6, C28H32O15, had 1H and 13C NMR data similar
to those of 1, except that there were no signals for an HMG
moiety. Analysis of the 2D NMR spectra combined with acid
hydrolysis revealed compound 6 to be rhamnocitrin-3-O-α-L-
rhamnopyranosyl-(1→2)-β-D-galactopyranoside, and it was
named oxytroflavoside F.
1
Compound 7, C34H42O19, had H and 13C NMR data that
resembled those of 6, except for the presence of an additional
α-L-rhamnose unit, as was confirmed by the acid hydrolysis
experiment. Furthermore, compared with the 13C NMR data of
6, a downfield shift of 5.1 ppm for C-6″ as well as an upfield
shift of 2.2 ppm for C-5″ indicated glycosylation of the C-6 OH
of galactose. An HMBC correlation between δ 4.35 (H-1″″) and
δ 65.2 (C-6″) confirmed the attachment. Thus, compound 7
was characterized as rhamnocitrin-3-O-α-L-rhamnopyranosyl-
(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside,
and it was named oxytroflavoside G.
The known compounds were identified as robinin (8),13
kaempferol-3-O-robinobioside (9),14 mauritianin (10),15 as-
trasikokioside I (11),13 kaempferol-3-O-α-L-rhamnopyranosyl-
(1→2)-β-D-galactopyranosyl-7-O-α-L-rhamnopyranoside
(12),13 kaempferol-3-O-[β-D-xylopyranosyl-(1→3)-α-L-rham-
nopyranosyl-(1→6)]-β-D-galactopyranoside (13),16 and
kaempferol-3-O-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyrano-
syl-(1→6)]-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyrano-
side (14)17 by comparison of the spectral data with those
previously reported in the literature. Compounds 9−14 were
isolated from the genus Oxytropis for the first time, and
compound 8 was obtained from this plant for the first time.
The anti-inflammatory effects of compounds 1−10 were
evaluated for the ability to inhibit NO production in LPS-
induced RAW 264.7 cells,18 but none of the compounds were
active, with inhibition rates of lower than 5% at 50 μM. As a
positive control, 2′,4′-dihydroxychalcone isolated from this
plant inhibited NO production by 40.1% at a concentration of
50 μM, which agreed with the result in a previous study2 of its
inhibitory effect on NO production. It was reported that 2′,4′-
dihydroxychalcone is one of the main constituents of this plant,
accounting for 7.28 0.13 mg/g in O. falcata2 and 6.1% of its
total flavonoids.1 Thus, this compound could explain, in part,
the in vivo anti-inflammatory activities of total flavonoids of O.
falcata reported previously.1 However, whether compounds 1−
10 contribute to the anti-inflammatory activities of O. falcata
will require further investigation using in vivo and other in vitro
models.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a Rudolph Research Analytical Autopol III automatic
polarimeter. UV spectra were measured on a Shimadzu UV-260
spectrometer. IR spectra were recorded on a Nicolet Nexus 470 FT-IR
spectrophotometer using KBr pellets. NMR spectra were recorded on
a Bruker AVANCE III-400 or a Varian INOVA-500 instrument.
Chemical shift values are given in δ (ppm) using the peak signals of
solvent DMSO-d6 (δH 2.50 and δC 39.5) as references, and coupling
constants are reported in Hz. HRMS data were recorded on a Agilent
QTOF 6538 or a Bruker APEX II FT-ICRMS spectrometer. ESIMS
data were obtained using an Abseciex QSTAR spectrometer. EIMS
data were measured on a Finnigan TRACE instrument. GC analysis
was performed on an Agilent 6890N GC system (Agilent Co., USA).
Column chromatography (CC) was performed on Diaion HP20
(200−300 mesh, Mitsubishi Chemical Co., Japan), MCI gel (20−45
μm, Fuji Silysia Chemical Co., Japan), ODS-A (50 μm, YMC Co. Ltd.,
Japan), Amberlite MB-3 (Organo, Tokyo, Japan), and Sephadex LH-
20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Analytical and
preparative TLC were carried out on precoated silica gel 60 F254
plates (Merck, Germany), and spots were visualized by 10% H2SO4/
EtOH reagent. All solvents used for extraction and isolation were of
analytical grade and purchased from Beijing Fine Chemicals (China)
without further purification. All chemicals, unless otherwise noted,
were purchased from J & K Co. Ltd. (China).
Plant Material. The whole plant of O. falcata was collected from
Guide County, Qinghai Province, in August 2008, and was
authenticated by Associate Professor Ying-Tao Zhang, Department
of Natural Medicines, School of Pharmaceutical Sciences, Peking
University Health Science Center. A voucher specimen (No.
20080801) was deposited in the Herbarium of Department of Natural
Medicines, School of Pharmaceutical Sciences, Peking University
Health Science Center.
Extraction and Isolation. The dried whole plant of O. falcata (4.0
kg) was pulverized and in turn percolated exhaustively with 95% EtOH
and 50% EtOH. After concentration, the 50% EtOH extract (401 g)
was successively partitioned with petroleum ether, CHCl3, EtOAc, and
The HMG group has been occasionally found conjugated in
natural products such as flavonoids,19 triterpenoids,20 and
steroids,21 with the majority of them being flavonoid glycosides.
An extensive literature survey showed that it tends to connect
with C-6 of glucose or galactose. This study is the first report of
the simultaneous isolation of three flavonol 3-O-glycoside
isomers (1−3) with the HMG moiety located respectively at C-
6, C-3, and C-4 of galactose. During the process of isolation we
found that the amount of compound 1 was significantly greater
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a
1
Table 1. H NMR Spectroscopic Data of Compounds 1−7 in DMSO-d6
position
1
2
3
4
5
6
7
6
6.35, d (2.0)
6.71, d (2.0)
8.07, d (8.8)
6.86, d (8.8)
3.85, s
6.39, d (2.0)
6.75, d (2.0)
8.12, d (9.0)
6.88, d (9.0)
3.87, s
6.36, d (2.0)
6.74 br, s
6.35, d (1.5)
6.72, d (1.5)
8.07, d (8.5)
6.87, d (8.5)
3.85, s
6.37 br, s
6.37, d (2.0)
6.74, d (2.0)
8.12, d (9.0)
6.87, d (9.0)
3.86, s
6.36, d (2.0)
6.72, d (2.0)
8.09, d (9.0)
6.85, d (9.0)
3.86, s
8
6.76 br, s
2′/6′
3′/5′
OMe-7
1″
8.08, d (9.0)
7.00, d (9.0)
3.86, s
8.15, d (9.0)
7.13, d (9.0)
3.86, s
5.58, d (8.0)
5.78, d (7.5)
4.00, dd (10.0, 7.5)
4.88, dd (10.0, 3.0)
3.89, d (3.0)
3.51, t (6.5)
5.67, d (7.5)
3.76, dd (9.5, 7.5)
3.81, m
5.31, d (7.5)
3.56, t (8.5)
3.39, dd (10.0, 3.0)
3.63, d (3.0)
3.60, t (6.0)
5.35, d (7.5)
3.53, t (9.0)
5.66, d (8.0)
3.80, dd (9.5, 8.0)
3.59 br, d (9.0)
3.65 br, s
5.57, d (8.0)
3.80, t (9.0)
b
2″
3.74−3.80
3.62−3.65
3.62−3.65
3.62−3.65
b
b
b
3″
3.38−3.40
3.64, d (2.5)
3.56−3.62
3.56−3.62
3.56−3.62
3.56−3.62
b
b
4″
5.08, d (3.0)
3.63, t (6.0)
3.15, m
b
b
5″
3.61, t (6.5)
3.36, t (5.5)
b
b
6″
4.00, dd (11.2, 8.0)
3.91, dd (11.2, 4.0)
3.39−3.43
3.98, dd (11.0, 7.0)
3.93, dd (11.0, 4.0)
3.97, dd (11.5, 7.5)
3.90, dd (11.5, 5.0)
3.43, dd (10.5, 6.0)
3.28, dd (10.5, 5.5)
3.27, dd (10.5, 5.0)
4.80, s
3.23, dd (13.0, 9.5)
5.06, s
1″′
2″′
3″′
4″′
5″′
6″′
5.06, s
4.98, s
5.02, d (7.5)
3.26, t (9.0)
5.06, s
b
3.74−3.80
3.59, br, s
3.70 br, s
3.74 br, s
3.74 br, s
b
b
3.48, dd (9.2, 2.8)
3.39−3.43
3.44, dd (9.5, 3.5)
3.11, t (9.5)
3.73, dq (9.5, 6.5)
0.75, d (6.5)
3.38−3.40
3.18, t (9.0)
3.47, dd (9.5, 3.0)
3.12, t (9.5)
3.73, dq (9.5, 6.0)
0.75, d (6.0)
3.50, dd (9.0, 3.0)
3.14, t (9.0)
3.14, t (9.2)
3.12 br, t (9.0)
3.72, dq (9.5, 6.0)
0.76, d (6.0)
b
3.74−3.80
3.31, m
3.78, dq (9.0, 6.0)
0.80, d (6.0)
0.79, d (6.0)
3.69, dd (12.0, 1.5)
3.48, dd (12.0, 5.5)
1″′′
2″″
4.35, s
b
2.37, d (14.0)
2.21, d (14.0)
2.77, d (14.5)
2.62, d (14.5)
2.64, d (13.5)
2.41, d (13.5)
2.29, d (14.0)
2.18, d (14.0)
2.22, d (13.0)
2.11, d (13.0)
3.34−3.35
3″′′
4″′′
3.28, dd (9.0, 3.0)
3.08, t (9.0)
2.23, s
2.55, s
2.16, d (14.0)
2.08, d (14.0)
2.14, d (15.0)
2.02, d (15.0)
2.04, d (15.0)
1.88, d (15.0)
b
5″′′
6″′′
3.34−3.35
0.99, s
1.34, s
1.21, s
0.97, s
0.92, s
1.05, d (6.0)
a
b
Measured at 400 MHz for 1 and 500 MHz for 2−7. Overlapping signals.
n-BuOH. The n-BuOH extract (136.0 g) was subjected to Dianion
HP20 macroporous absorbent resin CC using an EtOH/H2O gradient
(0, 20, 30, 50, 70, and 95% EtOH) to yield six fractions (I−VI).
Fraction III (eluted with 30% EtOH, 8.9 g) was subjected to MCI gel
CC with a gradient system of MeOH/H2O (0, 30, 50, 70, 100%
MeOH) to afford five subfractions (Da−De). Further purification of
Dc (2.3 g) by CC on Sephadex LH-20 (MeOH/H2O, 1:1), on ODS-A
(MeOH/H2O, 1:4), and again on Sephadex LH-20 (MeOH/H2O,
1:2) afforded compounds 5 (9 mg), 8 (13 mg), 10 (25 mg), 11 (22
mg), 12 (13 mg), 13 (16 mg), and 14 (27 mg). Fraction Dd (1.7 g)
was subjected to repeated Sephadex LH-20 CC eluted with MeOH/
H2O (1:2) to give compound 9 (18 mg). Fraction IV (eluted with 50%
EtOH, 19.4 g) was subjected to MCI gel CC using the same elution
system as fraction III to give a subfraction (eluted with 50% MeOH,
10.5 g), which was further separated on silica gel CC (EtOAc/EtOH/
H2O, 40:2:1−8:2:1) to yield five subfractions (Ga−Ge). Further
purification of Ga (0.7 g), Gb (3.2 g), and Gd (2.1 g) through repeated
Sephadex LH-20 CC (MeOH/H2O, 1:1) and preparative TLC (plate:
20 × 20 cm, developing solvents: EtOAc/EtOH/H2O/HCOOH,
16:2:1:1) afforded six compounds: 4 (16 mg) from Ga; 1 (320 mg), 3
(8 mg), and 2 (30 mg) from Gb; 7 (25 mg) and 6 (50 mg) from Gd.
Oxytroflavoside A (1): amorphous, yellow powder; [α]26D −78.3 (c
0.18, MeOH); UV (MeOH) λmax 267, 348 nm; IR (KBr) νmax 3408,
2930, 1723, 1656, 1597, 1499, 1447, 1349, 1282, 1213, 1168, 1136,
Tables 1 and 2; HRESIMS m/z 753.2235 [M + H]+ (calcd for
C34H41O19, 753.2236).
Oxytroflavoside D (4): amorphous, yellow powder; [α]26D −17.1 (c
0.14, MeOH); UV (MeOH) λmax 268, 348 nm; IR (KBr) νmax 3271,
2926, 1732, 1657, 1594, 1498, 1449, 1349, 1286, 1213, 1169, 1087,
1
1020 cm−1; H NMR and 13C NMR (DMSO-d6) data, see Tables 1
and 2; HRESIMS m/z 607.1668 [M + H]+ (calcd for C28H31O15,
607.1657).
Oxytroflavoside E (5): amorphous, yellow powder; [α]26D −76.5 (c
0.17, MeOH); UV (MeOH) λmax 268, 323 nm; IR (KBr) νmax 3367,
2927, 1726, 1657, 1598, 1501, 1452, 1352, 1303, 1247, 1214, 1168,
1
1074 cm−1; H NMR and 13C NMR (DMSO-d6) data, see Tables 1
and 2; HRESIMS m/z 769.2187 [M + H]+ (calcd for C34H41O20,
769.2185).
Oxytroflavoside F (6): amorphous, yellow powder; [α]26D −84.2 (c
0.24, MeOH); UV (MeOH) λmax 267, 348 nm; IR (KBr) νmax 3394,
2930, 1657, 1596, 1499, 1447, 1348, 1283, 1213, 1167, 1139, 1085
1
cm−1; H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2;
HRSIMS m/z 609.1811 [M + H]+ (calcd for C28H33O15, 609.1814).
Oxytroflavoside G (7): amorphous, yellow powder; [α]26D −86.2 (c
0.13, MeOH); UV (MeOH) λmax 267, 348 nm; IR (KBr) νmax 3392,
2927, 1657, 1596, 1499, 1448, 1349, 1283, 1214, 1168, 1137, 1052
1
cm−1; H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2;
HRSIMS m/z 755.2400 [M + H]+ (calcd for C34H43O19, 755.2393).
Acid Hydrolysis of Compounds 1−7 (ref 22). Compounds 1−7
(each 1.0 mg) were hydrolyzed with 1 N HCl (1.0 mL) for 6 h at 100
°C. After cooling, the yellow precipitate was obtained by filtration and
proved to be rhamnocitrin by NMR.
1
1087 cm−1; H NMR and 13C NMR (DMSO-d6) data, see Tables 1
and 2; HRESIMS m/z 753.2238 [M + H]+ (calcd for C34H41O19,
753.2236).
Oxytroflavoside B (2): amorphous, yellow powder; [α]26D −68.8 (c
0.17, MeOH); UV (MeOH) λmax 267, 348 nm; IR (KBr) νmax 3385,
2930, 1657, 1596, 1499, 1447, 1348, 1283, 1213, 1169, 1139, 1085
1
Rhamnocitrin: yellow solid; H NMR (DMSO-d6, 400 MHz) δ
12.5 (1H, s, OH-5), 10.2 (1H, s, OH-4′), 9.50 (1H, s, OH-3), 8.09
(2H, d, J = 8.8 Hz, H-2′, H-6′), 6.94 (2H, d, J = 8.8 Hz, H-3′, H-5′),
6.75 (1H, d, J = 2.0 Hz, H-8), 6.36 (1H, d, J = 2.0 Hz, H-6), 3.87 (3H,
s, OCH3); 13C NMR (DMSO-d6, 100 MHz) δ 176.0 (C, C-4), 164.9
(C, C-7), 160.4 (C, C-5), 159.3 (C, C-4′), 156.1 (C, C-9), 147.2 (C,
C-2), 135.9 (C, C-3), 129.5 (CH, C-2′, C-6′), 121.5 (C, C-1′), 115.4
(CH, C-3′, C-5′), 104.0 (C, C-10), 97.4 (CH, C-6), 92.0 (CH, C-8),
56.0 (CH3, OCH3).
1
cm−1; H NMR and 13C NMR (DMSO-d6) data, see Tables 1 and 2;
HRESIMS m/z 753.2238 [M + H]+ (calcd for C34H41O19, 753.2236).
Oxytroflavoside C (3): amorphous, yellow powder; [α]26D −80.0 (c
0.20, MeOH); UV (MeOH) λmax 267, 348 nm; IR (KBr) νmax 3367,
2936, 1731, 1658, 1595, 1498, 1448, 1349, 1285, 1214, 1169, 1136,
1058, 1025 cm−1; 1H NMR and 13C NMR (DMSO-d6) data, see
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Journal of Natural Products
Table 2. 13C NMR Spectroscopic Data of Compounds 1−7 in DMSO-d6
Article
a
position
1
2
3
4
5
6
7
2
156.7
132.9
177.4
160.9
97.9
156.5
132.7
177.3
160.9
97.9
157.7
132.2
177.5
160.9
97.9
156.8
133.4
177.6
160.9
97.9
156.4
134.0
177.6
160.8
98.0
156.4
133.0
177.5
160.9
97.9
156.9
3
132.8
177.4
160.9
97.9
4
5
6
7
165.1
92.3
165.1
92.3
165.0
92.3
165.1
92.3
165.2
92.4
165.0
92.2
165.0
92.3
8
9
156.3
105.0
120.7
130.9
115.1
160.0
56.1
156.2
104.9
120.6
130.9
115.1
160.1
56.1
156.3
104.9
119.5
130.5
115.6
161.5
56.1
156.3
104.9
120.4
130.9
115.2
160.5
56.1
156.1
105.0
123.5
130.6
115.8
159.3
56.1
156.2
104.9
120.7
130.9
115.1
160.1
56.1
156.3
104.9
120.3
130.9
115.3
160.9
56.1
10
1′
2′/6′
3′/5′
4′
OMe-7
1″
98.6
98.5
98.3
101.7
71.0
101.6
71.0
98.7
99.0
2″
74.8
73.2
76.2
75.1
74.8
3″
73.6
76.1
71.8
72.8
73.2
74.0
73.9
4″
68.7
65.2
70.5
68.2
67.9
68.5
68.6
5″
72.6
75.0
73.7
72.8
72.7
75.6
73.4
6″
63.0
59.5
59.9
63.0
62.6
60.1
65.2
1″′
2″′
3″′
4″′
5″′
6″′
1″″
2″″
3″″
4″″
5″″
6″″
100.6
70.64
70.56
71.9
101.0
70.7
100.7
70.59
70.63
71.8
100.1
73.2
100.5
70.64
70.57
71.8
100.6
70.7
70.4
77.1
70.6
71.6
69.6
71.9
68.2
68.6
68.2
76.5
68.2
68.2
17.3
17.1
17.1
60.6
17.2
17.3
170.1
45.3
170.1
45.6
170.3
45.8
170.3
45.9
68.7
46.2
174.5
27.4
170.3
46.2
100.1
70.4
68.7
69.0
69.2
68.6
70.6
45.2
45.5
49.0
46.4
71.9
172.6
27.1
172.5
27.2
175.8
27.4
174.4
27.6
68.3
18.0
a
Measured at 100 MHz for 1 and 125 MHz for 2−7.
The combined filtrate was neutralized by passing through an
Rhamnocitrin-3-O-{(3S)-hydroxy-3-methyl-5-[(S)-
phenylethylamino]glutaryl-(1→6)}-[α-L-rhamnopyranosyl-(1→2)]-
Amberlite MB-3 ion-exchange resin column. The H2O eluent was
reduced to dryness and then analyzed by TLC over silica gel (EtOAc/
EtOH/H2O/HCOOH, 6:4:1:1) by comparison with authentic sugars.
The sugar residue and 1.0 mg of L-cysteine methyl ester hydrochloride
(TCI Co. Ltd.) were dissolved in 0.1 mL of anhydrous pyridine, and
the resulting mixture was stirred at 60 °C for 2 h. After drying under
vacuum, the reaction mixture was trimethylsilylated with 0.3 mL of
HMDS/TMCS/pyridine (3:1:9, Sigma-Aldrich Co. Ltd.) at 60 °C for
1.5 h. The mixture was then concentrated and partitioned between n-
hexane and H2O. The hexane extract was analyzed by GC under the
following conditions: instrument, Agilent 6890N GC; column, HP-5
(30 m × 0.32 mm × 0.25 μm); detector, FID; column temperature,
230 °C; detector temperature, 280 °C; injector tempetature, 260 °C;
carrier gas, He (1.0 mL/min). The tR values (min) of standard D-Glc,
L-Glc, L-Rha, and D-Gal derivatives prepared in a similar way were
12.51, 13.49, 8.95, and 13.63 respectively. D-Gal and L-Rha were
detected from compounds 1−4, 6, and 7, and D-Glc and D-Gal were
identified from compound 5.
Determination of the Absolute Configuration of Com-
pounds 1−5 (ref 10). (S)-1-Phenylethylamine (19 μL, 150 μmol),
Et3N (32 μL, 225 μmol), PyBOP (58.5 mg, 115 μmol), and HOBt
(20.0 mg, 150 μmol) were added to a solution of compound 1 (56.5
mg, 75 μmol) in 0.5 mL of DMF under ice-cooling, and the mixture
was stirred at room temperature for 9 h. The reaction was quenched
with dilute aqueous HCl and then dried under vacuum to afford a
yellow residue, which was purified by Sephadex LH-20 CC eluting
with 50% MeOH to give amide S1 (41.0 mg).
1
β-D-galactopyranoside (S1): amorphous, yellow powder; H NMR
(CD3OD, 400 MHz) δ 8.10 (2H, d, J = 8.8 Hz, H-2′, H-6′), 7.19−7.31
(5H, m, C6H5-1″″′), 6.90 (2H, d, J = 8.8 Hz, H-3′, H-5′), 6.54 (1H, d, J
= 2.0 Hz, H-8), 6.25 (1H, d, J = 2.0 Hz, H-6), 5.71 (1H, d, J = 7.6 Hz,
H-1″), 5.21 (1H, s, H-1″′), 4.93 (1H, m, H-1″″′), 4.19 (1H, dd, J = 11.6,
7.6 Hz, H-6a″), 4.13 (1H, dd, J = 11.6, 4.4 Hz, H-6b″), 3.84 (3H, s,
OCH3), 2.26−2.42 (4H, m, H-2″′′, H-4″′′), 1.39 (3H, d, J = 6.8 Hz, H-
2″″′), 1.08 (3H, s, H-6″″), 0.95 (3H, d, J = 6.0 Hz, H-6″′); ESIMS m/z
856.227 [M + H]+, 854.311 [M − H]−.
LiBH4 (14.0 mg, 643 μmol) was added to a solution of S1 (37.0 mg,
43 μmol) in THF (0.5 mL) under ice-cooling. After stirring for 24 h at
room temperature, the reaction was quenched with dilute aqueous
HCl, and the resulting material was extracted with EtOAc. The
product was then purified by preparative TLC (EtOAc/HCOOH,
20:1) to give a colorless oil, which was acetylated with Ac2O (25 μL,
227 μmol) in pyridine (100 μL). The mixture was stirred for 24 h at
room temperature. The reaction mixture was then diluted with water,
extracted with EtOAc, and concentrated to afford S2 (7.5 mg) as a
colorless oil. Its 1H NMR spectrum was consistent with that of (3R)-5-
O-acetyl-1-[(S)-phenylethyl]mevalonamide, rather than the (3S)
isomer reported in the literature.
(3R)-5-O-Acetyl-1-[(S)-phenylethyl]mevalonamide (S2): colorless
oil; 1H NMR (CDCl3, 400 MHz) δ 7.27−7.37 (5H, m, C6H5-1′), 6.11
(1H, d, J = 7.2 Hz, NH), 5.14 (1H, quintet, J = 7.2 Hz, H-1′), 4.23
(2H, t, J = 6.8 Hz, H-5), 2.41, 2.28 (each 1H, d, J = 14.4 Hz, H-2),
2.04, (3H, s, CH3COO), 1.85 (2H, m, H-4), 1.50 (3H, d, J = 6.8 Hz,
H-2′), 1.23 (3H, s, H-6); EIMS m/z (%) 293 (M+, 2).
1363
dx.doi.org/10.1021/np300292f | J. Nat. Prod. 2012, 75, 1359−1364

Journal of Natural Products
Article
The absolute configuration of the HMG moiety in compounds 2−5
(each 6.0 mg) was determined using the same method, and all the final
products were proved to be (3R)-5-O-acetyl-1-[(S)-phenylethyl]-
(10) Hattori, Y.; Horikawa, K.-H.; Makabe, H.; Hirai, N.; Hirota, M.;
Kamo, T. Tetrahedron: Asymmetry 2007, 18, 1183−1186.
(11) (a) Kamo, T.; Hirai, N.; Matsumoto, C.; Ohigashi, H.; Hirota,
M. Phytochemistry 2004, 65, 2517−2520. (b) Fujimoto, H.; Nakamura,
E.; Kim, Y. P.; Okuyama, E.; Ishibashi, M.; Sassa, T. J. Nat. Prod. 2001,
64, 1234−1237. (c) Hirai, N.; Koshimizu, K. Phytochemistry 1981, 20,
1867−1869.
(12) (a) Marco, J. A.; Barbera, O.; Sanz, J. F.; Sanchez-Parareda, J. J.
Nat. Prod. 1986, 49, 151−152. (b) Marco, J. A.; Barbera, O.; Sanz, J.
F.; Sanchez-Parareda, J. Phytochemistry 1985, 24, 2471−2472.
(13) Yahara, S.; Kohjyouma, M.; Kohoda, H. Phytochemistry 2000,
53, 469−471.
(14) Brasseur, T.; Angenot, L. Phytochemistry 1986, 25, 563−564.
(15) Yasukawa, K.; Takido, M. Phytochemistry 1987, 26, 1224−1226.
(16) Semmar, N.; Fenet, B.; Gluchoff-Fiasson, K.; Hasan, A.; Jay, M.
J. Nat. Prod. 2002, 65, 576−579.
(17) Semmar, N.; Fenet, B.; Gluchoff-Fiasson, K.; Comte, G.; Jay, M.
Chem. Pharm. Bull. 2002, 50, 981−984.
(18) Kwon, O. K.; Lee, M. Y.; Yuk, J. E.; Oh, S. R.; Chin, Y. W.; Lee,
H. K.; Ahn, K. S. J. Ethnopharmacol. 2010, 130, 28−34.
(19) (a) Song, S.; Zheng, X. P.; Liu, W. D.; Du, R. F.; Bi, L. F.;
Zhang, P. C. Chem. Pharm. Bull. 2010, 58, 1587−1590. (b) Nakagawa,
H.; Takaishi, Y.; Tanaka, N.; Tsuchiya, K.; Shibata, H.; Higuti, T. J.
Nat. Prod. 2006, 69, 1177−1179. (c) Fu, X.; Li, X. C.; Wang, Y. H.;
Avula, B.; Smillie, T. J.; Mabusela, W.; Syce, J.; Johnson, Q.; Folk, W.;
Khan, I. A. Planta Med. 2010, 76, 178−181. (d) Donna, L. D.; Luca, G.
D.; Mazzotti, F.; Napoli, A.; Salerno, R.; Taverna, D.; Sindona, G. J.
Nat. Prod. 2009, 72, 1352−1354.
(20) (a) Kamo, T.; Asanoma, M.; Shibata, H.; Hirota, M. J. Nat. Prod.
2003, 66, 1104−1106. (b) Fang, R.; Veitch, N. C.; Kite, G. C.; Howes,
M.-J. R.; Porter, E. A.; Simmonds, M. S. J. Chem. Pharm. Bull. 2011, 59,
124−128.
(21) Kawashima, K.; Mimaki, Y.; Sashida, Y. Phytochemistry 1993, 32,
1267−1272.
1
mevalonamide by comparison with H NMR data in the literature.
Anti-inflammatory Effects on LPS-Induced RAW 264.7 Cells
(ref 18). The murine macrophage cell line, RAW 264.7 (ATCC,
USA), was incubated in Dulbecco’s modified Eagle’s medium
containing 0.1% sodium bicarbonate, 2 mmol/L L-glutamine, 100 U/
mL penicillin G, 100 μg/mL streptomycin, and 10% fetal bovine
serum and maintained at 37 °C in a humidified incubator containing
5% CO2. After preincubation for 8 h, RAW 264.7 cells (5 × 105/mL)
were treated with test compounds (1−10) and 2′,4′-dihydroxychalcone
(positive control) of 10 and 50 μM in a 96-well microplate (100 μL/
well) for 4 h, followed by addition of LPS with a final concentration of
1 μg/mL. After stimulation with LPS for 24 h, NO levels in cell culture
were measured using the Griess reaction described as follows: 50 μL of
culture supernatant was mixed with an equal volume of Griess reagent
(1% sulfanilamide and 0.1% N-[1-naphthyl]ethylenediamine dihydro-
chloride in 5% phosphoric acid). The absorbance was measured at 570
nm in a microplate reader, and the nitrite concentration was
determined using sodium nitrite as a standard.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
1
The H and 13C NMR, H−1H COSY, HSQC, and HMBC
spectra of compounds 1−3 and H and 13C NMR spectra of
1
compounds 4−7 are available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +86-10-82801725. Fax: +86-10-82801725. E-mail:
qyzhang@hsc.pku.edu.cn.
(22) (a) Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1986,
34, 1843−1845. (b) Tao, H. W.; Hao, X. J.; Liu, J. G.; Ding, J.; Fang,
Y. C.; Gu, Q. Q.; Zhu, W. M. J. Nat. Prod. 2008, 71, 1998−2003.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We sincerely thank Drs. Q. Li and X.-L. Sun at Peking
University Health Science Center for measuring the NMR data.
This project was financially supported by National Natural
Science Foundation of China (200872006, 21172008) and the
National Science and Technology Major Project “Key New
Drug Creation and Manufacturing Program”.
REFERENCES
■
(1) Chen, Z. P.; Qu, M. M.; Chen, H. X.; Liu, D.; Xiao, Y. Y.; Chen,
J.; Lu, T. L.; Cai, B. C. Fitoterapia 2011, 82, 426−433.
(2) Wang, D.; Tang, W.; Yang, G. M.; Cai, B. C. Chin. J. Nat. Med.
2010, 8, 461−465.
(3) Lou, C. H.; Yang, G. M.; Cai, H.; Zou, M. C.; Xu, Z. S.; Li, Y.;
Zhao, F. M.; Li, W. D.; Tong, L.; Wang, M. Y.; Cai, B. C. Toxicol. In
Vitro 2010, 24, 1333−1337.
(4) (a) Lv, F.; Xu, X. J. Zhongyaocai 2006, 29, 1303−1304. (b) Lv, F.;
Xu, X. J. Zhongguo zhongyao Zazhi 2007, 32, 318−320. (c) Que, S.;
Zhang, Y. S.; Zhao, Y. Y. Zhongyaocao 2007, 38, 1458−1460.
(5) Chen, W. H.; Wang, R.; Shi, Y. P. J. Nat. Prod. 2010, 73, 1398−
1403.
(6) Chen, W. H.; Qi, H. Y.; Shi, Y. P. J. Nat. Prod. 2009, 72, 1410−
1413.
(7) Chen, W. H.; Wu, Q. X.; Wang, R.; Shi, Y. P. Phytochemistry
2010, 71, 1002−1006.
(8) Barbera, O.; Sanz, J. F.; Sanchez-Parareda, J.; Marco, J. A.
Phytochemistry 1986, 25, 2361−2365.
(9) Sari, E.; Heikki, K.; Sampo, M.; Ari, T. J. Agric. Food Chem. 2006,
54, 9834−9842.
1364
dx.doi.org/10.1021/np300292f | J. Nat. Prod. 2012, 75, 1359−1364