Constituents of Vigna angularis and their in vitro anti-inflammatory activity

Article abstract of DOI:10.1016/j.phytochem.2014.08.011

Nine non-phenolic compounds, including four furanylmethyl glycosides, angularides A-D, one ent-kaurane diterpene glycoside, angularin A, and four triterpenoid saponins, angulasaponins A-D, were isolated from seeds of Vigna angularis, together with eight known compounds. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic analysis as well as chemical methods. Angularin A, angulasaponins A-C, and azukisaponins III and VI showed inhibition of nitric oxide production in LPS-activated RAW264.7 macrophages, with IC₅₀ values ranging from 13 μM to 24 μM.

Full text of DOI:10.1016/j.phytochem.2014.08.011

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Constituents of Vigna angularis and their in vitro anti-inflammatory
activity
a
b
c
Yong Jiang a,b, , Ke-Wu Zeng , Bruno David , Georges Massiot
⇑
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
b Pôle Actifs Végétaux, Institut de Recherche Pierre Fabre, 3 avenue Hubert Curien, 31035 Toulouse Cedex 1, France
c USR CNRS-Pierre Fabre No. 3388 ETaC, Centre de Recherche et Développement Pierre Fabre, 3 avenue Hubert Curien, 31035 Toulouse Cedex 01, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Nine non-phenolic compounds, including four furanylmethyl glycosides, angularides A–D, one ent-kaura-
ne diterpene glycoside, angularin A, and four triterpenoid saponins, angulasaponins A–D, were isolated
from seeds of Vigna angularis, together with eight known compounds. Their structures were elucidated
on the basis of extensive 1D and 2D NMR spectroscopic analysis as well as chemical methods. Angularin
A, angulasaponins A–C, and azukisaponins III and VI showed inhibition of nitric oxide production in LPS-
Received 24 February 2014
Received in revised form 7 July 2014
Available online xxxx
Keywords:
Vigna angularis
Leguminosae
activated RAW264.7 macrophages, with IC50 values ranging from 13
l
M to 24
lM.
Ó 2014 Published by Elsevier Ltd.
Furanylmethyl glycosides
ent-Kaurane diterpene glycosides
Triterpene saponins
Anti-inflammatory activity
1. Introduction
plant, a systematical phytochemical analysis of V. angularis was
performed and the products were subjected to bioassays. Nine
new compounds (Fig. 1), including four new furanylmethyl glyco-
sides, angularides A–D (1–4), one ent-kaurane diterpene glycoside,
angularin A (5), and four new triterpenoid saponins, angulasapo-
nins A–D (6–9), were thus isolated, along with eight known com-
pounds (10–17). Herein, the isolation and structural elucidation
of these new compounds 1–9 are described, as well as their inhib-
itory effects on the production of nitric oxide (NO) induced by lipo-
polysaccharide (LPS) in RAW264.7 cells.
The seeds of Vigna angularis (Willd.) Ohwi & H. Ohashi (Phaseo-
lus angularis (Willd.) W. Wight, Leguminosae), also called Xiaodou
(red beans) in Chinese, or Azuki beans in Japanese, are one of the
commonly used food materials for preparation of porridges, soups,
or confections in the Orient. They are also widely used as a tradi-
tional Chinese medicine for their diuretic action, detoxification
properties, and to promote the drainage of pus (Chinese
Pharmacopoeia Commission, 2010). Previous investigations on this
plant have led to the isolation of 3-furanylmethyl b-D-glucopyran-
oside, flavonoids, saponins, dimeric proanthocyanidins, and glyc-
eroglycolipids (Ariga and Asao, 1981; Kitagawa et al., 1983a,b,c;
Kojima et al., 1991; Yumiko et al., 2009). The polyphenolic constit-
uents of V. angularis are associated with many bioactivities, such as
antioxidant, renal cortex protective, hypoglycemic, anti-hypoten-
sive, and hepatoprotective effects (Han et al., 2004; Itoh et al.,
2004, 2009; Mukai and Sato, 2009; Shin et al., 2005; Yumiko
et al., 2009). Beside these reports, there is little known on the
action of other types of components from V. angularis. Thus, in
order to find non-phenolic bio-active constituents occurring in this
2. Results and discussion
The EtOH extract of the seeds of V. angularis was suspended in
H2O and partitioned with EtOAc. The aqueous layer was subjected
to passage over a XAD-16 resin column chromatography, eluting
with aqueous ethanol in a gradient manner. The 20%, 40% and
70% aqueous ethanol eluates, along with the EtOAc extract, were
purified using a combination of silica gel, ODS, and Sephadex LH-
20 chromatographic steps, and preparative HPLC to give nine
new (1–9), and eight known (10–17) compounds (Fig. 1). Their
structures were determined by 1D and 2D NMR spectroscopic anal-
ysis and mass spectrometry.
⇑
Corresponding author at: State Key Laboratory of Natural and Biomimetic
Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
Tel./fax: +86 10 82802719.
Angularides A–C (1–3) were obtained as hygroscopic powders,
and determined to have the same C13H18O8 molecular formulae
E-mail address: yongjiang@bjmu.edu.cn (Y. Jiang).
http://dx.doi.org/10.1016/j.phytochem.2014.08.011
0031-9422/Ó 2014 Published by Elsevier Ltd.
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2
Y. Jiang et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 1. Structures of compounds 1–12, 15 and 17.
by HRESIMS (m/z 325.0894, 325.0885 and 325.0880 [M+Na]+ for 1,
2 and 3, respectively). The 1H and 13C NMR spectra of 1 displayed
signals for a 3-substituted furan ring [dH 6.49 (1H, d, J = 1.5 Hz),
7.45 (1H, t, J = 1.5 Hz), 7.52 (1H, m), dC 111.7, 123.1, 142.5,
144.5], an oxygenated methylene [dH 4.56 and 4.71 (2H, d,
J = 12.0 Hz); dC 63.3], and one anomeric resonance [dH 4.34 (1H,
d, J = 8.0 Hz), dC 103.0], which were very similar to those of 10
(Kitagawa et al., 1983a) except for the presence of an additional
acetyl group [dH 2.07; dC 20.7, 172.8]. Acid hydrolysis of 1 gave
and two anomeric carbons (dC 101.4 and 110.8). Thorough analysis
of these data with the help of 2D NMR experiments allowed iden-
tification of the aglycone as carboxyatractyligenin, an ent-kaurane
characterized by two acid functions at C-4 (Lang et al., 2013;
Piacente et al., 1996). Acid hydrolysis of 5 offered glucose (Glc)
and apiose (Api), characterized as D according to optical rotation
measurement after HPLC separation. The J value of the anomeric
proton of glucose was characteristic for a b-
pyranosyl form, and the comparison of 13C NMR spectroscopic data
with literature values was in agreement with a b- apiose in its
furanosyl form (Çalisß et al., 1993). Compound 5 and the known
2-O-b- -glucopyranosyl-carboxyatractyligenin (12) (Lang et al.,
D configuration in the
D-glucose after HPLC separation and optical rotation measurement.
D
The anomeric configuration was determined to be b- according to
D
the JH1–H2 value (8.0 Hz). The acetylated position was determined
by observation of an HMBC correlation between glucose H-60 (dH
4.21 and 4.41) and the acetyl carbonyl (dC 172.8). Thus, 1 was
D
2013) differed by the presence of a supplementary apiofuranosyl
at the C-2 position of glucopyranosyl, demonstrated by HMBC cor-
relation of dH 5.36 (H-1 of Api) with dC 79.1 (C-2 of Glc) and NOESY
correlation of dH 5.36 (H-1 of Api) with dH 3.31 (1H, overlapped, H-
2 of Glc). The relative configuration of 5 was determined by obser-
vation of NOESY correlations (Fig. 2), in accordance with those
observed for carboxyatractyligenin and its glycoside (Konopleva
et al., 2006; Piacente et al., 1996). The isolation of ent-kauranes
11 and 12 of known absolute configuration from this plant sug-
determined to be 3-furanylmethyl-60-O-acetyl b-
D-glucopyrano-
side. The acetylation positions of 2 and 3 were determined by
the observation of obvious downfield shifts of acetylated positions
(1.58 ppm for 2, and 1.46 ppm for 3) through comparison with 10,
which are same with the results reported in the literature (Jansson
et al., 1987). Thus, 2 was elucidated as 3-furanylmethyl-30-O-acetyl
b-
D
-glucopyranoside, and 3 was deduced as 3-furanylmethyl-40-O-
-glucopyranoside.
acetyl b-
D
gested that 5 belonged to the same series, thus being: 2 b-[(b-
apiofuranosyl(1?2)-b- -glucopyranosyl)oxy]-15 -hydroxy-ent-
kaur-l6-en-18,19-dioic acid.
D-
Angularide D (4) was obtained as a hygroscopic powder, and its
molecular formula was deduced to be C15H20O8 according to HRE-
SIMS (molecular ion at m/z 351.1042 [M+Na]+, calcd. 351.1050).
The 1H and 13C NMR spectroscopic data of 4 were similar to those
of 1, except that the acetyl group was replaced by a crotonyl group
[dH 1.90 (3H, dd, J = 7.0, 1.5 Hz), 5.92 (1H, dq, J = 15.5, 1.5 Hz) and
7.04 (1H, dq, J = 15.5, 7.0 Hz); dC 18.0, 123.3, 146.8, 168.0]. The
HMBC correlations of glucose H-60 (dH 4.28 and 4.46) with the
crotonyl carbonyl signal (dC 168.0) proved the linkage position of
the crotonyl group. Thus, compound 4 was deduced to be 3-fura-
D
a
Compounds 6–9 were obtained as white amorphous powders.
Angulasaponin A (6) was assigned a C42H66O14 molecular formula
from its HRESIMS (m/z 793.4370 [MꢀH]ꢀ). The 1H NMR spectrum
of 6 showed signals for seven tertiary methyls at dH 0.90, 0.97,
0.99, 1.15, 1.27, 1.31 and 1.50, an olefinic proton at dH 5.30 (triplet
like), and two anomeric protons at dH 5.02 (1H, d, J = 8.0 Hz) and
5.42 (1H, d, J = 8.0 Hz). The 13C NMR spectrum presented two car-
bonyl resonances at dC 173.2 and 181.8, two olefinic carbon signals
at dC 123.1 and 145.2, and two anomeric carbon resonances at dC
106.7 and 105.9. The 1H and 13C NMR spectroscopic data suggested
that 6 was a pentacyclic triterpene substituted by two sugars. In
the HMBC spectrum, the methyl proton at dH 1.50 had correlations
with signals at dC 30.5 (C-21), 42.1 (C-19), 43.5 (C-20), and 181.8
(C@O), suggesting that one of the methyls at C-20 was oxidized
into a carboxylic acid. The characteristic 13C NMR chemical shifts
of the methyl and carboxyl (Mahato and Kumdo, 1994) groups
favored the C-29 position for the carboxylic acid, indicating 3-epik-
atonic acid as the aglycone (Silva et al., 2002). Acid hydrolysis of 6
nylmethyl-60-crotonyl-b-
D-glucopyranoside.
Angularin A (5) was isolated as an amorphous powder, and its
molecular formula was determined to be C31H46O15 from the
molecular ion peak [M+NH4]+ at m/z 676.3194 (calcd 676.3175)
in the positive-ion HRESIMS. The 1H NMR spectrum showed one
tertiary methyl singlet at dH 1.02, two exocyclic methylene protons
at dH 5.08 (1H, br s) and 5.19 (1H, br s), and two anomeric protons
at dH 4.48 (1H, d, J = 8.0 Hz) and 5.36 (1H, d, J = 1.5 Hz). The 13C
NMR spectrum showed the presence of signals for two carbonyls
(dC 175.4 and 175.8), two olefinic carbons (dC 109.0 and 160.4),
two oxygenated carbons (dC 73.1 and 83.5), one methyl (dC 17.8),
yielded D-glucose and D-glucuronic acid characterized by optical
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Y. Jiang et al. / Phytochemistry xxx (2014) xxx–xxx
3
an olefinic proton at dH 5.17 (triplet like). The 13C NMR spectrum
presented signals for one carbonyl at dC 178.1, two olefinic carbons
at dC 123.4 and 144.7, one oxygenated methine carbon at dC 83.1,
and one oxygenated methylene carbon at dC 65.3. The 1H and 13C
NMR spectroscopic data of 8 were reminiscent of those of 3b, 23-
dihydroxyolean-12-en-29-oic acid (Van Le et al., 2009), except that
C-3 and C-29 showed obvious glycosidation shifts. The HMQC
spectrum of 8 showed correlations corresponding to four anomeric
signals [dH 5.01 (1H, d, J = 8.0 Hz), dC 105.6; dH 5.22 (1H, d,
J = 6.0 Hz), dC 104.6; dH 5.43 (1H, d, J = 8.0 Hz), dC 106.3; dH 6.30
(1H, d, J = 8.0 Hz), dC 96.3]. After acid hydrolysis of 8, the detection
of hydrolysate only gave glucose, which was in the b-D-configura-
tion according to the J values of the anomeric protons and optical
rotation. The linkage positions and sequence of the sugar moieties
were established by observation of HMBC correlations between H-
1 (dH 5.22) of Glc and C-3 (dC 83.1) of the aglycone, between H-1
(dH 5.43) of Glc0 and C-2 (dC 83.9) of Glc, between H-1 (dH 6.30)
of Glc00 and C-29 (dC 178.1) of the aglycone, and between H-1 (dH
5.01) of Glc000 and C-6 (dC 69.9) of Glc00. The reverse HMBC
correlations of these signals further proved the linkage positions.
Therefore, structure of 8 was elucidated as 3-O-[b-
syl(1?2)-b- -glucopyranosyl]-3b,23-dihydroxyolean-12-en-29-
oic acid-29-O-b- -glucopyranosyl(1?6)-b- -glucopyranoside.
D-glucopyrano-
D
Fig. 2. Key NOESY correlations of 5 (geometry-optimized conformation).
D
D
The HRESIMS of angulasaponin D (9) presented a quasi-molec-
ular ion at m/z 809.4356 [MꢀH]ꢀ, in accordance with a C42H66O15
molecular formula. The 1H NMR spectrum of 9 showed seven ter-
tiary methyl groups at dH 0.92, 1.05, 1.16, 1.29, 1.31 ꢁ 2, and
1.85, and an olefinic proton at dH 5.42 (triplet like); its 13C NMR
spectrum presented signals for one carbonyl at dC 181.8, two ole-
finic carbons at dC 123.4 and 144.7, and two oxygenated methines
at dC 89.6 (C-3) and 75.8 (C-22). The 1H and 13C NMR spectroscopic
data of the aglycone moiety of 9 were almost superimposable with
those of 3b, 22b-dihydroxyolean-12-en-29-oic acid (Kutney et al.,
1992), except that C-3 showed obvious glycosidation shift
rotation measurement after HPLC separation. 1D and 2D NMR
spectroscopic data analysis showed that, in the intact saponin,
the sugars were in the pyranose form and in a b-D configuration
with JH1–H2 = 8.0 Hz for glucose and 8.0 Hz for glucuronic acid
(Glc A). The sequence of the sugars was established by an
HMBC experiment which showed correlations between the
glucuronic acid anomeric proton (dH 5.42) and C-3 of the aglycone
(dC 89.7), and between the Glc anomeric proton (dH 5.02) and
C-2 of the Glc
elucidated as 3-epikatonic acid 3-O-b-
-glucuronopyranoside.
The molecular formula of angulasaponin B (7) was deduced to
A
(dC 83.6). Thus, the structure of
6 was
D-glucopyranosyl (1?2)-b-
(DdC + 11.7 ppm). Configuration of the hydroxyl at C-22 position
D
was established as b to account for the coupling constant of H-22
(1H, dd, J = 7.0, 2.0 Hz); in the opposite configuration, a much lar-
ger trans diaxial coupling constant would have been observed.
The HMQC spectrum of 9 showed correlations corresponding to
two anomeric signals [dH 5.01 (1H, d, J = 7.5 Hz), dC 105.6; dH
5.41 (1H, d, J = 8.0 Hz), dC 106.3]. Just as the above compounds,
be C54H86O24 from the quasi-molecular ion at m/z 1117.5436
[MꢀH]ꢀ in the HRESIMS. The 1H NMR spectrum showed signals
for seven tertiary methyl groups at dH 0.89 ꢁ 2, 0.95, 1.14, 1.15,
1.31 and 1.39, an olefinic proton at dH 5.18 (triplet like), and four
anomeric protons at dH 5.01 (1H, d, J = 8.0 Hz), 5.04 (1H, d,
J = 7.5 Hz), 5.42 (1H, d, J = 8.0 Hz) and 6.31 (1H, d, J = 8.0 Hz). The
13C NMR spectrum presented two carbonyl resonances at dC
172.8 and 178.2, two olefinic carbons at dC 123.5 and 144.7, and
four anomeric carbons at dC 96.4, 105.7 ꢁ 2 and 106.5. The 1H
and 13C NMR spectra of 6 and 7 were almost superimposable,
except that the carbonyl signal (C-29) showed an upfield shift from
dC 181.8 to 178.2, and two additional glucopyranosyl resonances
(dH 5.01, dC 105.7; dH 6.31, dC 106.5) were observed (Table 2).
The deshielded chemical shift of the anomeric proton at d 6.31 sug-
gested that it corresponded to a sugar linked by an ester function,
therefore glycosidation took place at C-29 of the aglycone. Acid
the sugars were identified as b-D-glucose and b-D-glucuronic acid
on the basis of acid hydrolysis and analysis of the NMR spectro-
scopic data. The sequence of the sugars was established by obser-
vation of HMBC correlations between H-1 of Glc A (dH 5.01) and C-
3 (dC 89.6) and between H-1 of Glc (dH 5.41) and C-2 of Glc A (dC
83.2). The reverse HMBC correlations of these signals further
proved the linkage positions. Therefore, 9 was elucidated as 3-O-
[b-D-glucopyranosyl (1?2)-b-D-glucuronopyranosyl]-3b, 22b-
dihydroxyolean-12-en-29-oic acid.
Further to these, eight known compounds were identified
through comparison of their spectroscopic data with those
reported in the literature: 3-furanylmethyl b-
(10) (Kitagawa et al., 1983a), b-O-b- -glucopyranosyl-15
hydroxykaur-l6-en-18-carboxylic acid (11) (Lang et al., 2013;
Vargas et al., 1988), 2b-O-b- -glucopyranosyl-15 -hydroxykaur-
D-glucopyranoside
hydrolysis of 7 gave
D
-glucose and
D-glucuronic acid, whose config-
2
D
a-
urations were determined in the same way as in 6. The attachment
of sugars to the aglycone and their sequences were established by
observation of HMBC correlations and in particular, between H-1
(dH 6.31) of inner Glc0 and C-29 (dC 178.2) of the aglycone, and
between H-1 (dH 5.01) of the terminal Glc00 and C-6 (dC 69.9) of
the inner Glc0. Therefore, structure of 7 was elucidated to be 3-O-
D
a
l6-en-18,19-dicarboxylic acid (12) (Lang et al., 2013; Piacente
et al., 1996), and azukisaponins I–III, V and VI (13–17) (Kitagawa
et al., 1983a,b,c).
Among the isolated compounds, compounds 1–3, are the acety-
[b-
D-glucopyranosyl(1?2)-b-
D-glucuronopyranosyl]-3-epikatonic
lated products of 3-furanylmethylb-D-glucopyranoside (10) at the
acid-29-O-b-
D
-glucopyranosyl(1?6)-b-
D
-glucopyranoside.
different positions of glucose, except position C-2. Concerned by
the possibility that they could be artefacts produced by random
migration of the acetyl under storage or under extraction condi-
tions, a rapid extraction was performed anew and the extract
was swiftly examined by LC/MS, which unambiguously showed
The molecular formula of angulasaponin C (8) was determined
to be C54H88O24 from its quasi-molecular ion at m/z 1119.5588
[MꢀH]ꢀ in the HRESIMS. The 1H NMR spectrum showed six ter-
tiary methyl groups at dH 0.88, 0.97, 0.97, 1.09, 1.13 and 1.38 and
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4
Y. Jiang et al. / Phytochemistry xxx (2014) xxx–xxx
the presence of such compounds in V. angularis (see Supplemen-
tary material for experimental conditions). Compounds 5, 11 and
12 are three ent-kaurane diterpenoids, with atractyligenin or carb-
oxyatractyligenin as aglycone, which are isolated for the first time
from the Vigna genus. The sulfates and esters of 11 and 12, atract-
yloside and carboxyatractyloside were reported to be toxic to kid-
ney or liver through inhibition of the adenine nucleotide
translocase (ANT) in mitochondria (Stewart and Steenkamp,
2000; Vignais et al., 1978). Moreover, compound 12 isolated from
raw coffee beans was recently reported to present a similar toxic
activity, although this can be suppressed by degradation during
roasting. Even so, there is a recommendation to control the content
of 12 in raw coffee extracts when being used as the additives for
food products (Lang et al., 2013). The seeds of V. angularis are
one kind of widely consumed edible beans in Asia, and as a conse-
quence, the presence of carboxyatractyligenin-glycosides in this
plant deserves notice and precaution. Since the toxic carboxyatrac-
tyligenin-glycoside 12 decarboxylated to nontoxic 11 during cof-
fee-roasting (Lang et al., 2013), it would be worth investigating
the cooking procedure for the detoxification of red beans.
NO, one of the important pro-inflammatory mediators, is
involved in some inflammatory disorders, such as rheumatoid
arthritis, chronic hepatitis, and pulmonary fibrosis (Isomaki and
Punnonen, 1997; Kanwar et al., 2009; Tilg et al., 1992). Suppression
of the release of NO and other inflammatory mediators plays a crit-
ical role in the treatment of such diseases. Therefore, the in vitro
NO production inhibitory model has been widely used for screen-
ing bioactive components with potential anti-inflammatory effect
from natural medicines (Hsu et al., 2012; Li et al., 2012; Wang
et al., 2013). Yu et al. (2011) reported that V. angularis ethanol
extract dose-dependently suppressed the release of PGE2 and NO
in LPS-, poly(I:C)-, and pam3CSK activated macrophages. This plant
was also incorporated as part of an anti-inflammatory diet to pre-
vent and reduce risk factors for heart disease (Sato et al., 2008).
However, the anti-inflammatory constituents are still unknown.
Thus, in order to search for the potential anti-inflammatory com-
ponents from this plant, the compounds isolated here were
assessed for their inhibitory effects against LPS-induced NO pro-
duction in RAW264.7 macrophages. Compounds 5–8, 15 and 17
exhibited the most potent inhibitory activities with IC50 values
relatively toxic carboxyatractyligenin-glycosides in V. angularis
during the processing deserves further study.
4. Experimental
4.1. General experimental procedures
Column chromatography (CC) was performed on silica gel 60
(40–63 lm, Merck) with Buchi pump manager C-615, pump mod-
ule C-601 and Buchi fraction collector C-660. Preparative RP-18 CC
was performed on a prefilled column for packing stand NM 50 with
Lichrospher 100 RP-18 (12
lm, Merck), controlled by a La Prep-
system, including fraction valve P 206, P110 pump and P311 detec-
tor (VWR International). Amberlite XAD-16 and Sephadex LH-20
(25–100 lm) were purchased from Sigma. TLC was carried out
on pre-coated silica gel 60 GF254 plates (Merck), and detected by
spraying with 1% vanillin H2SO4 ethanol solution, and then heating
at 110 °C for 5 min. UV spectra were measured on a Lambda Perkin
Elmer UV/vis spectrometer. Preparative HPLC (prep. HPLC) was
performed on a Merck Hitachi chromatograph with a UV–vis
detector L-7420, an interface D-7000, an autosampler L-7200 and
a pump L-7150N. The column used for prep. HPLC is a Waters sym-
metry shield RP-18 column (7
lm, 19 ꢁ 300 mm). Lyophilization
was run on a Christ lyophilizator (Germany). Optical rotations
were measured on an Autopol-IV polarimeter (Rudolph Research,
USA). FT-IR spectra were recorded on a Perkin-Elmer Spectrum
100 FT-IR spectrometer. NMR spectra were recorded on a Bruker
Avance II-500 NMR spectrometer. 1H–1H COSY, HMQC, HMBC
and NOESY NMR spectra were obtained. MS and HRESIMS were
measured on a Bruker Daltonics Apex IV FT mass spectrometer or
a Waters Xevo G2 Q Tof mass spectrometer.
4.2. Plant material
The seeds of V. angularis were purchased from Zizhou County,
Shaanxi Province, China, in May 2009, and identified by one of us
(YJ). A herbarium specimen is deposited in the Herbarium of the
Pierre Fabre Research Institute (CBPF, Soual, France) under refer-
ence V600273.
ranging from 13 lM to 24 lM and the diterpenoids and saponins
showed much better NO inhibition effects than the furanylmethyl
glycosides (Table 3). In the screened diterpenoids, the diglycoside
(compound 5) showed a stronger inhibitory effect than the
mono-glycoside (compound 12), which suggests that the sugar
moiety is related to the activity of the diterpenoids. While for sap-
onins, not only the sugar moiety but also the aglycone has influ-
ence on the activity, and that of the carboxyl at C-29 may be
prominent. When the C-29 carboxyl was reduced to a methyl
group, the activity decreased, for example, the IC50 values for 13,
4.3. Extraction and isolation
The seeds of V. angularis (4.0 kg) were extracted with EtOH: H2O
(7.5 L ꢁ 4, 96:4, v/v) under reflux conditions, each time for 1.5 h,
and a residue (161.6 g) was produced. This extract was suspended
in H2O and partitioned with EtOAc to give the EtOAc extract
(37.2 g). The aqueous layer was subjected to XAD-16 resin CC,
and eluted with a gradient of H2O and EtOH to give an aqueous elu-
ate (89.5 g), a 20% aqueous EtOH eluate (5.5 g), 40% aqueous EtOH
eluate (1.9 g) and 70% aqueous ethanol eluate (4.5 g).
14 and 16 were all larger than 50 lM.
Part of the EtOAc extract (32.1 g) was subjected to silica gel CC,
and eluted with n-hexane–EtOAc (10:1 ? 0:1) to obtain nine frac-
tions. Fr. 9 (8.2 g) was further separated on silica gel CC, with
CHCl3–MeOH–H2O (80:20:2 and 70:30:5) as eluents to afford five
subfractions. Subfraction 2 (0.35 g) was purified by silica gel CC
[eluting with CHCl3-acetone (6:4)] and prep. HPLC [eluting with
MeCN-0.05% TFA aqueous solution (12:88)] to furnish 2 (5.7 mg)
and 3 (6.4 mg).
The 20% aqueous EtOH eluate (5.2 g) was submitted to silica gel
CC, and eluted with CHCl3–MeOH–H2O gradients (70:30:10 and
65:35:10, lower phase) to give five fractions. Fr. 1 (0.82 g) was
purified by Sephadex LH-20 CC (eluting with EtOH: H2O, 8:2) to
afford three subfractions. Subfraction 2 (0.21 g) was separated on
silica gel CC (eluting with CHCl3-acetone from 6:4 to 2:8) and prep.
HPLC [eluting with MeCN–H2O (25:75)] to yield 4 (1.6 mg) and 1
3. Conclusions
Seventeen compounds, including four new furanylmethyl gly-
cosides, angularides A –D (1–4), one new ent-kaurane diterpene
glycoside, angularin A (5), and four new triterpenoid saponins,
angulasaponins A–D (6–9), together with eight known compounds
(10–17), were obtained from the 96% ethanol extract of the seeds
of V. angularis. Compounds 5–8, 15 and 17 showed potent NO pro-
duction inhibitory effects in LPS-activated macrophages, with IC50
values ranging from 13 lM to 24 lM. These data suggest that not
only the phenolic constituents, but also some non-phenolic com-
pounds, such as saponins and diterpenoids are the active compo-
nents of the red beans. Moreover, the quantity variance of the
Please cite this article in press as: Jiang, Y., et al. Constituents of Vigna angularis and their in vitro anti-inflammatory activity. Phytochemistry (2014), http://
dx.doi.org/10.1016/j.phytochem.2014.08.011

Y. Jiang et al. / Phytochemistry xxx (2014) xxx–xxx
5
Table 1
NMR spectroscopic data of compounds 1–4 (in CD3OD, 500 MHz for 1H and 125 MHz for 13C, d in ppm and J in Hz).
No.
1
2
3
4
dH
dC
dH
dC
dH
dC
dH
dC
2
3
4
5
7.52 (m)
142.5 7.53 (m)
123.1
142.5 7.53 (m)
123.1
142.5 7.51 (m)
123.1
111.7 6.49 (d, 1.5)
144.5 7.44 (t, 1.5)
142.5
123.1
111.7
144.5
63.3
103.0
75.0
77.9
6.49 (d, 1.5)
7.45 (t, 1.5)
4.71 (d, 12.0) 4.56 (d, 12.0)
4.34 (d, 8.0)
111.7 6.49 (d, 1.5)
144.5 7.45 (t, 1.5)
63.3 4.78 (d, 12.0) 4.59 (d, 12.0)
103.0 4.42 (d, 9.0)
75.3 3.46 (t, 9.0)
77.9 4.91 (t, 9.0)
71.7 3.35 (m)
75.0 3.33 (m)
64.8 3.91 (dd, 2.0, 12.0) 3.70 (dd,
5.5, 12.0)
111.6 6.50 (d, 1.5)
144.5 7.45 (t, 1.5)
63.3 4.78 (d, 12.0) 4.60 (d, 12.0)
102.8 4.37 (d, 8.0)
6
63.3
4.69 (d, 12.0) 4.56 (d, 12.0)
10
20
30
40
50
60
103.0 4.34 (d, 8.0)
a
3.21 (dd, 8.0, 9.0)
3.35 (t, 9.0)
73.3 3.30 (o)
75.0
77.9
71.7
75.4
3.21 (t, 8.0)
3.35 (t, 8.0)
3.31 (o)
79.1 3.53 (t, 9.5)
69.9 4.75 (t, 9.5)
77.8 3.43 (m)
a
a
3.32 (o)
71.7
75.4
64.6
3.44 (m)
4.41 (dd, 2.0, 12.0) 4.21 (dd,
6.0, 12.0)
3.47 (m)
62.5 3.63 (dd, 2.5, 12.0) 3.54 (dd, 64.6
4.46 (dd, 2.0, 12.0) 4.28 (dd,
6.0, 12.0)
168.0
123.3 5.92 (dq, 15.5, 1.5)
146.8 7.04 (dq, 15.5, 7.0)
18.0 1.90 (dd, 1.5, 7.0)
6.0, 12.0)
70
168.0
123.3
146.8
18.0
80
90
100
2.07 (s)
20.7 2.10 (s)
172.8
21.1 2.09 (s)
172.7
CH3CO-
CH3CO-
a
Overlapped signal.
18
(37.1 mg). Fr. 2 (1.82 g) was recrystallized with MeOH to produce
10 (1.28 g). Fr. 5 (0.54 g) was first separated on Sephadex LH-20
CC (eluting with MeOH), and then on RP-18 CC (eluting with
MeCN:0.05%TFA aqueous solution from 10:90 to 100:0), and finally
on prep. HPLC [eluting with MeCN:H2O (25:75)] to afford 5
(7.7 mg).
The 40% aqueous EtOH eluate (1.9 g) was purified using Sepha-
dex LH-20 (MeOH: H2O, 80:20, as eluent) and silica gel CC (eluting
with CHCl3–MeOH–H2O, 80:20:2, 70:30:5 and 60:40:10) to give 5
fractions. Fr.1 (0.42 g) was further isolated on Sephadex LH-20 CC
(MeOH: H2O, 80:20, as eluent), and prep. HPLC (MeOH:0.05%TFA,
40:60) to give 11 (118.8 mg). Fr. 5 (0.23 g) was separated on Sepha-
dex LH-20 CC (MeOH: H2O, 80:20, as eluent) to furnish 12
(16.2 mg).
The 70% aqueous EtOH eluate (6.0 g) was subjected to silica gel
CC, and eluted with CHCl3–MeOH–H2O (70:30:5 and 60:40:10) to
give 5 fractions. Fr. 2 (0.28 g) was isolated by repeated prep. HPLC
(MeCN:0.05%TFA, 29:71) to give 6 (3.5 mg), 9 (1.7 mg), 13 (8.7 mg),
14 (18.5 mg), 15 (7.2 mg) and 16 (13.9 mg), respectively. Fr. 4
(1.21 g) was separated on prep. HPLC (MeCN:0.05%TFA, 29:71) to
Angularin A (5): amorphous powder; [
a
]
ꢀ91 (c 0.01, MeOH);
D
IR (KBr) mmax 3401, 2932, 1657, 1059 cmꢀ1
;
1H NMR (CD3OD,
500 MHz), d 5.36 (1H, d, J = 1.5 Hz, Api-1), 5.19 (1H, br s, H-17a),
5.08 (1H, br s, H-17b), 4.48 (1H, d, J = 8.0 Hz, Glc-1), 4.21 (1H, m,
H-2), 4.01 (1H, d, J = 9.5 Hz, Api-4a), 3.93 (1H, d, J = 1.5 Hz, Api-2),
3.84 (1H, dd, J = 12.0, 2.0 Hz, Glc-6a), 3.79 (1H, s, H-15), 3.72 (1H,
d, J = 9.5 Hz, Api-4b), 3.67 (1H, dd, J = 12.0, 5.5 Hz, Glc-6b), 3.62
(2H, d, J = 2.0 Hz, Api-5), 3.48 (1H, t, J = 9.0 Hz, Glc-3), 3.31 (1H, over-
lapped, Glc-2), 3.29 (1H, overlapped, Glc-4), 3.27 (1H, m, Glc-5), 2.73
(1H, m, H-3a), 2.72 (1H, m, H-13), 2.36 (1H, dd, J = 12.0, 3.5 Hz, H-
1a), 1.88 (1H, d, J = 11.0 Hz, H-14a), 1.87 (1H, m, H-6a), 1.86 (1H,
m, H-5), 1.69 (1H, m, H-11a), 1.68 (1H, m, H-6b), 1.66 (1H, m,
H-7a), 1.64 (1H, m, H-12a), 1.51 (1H, m, H-11a), 1.48 (1H, m, H-
12b), 1.46 (1H, m, H-7b), 1.42 (1H, m, H-3b), 1.40 (1H, dd, J = 11.0,
5.0 Hz, 14b), 1.15 (1H, d, J = 7.5 Hz, H-9), 1.02 (3H, s, H-20), 0.88
(1H, t, J = 12.0 Hz, H-1b). 13C NMR (CD3OD, 125 MHz), d 175.8 (C-
18), 175.4 (C-19), 160.4 (C-16), 110.8 (Api-1), 109.0 (C-17), 101.4
(Glc-1), 83.5 (C-15), 80.7 (Api-3), 79.1 (Glc-2), 78.7 (Glc-3), 78.2
(Api-2), 77.7 (Glc-5), 75.4 (Api-4), 73.1 (C-2), 71.7 (Glc-4), 66.3
(Api-5), 62.7 (Glc-6), 59.3 (C-4), 55.0 (C-9), 52.3 (C-5), ꢂ49.0 (C-1,
C-8, overlapped with signal of CD3OD), 43.7 (C-13), 41.4 (C-10),
41.0 (C-3), 37.3 (C-14), 36.3 (C-7), 33.7 (C-12), 24.2 (C-6), 19.3 (C-
11), 17.8 (C-20); positive ESIMS m/z 681 [M+Na]+; negative ESIMS
m/z 657 [MꢀH]ꢀ, 613 [MꢀHꢀCOO]ꢀ; HRESIMS m/z 676.3196
give 7 (48.5 mg), 8 (26.8 mg) and 17 (372.7 mg).
18
Angularide A (1): hygroscopic powder, [
a]
ꢀ45 (c 0.13, MeOH);
D
UV (MeOH) kmax (log e) 210 (3.68) nm; IR (KBr) mmax 3396, 2889,
1735, 1250, 1082, 1040 cmꢀ1; For 1H NMR (CD3OD, 500 MHz)
and13C NMR (CD3OD, 125 MHz) spectroscopic data, see Table 1;
positive ESIMS m/z 325 [M+Na]+; HRESIMS m/z 325.0884 [M+Na]+
[M+NH4]+ (calcd for C31H50NO15, 676.3175).
18
Angulasaponin A (6): amorphous powder; [
a]
ꢀ15 (c 0.08,
D
(calcd for C13H18O8Na, 325.0894).
MeOH); IR (KBr) mmax 3391, 2927, 1686, 1204 cmꢀ1; for 1H NMR
(pyridine-d5, 500 MHz) and13C NMR (pyridine-d5, 125 MHz) spectro-
scopic data, see Table 2; negative ESIMS m/z 793 [MꢀH]ꢀ; HRESIMS
18
Angularide B (2): hygroscopic powder; [
a
]
ꢀ42 (c 0.05,
D
MeOH); UV (MeOH) kmax (loge
) 210 (3.45) nm; for 1H NMR (CD3OD,
500 MHz) and 13C NMR (CD3OD, 125 MHz) spectroscopic data, see
m/z 793.4370 [MꢀH]ꢀ (calcd for C42H65O14, 793.4374).
Table 1; positive ESIMS m/z 325 [M+Na]+, 627 [2M+Na]+; HRESIMS
Angulasaponin B (7): amorphous powder; [
a]
ꢀ22 (c 0.09,
18
D
m/z 325.0885 [M+Na]+ (calcd for C13H18O8Na, 325.0894).
MeOH); IR (KBr) mmax 3343, 2944, 1739, 1620, 1031 cmꢀ1; For 1H
NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz)
spectroscopic data, see Table 2; positive ESIMS m/z 1141 [M+Na]+;
negative ESIMS m/z 1117 [MꢀH]ꢀ; HRESIMS m/z 1117.5436 [MꢀH]ꢀ
18
Angularide C (3): hygroscopic powder; [
a]
ꢀ44 (c 0.03,
D
MeOH); UV (MeOH) kmax (loge
) 210 (3.62) nm; for 1H NMR (CD3OD,
500 MHz) and 13C NMR (CD3OD, 125 MHz) spectroscopic data, see
Table 1; positive ESIMS m/z 325 [M+Na]+, 627 [2M+Na]+; HRESIMS
(calcd for C54H85O24, 1117.5431).
m/z 325.0880 [M+Na]+ (calcd for C13H18O8Na, 325.0894).
Angulasaponin C (8): amorphous powder; [
a
]
ꢀ35 (c 0.09,
18
D
18
Angularide D (4): hygroscopic powder; [
MeOH); UV (MeOH) kmax (log
a
]
ꢀ56 (c 0.04,
MeOH); IR (KBr) mmax 3400, 2932, 1717, 1629, 1032 cmꢀ1; for 1H
NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz)
spectroscopic data, see Table 2; negative ESIMS m/z 1119 [MꢀH]ꢀ;
D
e
) 210 (3.81) nm; For 1H NMR (CD3OD,
500 MHz) and13C NMR (CD3OD, 125 MHz) spectroscopic data, see
Table 1; positive ESIMS m/z 351 [M+Na]+, 679 [2M+Na]+; negative
ESIMS m/z 327 [MꢀH]ꢀ, 655 [2 MꢀH]ꢀ; HRESIMS m/z 351.1042
[M+Na]+ (calcd for C15H20O8Na, 351.1050).
HRESIMS m/z 1119.5588 [MꢀH]ꢀ (calcd for C54H88O24, 1119.5587).
18
Angulasaponin D (9): amorphous powder; [
a
]
ꢀ18 (c 0.08,
D
MeOH); IR (KBr) mmax 3393, 2927, 1645, 1025 cmꢀ1; For 1H NMR
Please cite this article in press as: Jiang, Y., et al. Constituents of Vigna angularis and their in vitro anti-inflammatory activity. Phytochemistry (2014), http://
dx.doi.org/10.1016/j.phytochem.2014.08.011

6
Y. Jiang et al. / Phytochemistry xxx (2014) xxx–xxx
Table 2
NMR spectroscopic data of compounds 6–9 (in pyridine-d5, 500 MHz for 1H and 125 MHz for 13C, d in ppm and J in Hz).
No.
6
7
8
9
dH
dC
dH
dC
dH
dC
dH
dC
1
2
1.44 (m) 0.87 (o)
2.27 (m) 1.91 (m)
39.4
27.2
1.45 (dd, 10.5, 3.0) 0.88 (o)
2.27 (dd, 13.0, 3.0) 1.92 (dd,
13.0, 1.0)
39.2 1.52 (m) 0.99 (o)
27.0 2.35 (br d, 11.0) 2.04 (o)
39.2 1.48 (br d, 12.0) 0.88 (o)
26.4 2.27 (dd, 13.0, 4.0) 1.91 (o)
39.2
27.0
3
4
5
6
7
8
9
10
11
12
13
14
15
3.30 (dd, 12.0, 4.5)
89.7
40.2
56.3
19.1
33.5
40.7
48.4
37.4
24.4
3.36 (dd, 12.0, 4.5)
89.6 4.24 (dd, 9.0,4.0)
40.0
56.1 1.55 (o)
18.8 1.55 (o) 1.40 (o)
33.3 1.56 (o) 1.23 (o)
40.5
48.1 1.62 (o)
37.2
24.2 1.82 (m)
123.5 5.17 (t-like)
144.7
83.1 3.32 (dd, 11.5, 4.0)
43.9
48.3 0.76 (d, 11.0)
18.6 1.54 (m) 1.36 (o)
33.0 1.50 (m) 1.35 (o)
40.5
48.2 1.62 (dd, 8.0, 9.5)
37.1
24.2 1.89 (o)
123.4 5.42 (br s)
144.7
89.6
39.9
56.2
18.9
33.6
40.4
48.3
37.2
24.2
123.4
144.7
43.0
0.74 (d, 11.0)
0.73 (d, 12.0)
1.51 (o) 1.33 (o)
1.42 (m) 1.24 (dd,13.0, 3.0)
1.52 (o)a 1.34 (m)
1.47 (m) 1.29 (o)
1.59 (o)
1.51 (o)
1.87 (m)
5.30 (t-like)
1.81 (m)
123.1 5.18 (t-like)
145.2
42.5
42.2
42.2
1.80 (dd, 13.5, 3.0) 0.98 (o)
2.18 (m) 0.89 (o)
27.1
1.72 (dd, 13.0, 4.0) 0.90 (o)
26.7 1.68 (m) 0.82 (m)
26.7 2.04 (td, 12.0, 2.5) 1.48 (br d, 29.3
12.0)
27.4 1.90 (o) 1.06 (o)
16
27.9
1.99 (td,13.5, 4.0) 0.78 (d,
13.5)
27.5 1.96 (td, 13.0, 3.0) 0.73 (d,
26.7
13.0)
17
18
19
20
21
33.4
47.1
33.0
33.0
38.4
45.0
2.18 (m)
2.06 (dd, 13.5, 4.0)
2.48 (t, 13.5) 1.66 (br d, 13.5)
46.7 2.04 (dd, 13.0, 4.0)
41.0 2.45 (t, 13.0) 1.64 (o)
43.5
46.7 2.54 (br d, 13.5)
40.9 2.83 (t, 13.5) 1.72 (br d, 13.5) 41.9
43.4
29.8 2.66 (dd, 14.0, 2.5) 2.16 (dd,
14.0, 7.0)
2.60 (t, 13.0) 1.74 (br d, 13.0) 42.1
43.5
2.30 (dd, 13.0, 3.0) 1.74 (br d, 30.5
13.0)
42.8
38.2
2.14 (td, 13.5, 3.5) 1.64 (br d,
13.5)
29.8 2.10 (td, 13.0, 3.5) 1.62 (o)
22
23
24
25
26
27
28
29
30
Glc A
1
2
3
4
5
6
Glc
1
2
3
1.59 (o) 1.40 (m)
1.31 (s)
1.15 (s)
0.90 (s)
0.99 (s)
37.1
28.7
17.4
16.2
17.6
26.7
29.0
181.8
20.6
1.44 (dd, 13.5, 3.0) 1.28 (o)
36.5 1.43 (m) 1.26 (m)
28.5 3.79 (d, 11.0) 4.38 (d, 11.0)
17.2 1.13 (s)
16.0 0.97 (s)
17.4 0.97 (s)
26.5 1.09 (s)
28.6 0.88 (s)
178.2
19.9 1.38 (s)
36.5 4.03 (dd, 7.0, 2.0)
65.3 1.31 (s)
13.8 1.16 (s)
16.6 0.92 (s)
17.4 1.05 (s)
26.5 1.31 (s)
28.5 1.29 (s)
178.1
75.8
28.5
17.2
16.1
17.7
25.9
21.3
181.8
25.3
1.31 (s)
1.15 (s)
0.89 (s)
0.95 (s)
1.14 (s)
0.89 (s)
1.27 (s)
0.97 (s)
1.50 (s)
1.39 (s)
Glc A
19.9 1.85 (s)
Glc A
Glc
5.02 (d, 8.0)
4.35 (dd, 8.0)
4.40 (t, 8.0)
4.57 (t, 8.0)
4.62 (d, 8.0)
105.9 5.04 (d, 7.5)
105.7 5.22 (d, 6.0)
83.4 4.29 (o)
78.1 4.15 (t, 8.0)
73.5 4.20 (o)
104.6 5.01 (d, 7.5)
83.9 4.34 (o)
78.4 4.40 (t, 9.0)
72.0 4.55 (m)
78.4 4.60 (d, 9.5)
63.0
105.6
83.2
78.2
73.5
77.7
173.1
83.6
78.4
73.8
77.9
173.2
4.35 (o)
4.40 (t, 9.0)
4.57 (t, 9.0)
4.61 (d, 9.0)
77.8 4.31 (o)
172.8 4.52 (dd,11.5, 2.5) 4.46 (o)
Glc
Glc0
Glc
5.42 (d, 8.0)
4.14 (t, 8.0)
4.27 (t, 8.0)
4.35 (t, 8.0)
3.95 (m)
4.52 (dd, 12.0, 3.0) 4.48 (dd,
12.0, 5.0)
106.7 5.42 (d, 8.0)
106.5 5.43 (d, 8.0)
77.5 4.12 (t, 8.0)
78.4 4.24 (o)
72.2 4.19 (t, 7.0)
78.8 3.94 (m)
106.3 5.41 (d, 8.0)
77.2 4.14 (t, 8.0)
78.8 4.26 (t, 8.0)
71.9 4.33 (t, 8.0)
78.8 3.95 (m)
63.1 4.52 (dd, 12.0, 3.0) 4.48 (dd,
12.0, 4.5)
106.3
77.4
78.3
72.2
78.6
63.2
77.7
78.6
72.4
78.8
63.4
4.14 (o)
4.26 (t, 9.0)
4.34 (o)
3.95 (m)
4.49 (dd, 12.0, 3.0) 4.34 (o)
4
5
6
63.2 4.46 (o) 4.35 (dd, 12.0, 5.0)
Glc0
1
2
3
4
Glc0
6.31 (d, 8.0)
4.13 (o)
4.19 (t, 8.0)
4.31 (t, 8.0)
4.26 (m)
Glc00
96.4 6.30 (d, 8.0)
74.5 4.12 (t, 8.0)
79.0 4.20 (o)
71.4 4. 31 (o)
78.4 4.30 (o)
96.3
74.5
79.0
71.4
78.4
69.9
5
6
4.72 (dd, 12.0, 3.0) 4.35 (o)
69.9 4.72 (dd, 10.0, 2.0) 4.34 (dd,
11.0, 5.0)
Glc00
1
2
3
4
Glc00
Glc000
5.01 (d, 8.0)
3.99 (t, 8.0)
4.18 (t, 8.0)
4.35 (o)
3.87 (m)
4.49 (dd, 12.0, 3.0) 4.34 (o)
105.7 5.01 (d, 8.0)
75.5 3.99 (t, 8.0)
78.6 4.17 (o)
72.0 4.20 (o)
78.8 3.87 (m)
105.6
75.5
78.8
71.9
78.7
63.1
5
6
63.1 4.46 (o) 4.35 (dd, 12.0, 5.0)
a
Overlapped signal.
(pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spec-
troscopic data, see Table 2; HRESIMS m/z 809.4356 [MꢀH]ꢀ (calcd
for C42H65O15, 809.4323), 833.4294 [M+Na]+ (calcd for C42H66O15Na,
833.4299).
4.4. Acid hydrolysis of compounds 1 and 5–8
Compounds 1, 5–8 (2 mg for each compound) were heated in
2 mol/L aqueous CF3COOH (5 mL) at 110 °C for 6 h in a sealed tube.
Please cite this article in press as: Jiang, Y., et al. Constituents of Vigna angularis and their in vitro anti-inflammatory activity. Phytochemistry (2014), http://
dx.doi.org/10.1016/j.phytochem.2014.08.011

Y. Jiang et al. / Phytochemistry xxx (2014) xxx–xxx
7
Table 3
her stay in France. This work was also financially supported by
National Science Fund for Excellent Young Scholars of China (No.
81222051), and National Key Technology R&D Program ‘‘New Drug
Innovation’’ of China (Nos. 2012ZX09301002-002-002 and 2012ZX
09304-005).
NO inhibitory activities of the isolated compoundsa,b
.
Compounds
IC50
/
l
M
Compounds
IC50/lM
5
6
7
8
22
22
14
13
9
15
17
13
15
14
7
c
Indomethacin
Appendix A. Supplementary data
a
Inhibition of the 96% ethanol crude extract of V. angularis was only 21.46% at
50
l
g/mL.
b
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
08.011.
The data of 1, 3 and 4 were not reached due to quantity limitation, and the IC50
values of 2, 10–14, and 16 were more than 50 lM.
c
Positive control.
Then, the sugars and aglycone were separated by liquid–liquid
partitioning between CHCl3 and H2O. The aqueous layer was
repeatedly concentrated with MeOH until neutral, and the residue
was separated by Sep-Pak C18 cartridge column (H2O ? MeOH).
The H2O-eluted fraction was subjected to HPLC analysis under fol-
lowing conditions: HPLC column, Alltech Prevail carbohydrate ES,
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