Three new phenolic glycosides from the whole plant of Aconitum tanguticum (Maxim.) Stapf

Article abstract of DOI:10.1016/j.phytol.2015.01.020

Three new phenolic glycosides 2-(3-O-β-d-glucopyranosyl-4-hydroxyphenyl) ethanol 1-O-β-d-glucopyranoside (1), 2-(4-O-β-d-fructopyranosylphenyl) ethanol 1-O-β-d-galactopyranoside (2) and 3-methoxy-4-O-β-d-allopyranosyl acetophenone (3), along with nine known compounds (4-12), were isolated from the ethanol extract of the whole plant of Aconitum tanguticum (Maxim.) Stapf. Their structures were elucidated by analysis of spectroscopic data including 1D-, 2D-NMR and HRESIMS, and the reported literature data comparison. All the compounds were evaluated for their potential anti-inflammatory effects by the inhibition of TNF-α production on LPS-stimulated RAW264.7 macrophages. Compounds 1, 3, 5 and 7-9 showed certain inhibition activity and their IC₅₀ values were 38.18, 27.64, 3.25, 84.45, 12.76 and 18.44 μg/mL, respectively.

Full text of DOI:10.1016/j.phytol.2015.01.020

Phytochemistry Letters 11 (2015) 311–315
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Three new phenolic glycosides from the whole plant of Aconitum
tanguticum (Maxim.) Stapf
a,b
a
a
a
c
a
Yan-Rong Li , Ting Liu , Ren-Yi Yan , Lian-Qiang Hui , Li-mei Lin , Chun-Yu Cao ,
a
a,b
a,b
a,b,
a,b,
Shan-shan Guo , Li-Xin Yang , Wei-Hong Feng , Chun Li *, Zhi-Ming Wang
*
a
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
b
National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines, Beijing 100700, China
c
School of Pharmaceutical Science, Hunan University of Chinese Medicine, Changsha 410208, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new phenolic glycosides 2-(3-O-
b
-
D
-glucopyranosyl-4-hydroxyphenyl) ethanol 1-O-
-galactopyranoside (2) and 3-
b-D-allopyranosyl acetophenone (3), along with nine known compounds (4–12), were
b-D-gluco-
Received 12 August 2014
pyranoside (1), 2-(4-O-
methoxy-4-O-
b-D-fructopyranosylphenyl) ethanol 1-O-b-D
Received in revised form 25 December 2014
Accepted 30 January 2015
Available online 10 February 2015
isolated from the ethanol extract of the whole plant of Aconitum tanguticum (Maxim.) Stapf. Their
structures were elucidated by analysis of spectroscopic data including 1D-, 2D-NMR and HRESIMS, and
the reported literature data comparison. All the compounds were evaluated for their potential anti-
Keywords:
inflammatory effects by the inhibition of TNF-
Compounds 1, 3, 5 and 7–9 showed certain inhibition activity and their IC50 values were 38.18, 27.64,
.25, 84.45, 12.76 and 18.44 g/mL, respectively.
a production on LPS-stimulated RAW264.7 macrophages.
Aconitum tanguticum
Phenolic acid glycosides
Anti-inflammatory activity
3
m
ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1
. Introduction
on the non-alkaloid constituents in it (Luo et al., 2012; Xu et al.,
2013a,b,c; Li et al., 2014a,b).
Aconitum tanguticum (Maxim.) Stapf (Ranunculaceae) is a
perennial herb and mainly distributed around the area of 3800–
000 m altitude of Qinghai, Gansu, Sichuan, Yunnan and Tibet
In the course of ongoing search for the bioactive compounds, a
chemical investigation of the anti-inflammatory active fractions of
A. tanguticum was undertook. As a result, three new phenolic
5
provinces in China (Chinese Materia Medica editorial board, 2002).
Its whole plant, with a Tibetan name ‘‘Ponka’’, has been used for
thousands of years in Tibetan fork medicine for treating gastritis,
hepatitis, pneumonia, influenza and other diseases (Luo, 1997).
Previous phytochemical studies have revealed the presence of a
large number of diterpenoid alkaloids in the plant (Chen and Song,
glycosides 2-(3-O-
1-O- -glucopyranoside (1), 2-(4-O-
ethanol 1-O- -galactopyranoside (2) and 3-methoxy-4-O-
allopyranosyl acetophenone (3) (Fig. 1), and nine known com-
pounds 2-(3,4-dihydroxyphenyl) ethanediol 1-O- -glucopyra-
noside (4) (Wu et al., 2011), 4-O- -glucopyranosyl-(E)-ferulic
acid methyl ester (5) (Li et al., 2008), (2-methoxy-4-O-
glucopyranosyl) phenol 1-O- -glucopyranoside (6) (Lin et al.,
2007), p-hydroxybenzoyl-O- -galactopyranoside (7) (Kiso et al.,
b
-
D
-glucopyranosyl-4-hydroxyphenyl) ethanol
b
-D
b-D
-fructopyranosylphenyl)
b
-D
b-D-
b-D
b
-D
1
2
985; Joshi et al., 1991, 1993; Wang et al., 2002a,b, 2005; Li et al.,
004; Lin and Chen, 2009; Lin et al., 2009, 2010; Qu et al., 2011),
b-D-
b-D
but few non-alkaloid components were reported. However,
preliminary study results showed in addition to the basic parts,
acidic and neutral fractions of the ethanol extract of A. tanguticum
also had good anti-inflammatory and analgesic activities. So, since
b-D
2000), 4-hydroxy-3-methoxy-benzaldehyde (8) (Wang et al.,
2011), Loliolide (9) (Gao et al., 2006), (3R,5S,6S,7E,9R)-megastig-
man-7-ene-3,5,6,9-tetrol-9-O-
et al., 2006), (+)-pinoresinol (11) (Leng et al., 2003) and (2E,6R)-
,6-dimethyl-2,7-octadiene-1,6-diol (12) (Wang et al., 2002a,b)
b-D-glucopyranoside (10) (Sueyoshi
2
010 our research interest has focused on this species, particularly
2
were isolated from the ethanol extract of the plant. The structures
of new compounds were elucidated by means of spectroscopic
methods and the known compounds were identified by compari-
son with data reported in literature. All the known compounds
were isolated from this plant for the first time. According to the
medicinal use of the plant, all the isolated compounds were
*
Corresponding authors at: Institute of Chinese Materia Medica, China Academy
of Chinese Medical Sciences, Beijing 100700, China. Tel.: +86 10 64014411x2984;
fax: +86 10 64013996.
E-mail addresses: lichun@icmm.ac.cn (C. Li), zhimw123@263.net (Z.-M. Wang).
http://dx.doi.org/10.1016/j.phytol.2015.01.020
1
874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

3
12
Y.-R. Li et al. / Phytochemistry Letters 11 (2015) 311–315
HRESIMS, which was supported by its NMR data. The IR spectrum
ꢀ1
showed the presence of hydroxyl group (3370 cm ) and benzene
ꢀ
1
ring (1601 and 1516 cm ). The presence of a 3,4-dihydroxyphenyl
1
13
ethanol moiety was elucidated by its H, and C NMR spectra:
.13 (1H, d, J = 1.8 Hz), 6.76 (1H, d, J = 7.8 Hz) and 6.82 (1H, dd,
J = 1.8, 7.8 Hz), 4.08 (1H, dd, J = 2.4, 7.2 Hz), 3.72–3.74 (1H, m), 2.84
2H, d, J = 7.2 Hz); 130.4, 118.1, 145.1, 145.3, 115.6, 123.6,
0.4 and 35.1(Yu et al., 2012). The twelve carbon signals [ 102.9
2C), 76.9, 76.7, 76.6, 76.3, 73.7, 73.5, 70.3, 70.1, 61.4 and 61.2] and
two anomeric proton signals at 4.30 (1H, d, J = 7.8 Hz) and 4.77
1H, d, J = 7.2 Hz) were in agreement with two -glucopyranosyl
residues (Muhammad et al., 2012). In the HMBC spectrum, long
d
H
7
(
d
C
7
d
C
(
d
H
(
b-D
0
0
range correlations between
d
H
4.30 (H-1 ) and
d
C
70.4 (C-1), as well
000
0
as
d
H
4.77 (H-1 ) and
d
C
145.1 (C-3 ), demonstrated the linkage
of sugar units (Fig. 2). Therefore, compound 1 was elucidated as
-(3-O- -glucopyranosyl-4-hydroxyphenyl) ethanol 1-O-
glucopyranoside.
Compound 2 was isolated as a white amorphous powder. Its
molecular formula was established as C20 12 by HRESIMS (m/z
12Na 485.1629). The IR
2
b
-D
b-D-
30
H O
+
4
85.1622 [M+Na] , calcd. for C20
30
H O
ꢀ
1
spectrum displayed the presence of hydroxyl group (3368 cm
and benzene ring (1605 and 1516 cm ). The presence of a
)
ꢀ1
Fig. 1. Structures of compounds 1–3.
1
4
-dihydroxyphenyl ethanol moiety was elucidated by its H, and
1
3
C NMR spectra:
H
d 7.07 (2H, d, J = 8.4 Hz), 6.72 (2H, d, J = 8.4 Hz),
evaluated for their potential anti-inflammatory effects by the
inhibition of tumor necrosis factor alpha (TNF- ) production on
lipopolysaccharides (LPS)-induced RAW264.7 macrophages. Here-
in described are the isolation, structural elucidation, and biological
investigations of these compounds.
3.97 (1H, d, J = 7.2 Hz), 3.70–3.72 (1H, m) and 2.84–2.86 (2H, m); d
C
a
128.9, 114.5, 155.2, 70.5 and 35.1 (Li et al., 2014b). The rest of the
1
carbon signals in association with H NMR signals (Table 1)
suggested the presence of two hexose residues. The sugar residues
1
were deduced to be fructose and galactose by comparing the
H
1
3
and C NMR data with reported literature data (Li et al., 2014b;
Straubinger et al., 1997). Further acid hydrolysis of compound 2,
2
. Results and discussion
confirmed the sugar residues of D-fructose and D-galactose by gas
chromatography (GC) analysis, comparison of retention times of
sugars from hydrolysate with those of the authentic sugars. The
2.1. Structure elucidation
linkage of
D-galactosyl at C-1 was established based on HMBC
00
Compound 1 was obtained as a white amorphous powder. Its
correlation between H-1 (dH 4.30) with C-1 ( C
3.97 and 3.71) with C-1 (
could only be linked to C-4 (dC 155.2) by the NMR data analysis
(Fig. 2). On the basis of all the above evidences, compound 2 was
d
70.5) and H-1 ( H
d
00
molecular formula was determined as C20
H
30
O
13 with six degrees
d
103.1). The D-fructopyranosyl group
C
0
of unsaturation based on the observed pseudomolecular ion at
+
30
m/z 501.1564 [M+Na] (calcd. for C20H O13Na 501.1579) by
assigned as 2-(4-O-b-D-fructopyranosylphenyl) ethanol 1-O-b-D-
galactopyranoside.
20 8
Compound 3 was assigned the molecular formula C15H O
+
based on the HRESIMS at m/z 351.1054 [M+Na] (calcd. for
Na 351.1052). Its IR spectrum displayed the presence of
15 20 8
C H O
ꢀ
1
ꢀ1
13
hydroxyl (3433 cm ), carbonyl (1710 cm ) and benzene ring
ꢀ1
1
(
1603 and 1516 cm ) groups. The H and C NMR spectrum of 3
together with an HMQC experiment showed the presence of a
,3,4-trisubstituted benzene ring [ 7.47 (1H, d, J = 1.8 Hz), 7.13
1H, d, J = 9.0 Hz) and 7.55 (1H, dd, J = 1.8, 9.0 Hz)], one methoxyl
signal at [ 3.80 (3H, s) corresponding to 56.8], one methyl
2.47 (3H, s) corresponding to 26.0.], and one ketone
199.7). In addition, one anomeric proton resonance
5.28 (1H, d, J = 7.8 Hz), which correlated with signal
99.7 in the HMQC spectrum. These data suggested compound
was an apocynin glycoside (Zhang et al., 2006). A series of signals
1
(
d
H
d
H
d
C
signal at [
carbonyl (
d
H
d
C
d
C
appeared at
at
d
H
d
C
3
1
in H NMR at
3
3
d
H
5.28 (1H, d, 7.8 Hz), 3.56–3.68 (1H, m), 4.06 (1H, t,
.0), 3.52 (1H, dd, 3.0, 10.2), 3.77–3.78 (1H, m), 3.76 (1H, m) and
.57 (1H, m) indicated a rarely occurred -allopyranosyl unit in the
b
structure of 3. Also, it was confirmed by comparison NMR features
of those of 3,4-dimethoxy-trans-cinnamic acid-9-O- -allopyr-
anoside (Xu et al., 2013a) isolated from A. tanguticum, which
contained -allopyranose. Acidic hydrolysis afforded monosac-
charide component identified as -allose by the GC analysis. An
b
-D
b
D
unambiguous determination of the sequence and linkage sites was
obtained from the HMBC correlations. Thus, the following cross-
Fig. 2. The key HMBC correlations in compounds 1–3.
H
peaks were observed: the anomeric proton of allose (d 5.28) with

Y.-R. Li et al. / Phytochemistry Letters 11 (2015) 311–315
313
Table 1
1
13
3
H NMR (600 MHz) and C NMR (150 MHz) spectral data of compounds 1–3 (CD OD, d/ppm, J/Hz).
Position
1
2
3
d
H
d
C
d
H
d
C
d
H
d
C
1
2
4.08, 1H, dd (2.4, 7.2)
70.4
3.97, 1H, d (7.2)
3.70–3.72, 1H, m
2.82–2.84, 2H, m
70.5
2.47, 3H, s
26.0
3.72–3.74, 1H, m
2.84, 2H, t (7.2)
35.1
35.1
199.7
132.3
112.2
152.9
150.2
116.1
124.2
99.7
0
1
130.4
118.1
145.1
145.3
115.6
123.6
102.9
73.5
128.9
128.9
114.5
155.2
114.5
128.9
103.1
73.5
0
2
7.13, 1H, d (1.8)
7.07, 1H, d (8.4)
6.72, 1H, d (8.4)
7.47, 1H, d (1.8)
0
3
0
4
0
5
6.76, 1H, d (7.8)
6.72, 1H, d (8.4)
7.07, 1H, d (8.4)
4.30, 1H, d (7.8)
3.18–3.20, 1H, m
3.31–3.33, 1H, m
3.75–3.77, 1H, m
3.37–3.39, 1H, m
3.75–3.77, 2H, m
7.13, 1H, d (8.4)
0
6
6.82, 1H, dd (1.8, 7.8)
4.30, 1H, d (7.8)
7.55, 1H, dd (1.8, 8.4)
5.28, 1H, d (7.8)
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
3.18, 1H, dd (7.8, 9.0)
3.33–3.35, 1H, m
3.26–3.28, 1H, m
3.46–3.48, 1H, m
3.88, 1H, dd (1.2, 12.0)
3.56–3.58, 1H, m
4.06, 1H, t (3.0)
72.0
76.3
74.8
73.1
70.1
70.3
3.52, 1H, dd (3.0, 10.2)
3.77–3.79, 1H, m
3.75–3.77, 1H, m
3.56–3.58, 1H, m
68.5
76.7
76.3
75.8
61.4
61.8
62.6
3.71, 1H, dd (1.2, 12.0)
0
00
1
4.77, 1H, d (7.2)
102.9
4.04, 1H, d (12.0)
63.9
3
.56, 1H, d (12.0)
0
0
0
0
0
00
00
00
00
00
2
3
4
5
6
3.48–3.50, 1H, m
3.33–3.35, 1H, m
3.33–3.40, 1H, m
3.46–3.48, 1H, m
3.91, 1H, dd (2.4, 12.0)
73.7
76.6
70.3
76.9
61.2
100.5
69.0
70.3
69.9
59.6
3.90, 1H, d (9.6)
3.79–3.81, 1H, m
3.48–3.50, 1H, m
3.85, 1H, dd (1.8, 10.8)
3.73–3.75, 1H, m
3.66, 1H, dd (2.4, 12.0)
0
3
-OMe
3.80, 3H, s
56.8
0
0
0
d
C-4 (
d
C
150.2), H-2 (
d
H
7.47) and H-6 (
H
7.55) with C-2 (
d
C
199.7)
standard. ESIMS spectra were measured on an Agilent 6130SQ-
MSD, and HRESIMS on an Agilent 6250 Accurate-Mass Q-TOF
LC/MS spectrometer. Optical rotations were acquired using an
Autopol V plus polarimeter. IR spectra were recorded on a
Nicolet 5700 FT-IR spectrometer. GC data were recorded on an
Agilent 7890A instrument with a flame ionization detector.
Materials for column chromatography (CC) were silica gel
0
0
and C-4 (
proton of methoxyl (
the structure of compound 3 was established as 3-methoxy-4-O-
-allopyranosyl acetophenone.
d
C
150.2), H-1 (
d
H
2.47) and C-1 (
d
C
132.3), and the
0
d
H
3.80) with C-3 (
d
C
152.9) (Fig. 2). Therefore,
b-
D
2
.2. Anti-inflammatory activity
(
Qingdao Marine Chemical Ltd., China), octadecylsilyl (ODS)
silica gel (50 m, Fuji Silysia Chemical Ltd., Japan), Sephadex
LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and
MCI gel (50–75 m, Mitsubishi chemical corporation, Japan).
All the isolated compounds were evaluated for their inhibitory
activities against TNF- production on LPS-induced RAW264.7
macrophages. As a result, six compounds including 1, 3, 5 and 7–9
m
a
m
showed certain inhibitory activity and their IC50 values were 38.18,
Preparative HPLC was performed on a LC 3000 instrument
(Beijing Chuangxintongheng Science and Technology Co., Ltd.,
2
7.64, 3.25, 84.45, 12.76 and 18.44
mg/mL, respectively, while the
other compounds are basically invalid (IC50 >200
mg/mL) (Table 2).
Beijing, China; n = 10 mL/min) connected to a UV 3000 detector,
using an Inertsil C18 column (20 mm ꢁ 250 mm, 10
mm; GL
Sciences Inc., Tokyo, Japan).
3
. Experimental
.1. General experimental procedures
D and 2D NMR spectra were recorded on a Bruker Advance
3.2. Plant material
3
The whole plants of A. tanguticum were purchased from Xining
1
Sanjiangbao Trade Co., Ltd., Xining city, Qinghai province, China, in
August 2010, and identified by Prof. Xue-feng Feng. Voucher
specimens (No. 20100813) were kept in the Institute of Chinese
Materia Medica, China Academy of Chinese Medical Sciences.
6
3 3
00 spectrometer in CD OD, CDCl or DMSO with TMS as internal
Table 2
Inhibitory activities of compounds 1–12 against the
TNF-a
production on LPS-stimulated RAW 264.7 cells.
3.3. Extraction and isolation
a
Compound
IC50 (mg/mL)
1
2
3
4
5
6
7
8
9
38.18 ꢂ 0.4
The air-dried and powdered whole plants of A. tanguticum
a
–
(
10 kg) were extracted three times with 95% EtOH (200 L ꢁ 2 h) at
80 8C. After evaporation of the solvent under reduced pressure, the
residue was suspended in and then partitioned with
3.25 ꢂ 0.1
a
–
2
H O
27.64 ꢂ 0.2
a
–
petroleum ether (PE) and ethyl acetate (EtOAc) sequentially. The
two organic partitions were separately evaporated to obtain the
PE-soluble fraction (256 g) and EtOAc-soluble fraction (130 g),
respectively. The residual aqueous solution was chromatographed
on AB-8 macroporous resin and then successively eluted with
different concentration of EtOH (0, 30%, 60% and 95% EtOH). This
procedure yielded 605 g, 407 g, 107 g and 1.23 g of the water-, 30%
EtOH-, 60% EtOH- and 95% EtOH -soluble fractions.
84.45 ꢂ 0.7
12.76 ꢂ 0.3
18.44 ꢂ 0.2
a
1
1
1
0
1
2
–
a
–
a
–
Baicalein
13.42 ꢂ 0.3
a
IC50 > 200 mg/mL.

3
14
Y.-R. Li et al. / Phytochemistry Letters 11 (2015) 311–315
The water-soluble fraction (605 g) was subjected to column
chromatography (CC) over silica gel eluting with a gradient of
CHCl –MeOH–H O (8:2:0.125, 7:3:0.5, 6:4:1, 5:5:1) to afford eight
fractions (Fr.1–Fr.8). Fr. 4 (200 g) was further separated on silica
gel CC eluting with CHCl –MeOH gradient (9:1–7:3) to give six
subfractions. Fr. 4–2 (5 g) was further purified by preparative HPLC
MeOH–H O, 16:84) to yield 4 (t 14.2 min, 13 mg) and 6 (t
3.0 min, 8 mg). Fr. 4–5 (22 g) was subjected to an ODS column
using a gradient elution of MeOH–H O (1:9–9:1) into six fractions.
Fr. 4-5-3 (2.2 g) was further purified by preparative HPLC (MeOH–
O, 1:9) to give 1 (t 28.2 min, 20 mg). Fr. 4-5-4 (1.1 g) was further
separated by Sephadex LH-20 (CHCl –MeOH, 1:1) to afford 2
7 mg) and 10 (13 mg).
A portion of the EtOAc-soluble fraction (78 g) was subjected to
silica gel CC using CHCl –MeOH gradient elution (25:1, 10:1, 8:2,
:5) into 10 fractions (Fr.1–Fr.10). Fr.1 (10 g) was further separated
by silica gel CC eluting with a gradient of PE–acetone (50:1, 25:1,
0:1, 8:2) to give seven subfractions. Fr.1–2 (200 mg) was further
separated on MCI (CHCl –MeOH, 1:1) and preparative HPLC
MeOH–H O, 25:75) to afford 8 (t 28.1 min, 5 mg). Fr.1–3 (1 g)
was successively separated by MCI (CHCl –MeOH, 1:1) and
preparative HPLC (MeOH–H O, 30:70) to give 7 (t 29.0 min,
0 mg). Fr.1–4 (800 mg) was separated by MCI (CHCl –MeOH, 1:1),
then chromatographed on Sephadex LH-20 (CHCl –MeOH, 1:1),
finally purified by preparative HPLC (MeOH–H O, 12:88) to obtain
(t 25.2 min, 50 mg). Fr.1–5 (1.2 g) was successively purified on
Sephadex LH-20 (CHCl –MeOH, 1:1) and preparative HPLC
MeOH–H O, 12:88) to give 11 (t 15.7 min, 13 mg). Fr.5 (8 g)
was successively separated on MCI (CHCl –MeOH, 1:1), Sephadex
LH-20 (CHCl –MeOH, 1:1) and preparative HPLC (MeOH–H
2:88) to afford 12 (t 25.0 min, 25 mg). Fr.7 (5.5 g) was eluted
with a gradient of CHCl –MeOH (1:9–10:0) on MCI to furnish three
subfractions. Fr.7-1 (1.2 g) was passed over a Sephadex LH-20
column (CHCl –MeOH, 1:1) to obtain 3 (5 mg). Fr.7-2 (900 mg) was
purified on preparative HPLC (MeOH–H
2.3 min, 7 mg).
evaporated to give the monosaccharide residue. The residue was
dissolved in pyridine (100 L), to which 100 L of 0.06 mol/L
-cysteine methyl ester hydrochloride pyridine solution was
added. This mixture was heated at 60 8C for 1 h. After cooled to
room temperature, 150 L of HMDS-TMCS (hexamethyldisila-
zane/ trimethylchorosilane, 1:1) was added to the mixture. The
resultant reaction mixture was kept at 60 8C for another 0.5 h.
The mixture was centrifuged and the supernatant was analyzed
by GC under the following conditions: capillary column, HP-5
m
m
3
2
L
3
m
(
1
2
R
R
2
(30 m ꢁ 0.32 mm ꢁ 0.25
ture, 250 8C; injection temperature, 250 8C; column temperature
230 8C and the temperature was maintained for 30 min; carrier, N
mm); detection, FID; detector tempera-
H
2
R
3
2
(
gas; flow rate, 1.0 mL/min. Peaks of the hydrolysates of 2 and 3
were detected by comparison with retention times of authentic
3
samples of
D
-fructose (t
-galactose (t
12.696) after being treated simultaneously in the same manner.
R
10.000),
L
-fructose (t
R
9.744),
13.099) and
D
-galactose
5
(t
(t
R
R
12.651),
L
R
12.689),
D
-allose (t
R
L-allose
1
3
3.5. Inhibitory activities against TNF-
RAW 264.7 macrophages
a production on LPS-stimulated
(
2
R
3
2
R
As literature reported (Yang et al., 2008), RAW 264.7 cells were
4
1
3
seeded onto 96-well plates at a density of 1 ꢁ 10 cells per well and
3
2
incubated at 37 8C with 5% CO for 24 h. Then, the cells were
2
divided into four groups, namely control group, sample group,
LPS group and LPS + sample group. The sample group cells
were treated with different concentrations ranging from 1.6 to
9
R
3
(
2
R
1000
treated with different concentrations of samples, followed by
stimulation with LPS (final concentration was 1 g/mL). LPS group
cells were only treated with LPS. After co-incubation for 24 h,
100 L of cell free supernatant were collected for ELISA in the
LPS + sample group. For the sample group cells, the culture
medium were removed and then 50 L of MTT (1 mg/mL) was
mg/mL of tested samples, and LPS + sample group cells were
3
3
2
O
m
1
R
3
m
3
m
2
O, 3:7) to give 5 (t
R
added to each well, next the cells were further incubated for
additional 4 h. The supernatant was removed and cells were
2
lysed with 200
mL/well DMSO. Cell viability was determined by
3
.3.1. 2-(3-O-
b
-
D
-glucopyranosyl-4-hydroxyphenyl) ethanol 1-O-
b
-
measuring the optical density at 570 nm using a microplate
reader (Kendro, Germany). The supernatant without obvious
D
-glucopyranoside (1)
2
D
0
White amorphous powder; ½
aꢃ
ꢀ39.9 (c 0.05, MeOH); UV
cell growth inhibitory effect were selected for TNF-
a analysis.
MeOH
max
KBr
max
1
ꢀ1
1
l
: 280 nm; IR
n
: 3370, 1601, and 1516 cm
;
H NMR
TNF- was determined using the murine TNF- ELISA kit (BD
a
a
3
(CD
3
OD, 600 MHz) and C NMR (CD
3
OD, 150 MHz) spectral data
Biosciences, USA) following the manufacture’s instruction.
ꢀ
ꢀ
see Table 1; (ꢀ) ESIMS m/z 513 [M+Cl] , 477 [MꢀH] ; (+) HRESIMS
30
+
m/z 501.1572 [M+Na] (calcd. for C20
H
O
13Na, 501.1579).
3.6. Statistical assessment for the bioassays
3
.3.2. 2-(4-O- -fructopyranosyl phenyl) ethanol 1-O-
b
-
D
b
-
D
-
The bioassay was performed three times. The data were fed to
the microcomputer program Statistical Package for Social Science
SPSS (version 13.0). The statistical reliability were expressed as
mean ꢂ SD.
galactopyranoside (2)
2
D
0
White amorphous powder; ½
aꢃ
ꢀ60.0 (c 0.05, MeOH); UV
ꢀ1 1
MeOH
max
KBr
max
3
l
: 220, 275 nm; IR
n
: 3368, 1605, and 1516 cm ; H NMR
OD, 150 MHz) spectral data
1
(CD
3
OD, 600 MHz) and C NMR (CD
3
+
Acknowledgments
see Table 1; (+) ESIMS m/z 485 [M+Na] ; (+) HRESIMS m/z
4
+
85.1622 [M+Na] (calcd. for C20
30
H O12Na, 485.1629).
This work was jointly financially supported by the Natural
Science Foundation of Beijing (7132152), the Fundamental
Research Funds for the Central Public Welfare Research Institutes
(ZZ070828) and the National Basic Research Program of China
3
.3.3. 3-Methoxy-4-O- -allopyranosyl acetophenone (3)
b-D
2
0
White amorphous powder; ½
a
ꢃ
ꢀ28.0 (c 0.05, MeOH); UV
ꢀ1 1
D
MeOH
max
KBr
max
l
: 282 nm; IR
n
: 3433, 1710, 1603, and 1516 cm
; H
13
(
973 Program) (2012CB724001-21).
NMR (CD
3 3
OD, 600 MHz) and C NMR (CD OD,150 MHz) spectral
+
data see Table 1; (+) ESIMS m/z 351 [M+Na] ; (+) HRESIMS m/z
3
+
Appendix A. Supplementary data
20 8
51.1054 [M+Na] (calcd. for C15H O Na, 351.1052).
Supplementary data associated with this article can be found, in
theonline version, at http://dx.doi.org/10.1016/j.phytol.2015.01.020.
3.4. Acid hydrolysis of compounds 2 and 3
Compounds 2 and 3 (each 2.0 mg) were separately hydrolyzed
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