Triterpenoids and Flavonoids from the Leaves of Astragalus membranaceus and Their Inhibitory Effects on Nitric Oxide Production

Article abstract of DOI:10.1002/cbdv.201400371

Four new cycloartane triterpenes, named huangqiyegenins V and VI and huangqiyenins K and L (1-4, resp.), together with nine known triterpenoids, 5-13, and eight flavonoids, 14-21, were isolated from a 70%-EtOH extract of Astragalus membranaceus leaves. The structures of the new compounds were elucidated by detailed spectroscopic analyses, and the compounds were identified as (9β,11α,16β,20R,24S)-11,16,25-trihydroxy-20,24-epoxy-9,19-cyclolanostane-3,6-dione (1), (9β,16β,24S)-16,24,25-trihydroxy-9,19-cyclolanostane-3,6-dione (2), (3β,6α,9β,16β,20R,24R)-16,25-dihydroxy-3-(β-D-xylopyranosyloxy)-20,24-epoxy-9,19-cyclolanostan-6-yl acetate (3), and (3β,6α,9β,16β,24E)-26-(β-D-glucopyranosyloxy)-16-hydroxy-3-(β-D-xylopyranosyloxy)-9,19-cyclolanost-24-en-6-yl acetate (4). All isolated compounds were evaluated for their inhibitory activities against LPS-induced NO production in RAW264.7 macrophage cells. Compounds 1-3, 14, 15, and 18 exhibited strong inhibition on LPS-induced NO release by macrophages with IC₅₀ values of 14.4-27.1μM.

Full text of DOI:10.1002/cbdv.201400371

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1575
Triterpenoids and Flavonoids from the Leaves of Astragalus membranaceus
and Their Inhibitory Effects on Nitric Oxide Production
a
d
a
a
b
d
c
by Zhi-Bin Wang ) ), Ya-Dong Zhai ), Zhen-Ping Ma ), Chun-Juan Yang ) ), Rong Pan ),
a
a
a
a
Jia-Li Yu ), Qiu-Hong Wang ), Bing-You Yang ), and Hai-Xue Kuang* )
a) Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of
Chinese Medicine, 24 Heping Road, Harbin 150040, P. R. China
(
phone: þ86-451-82193001; fax: þ86-451-82110803; e-mail: hxkuang@yahoo.com)
b
)
College of Pharmacy, Harbin Medical University, Harbin 150081, P. R. China
c
)
Austar Pharma LLC, 18 Mayfield Ave, Edison, NJ 08837, USA
d) Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of
New Jersey, Piscataway, NJ 08854, USA
Four new cycloartane triterpenes, named huangqiyegenins V and VI and huangqiyenins K and L
(1–4, resp.), together with nine known triterpenoids, 5–13, and eight flavonoids, 14–21, were isolated
from a 70%-EtOH extract of Astragalus membranaceus leaves. The structures of the new compounds
were elucidated by detailed spectroscopic analyses, and the compounds were identified as
(
1
9b,11a,16b,20R,24S)-11,16,25-trihydroxy-20,24-epoxy-9,19-cyclolanostane-3,6-dione (1), (9b,16b,24S)-
6,24,25-trihydroxy-9,19-cyclolanostane-3,6-dione (2), (3b,6a,9b,16b,20R,24R)-16,25-dihydroxy-3-(b-d-
xylopyranosyloxy)-20,24-epoxy-9,19-cyclolanostan-6-yl acetate (3), and (3b,6a,9b,16b,24E)-26-(b-d-
glucopyranosyloxy)-16-hydroxy-3-(b-d-xylopyranosyloxy)-9,19-cyclolanost-24-en-6-yl acetate (4). All
isolated compounds were evaluated for their inhibitory activities against LPS-induced NO production in
RAW264.7 macrophage cells. Compounds 1–3, 14, 15, and 18 exhibited strong inhibition on LPS-induced
NO release by macrophages with IC50 values of 14.4–27.1 mm.
Introduction. – Dried roots of Astragalus membranaceus have been well used in
traditional Chinese medicine as antiperspirants, diuretics, tonics, etc. [1]. A previous
chemical investigation of this plant revealed that the major chemical constituents were
saponins, flavonoids, and polysaccharides [2]. It also includes components such as
alkaloids, amino acids, b-sitosterol, and metallic elements. In addition, the leaves of A.
membranaceus have many biological functions including anticaducity, anti-inflamma-
tory, and antioxidation activities [3], and protection against pancreatic injury [4]. Dried
leaves of A. membranaceus (named beiqishencha) were authorized as functional food
by the State Food and Drug Administration of P. R. China in 1998.
The structure of 9,19-cycloartane-type saponins has drawn more and more attention
of chemists ever since cycloartane triterpenoids were first discovered in Astragalus
plants [5]. Up to now, more than 150 uncommon cycloartane triterpenoids have
been isolated from Astragalus plants [6]. The Astragalus saponins have been studied
extensively during the past 30 years and have been reported to have a wide range of
biological properties [7], such as hepatoprotection [7a], cardioprotection [7b],
antidiabetic nephropathy [7c], anti-inflammatory [7d], gastroprotection [7e], and
neuroprotection activities [7f]. In the process of our continuing efforts to study this
ꢀ
2015 Verlag Helvetica Chimica Acta AG, Zürich

1
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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
source, four new cycloartane-type triterpenoids, huangqiyegenin V (1), huangqiyege-
nin VI (2), huangqiyenin K (3), and huangqiyenin L (4), together with 17 known com-
pounds, 5–21 (Fig. 1), were isolated from a 70%-EtOH extract of A. membranaceus
leaves. These isolated compounds were evaluated for their inhibitory effects on
lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells. Here, we
describe the structural elucidation of the four new cycloartane triterpenoids and NO
inhibitory effects of all isolated compounds.
Results and Discussion. – Structure Elucidation. Huangqiyegenin V (1) was
obtained as white amorphous powder. The molecular formula, C H O , was
30
46
6
þ
determined by positive-ion mode HR-ESI-MS (m/z 525.3194 ([MþNa] ; calc.
À1
5
(
25.3187)). The IR spectrum of 1 exhibited absorptions at 3393 (OH) and 1705 cm
1
C¼O). The H-NMR spectrum (Table 1) of 1 revealed the presence of seven Me
groups resonating at d(H) 1.80, 1.59, 1.54, 1.34, 1.29, 1.26, and 0.94, and three CHÀO
groups at 5.08 (ddd, J¼14.0, 7.7, 2.8), 4.00 (br. d), and 3.85 (dd, J¼9.0, 5.0). The
13
C-NMR spectrum (Table 1) of 1 exhibited signals of two C¼O groups at d(C) 213.9
and 211.2. A comparative study of the NMR data (Table 1) of 1 with those of
huangqiyegenin I indicated that the structures of these two compounds are very similar
[
8]. In the HMBC spectrum (Fig. 2), the correlations of CH (1) (d(H) 1.78–1.86 and
2
1
.98–2.07) with C(3) (d(C) 213.9) and of HÀC(8) (d(H) 3.20) with C(6) (d(C) 211.2)
indicated that the two C¼O groups are located at C(3) and C(6). Moreover, the
correlation of CH (19) (d(H) 1.22) with C(11) (d(C) 68.2) in the HMBC spectrum
2
(
Fig. 2) showed that a OH group is attached to C(11). In the NOESY spectrum, the
correlation between Me(21) (d(H) 1.34 (s)) and HÀC(17) (2.60 (d, J¼7.7)), and the
correlation between HÀC(17) and HÀC(24) (3.85 (dd, J¼9.0, 5.0)) indicated that
HÀC(17) is a-oriented. Furthermore, the cross-peaks HÀC(11) (d(H) 4.00)/Me(18)
(
1.80) and HÀC(16) (5.08)/Me(30) (0.94) indicated that HÀC(11) is b- and HÀC(16) is
a-oriented. Therefore, 1 was established to be (9b,11a,16b,20R,24S)-11,16,25-trihy-
droxy-20,24-epoxy-9,19-cyclolanostane-3,6-dione.
Huangqiyegenin VI (2) was obtained as white amorphous powder with a molec-
ular formula established as C H O on the basis of HR-ESI-MS (m/z 511.3396
3
0
48
5
þ
(
(
[MþNa] ; calc. 511.3394)). The IR spectrum revealed the presence of OH
3404 cm ) and C¼O groups (1728 cm ). The H-NMR spectrum (Table 1) of 2
À1
À1
1
revealed the presence of six quaternary Me groups resonating at d(H) 1.60, 1.48, 1.45,
13
1
.27, 1.06, and 0.86, and two CHÀO groups at 4.69–4.70 and 3.93. The C-NMR
spectrum (Table 1) of 2 exhibited signals of two C¼O groups at d(C) 214.0 and 211.0.
The NMR data were similar to those of huangqiyegenin II [8], except for the presence
of an additional C¼O group in 2. In the HMBC spectrum (Fig. 2), the correlations of
CH (1) (d(H) 2.05–2.08 and 1.53–1.58) with C(3) (d(C) 214.0) and of HÀC(8) (d(H)
2
2
.68) with C(6) (d(C) 211.0) indicated that the two C¼O groups are located at C(3) and
C(6). Thus, 2 was assigned as (9b,16b,24S)-16,24,25-trihydroxy-9,19-cyclolanostane-
,6-dione.
Huangqiyenin K (3), obtained as white amorphous powder, has the molec-
ular formula of C H O , as established on the basis of HR-ESI-MS data (m/z
3
3
þ
7
60 10
6
(
65.4261 ([MþH] ; calc. 665.4259)). The IR spectrum indicated the presence of OH
À1
À1
3393 cm ) and C¼O groups (1726 cm ). The NMR data of 3 (Table 2) were in good

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1577
Fig. 1. Structures of 1–21

1
578
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 1. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; in C D N) of 1 and 2. d in ppm, J in Hz.
5
5
Position
1
2
d(H)
d(C)
d(H)
d(C)
1
2
3
4
5
6
7
1.78–1.86 (m), 1.98–2.07 (m)
2.57–2.64 (m)
31.2
36.0
213.9
50.2
56.5
211.2
41.5
1.53–1.58 (m), 2.05–2.08 (m)
2.61–2.63 (m)
30.6
36.1
214.0
50.1
56.4
211.0
41.4
2.96 (s)
2.89 (s)
2.39 (dd, J¼17.0, 7.8),
2.30–2.31 (m), 2.61–2.63 (m)
2.68 (dd, J¼7.5, 3.4)
2
.42 (dd, J¼17.0, 3.4)
8
9
3.20 (dd, J¼7.8, 3.4)
40.6
29.5
30.0
68.2
42.5
45.4
47.4
44.2
73.1
57.9
21.3
19.3
87.3
28.7
34.9
26.5
81.6
71.4
28.2
27.1
25.7
20.6
19.5
42.1
22.4
28.6
26.6
33.2
46.0
47.6
45.7
71.3
56.5
18.5
20.5
28.8
15.5
32.7
28.0
77.3
72.5
25.8
26.5
25.6
20.6
19.0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4.00 (br. d)
1.98–2.07 (m), 2.19–2.25 (m)
1.34–1.35 (m), 1.56–1.57 (m)
1.56–1.57 (m), 2.22–2.24 (m)
1.85 (d, J¼6.4), 1.98–2.07 (m)
5.08 (ddd, J¼14.0, 7.7, 2.8)
2.60 (d, J¼7.7)
1.78–1.79 (m), 2.05–2.08 (m)
4.69–4.70 (m)
1.78–1.79 (m)
1.80 (s)
1.22 (s)
1.06 (s)
0.24 (d, J¼4.8), 0.97 (d, J¼4.8)
2.30–2.33 (m)
1.34 (s)
1.27 (s)
1.65–1.66 (m), 3.10–3.13 (m)
1.98–2.07 (m), 2.22–2.25 (m)
3.85 (dd, J¼9.0, 5.0)
1.38–1.39 (m), 1.56–1.57 (m)
1.78–1.79 (m), 1.94–1.96 (m)
3.93 (dd, J¼10.8, 2.4)
1.54 (s)
1.26 (s)
1.29 (s)
1.59 (s)
0.94 (s)
1.48 (s)
1.45 (s)
1.27 (s)
1.60 (s)
0.86 (s)
agreement with those of huangqiyenin D [9] except for the configurations at C(24) and
1
13
the sugar moiety positioned at C(3). The H- and C-NMR spectra of 3 (Table 2)
indicated the presence of a pentose moiety with the anomeric H-atom resonating at
d(H) 4.85 (d, J¼7.6). Acid hydrolysis of 3 afforded d-xylose detected by GC [10]. The
coupling constant (J¼7.6) of the anomeric H-atom suggested that the anomeric C-
atom of the d-xylopyranose moiety is b-configured. In the HMBC spectrum (Fig. 2),
the long-range correlations of HÀC(6) (d(H) 4.97–5.00) with the ester C¼O C-atom
(
d(C) 170.4) indicated that the AcO group is linked to C(6), and the significant HMB
correlation of the anomeric H-atom resonating at d(H) 4.85 (d, J¼7.6) with C(3) (d(C)
8
7.6) indicated that the sugar unit is located at C(3). In the NOESY spectrum, a cross-
peak between Me(21) (d(H) 1.42 (s)) and Me(26) (1.28 (s)) was observed. Moreover,
the NOESY correlation between Me(21) and HÀC(17) (2.28 (d, J¼7.7)), as well as
comparison with the NMR data of huangqiyenin D, suggested that the side chain at

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1579
1
1
Fig. 2. Key H, H-COSY (—) and selected HMB (H!C) correlations of 1–4
C(17) is b-oriented. Thus, the structure of 3 was elucidated as (3b,6a,9b,16b,20R,24R)-
1
6,25-dihydroxy-3-(b-d-xylopyranosyloxy)-20,24-epoxy-9,19-cyclolanostan-6-yl acetate.
Huangqiyenin L (4) was obtained as white amorphous powder. Its molecular
formula was determined as C H O by positive-ion mode HR-ESI-MS (m/z 811.4847
4
3
70 14
þ
(
[MþH] ; calc. 811.4838)). The IR spectrum indicated the presence of an ester C¼O
group (1728 cm ) and an olefinic moiety (1634 cm ). The C-NMR spectrum
Table 2) of 4 revealed a total of 43 C-atom signals due to the aglycon moiety (32 C-
atom signals) along with two sugar units (eleven C-atom signals). The NMR data
Table 2) of 4 were similar to those of kahiricoside V [11], except for the sugar moiety
À1
À1
13
(
(
and the signals of one additional AcO group. Acid hydrolysis of 4 afforded d-xylose
and d-glucose (GC evidence) [10]. The HMBCs (Fig. 2) of HÀC(3) (d(H) 3.53 (dd, J¼
1
1.0, 4.0)) with C(1’) (d(C) 107.8) and of CH (27) (d(H) 4.25 (d, J¼11.6) and 4.46 (d,
2
J¼11.6)) with C(1’’) (d(C) 103.5) suggested that d-xylopyranose and d-glucopyranose
are located at C(3) and C(27), respectively. Moreover, it is obvious that the AcO group
is located at C(6), based on comparison with the NMR data of 3. Hence, 4 was
elucidated as (3b,6a,9b,16b,24E)-26-(b-d-glucopyranosyloxy)-16-hydroxy-3-(b-d-
xylopyranosyloxy)-9,19-cyclolanost-24-en-6-yl acetate.
The known triterpenoids were identified as huangqiyesaponin C (5) [12],
astragaloside II (6) [13], huangqiyegenin I (7) [8], astragaloside IV (8) [14],
soyasapogenol E (9) [15], (3b,21a)-olean-12-ene-3,21,24-triol (10) [16], (3b,22b)-
olean-12-ene-3,22,24,29-tetrol (11) [17], soyasapogenol B (12) [18], and soyasapogenol
B
3-O-a-l-rhamnopyranosyl-(1!2)-b-d-glucopyranosyl-(1!4)-b-d-glucopyranosi-

1
580
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 2. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; in C D N) of 3 and 4. d in ppm, J in Hz.
5
5
Position
3
4
d(H)
d(C)
d(H)
d(C)
1
2
3
4
5
6
7
8
9
1.20–1.22 (m), 1.64–1.66 (m)
1.96–1.97 (m), 2.35–2.41 (m)
3.53 (dd, J¼11.3, 3.3)
32.1
30.1
87.6
42.3
50.2
70.8
33.3
45.4
20.9
28.6
25.6
33.4
46.5
46.8
48.8
73.0
56.6
20.9
29.0
87.7
29.3
38.4
26.1
87.7
70.3
26.4
27.2
27.0
16.6
20.2
107.8
75.6
78.7
71.3
67.2
1.23–1.26 (m), 1.62–1.64 (m)
1.94–1.97 (m), 2.37–2.42 (m)
3.53 (dd, J¼11.0, 4.0)
32.1
30.1
87.7
42.3
50.1
70.8
33.4
45.5
21.1
28.3
26.0
33.1
45.8
46.7
48.9
71.1
56.8
18.7
28.6
30.7
18.2
36.6
25.6
129.5
131.9
14.3
75.2
27.0
16.6
20.0
107.8
75.6
78.7
71.2
67.2
1.80 (d, J¼5.5)
1.85 (d, J¼9.0)
4.97–5.00 (m)
4.99–5.01 (m)
1.44–1.45 (m), 1.74–1.76 (m)
1.99 (dd, J¼10.4, 4.2)
1.48–1.53 (m), 1.71–1.73 (m)
1.94–1.97 (m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1
2
3
4
5
1.85–1.87 (m), 2.09–2.15 (m)
1.44–1.45 (m), 1.73–1.74 (m)
1.23–1.26 (m), 1.85 (d, J¼9.0)
1.62–1.64 (m)
1.77–1.79 (m), 2.07–2.09 (m)
4.69–4.73 (m)
1.69–1.70 (m), 2.08–2.11 (m)
4.57–4.63 (m)
1.80–1.82 (m)
2.28 (d, J¼7.7)
1.49 (s)
1.32 (s)
0.23 (d, J¼4.3), 0.50 (d, J¼4.3)
0.22 (d, J¼4.0), 0.50 (d, J¼4.0)
2.22–2.27 (m)
1.42 (s)
1.03 (d, J¼6.6)
1.83–1.85 (m), 2.57–2.62 (m)
1.90–1.93 (m), 2.12–2.15 (m)
4.10 (dd, J¼10.4, 5.7)
1.24–1.25 (m), 2.08–2.11 (m)
2.13–2.15 (m), 2.22–2.27 (m)
5.72 (t, J¼6.0)
1.28 (s)
1.41 (s)
1.16 (s)
1.39 (s)
1.80 (s)
4.25 (d, J¼11.6), 4.46 (d, J¼11.6)
1.17 (s)
1.42 (s)
0.99 (s)
0.96 (s)
’
’
’
’
’
4.85 (d, J¼7.6)
4.03–4.05 (m)
4.16–4.18 (m)
4.23–4.28 (m)
3.76 (dd, J¼11.0, 10.1),
4.85 (d, J¼7.5)
4.02–4.08 (m)
4.14–4.18 (m)
4.22–4.28 (m)
3.77 (dd, J¼11.0, 10.1),
4.38 (dd, J¼11.0, 5.0)
4.89 (d, J¼7.8)
4.22–4.28 (m)
4.22–4.28 (m)
4.22–4.28 (m)
3.93–3.98 (m)
4.38 (dd, J¼11.0, 5.0),
4
.38 (dd, J¼11.0, 5.0)
1
2
3
4
5
6
’’
’’
’’
’’
’’
’’
103.5
75.3
78.7
71.8
78.5
62.9
4
.58 (d, J¼11.0)
AcO
2.04 (s)
21.8,
2.03 (s)
21.8,
1
70.4
170.3

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1581
Table 3. Inhibitory Effects of Compounds on NO Production in LPS-Stimulated RAW264.7 Cells
Compound
IC ÆSD [mm]
50
Compound
IC ÆSD [mm]
50
1
2
3
4
5
9
21.5Æ4.5
14.7Æ2.3
27.1Æ3.0
64.9Æ7.6
73.6Æ10.0
63.0Æ11.6
98.5Æ12.9
95.6Æ11.6
14
15
17
18
20
21
14.8Æ3.8
14.4Æ2.5
31.2Æ3.6
21.2Æ4.8
44.4Æ11.0
50.0Æ12.6
23.1Æ4.9
>100
1
1
0
2
Hydrocortisone
Other compounds )
a
a) Specifically 6–8, 11, 13, 16, and 19.
duronic acid (13) [19], and the eight known flavonoids were rhamnocitin 3-O-b-d-
glucopyranoside (14) [9], quercetin (15) [20], quercetin 3-O-b-d-glucopyranoside (16)
[
21], rhamnocitin 3-O-b-neohesperidoside (17) [22], complanaruside (18) [23],
calycosin 7-O-b-d-glucopyranoside (19) [24], 4’,7-dihydroxy-3’-methoxyisoflavone
(
20) [25], and formononetin (21) [26] (Fig. 1).
Biological Assays. All isolated compounds were examined for their inhibitory
effects on the release of NO from macrophages using LPS-induced RAW264.7 cells as
model system. Firstly, the noncytotoxic concentrations of the compounds toward
macrophage RAW264.7 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay. Compounds 1–3, 14, 15, and 18
exhibited strong inhibitory activities against NO production in noncytotoxic concen-
trations with IC50 values of 14.4–27.1 mm (Table 3), with hydrocortisone (IC50 23.1Æ
4
.9 mm) being used as positive control [27].
Conclusions. – Investigation of the EtOH extract of A. membranaceus leaves led to
the isolation of 13 triterpenoids, 1–13, and eight flavonoids, 14–21, of which four, i.e.,
huangqiyegenin V (1), huangqiyegenin VI (2), huangqiyenin K (3), and huangqiyenin
L (4), are new cycloartane triterpenes. Compounds 1–3, 14, 15, and 18 possess strong
inhibitory effects on LPS-induced NO production in RAW264.7 cells. These results
indicate that aglycons seem to show stronger activities than glycosides on the inhibition
of NO production. Thus, this work provided valuable information on the structural
features of cycloartane triterpenes and the potential anti-inflammatory activity of A.
membranaceus leaves.
This research program was supported financially by the National Science and Technology Major
Project (2009ZX09308-004), Program for Excellent Talents in Heilongjiang University of Chinese
Medicine (2012cj02), and the grant from Heilongjiang University of Chinese Medicine (2012RCD05).
Experimental Part
General. Thin layer chromatography (TLC): precoated silica gel F254 plates (SiO ; Merck). Column
2
chromatography (CC): SiO (200–300 mesh; Qingdao Marine Chemical Industry), ODS (50 mm; YMC),
2
and Sephadex LH-20 (Pharmacia). Semi-prep. HPLC: Sunfire C18 column (10Â250 mm, 5 mm); Waters
À1
Delata-600 pump; Waters RID-2414 detector; flow rate, 3.0 ml min . GC: DM-5 column (30 mÂ

1
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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
0
2
.25 mm, 0.25 mm; Dikma, P. R. China); Fuli-9790 hydrogen flame detector. Optical rotations: Jasco P-
À1
1
000 polarimeter. IR Spectra: Shimadzu FTIR-8400S infrared spectrometer; KBr pellets; n˜in cm . H-
13
and C-NMR spectra: Bruker DPX-400 spectrometer (400 and 100 MHz, resp.); in C D N; at r.t.; d in
5
5
ppm rel. to Me Si as internal standard, J in Hz. HR-ESI-MS (pos.): Waters Xero Q Tof MS spectrometer;
4
in m/z. Microplate reader: PerkinElmer VICTORꢀ X3 multilabel plate reader.
Plant Material. Leaves of A. membranaceus were collected in July 2010 from Daxinganling,
Heilongjiang Province, P. R. China. The botanical identification was made by Prof. Zhen-Yue Wang,
School of Pharmacy, Heilongjiang University of Chinese Medicine. A voucher specimen (Herbarium
No. 20100706) was deposited with the School of Pharmacy, Heilongjiang University of Chinese Medicine,
P. R. China.
Extraction and Isolation. Dried leaves of A. membranaceus (10.0 kg), which were divided into four
equal parts, were extracted three times under reflux conditions with 75% aq. EtOH (each 3.0 l, 2 h,
2
(
(
.5 kg). The combined EtOH extracts were concentrated in vacuo to yield a syrup-like residue
1395.0 g), which was dissolved in H O (5.0 l) and then portioned between petroleum ether-soluble
50.0 g), AcOEt-soluble (190.0 g), BuOH-soluble (195.0 g), and H O-soluble (960.0 g) portions. The
2
2
BuOH-soluble portion (120.0 g) was subjected to CC (SiO ; CH Cl /MeOH 100 :1!0 :1, gradient) to
2
2
2
furnish ten fractions, Frs. 1–10. Fr. 2 (5.8 g) was successively subjected to CC (ODS; MeOH/H O 3 :7!
2
7
:3) to yield four fractions, Frs. 2.1–2.4. Fr. 2.2 (1.6 g) was further separated by CC (Sephadex LH-20;
MeOH) to afford 19 (12.5 mg) and 21 (23.2 mg). Fr. 3 (7.5 g) was further subjected to CC (ODS; MeOH/
H O 3 :7!7:3) to afford six fractions, Frs. 3.1–3.6. Compound 14 (200.0 mg) was crystallized directly
2
with MeOH from Fr. 3.3 (1.8 g). The mother liquor of Fr. 3.3 was purified by semi-prep. HPLC (MeOH/
H O 68 :32) to give 2 (12.5 mg). Fr. 3.4 (680.0 mg) was further separated by semi-prep. HPLC (MeOH/
2
H O 3 :1) to afford 20 (15.8 mg). Fr. 3.5 (527.0 mg) was further purified by semi-prep. HPLC (MeOH/
2
H O 53 :47) to give 3 (19.0 mg), 6 (12.5 mg), and 8 (7.1 mg). Fr. 5 (3.6 g) was successively subjected to
2
CC (ODS; MeOH/H O 3 :7!7:3) to give five fractions, Frs. 5.1–5.5. Fr. 5.2 (830.0 mg) was further
2
separated by semi-prep. HPLC (MeOH/H O 3 :1) to afford 9 (15.8 mg) and 12 (18.5 mg). Compounds 11
2
(
(
7.1 mg) and 7 (18.0 mg) were obtained from Fr. 5.3 (395.0 mg) after purification by semi-prep. HPLC
MeOH/H O 7:3). Fr. 6 (1.2 g) was subjected to CC (ODS; MeOH/H O 3 :7!7:3) to afford six
2
2
fractions, Frs. 6.1–6.6. Fr. 6.3 (450.0 mg) was further separated by CC (Sephadex LH-20; MeOH) to
afford 15 (7.5 mg), 16 (8.9 mg), and a mixture, which was purified by semi-prep. HPLC (MeOH/H O
2
7
3 :27) to give 5 (7.9 mg). Fr. 6.5 (228.0 mg) was successively subjected to semi-prep. HPLC (MeOH/
H O 55 :45) to obtain 1 (15.6 mg) and 10 (8.5 mg). Fr. 8 (1.0 g) was successively separated by CC (ODS;
2
MeOH/H O 3 :7!7:3) to give seven fractions, Frs. 8.1–8.7. Fr. 8.3 (350.0 mg) was further separated by
2
CC (Sephadex LH-20; MeOH) to afford 18 (39.7 mg) and a mixture. The mixture was subsequently
purified by semi-prep. HPLC (MeOH/H O 73 :27) to yield 13 (7.9 mg). Fr. 8.5 (230.0 mg) was purified by
2
semi-prep. HPLC (MeOH/H O 7:3) to afford 4 (8.4 mg) and 17 (17.7 mg).
2
Huangqiyegenin V (¼(9b,11a,16b,20R,24S)-11,16,25-Trihydroxy-20,24-epoxy-9,19-cyclolanostane-
2
5
3
1
5
,6-dione; 1). White amorphous powder. [a] ¼ þ46.7 (c¼0.39, MeOH). IR: 3393, 2988, 1705, 1425,
D
1
13
þ
þ
377, 1356, 1038. H- and C-NMR: see Table 1. HR-ESI-MS: 525.3194 ([MþNa] , C H NaO ; calc.
30
46
6
25.3187).
Huangqiyegenin VI (¼(9b,16b,24S)-16,24,25-Trihydroxy-9,19-cyclolanostane-3,6-dione; 2). White
2
5
amorphous powder. [a] ¼ þ41.5 (c¼0.40, MeOH). IR: 3404, 2999, 1728, 1481, 1443, 1377, 1366, 1250,
D
217, 1146. 1H- and C-NMR: see Table 1. HR-ESI-MS: 511.3396 ([MþNa] , C H NaO
13
þ
þ
; calc.
1
5
30
48
11.3394).
Huangqiyenin K (¼(3b,6a,9b,16b,20R,24R)-16,25-Dihydroxy-3-(b-d-xylopyranosyloxy)-20,24-ep-
2
5
oxy-9,19-cyclolanostan-6-yl Acetate; 3). White amorphous powder. [a] ¼ þ4.9 (c¼0.37, MeOH). IR:
D
1
13
3
6
393, 2968, 2939, 2872, 1726, 1439, 1379, 1362, 1032. H- and C-NMR: see Table 2. HR-ESI-MS:
þ
þ
65.4261 ([MþH] , C H O10 ; calc. 665.4259).
37
61
Huangqiyenin L (¼(3b,6a,9b,16b,24E)-26-(b-d-Glucopyranosyloxy)-16-hydroxy-3-(b-d-xylopyra-
2
5
nosyloxy)-9,19-cyclolanost-24-en-6-yl Acetate; 4). White amorphous powder. [a]
D
¼ þ38.18 (c¼0.33,
1
13
MeOH). IR: 3380, 3060, 3055, 3034, 1728, 1634, 1441, 1252, 1032. H- and C-NMR: see Table 2. HR-
ESI-MS: 811.4847 ([MþH] , C H O ; calc. 811.4838).
þ
þ
43
71 14

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1583
Acid Hydrolysis and Determination of Sugar Components. Compounds 3 and 4 (each 2.0 mg) were
hydrolyzed in 1m aq. HCl soln. (1.0 ml) for 2 h at 858. The mixture was cooled and partitioned between
CHCl (2.0 ml) and H O (2.0 ml). The aq. layer was washed with CHCl (3Â3.0 ml), neutralized with
3
2
3
Ba(OH) , filtered, and evaporated under reduced pressure. The residue was dissolved in pyridine
2
(
1.0 ml) and 0.1m l-cysteine methyl ester hydrochloride in pyridine (2.0 ml) was added. The mixture was
heated at 608 for 1 h. An equal volume of Ac O was added and heating was continued for 1 h. The
2
acetylated thiazolidine derivatives were analyzed by GC using authentic samples as standards.
Temperatures of injector and detector were both 2808. A temp. gradient system was used for the oven,
starting at 1608 and increasing up to 1958 at a rate of 58/min. Identification of d-xylose from 3 and d-
xylose and d-glucose from 4 present in the aq. layer was carried out by comparison of t values with those
R
of authentic samples (NICPBP, P. R.China; tR1 10.2 min (d-xylose), tR2 13.9 min (d-glucose)).
Determination of NO Production and Cell Viability Assay. Mouse monocyte-macrophage RAW264.7
cells (ATCC TIB-71) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai,
P. R. China). RPMI 1640 medium, penicillin, streptomycin, and fetal bovine serum (FBS) were
purchased from HyClone (N.Y., USA). LPS, DMSO, MTT, and hydrocortisone were purchased from
À1
Sigma Co. The cells were suspended in RPMI 1640 medium supplemented with penicillin (100 U ml ),
À1
streptomycin (100 U ml ), and 10% heat-inactivated FBS under a humidified atmosphere of 5% CO at
2
3
78. The cells were harvested with trypsin until they attained confluence and were used for assays during
the exponential growth phase.
4
The cells were seeded in 96-well plates with 6Â10 cells/well. After 1 h incubation, cells were treated
À1
with 1 mg ml LPS and various concentrations of test compounds for 24 h [28]. Each compound was
dissolved in DMSO, which was applied at a final concentration of 0.1% (v/v) in cell culture supernatants.
Control groups received an equal amount of DMSO. NO Production was determined by adding 100 ml
Griess reagent (1% sulfanilamide and 0.1% N-(naphthalen-1-yl)ethane-1,2-diamine in 5% H PO ) to
3
4
1
00 ml supernatant from LPS or the compound-treated cells in triplicate. After 5 min of incubation, the
absorbance was measured at 540 nm with a microplate reader. Cytotoxicity was determined by MTT
À
colorimetric assay, after 24 h of incubation with test compounds. Concentrations of NO2 in the
supernatant were calculated by a working line from 0, 1, 2, 5, 10, 20, 50, and 100 mm NaNO solns. The
2
inhibitory rate was calculated according to the formula:
À
À
½
NO Š À ½NO Š
À À
2
LPS
2 LPSþsample
Inhibitory rate ½%Š ¼ 100 Á
½
NO Š À ½NO Š
2
LPS
2 untreated
Every experiment was performed in triplicate; data are expressed as meanÆSD of three
independent experiments.
REFERENCE
[
[
[
[
[
[
1] X. Li, L. Qu, Y. Dong, L. Han, E. Liu, S. Fang, Y. Zhang, T. Wang, Molecules 2014, 19, 18850.
2] H.-H. Chen, S.-X. Gong, T.-J. Zhang, T.-T. Xiao, S.-L. Wang, Drug Eval. Res. 2011, 34, 134.
3] Y. Zhang, Z.-K. Han, L.-C. Liu, Chin. Wild Plant Resour. 2003, 22, 40.
4] Y. Zhang, J.-J. Li, H.-Y. Sun, M. Su, J. Chin. Med. Mater. 2007, 30, 1433.
5] R. P. Mamedova, M. I. Isaev, Chem. Nat. Compd. 2004, 40, 303.
6] R. A. Hill, J. D. Connolly, Nat. Prod. Rep. 2013, 30, 1028; H. Kuang, Y. Okada, B. Yang, Z. Tian, T.
Okuyama, Helv. Chim. Acta 2009, 92, 950; H.-X. Kuang, Q.-H. Wang, B.-Y. Yang, Z.-B. Wang, Y.
Okada, T. Okuyama, Helv. Chim. Acta 2011, 94, 2239.
[
7] a) H. Liu, W. Wei, W.-Y. Sun, X. Li, J. Ethnopharmacol. 2009, 122, 502; b) W.-D. Zhang, H. Chen, C.
Zhang, R.-H. Liu, H.-L. Li, H.-Z. Chen, Planta Med. 2006, 72, 4; c) X. Yin, Y. Zhang, H. Wu, X.
Zhu, X. Zheng, S. Jiang, H. Zhuo, J. Shen, L. Li, J. Qiu, J. Pharmacol. Sci. (Tokyo, Jpn.) 2004, 95,
2
56; d) W.-J. Zhang, P. Hufnagl, B. R. Binder, J. Wojta, Thromb. Haemostasis 2003, 90, 904;
e) A.Navarrete, J. Arrieta, L. Terrones, H. Abou-Gazar, I. Calis, J. Pharm. Pharmacol. 2005, 57,
1
2
059; f) Y. Luo, Z. Qin, Z. Hong, X. Zhang, D. Ding, J.-H. Fu, W.-D. Zhang, J. Chen, Neurosci. Lett.
004, 363, 218.

1
584
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
[
8] Y. Ma, Z. Tian, H. Kuang, C. Yuan, C. Shao, K. Ohtani, R. Kasai, O. Tanaka, Y. Okada, T. Okuyama,
Chem. Pharm. Bull. 1997, 45, 359.
[
9] H.-X. Kuang, N. Zhang, Z.-K. Tian, P. Zhang, Y. Okada, T. Okuyama, J. Nat. Med. 1997, 51, 358.
[
[
10] C. Yang, Q. An, Z. Xiong, Y. Song, K. Yu, F. Li, Planta Med. 2009, 75, 656.
11] M. M. Radwan, N. A. El-Sebakhy, A. M. Asaad, S. M. Toaima, D. G. I. Kingston, Phytochemistry
2
004, 65, 2909.
[
[
[
[
[
[
[
[
[
[
[
[
[
[
12] Y.-L. Ma, Z.-K. Tian, H.-X. Kuang, C.-S. Yuan, L.-J. Liu, Acta Bot. Sin. 1993, 35, 480.
13] F. Pan, J.-S. Yang, Y.-X. Feng, Acta Bot. Sin. 1996, 38, 836.
14] F. Fathiazad, M. K. Khosropanah, A. Movafeghi, Nat. Prod. Res. 2010, 24, 1069.
15] T. Konoshima, M. Kozuka, M. Haruna, K. Ito, J. Nat. Prod. 1991, 54, 830.
16] J.-F. Hu, Z.-L. Ye, F.-J. Shen, Acta Pharmacol. Sin. 1995, 30, 27.
17] R.-Q. Sun, Z.-J. Jia, Phytochemistry 1990, 29, 2032.
18] T. Konoshima, M. Kozuka, M. Haruna, K. Ito, T. Kimura, Chem. Pharm. Bull. 1989, 37, 1550.
19] R.-Q. Sun, Z.-J. Jia, D.-L. Cheng, Phytochemistry 1991, 30, 2707.
20] M. A. Aderogba, A. R. Ndhlala, K. R. R. Rengasamy, J. Van Staden, Molecules 2013, 18, 12633.
21] J.-H. Yang, H. Lu, R. Huang, Y.-S. Wang, Chem. Nat. Compd. 2011, 47, 112.
22] A. Walter, U. SØquin, Phytochemistry 1990, 29, 2561.
23] Z.-M. Bi, Q.-T. Yu, P. Li, Y. Lin, X.-D. Gao, Chin. J. Nat. Med. 2007, 5, 263.
24] X. Du, Y. Bai, H. Liang, Z. Wang, Y. Zhao, Q. Zhang, L. Huang, Magn. Reson. Chem. 2006, 44, 708.
25] K. Hirakura, M. Morita, K. Nakajima, K. Sugama, K. Takagi, K. Niitsu, Y. Ikeya, M. Maruno, M.
Okada, Phytochemistry 1997, 46, 921.
[
[
26] S. Zhao, L. Zhang, P. Gao, Z. Shao, Food Chem. 2009, 114, 869.
27] L. Qiu, F. Zhao, Z.-H. Jiang, L.-X. Chen, Q. Zhao, H.-X. Liu, X.-S. Yao, F. Qiu, J. Nat. Prod. 2008,
7
1, 642.
28] L. Qiu, F. Zhao, H. Liu, L. Chen, Z. Jiang, H. Liu, N. Wang, X. Yao, F. Qiu, Chem. Biodiversity 2008,
, 758.
[
5
Received October 7, 2014