Cycloartane-type triterpenoids from the root of Astragalus membranaceus var. mongholicus

Article abstract of DOI:10.1080/10286020.2020.1787994

Three new (1–3) and 11 known (4–14) cycloartane-type triterpenoids were isolated from the root of Astragalus membranaceus var. mongholicus. Their structures were determined by spectroscopic analyses and chemical methods. Cycloartane-type triterpenoids are a class of major bioactive constituents in the root of A. membranaceus var. mongholicus, and the discovery of compounds 1–3 added new members of this kind of natural product.

Full text of DOI:10.1080/10286020.2020.1787994

Journal of Asian Natural Products Research
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Cycloartane-type triterpenoids from the root of
Astragalus membranaceus var. mongholicus
Luo-Min Xiao , Pei-Hong Cao , Zhi-Hui Luo , Xue-Feng Bao , Zheng-Qun Zhou ,
Shuang Li , Sen-Sen Zhao , Jun-Xia Zheng , Hao Gao & Hui Zhi
To cite this article: Luo-Min Xiao , Pei-Hong Cao , Zhi-Hui Luo , Xue-Feng Bao , Zheng-Qun
Zhou , Shuang Li , Sen-Sen Zhao , Jun-Xia Zheng , Hao Gao & Hui Zhi (2020): Cycloartane-type
triterpenoids from the root of Astragalusꢀmembranaceus var. mongholicus , Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2020.1787994
To link to this article: https://doi.org/10.1080/10286020.2020.1787994
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1787994
Cycloartane-type triterpenoids from the root of
Astragalus membranaceus var. mongholicus
a
b
c
c
ꢀ
ꢀ
Luo-Min Xiao , Pei-Hong Cao , Zhi-Hui Luo , Xue-Feng Bao , Zheng-Qun
c
b
c
d
b,c
a
Zhou , Shuang Li , Sen-Sen Zhao , Jun-Xia Zheng , Hao Gao and Hui Zhi
a
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006,
b
China; College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China; Institute of Traditional Chinese Medicine and Natural Products, College of
c
Pharmacy/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New
d
Drugs Research, Jinan University, Guangzhou 510632 China; School of Biomedical and
Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China
ABSTRACT
ARTICLE HISTORY
Three new (1–3) and 11 known (4–14) cycloartane-type triterpe-
noids were isolated from the root of Astragalus membranaceus
var. mongholicus. Their structures were determined by spectro-
scopic analyses and chemical methods. Cycloartane-type triterpe-
noids are a class of major bioactive constituents in the root of A.
membranaceus var. mongholicus, and the discovery of compounds
Received 28 March 2020
Accepted 23 June 2020
KEYWORDS
Leguminosae; Astragalus
membranaceus var.
mongholicus; cycloartane-
type triterpenoids;
natural product
1–3 added new members of this kind of natural product.
1. Introduction
Astragalus membranaceus var. mongholicus, a perennial herb in the Leguminosae fam-
ily, is primarily distributed in Northeast, North, and Northwest China [1]. The root
of A. membranaceus var. mongholicus, named “Huang-Qi” in Chinese, has been
CONTACT Hao Gao
tghao@jnu.edu.cn
College of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang 110016, China; Institute of Traditional Chinese Medicine and Natural Products,
College of Pharmacy/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs
Research, Jinan University, Guangzhou 510632, China; Hui Zhi
zhihui@gzucm.edu.cn
School of Chinese
Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
ꢀ
These authors contributed equally to this work.
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1787994.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
L.-M. XIAO ET AL.
Figure 1. Chemical structures of compounds 1–3.
utilized as a famous traditional Chinese medicine (TCM) to reinforce “Qi” (vital
energy) [2–4]. Recent pharmacological studies showed that it exhibited a series of bio-
logical activities, such as antidiabetic, anticancer, antiinflammatory, and immunoregu-
latory effects [5–9]. Previous studies demonstrated that polysaccharides, flavonoids,
and triterpenoids were the major constituents in the root of A. membranaceus var.
mongholicus [9–11].
The present phytochemical study on the root of A. membranaceus var. mongholicus
led to the discovery of fourteen cycloartane-type triterpenoids, including three new
ones (1ꢁ3) (Figure 1). Details of the isolation and structural elucidation of 1–14 are
reported in this paper.
2. Results and discussion
Compound 1 was obtained as white amorphous powders. The quasi-molecular ion at
þ
m/z 825.4620 [M þ Na] by HRESIMS indicated that the molecular formula of 1 was
1
3
C H O (7 degrees of unsaturation). The C NMR spectrum of 1 showed 41 car-
4
1
70 15
3
bons. Based on the DEPT-135 data, these carbons could be categorized into seven sp
quaternary carbons (including two oxygenated carbons), 16 sp methine carbons
including 13 oxygenated carbons), 11 sp methylene carbons (including two oxygen-
ated carbons), and seven methyl carbons. The H NMR spectrum showed that 1 had
a set of typical cyclopropane methylene protons and 7 sets of methyl protons. The
proton signals were associated with the directly attached carbon atoms in the HSQC
3
3
(
1
1
1
experiment. The analysis of the Hꢁ H COSY experiment and the coupling values of
the protons exhibited the presence of 7 subunits (H /H -1 ꢁ H /H -2 ꢁ H-3, H-
a
b
a
b
5
2
ꢁ H-6 ꢁ H /H -7 ꢁ H-8, H /H -11 ꢁ H /H -12, H /H -15 ꢁ H-16 ꢁ H-17, H /H -
a
b
a
b
0
a
b
0
a
b
a
b
00
0
0
0
00
00
00
2 ꢁ H /H -23 ꢁ H-24, H-1 ꢁH-2 ꢁH-3 ꢁH-4 ꢁH /H -5 , and H-1 –H-2 –H-3 –H-4 –
a
b
a
b
00
00
H-5 –Ha/Hb-6 ) (Figure 2A). Combined with these deduced subunits, molecular for-
mula, and degrees of unsaturation, the key HMBC correlations shown in Figure 2A
deduced the planar structure of compound 1. The assignments of all proton and carbon
resonances are provided in Table 1.
The key ROESY correlations shown in Figure 2B/C and the nature of b-orienta-
tions for C-18 and C-19 exhibited that the partial absolute configurations of 1 were
(3S, 5 R, 6S, 8S, 9S, 10 R, 13 R, 14S, 16S, 17 R, 20 R) [12]. Considering the nature of
the 24S configuration for astragalosides in the genus Astragalus [13, 14], the absolute
configurations of the aglycone of 1 were (3S, 5 R, 6S, 8S, 9S, 10 R, 13 R, 14S, 16S,
17 R, 20 R, 24S).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. Key 2 D NMR correlations of compound 1.
The planar structure of 1 showed one pyranohexose unit and one pyranopentose
unit. Based on the coupling values (J1
0
0
¼ 6.8 Hz, J
0
0
0
¼ 8.8 Hz, J
0 0
4 -5 -Hb
¼
-2
2 -3'/3 -4
1
0.1 Hz, J100 00 ¼ 7.6 Hz, J 00 00 ¼ 8.7 Hz, J 00 00 ¼ 9.2 Hz, J 00 00 ¼ 9.6 Hz), the pyrano-
-2
2 -3
3 -4
4 -5
hexose and pyranopentose units were established as b-Glc and b-Xyl, respectively.
The absolute configurations of the glucopyranosyl and xylopyranosyl were determined
by HPLC analysis of products obtained from acid hydrolysis and derivatization reac-
tions by L-cysteine methyl ester hydrochloride and o-tolyl isothiocyanate [15].
Analytical HPLC was performed on the Cosmosil Packed C18 column with isocratic
elution of CH CN ꢁ H O ꢁ HCOOH (25:75:0.1, v/v/v) for 40 min at a flow rate of
3
2
1
.0 ml/min, and the peaks of the standard monosaccharide and sample derivatives were
recorded at t 14.6 (L-Glc), 15.7 (D-Glc), 17.3 (L-Xyl), 18.5 (D-Xyl), and 15.8/18.4 (1)
R
min, respectively. This evidence revealed that the glucopyranosyl and xylopyranosyl
were D-Glc and D-Xyl, respectively. Therefore, compound 1 was established as
(
3b,6a,16b,20 R,24S)-6,16,20,24,25-pentahydroxy-3-O-[b-D-glucopyranosyl-(1!2)-b-D-
xylopyranosyl]-9,19-cyclolanostan (named 20,24-non-epoxy-astragaloside III).
Compound 2 was obtained as white amorphous powders. The quasi-molecular ion
þ
at m/z 825.4603 [M þ Na] by HRESIMS indicated that the molecular formula of 2
was C H O (7 degrees of unsaturation), which was the same as that of 1. Except
4
1
70 15
0
for the significantly different chemical shifts of C-6 and C-2 , the NMR data of 2
were similar to those of 1. The key HMBC correlations from H-6 to C-4/C-5/C-8/
00
00
00
00
C-1 and from H-1 to C-6/C-3 /C-5 showed that the pyranohexose unit in 2 was
attached to the C-6 position. Thus, the planar structure of 2 was established, and the
detailed analyses of 1 D and 2 D NMR data of 2 (Table S2, Supporting Information)
confirmed the above deduction. The key ROESY correlations (between H-3 and H-5,
between H-6 and H-8/Ha-19/H -29, between H-8 and H -18/Ha-19, between Hb-15
3
3
and H -18, between H-16 and H -21/H -30, between HO-16 and H -18/H -21,
3
3
3
3
3
between H-17 and H -21/H -22/H -30, between H -18 and H -22/HO-20, and
3
2
3
3
2
between Ha-19 and H -29), the nature of b-orientations for C-18 and C-19, and the
3
nature of the 24S configuration for astragalosides in the genus Astragalus indicated
that the absolute configurations of the aglycone of 2 were (3S, 5 R, 6S, 8S, 9S, 10 R,
13 R, 14S, 16S, 17 R, 20 R, 24S) [12–14].
The planar structure of 2 showed one pyranohexose unit and one pyranopentose
1
3
unit. A precise comparison of C NMR data of the sugar units with those of glyco-
sides recorded in the literatures [16–18] and the coupling values (J1
¼
0
0
¼ 7.4 Hz, J 00 00
1 -2
-2
8.0 Hz indicated that the pyranohexose and pyranopentose units were b-Glc and
b-Xyl, respectively. Acid hydrolysis indicated that the absolute configurations of the

4
L.-M. XIAO ET AL.
1
13
Table 1. H (600 MHz) and C (150 MHz) NMR data of 1–3 in Pyr-d (d in ppm, J in Hz).
5
1
2
3
a
H
a
H
a
No.
1
d
C
d
d
C
d
d
C
d
H
32.5 1.64, Ha
.25, Hb
30.4 2.38, Ha
.02, Hb
32.2 1.61, Ha
1.26, Hb
30.2 2.39, Ha
1.99, Hb
31.9
1.51, t (13.1), Ha
1.21, Hb
2.17, br d (11.1), Ha
1.89, Hb
1
2
29.6
2
3
4
5
6
7
88.6 3.57, dd (11.7, 4.6)
42.8
54.1 1.74
67.7 3.77, td (9.1, 3.9)
38.4 1.82, Ha
88.6 3.54, dd (11.6, 4.2)
42.6
89.3
42.2
52.4
79.3
34.8
3.38, dd (11.4, 2.6)
52.6 1.93, d (8.8)
79.3 3.82
34.4 2.27, Ha
1.87, Hb
45.3 2.04
21.2
28.9
26.4 1.80, Ha
1.26, Hb
1.85
3.77, br t (8.0)
2.32, Ha
1.80, Hb
1.92
1
.66, Hb
8
9
1
46.4 2.02
21.1
29.3
46.0
21.1
28.9
26.1
0
1
1
26.4 1.93, Ha
1.83, Ha
1.23, Hb
1.62, Ha
1.57, Hb
1
.20, Hb
33.5 2.02, Ha
.85, Hb
1
2
33.7 2.05, Ha
1.80, Hb
33.3
1
1
1
1
3
4
5
46.8
47.1
48.8 2.18, Ha
.90, Hb
46.8
47.2
48.3 2.40, Ha
1.99, Hb
45.0
46.2
46.3
2.34, Ha
1.85, Hb
1
1
1
1
1
6
7
8
9
74.0 5.01
56.1 2.17
20.4 1.83, s
30.0 0.57, d (4.3), Ha
74.0 4.98
56.5 2.14, d (7.7)
20.1 1.84, s
28.6 0.60, d (2.8), Ha
0.19, d (3.9), Hb
77.0
73.4
58.2
21.2
29.2
4.99
2.52, d (7.2)
1.42, s
0.56, br s, Ha
0.21, br s, Hb
0
.26, d (4.3), Hb
2
2
2
0
1
2
77.0
28.8 1.73, s
39.9 2.69, Ha
87.2
28.5
34.9
28.6 1.73, s
39.9 2.32
1.31, s
3.13, q (10.1), Ha
1.68, Hb
2.32, Ha
2.05, Hb
3.89
1
.91, Hb
26.7 2.20, Ha
.09, Hb
2
3
26.6 2.41, Ha
1.96, Hb
79.9 3.91
26.4
2
2
2
2
2
2
2
3
1
2
3
4
5
4
5
6
7
8
9
80.1 3.85, br d (10.3)
72.8
25.9 1.55, s
26.3 1.58, s
28.8 1.95, s
16.6 1.44, s
20.3 1.05, s
105.6 4.93, d (6.8)
81.7
71.3
27.1
28.2
28.3
16.4
19.9
103.5
72.3
72.6
69.7
62.6
72.8
25.9 1.55, s
26.3 1.52, s
28.6 2.04, s
16.6 1.37, s
20.0 0.97, s
107.7 4.85, d (7.4)
75.6 4.05
78.5 4.15
71.3 4.23
67.1 4.36, dd (11.3, 5.3), Ha
3.70, t (11.0), Hb
105.2 4.93, d (8.0)
75.6 4.05
1.31, s
1.59, s
1.77, s
1.25, s
0
0.93, s
0
4.89, d (7.3)
5.49, td (9.0, 1.6)
5.74, td (9.4, 1.6)
5.39
4.37, dd (11.5, 5.1), Ha
3.66, t (9.9), Hb
4.92, d (7.6)
4.05, t (7.6)
4.22, t (8.8)
4.19, t (8.3)
3.92
0
0
0
0
83.4 4.25
77.9 4.23, t (8.8)
71.0 4.19
66.7 4.30, dd (11.5, 5.0), Ha
3
.65, t (10.1), Hb
1
2
3
4
5
6
"
"
"
"
"
"
106.1 5.41, d (7.6)
77.0 4.14, t (8.7)
78.0 4.26
71.7 4.35, t (9.2)
78.2 3.97, dt (9.6, 3.5)
62.8 4.52, dd (11.6, 3.3), Ha
105.2
75.6
79.2
71.8
78.2
63.0
79.2 4.23
71.9 4.20
78.2 3.90
63.1 4.47, br d (11.2), Ha
4.30, Hb
4.49, br d (11.4), Ha
4.32, dd (11.2, 5.1), Hb
4
.47, dd (11.5, 4.0), Hb
0
2
2
3
3
1
2
3
4
-OCOCH
-OCOCH
-OCOCH
-OCOCH
’’
’’
’’
’’
3
3
3
3
169.6
20.7
170.2
20.5
165.3
122.1
146.6
17.8
0
0
0
0
0
0
0
ꢀ
ꢀ
2
2
2.03, s
2.03, s
5.85, dq (15.6, 1.7)
7.01, dq (15.9, 6.7)
1.61, br d (6.8)
(continued)

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. Continued.
1
2
3
5.79, s
6.52, s
a
H
a
H
a
No.
d
C
d
d
C
d
d
C
d
H
1
2
2
2
2
4
2
3
4
6
6-OH
7.50, br s
6.09, s
5.79, d (4.6)
5.45, s
0-OH
4-OH
5-OH
’-OH
6.99, d (3.2)
7.06 , br s
ꢀ
1
’-OH
’’-OH
’’-OH
’’-OH
’’-OH
6.93, d (3.2)
ꢀ
1
7.10 , br s
ꢀ
1
7.10 , br s
6.03, br s
ꢀ
a
Assignment may be interchanged. The indiscernible signals due to overlap or having the complex multiplicity are
reported without designating multiplicity.
glucopyranosyl and xylopyranosyl were D-Glc and D-Xyl, respectively [15]. Therefore,
compound 2 was established as (3b,6a,16b,20 R,24S)-16,20,24,25-tetrahydroxy-3-O-b-D-
xylopyranosyl-6-O-b-D-glucopyranosyl-9,19-cyclolanostan (named 20,24-non-epoxy-
astragaloside IV).
Compound 3 was obtained as white amorphous powders. The quasi-molecular ion
þ
at m/z 959.4986 [M þ Na] by HRESIMS indicated that the molecular formula of 3
was C H O (12 degrees of unsaturation). Except for the appearance of an add-
4
9
76 17
itional crotonyl, the NMR data of 3 were similar to those of astragaloside I [16]. The
1
1
000
000
000
000
Hꢁ H COSY correlations between H-3 and H-2 /H3-4 and the key HMBC cor-
0
0
0
000
000
000
relations from H-4 to C-3 /C-5 /C-1 and from H-2 to C-1 /C-4 showed that the
crotonyl in 3 was attached to the C-4 position. Thus, the planar structure of 3 was
0
established, and the detailed analyses of 1 D and 2 D NMR data of 3 (Table S3,
Supporting Information) confirmed the above deduction. The key ROESY correla-
tions (between H-3 and H-5, between H-6 and H-8/Ha-19/H -29, between H-8 and
3
H -18/Ha-19, between Hb-15 and H -18, between H-16 and H -30, between HO-16
3
3
3
and Ha-22, between H-17 and H -21/H -30, between H -18 and Ha-22/Hb-22,
3
3
3
between Ha-19 and H -29, and between H -21 and Ha-12/Hb-12/H-24) and the
3
3
nature of b-orientations for C-18 and C-19 showed that the absolute configurations
of the aglycone of 3 were (3S, 5 R, 6S, 8S, 9S, 10 R, 13 R, 14S, 16S, 17 R, 20 R,
24S) [12].
The planar structure of 3 showed one pyranohexose unit and one pyranopentose
unit. Based on the coupling values (J1
0
0
¼ 7.3 Hz, J
0
0
¼ 9.0 Hz, J
0
0
¼ 9.4 Hz, J
0 0
-2
2 -3
3 -4
4 -5 -Hb
¼
9.9 Hz, J100 00 ¼ 7.6 Hz, J 00 00 ¼ 8.8 Hz, J 00 00 00 00 ¼ 8.3 Hz), the pyranohexose and
-2
2 -3
3 -4 /4 -5
pyranopentose units were established as b-Glc and b-Xyl, respectively. Acid hydroly-
sis demonstrated that the absolute configurations of the glucopyranosyl and xylopyra-
nosyl were D-Glc and D-Xyl, respectively [15]. Therefore, compound 3 was
established as (3b,6a,16b,20 R,24S)-16,25-dihydroxy-3-O-(2,3-O-diacetyl-4-O-trans-
crotonyl-b-D-xylopyranosyl)-6-O-b-D-glucopyranosyl-20,24-epoxy-9,19-cyclolanostan
0
(
named 4 -O-crotonyl-astragaloside I).
The known compounds, astragaloside IV (4, CAS: 84687-43-4) [16], astragaloside
II (5, CAS: 84676-89-1) [17], isoastragaloside II (6, CAS: 86764-11-6) [19], cycloce-
phaloside II (7, CAS: 215776-78-6) [20], astragaloside I (8, CAS: 84680-75-1) [16],

6
L.-M. XIAO ET AL.
isoastragaloside I (9, CAS: 84676-88-0) [17], acetylastragaloside I (10, CAS: 84687-47-
8
4
) [16], astragaloside III (11, CAS: 84687-42-3) [16], astragaloside V (12, CAS: 84687-
4-5) [21], astragaloside VI (13, CAS: 84687-45-6) [21], and astragaloside VII (14,
CAS: 84687-46-7) [22] were identified by spectroscopic analyses and comparison of
their reported spectroscopic data.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a JASCO P1020 digital polarimeter (Jasco
International Co. Ltd, Tokyo, Japan). UV data were recorded using a JASCO V-550
UV/vis spectrometer (Jasco International Co. Ltd). IR data were recorded on a
JASCO FT/IR-480 plus spectrometer (Jasco International Co. Ltd). The HRESIMS
were obtained on a Micromass Q-TOF mass spectrometer (Waters Corporation,
Milford, USA). 1 D and 2 D NMR spectra were acquired with a Bruker AV 600 spec-
trometer (Bruker BioSpin Group, Faellanden, Switzerland) using the solvent signals
(
Pyr-d : d 7.21/d 123.5) as internal standards. The analytical HPLC was performed
5 H C
on a Shimadzu HPLC system equipped with a LC-20AT pump, a SPD-M20A DAD, a
CTO-10AS VP column oven, a SIL-20A autosampler (Shimadzu Inc., Kyoto, Japan),
and an Alltech (Grace) 6000 ELSD (Alltech Co. Ltd, Portland, USA) or a Waters
e2695 Alliance HPLC system equipped with a Waters 1500 pump, a Waters 2998
PDA, a Waters Alliance series column compartment, a Waters 717plus autosampler
(
Waters Corporation, Milford, USA), and an Alltech 3300 ELSD (Alltech Co. Ltd)
2
using a Cosmosil Packed C18 column (4.6 ꢂ 250 mm , 5 lm) (Nacalai Tesque Inc.,
Kyoto, Japan). The preparative HPLC was performed on a Shimadzu LC-6-AD liquid
chromatography system with SPD-20A and RID-20A detectors (Shimadzu Inc.) or a
Waters Breeze 2 system equipped with a Waters 1515 isocratic HPLC pump and a
Waters 2489 UV/visible detector (Waters Corporation) using a Cosmosil Packed C18
2
column (20.0 ꢂ 250 mm , 5 lm) (Nacalai Tesque Inc.). Medium pressure liquid chro-
matography (MPLC) was equipped with a dual pump gradient system and an UV
preparative detector (Lisui E-Tech Co. Ltd, Shanghai, China) using an ODS column
(50 lm, YMC Co. Ltd, Tokyo, Japan). Column chromatography (CC) was performed
on D101 macroporous resin (Nankai University Resin Co. Ltd, Tianjin, China) and
silica-gel (200–300 mesh, Haiyang Chemical Co. Ltd, Qingdao, China).
3
.2. Plant material
The root of Astragalus membranaceus var. mongholicus was collected from Hunyuan
County, Datong, Shanxi Province, China in Nov. 2013. The plant materials were
identified by associate Prof. Jia-Yun Tong (School of Chinese Materia Medica,
Guangzhou University of Chinese Medicine, Guangzhou, China). A voucher specimen
(ASME-2013-SX) was deposited in the Institute of Traditional Chinese Medicine and
Natural Products, College of Pharmacy, Jinan University, Guangzhou, China.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
3.3. Extraction and isolation
The dried root of A. membranaceus var. mongholicus (8.4 kg) was extracted with
ꢃ
supercritical carbon dioxide at 45 C and 15–25 MPa using a HA121-50-05 supercrit-
ical fluid extraction system (Huaan Scientific Instrument Co. Ltd, Jiangsu, China).
Then, the residue was refluxed three times with 100 L of 60% EtOH ꢁ H O for 2 h
2
each time. After filtration, the EtOH was removed under reduced pressure to yield a
concentrated solution. The solution was passed through a D101 macroporous resin
2
column (20 ꢂ 100 cm ) using an elution of EtOH ꢁ H O to yield five frac-
2
tions (F1 ꢁ F5).
A portion (70.0 g) of fraction F3 (91.7 g) was subjected to open silica-gel CC
2
(
8.5 ꢂ 40 cm ), which was eluted with an elution of CH Cl ꢁMeOH ꢁ H O to yield
2
2
2
fractions 3.1–3.15. Fraction 3.12 (4.1 g) was isolated using ODS-MPLC/HPLC to yield
(3.2 mg) and 2 (13.8 mg).
Fraction F4 (63.8 g) was subjected to open silica-gel CC (6.5 ꢂ 80 cm ), which was
1
2
eluted with an elution of CH Cl ꢁMeOH ꢁ H O to yield fractions 4.1–4.12. Fraction
2
2
2
4
.7 was isolated using ODS-MPLC/HPLC to yield 12 (48.7 mg) and 13 (200.4 mg).
2
Fraction F5 (28.7 g) was subjected to open silica-gel CC (6.5 ꢂ 30 cm ), which was
eluted with an elution of CH Cl ꢁMeOH to yield fractions 5.1–5.9. Fractions 5.5/5.6/
2
2
5
.7 were isolated using ODS-MPLC/HPLC to yield 3 (9.4 mg), 4 (33.7 mg), 5
(98.4 mg), 6 (24.4 mg), 7 (3.7 mg), 8 (26.4 mg), 9 (30.4 mg), 10 (8.1 mg), 11 (3.7 mg),
and 14 (17.1 mg). The detailed separation procedures of 14 cycloartane-type triterpe-
noids see “Extraction and isolation” in Supporting Information.
3
.3.1. 20,24-Non-epoxy-astragaloside III (1)
2
5
White amorphous powders; ½aꢄ þ 25.6 (c ¼ 0.50, MeOH); IR (KBr) v
3423, 2934,
max
D
ꢁ
1
1
13
1456, 1040 cm ; H and C NMR spectral data see Table 1; HRESIMS (positive): m/
þ
z 825.4620 [M þ Na] (calcd for C H O Na, 825.4612).
4
1
70 15
3
.3.2. 20,24-Non-epoxy-astragaloside IV (2)
2
5
White amorphous powders; ½aꢄ þ 29.0 (c ¼ 0.50, MeOH); IR (KBr) v
3420, 2931,
max
D
ꢁ
1
1
13
1463, 1035 cm ; H and C NMR spectral data see Table 1; HRESIMS (positive): m/
þ
z 825.4603 [M þ Na] (calcd for C H O Na, 825.4612).
4
1
70 15
3
.3.3. 4’-O-Crotonyl-astragaloside I (3)
2
5
White amorphous powders; ½aꢄ þ 13.4 (c ¼ 0.50, MeOH); UV (MeOH) k
(log e)
D
max
ꢁ
1
1
13
205 (4.28) nm; IR (KBr) v
2938, 1757, 1365, 1235 cm ; H and C NMR spectral
max
þ
data see Table 1; HRESIMS (positive): m/z 959.4986 [M þ Na] (calcd for
C H O Na, 959.4980).
4
9
76 17
3.4. Acid hydrolysis
Acid hydrolysis was performed according to the previously described method with
minor modifications [15]. The compound (1.0 mg) was hydrolyzed with 2 M HCl for
ꢃ
1
h at 90 C. After extracted with EtOAc two times, the H O layer was evaporated in
2
vacuo to furnish a monosaccharide residue. The residue was dissolved in pyridine

8
L.-M. XIAO ET AL.
(
6
1.0 ml) containing L-cysteine methyl ester hydrochloride (1.0 mg) and heated at
ꢃ
0 C. After 1 h, 10 ll of o-tolyl isothiocyanate was added to the reaction mixture and
ꢃ
further reacted at 60 C for 1 h. Then, the reaction mixture was directly analyzed by
the Shimadzu HPLC system and detected by an UV detector (at 254 nm). Analytical
HPLC was performed with an isocratic elution of CH CN ꢁ H O ꢁ HCOOH
3
2
(
25:75:0.1, v/v/v, 1.0 ml/min) for 40 min. The standard monosaccharides of D-Glc, L-
Glc, D-Xyl, and L-Xyl were subjected to the same method.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was financially supported by grants from the National Natural Science Foundation
of China (81303201, 81925037, and 31761143016), Chang Jiang Scholars Program (Young
Scholar) from the Ministry of Education of China (Hao Gao, 2017), National High-level
Personnel of Special Support Program (2017RA2259), Guangdong Special Support Program
(
2016TX03R280), Local Innovative and Research Teams Project of Guangdong Pearl River
Talents Program (2017BT01Y036), Science and Technology Program of Guangzhou
201707010414), and K. C. Wong Education Foundation (Hao Gao, 2016). We appreciate the
(
identification of the plant material of Jia-Yun Tong, associate Prof. of School of Chinese
Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, China.
References
[1] Y. Li, S. Guo, Y. Zhu, H. Yan, D.W. Qian, H.Q. Wang, J.Q. Yu, and J.A. Duan,
Molecules 24, 434 (2019).
[
[
2] Q.T. Yu, P. Li, Z.M. Bi, J. Luo, and X.D. Gao, Chin. Chem. Lett. 18, 554 (2007).
3] A.G.W. Gong, R. Duan, H.Y. Wang, X.P. Kong, T.T.X. Dong, K.W.K. Tsim, and K.
Chan, Medicines 5, 46 (2018).
[4] H. Gao, and X.S. Yao, Chin. J. Nat. Med. 17, 1 (2019).
[5] K. Agyemang, L.F. Han, E. Liu, Y. Zhang, T. Wang, and X.M. Gao, Evid. Based
Complement. Alt. Med. 2013, 1 (2013).
[
[
6] Y. Jung, U. Jerng, and S. Lee, Chin. J. Integr. Med. 22, 225 (2016).
7] D.Y. Lee, H.J. Noh, J. Choi, K.H. Lee, M.H. Lee, J.H. Lee, Y. Hong, S.E. Lee, S.Y. Kim,
and G.S. Kim, Molecules 18, 3725 (2013).
[
[
8] W. Wang, Q.B. Liu, and W. Jing, Chin. J. Nat. Med. 17, 252 (2019).
9] C.H. Zhang, X. Yang, J.R. Wei, N.M.H. Chen, J.P. Xu, Y.Q. Bi, M. Yang, X. Gong, Z.Y.
Li, K. Ren, Q.H. Han, L. Zhang, X. Li, M.Y. Ji, C.C. Wang, and M.H. Li, Chin. J.
Integr. Med. (2019). DOI: 10.1007/s11655-019-3032-8.
[
[
10] K.K. Auyeung, Q.B. Han, and J.K. Ko, Am. J. Chin. Med. 44, 1 (2016).
11] J. Fu, Z.H. Wang, L.F. Huang, S.H. Zheng, D.M. Wang, S.L. Chen, H.T. Zhang, and
S.H. Yang, Phytother. Res. 28, 1275 (2014).
[
[
[
12] Q. Zhao, G.Q. Wang, G.D. Chen, D. Hu, X.X. Li, L.D. Guo, Y. Li, X.S. Yao, and H.
Gao, Steroids 102, 101 (2015).
13] X.X. Li, L. Qu, Y.Z. Dong, L.F. Han, E.W. Liu, S.M. Fang, Y. Zhang, and T. Wang,
Molecules 19, 18850 (2014).
14] I. Ionkova, A. Shkondrov, I. Krasteva, and T. Ionkov, Phytochem. Rev. 13, 343 (2014).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
[
15] Z.Q. Zhou, J. Xiao, H.X. Fan, Y. Yu, R.R. He, X.L. Feng, H. Kurihara, K.F. So, X.S.
Yao, and H. Gao, Food Chem. 214, 644 (2017).
[16] M. Hirotani, Y. Zhou, H. Lui, and T. Furuya, Phytochemistry 36, 665 (1994).
[17] M. Hirotani, Y. Zhou, H. Rui, and T. Furuya, Phytochemistry 37, 1403 (1994).
[18] H.Y. Zhang, W.C. Hu, G.X. Ma, N.L. Zhu, X.B. Sun, H.F. Wu, and X.D. Xu, J. Asian
Nat. Prod. Res. 20, 586 (2018).
[
19] I. Kitagawa, H.K. Wang, M. Saito, A. Takagi, and M. Yoshikawa, Chem. Pharm. Bull.
3
1, 698 (1983).
[
[
20] E. Bedir, I. Calis, R. Aquino, S. Piacente, and C. Pizza, J. Nat. Prod. 61, 1469 (1998).
21] I. Kitagawa, H.K. Wang, M. Saito, and M. Yoshikawa, Chem. Pharm. Bull. 31, 709
(
1983).
[
22] Y. Zhou, M. Hirotani, H. Rui, and T. Furuya, Phytochemistry 38, 1407 (1995).