Triterpenoids from Anchusa italica and their protective effects on hypoxia/reoxygenation induced cardiomyocytes injury

Article abstract of DOI:10.1016/j.bioorg.2020.103714

Six new triterpenoids (1–6) and 22 known analogues (7–28), were separated from the aerial parts of Anchusa italica Retz., a traditional Uygur medicine for treating cardiovascular and cerebrovascular diseases in the Xinjiang region, China. The possible effects of compounds 1–28 on hypoxia/reoxygenation (H/R) induced cardiomyocytes injury were assayed, and compounds 4, 6–17, 21–22 and 26–28 showed significant protective effects. Further, the representative new compound 6 significantly suppressed the levels of H/R-induced apoptosis and autophagy in neonatal rat cardiomyocytes, with the reversing of the downregulated expression of Bcl–2 and upregulated expression of Bax and Beclin–1 by compound 6 treatment in neonatal rat cardiomyocytes following H/R injury. In addition, compound 6 protected cardiomyocyte from H/R injury, and pretreatment with 6 could decrease CK and LDH levels. Compound 6 also alleviated H/R-induced phosphorylation of p38 MAPK in neonatal rat cardiomyocytes. Therefore, tripterpenoid 6 and its analogues may be the pharmacodyamic material of A. italica, and offer a promising therapeutic approach for treating cardiomyocyte injury induced by H/R.

Full text of DOI:10.1016/j.bioorg.2020.103714

Journal Pre-proofs
Triterpenoids from Anchusa italica and their protective effects on hypoxia/
reoxygenation induced cardiomyocytes injury
Bei-Chen Hu, Yang Liu, Meng-Zhu Zheng, Ruo-Yu Zhang, Ming-Xue Li,
Feng-Yan Bao, Hua Li, Li-Xia Chen
PII:
S0045-2068(20)30252-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103714
YBIOO 103714
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 February 2020
23 February 2020
27 February 2020
Please cite this article as: B-C. Hu, Y. Liu, M-Z. Zheng, R-Y. Zhang, M-X. Li, F-Y. Bao, H. Li, L-X. Chen,
Triterpenoids from Anchusa italica and their protective effects on hypoxia/reoxygenation induced
cardiomyocytes injury, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103714
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Triterpenoids from Anchusa italica and their protective effects on
hypoxia/reoxygenation induced cardiomyocytes injury
a,1
a,1
b,1
a
a
Bei-Chen Hu, Yang Liu, Meng-Zhu Zheng, Ruo-Yu Zhang, Ming-Xue Li,
a
a,b,
a,
Feng-Yan Bao, Hua Li, * Li-Xia Chen *
a
Wuya College of Innovation, School of pharmaceutical engineering, Shenyang
Pharmaceutical University, Shenyang 110016, People’s Republic of China.
b
School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, 430030, People’s Republic of China.
1
These authors contributed equally to this work.
*
Corresponding Authors.
E-mail addresses: li_hua@hust.edu.cn. (H. Li), syzyclx@163.com. (L.X. Chen).
1

ABSTRACT
Six new triterpenoids (1-6) and 22 known analogues (7-28), were separated from the
aerial parts of Anchusa italica Retz., a traditional Uygur medicine for treating
cardiovascular and cerebrovascular diseases in the Xinjiang region, China. The
possible effects of compounds 1-28 on hypoxia/reoxygenation (H/R) induced
cardiomyocytes injury were assayed, and compounds 4, 6-17, 21-22 and 26-28
showed significant protective effects. Further, the representative new compound 6
significantly suppressed the levels of H/R-induced apoptosis and autophagy in
neonatal rat cardiomyocytes, with the reversing of the downregulated expression of
Bcl-2 and upregulated expression of Bax and Beclin-1 by compound 6 treatment in
neonatal rat cardiomyocytes following H/R injury. In addition, compound 6 protected
cardiomyocyte from H/R injury, and pretreatment with 6 could decrease CK and LDH
levels. Compound 6 also alleviated H/R-induced phosphorylation of p38 MAPK in
neonatal rat cardiomyocytes. Therefore, tripterpenoid 6 and its analogues may be the
pharmacodyamic material of A. italica, and offer a promising therapeutic approach for
treating cardiomyocyte injury induced by H/R.
Keywords: Triterpenoids; Anchusa italica; Cardiomyocytes injury; Protective effects
1

1
. Introduction
The genus Anchusa (Boraginaceae family), including about fifty species around
the world, mainly grows in the Mediterranean, African, European and western Asian
countries [1,2]. Two species, Anchusa italica Retz. and A. officinalis L. were
cultivated in China [2], and the former is traditionally utilized in Uygur medicine for
treating cardiovascular and cerebrovascular diseases in the Xinjiang region, China [3].
The representative constituents, such as alkaloids, triterpenes, lignans, megastigmanes,
and flavonoids have been isolated from this plant [2,4]. Some compounds from A.
italica or its extract have been found to possess various pharmacological activities
including anticancer, antioxidant, antiviral, and memory-enhancing effects [5-9].
Ischemic heart disease is a major cause of morbidity and mortality threatening
human health. Reperfusion is a common method in rescuing salvageable myocardium,
but reperfusion may induce further myocardial injury, and affect the recovery of
cardiac function [10]. Although cell apoptosis, oxidative stress, calcium overload, and
inflammation
are
recognized
as
the
key
reasons
for
H/R
(hypoxia/reoxygenation)-induced myocardial damage, no effective drugs that can
abate myocardial H/R injury are being tested in clinical trials [11,12]. Therefore,
reducing myocardial H/R injury is of great significance to improve the curative effect
of cardiovascular diseases. Natural medicines acting on cardiovascular could be
considered as a promising source for the discovery and development of cardiovascular
drugs.
In spite of the traditional utility of A. italica as cardiovascular and cerebrovascular
agent in Uygur medicine, the effect of constituents from this plant on cardiovascular
hasn’t been investigated. Herein, 28 triterpenoids including 6 new (1-6) and 22 known
analogues (7-28) were isolated from the aerial parts of A. italica, and their structures
were determined by spectroscopic analyses. All these isolated compounds were
assayed for their possible effects on hypoxia/reoxygenation (H/R) induced
cardiomyocytes injury, and their structure-activity relationship is preliminarily
discussed. The representative new compound 6 was chosen to study its possible
mechanism for cardiomyocytes injury inducing by H/R.
2

2
. Results
Studies on constituents of the aerial parts of A. italica yielded 6 novel
compounds (1-6) and 22 known analogues (7-28), including ursolazuroside 1 (7) [13],
2
3
4-epi-pinfaensin (8) [14], 24-epi-niga-ichigoside F1 (9) [15], anserinoside (10) [16],
β,19ɑ,23,24-tetrahydroxyurs-12-en-28-oic acid (11) [17], ziyu-glycoside I (12) [18],
hyptatic acid B (13) [15], laevigin C (14) [19], euscaphic acid (15) [20],
ursolazuroside 2 (16) [13], officinoterpenoside B (17) [21], sweriyunnanoside A (18)
[22], 2ɑ,3β-dihydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyranoside (19) [23],
oleanazuroside 2 (20) [13], anchusosid-5 (21) [24], anchusosid-9 (22) [25],
anchusosid-8 (23) [26], anchusosid-11 (24) [26], maslinic acid (25) [27],
2
ɑ,3β,21β-trihydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyranoside (26) [23],
sericoside (27) [28], 21-O-β-D-glucopyranosyl-2ɑ,3β,21β,23-tetrahydroxyolean-12-
en-28-oic acid (28) [29] (Fig. 1).
Compound 1, amorphous powder, possesses a molecular formula of C H O
78 21
4
8
+
13
based on its HRESIMS ([M + Na] m/z 1013.4966) and C NMR data, indicating ten
indices of hydrogen deficiency. The 1D NMR data (Tables 1 and 2) revealed the
presence of six tertiary methyl groups at δ 1.39 (CH -29), 1.36 (CH -30), 1.11
H
3
3
(
CH -27), 1.00 (CH -25), 1.00 (CH -24), and 0.97 (CH -26), one olefinic proton at δ
3 3 3 3 H
5
1
.45 (1H, t, J = 3.6 Hz, H-12) with two typical olefinic carbon signals at δ 123.1 and
C
44.5, and one carboxyl carbon signal at δ 179.5, indicative of an oleanolic acid
C
1
3
derivative. The C NMR spectrum of 1 showed 48 carbon signals, 30 of which were
assigned to aglycone, and 18 to sugar moieties. Enzymatic hydrolysis of 1 with
cellulase liberated D-glucose, which was identified by HPLC with an optical rotation
detector (HPLC-OR). The H and 13C NMR data (Tables 1 and 2) of 1 were in
accordance with those of anchusoside-3 [30], except for the presence of two more
1
glucose units in 1, and the chemical shift value of C-23 was deshielded from δ 66.4
C
in anchusoside-3 to δ 74.3 in 1. In addition, the HMBC correlations observed from
C
δ 4.93 (Glc′′-H-1) to δ 74.3 (C-23), and δ 5.38 (Glc′′′-H-1) to δ 83.9 (Glc′′-C-2),
H
C
H
C
demonstrated that the disaccharide residue [β-D-glucopyranosyl-(1→2)-β-D-
glucopyranosyl] was linked at C-23 of the aglycone. Based on biosynthetic
3

considerations [4], H-5 was α-oriented, and H-18 and CH -25 were β-oriented for the
3
natural oleanane skeleton. In the NOESY spectrum, H-3 correlated to H-5, CH -24
3
correlated to H-2 and CH -25, and H-21 correlated to CH -29 (Fig. 3), indicating H-2
3
3
and CH -24 were β-oriented, while H-3 and CH -29 were α-oriented. Therefore, the
3
3
structure of (2α,3β,21β)-21-(β-D-glucopyranosyloxy)-23-{[β-D-glucopyranosyl(1→2)
-β-D-glucopyranosyl]oxy}-2,3-dihydroxyolean-12-en-28-oic acid was established.
Compound 2, isolated as an amorphous powder, shared the identical molecular
+
formula of C H O with 1 according to its HRESIMS at m/z 1013.4963 [M + Na]
4
8
78 21
1
3
(
calcd 1013.4927) and C NMR data, indicative of 10 indices of hydrogen deficiency.
Three anomeric proton signals were displayed in its NMR spectra at δ 5.04 (1H, d, J
H
=
7.6 Hz), 5.19 (1H, d, J = 7.2 Hz) and 5.38 (1H, d, J = 7.6 Hz); δ 105.2, 106.2 and
C
1
06.5. The IR and 1D NMR data of 2 were almost identical to those of 1. However,
detailed NMR analysis revealed that the chemical shift value of C-3 deshielded to δC
8.9, while C-23 shielded to δ 64.3 in 2, indicating that the disaccharide residue was
8
C
placed at C-3 of the aglycone instead of C-23. This assumption was validated by the
HMBC correlations from Glc′′-H-1 (δ 5.19) to C-3 (δ 88.9) and Glc′′′-H-1 (δ 5.38)
H
C
H
to Glc′′-C-2 (δ 83.9). Thus, the structure of 2 was determined as (2α,3β,21β)-3-
C
{
[β-D-glucopyranosyl(12)-β-D-glucopyranosyl]oxy}-21-(β-D-glucopyranosyloxy)-2,
3-dihydroxyolean-12-en-28-oic acid.
2
Compound 3 was obtained as a white, amorphous powder. The HRESIMS peak
+
13
at m/z 997.5019 [M + Na] , together with its C NMR data, indicated the molecular
formula of 3 to be C H O , with one oxygen atom less than that of 1. By careful
4
8
78 20
inspection of the 1D NMR data for 1 and 3, obvious upfield shifts of C-2 (δ 27.8 for
C
3
vs. δ 69.1 for 1) indicated that C-2 in 3 was reduced, which was further verified by
C
the HMBC correlations of H-1(δ 1.48) with C-2 (δ 27.8), as well as H -23 (δ
H
H
C
2
4
.05/4.00) and H -24 (δ 0.97) with C-3 (δ 72.6) in 3. Accordingly, the structure of 3
3 H C
was identified as (3β,21β)-21-(β-D-glucopyranosyloxy)-23-{[β-D-glucopyranosyl
(12)-β-D-glucopyranosyl]-oxy}-3-hydroxyolean-12-en-28-oic acid.
4

3
0
29
21
1
9
13
1
1
2
5
26
H17 C8OOH
2
1
9
H
COOH
OH
HO
HO
O
OH
H 7
15
OH
6'
OH
OH
H
3
5
27
O
O
6''
H
HO
O
HO
O
5
''
5'
HO
HO
HO
HO
O
O
24H
HO
O
HO
HO
O
H
O
HO
HO
O
23
HO
3''
3'
1
''
OH 1'
OH
OH
H
6'''
OH
5'''
HO
HO
HO
HO
O
O
O
3'''
OH 1'''
OH
1
3
6
R4
H
R3
O
H
COOH
OH
H
COOH
OH
R1O
R2O
HO
HO
R O
1
H
O
O
2
R O
H
H
H
O
R3
HO
HO
O
R1
R2
OH
R3
HO
HO
OR4
HO
HO
O
OR4
O
OR5
7
8
9
R1 = R2 = R4 = CH3, R3 = OH, R5= H
R1 = CHO, R2 = R4 = CH3, R3 = OH, R5 = H
R1 = CH2OH, R2 = R4 = CH3, R3 = OH, R5 = H
2
4
5
R1 = R4 = H, R2 = Glc', R3 = CH2OH
R1 = R4 = Glc, R2 = H, R3 = CH3
R1 = H, R2 = R4 = Glc, R3 = CH3
20 R1 = R2 = H, R3 = CH , R = Glc
3 4
22 R1 = R2 = H, R3 = CH OH, R = Glc
2 4
10 R1 = R2 = R3 = CH3, R4 = OH, R5= H
23 R1 = H, R2 = Rib, R3 = CH OH,R = Glc
2 4
1
1
6 R1 = R4 = CH3, R2 = CHO, R3 = OH, R5 = H
7 R1 = R2 = R4 = CH3, R3 = OH, R5 = Glc
O
HO
HO
O
HO
H
COOR4
HO
HO
H
COOH
HO
HO
H
COOH
HO
HO
OH
R3O
H
H
R1 R2
H
OHHO
O
H
OH
HO
O
OH
11 R1 = R2 = CH2OH, R3 = R4 = H
12 R1 = R2 =CH3, R3 = R4 = Glc
13
14
15
O
H
H
COOH
OH
OR2
O
HO
OH
H
1
R O
H
H
COOR1
HO
HO
O
HO
H
COOR3
HO
HO
O
OH
HO
HO
O
OH
HO
OH
H
H
O
R1
HO
HO
HO
HO
O
O
O
OH
OH
1
9 R1 = Glc
2
2
1 R1 = H
4 R1 = Rib
26 R1 = CH3, R2 = H, R3 = Glc
28 R1 = CH2OH, R2 = Glc, R3 = H
18
25 R1 = H
HO
OH
O
OH
O
H
HO
HO
HO
Glc = HO
HO
HO
O
OH
OH
H
Glc' =
OH
OH HO
HO
O
HO
HO
O
HO
Rib =
O
O
OH
HO
O
OH
OH
27
Fig. 1. Structures of compounds 1-28 from A. italica.
5

Compound 4 has a molecular formula of C H O , which was deduced from the
4
8
78 20
+
13
1
[
M + Na] ion peak at m/z 997.5018 in the HRESIMS and its C NMR data. The H
NMR spectrum of 4 showed signals attributable to seven tertiary methyl groups at δH
.90 (CH -25), 0.95 (CH -26), 1.01 (CH -24), 1.20 (CH -27), 1.24 (CH -23), 1.36
0
3
3
3
3
3
(
CH -30) and 1.42 (CH -29), two oxymethine protons at δ 4.13 (1H, m, H-2) and
3 3 H
3
.41 (1H, d, J = 9.4 Hz, H-3), and one olefinic proton at δ 5.50 (1H, t, J = 3.2 Hz,
H
H-12). Signals were also observed assignable to three anomeric protons of three sugar
units at δ 5.05 (1H, d, J = 7.6 Hz, Glc′-H-1), 5.39 (1H, d, J = 7.6 Hz, Glc′′-H-1) and
H
5
.10 (1H, d, J = 7.7 Hz, Glc′′′-H-1). Further, the β-D-glycosidic linkages for three
glucose units were confirmed by the coupling constants and HPLC with an optical
rotation detector (HPLC-OR). The NMR data (Tables 1 and 2) analysis of 4 indicated
the same aglycone moiety of 4 as oleanazuroside 2 (20) [13], but one more glucose is
present in 4. Further, the glucose was determined to be connected with C-2 of the
aglycone from the HMBC correlations from Glc′′′-H-1 (δ 5.10) to C-2 (δ 80.8), and
H
C
from H-3 (δ 3.41) to C-2 (δ 80.8), C-4 (δ 40.2), C-23 (δ 29.7), and C-24 (δ 18.1)
H
C
C
C
C
(
Fig. 2). The NOESY correlations of H-3 with H-5, and H-2 with CH -25 indicated
3
the α-orientation of H-3 and β-orientation of H-2 (Fig. 3). Thus, the structure of 4 was
elucidated as (2α,3β,21β)-2-(β-D-glucopyranosyloxy)-21-{[β-D-glucopyranosyl
12)-β-D-glucopyranosyl]oxy}-3-hydroxyolean-12-en-28-oic acid.
(
Compound 5, a white, amorphous powder, shared the same molecular formula
+
C H O as 4 based on the HRESIMS at m/z 997.4986 [M + Na] (calcd 997.4978)
4
8
78 20
1
3
13
and C NMR data. The exceedingly similar C NMR data (Table 2) of 4 and 5
manifested that their structural difference was restricted to the interchange of glucose
moiety at C-2 and C-3. The glucose moiety was attached to C-3 due to the HMBC
correlations from Glc′′′-H-1 (δ 4.97) to C-3 (δ 95.8). H-2 and H-3 were inferred to
H
C
be β- and α-oriented, respectively, in view of the NOESY correlations of H-2 with
CH -25, and H-3 with H-5 (Fig. 3). Consequently, the structure of 5 was elucidated as
3
(2α,3β,21β)-3-(β-D-glucopyranosyloxy)-21-{[β-D-glucopyranosyl(12)-β-D-
glucopyranosyl]oxy}-2-hydroxyolean-12-en-28-oic acid.
Compound 6 was isolated as a white, amorphous powder with a molecular
6

formula of C H O (eight indices of hydrogen deficiency) as deduced from the
3
0
46
6
+
13
HRESIMS data at m/z 503.3383 [M + H] (calcd for C H O 503.3367) and C
3
0
47
6
NMR data. The 1D NMR data (Tables 1 and 2) showed six methyl groups [δ 0.98
H
(
CH -26), 1.09 (CH -24), 1.09 (CH -25), 1.12 (CH -29), 1.26 (CH -30) and 1.53
3 3 3 3 3
(
CH -27); δ 17.6, 14.9, 18.3, 14.3, 28.5 and 26.2], one oxygenated methylene [δ
3
C
H
3
.74 (1H, d, J = 10.5 Hz) and 4.20 (1H, d, J = 9.7 Hz); δ 66.6], three oxygenated
C
methines [δ 4.20 (1H, d, J = 10.5 Hz, H-3), 4.27 (1H, m, H-2), and 4.39 (1H, d, J =
H
5
.3 Hz, H-21); δ 78.4, 69.3 and 82.5], an olefinic bond [δ 5.48 (1H, t, J = 3.4 Hz,
C H
H-12); δ 130.7 and 137.3], and an ester carbonyl (δ 183.7), indicative of an ursane
C
C
derivative. Its NMR data (Tables 1 and 2) demonstrated that 6 was highly similar to
α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid [31], except that the chemical shift of
C-21 was deshielded from δ 27.4 to δ 82.5 in 6, indicating the oxygenation of C-21.
2
C
C
The pivotal HMBC correlations from H-21 to C-19, C-20, C-22, C-28 and C-29, in
combination with eight indices of hydrogen deficiency, established the presence of a
γ-lactone unit. The coupling constant (J = 10.5 Hz) between H-2 and H-3 indicated
their trans-diaxial orientation, which was confirmed by the key NOESY cross-peaks
of H-2/CH -25 and H-3/H-5/CH -23. The NOESY correlation of H-21/CH -29
3
3
3
indicated an α-orientation for H-21 (Fig. 3). Consequently, the structure of 6 was
elucidated as 2α,3β,19α,23-tetrahydroxyurs-12-en-28,21β-olide.
1
a
Table 1 H NMR Data of Compounds 1-6 in Pyridine-d (600 MHz, J in Hz)
5
pos.
1
2
3
4
5
6
2
1
.21 dd (12.5, 3.9)
2.23 o
1.42 - 1.48 m
0.89 - 0.92 m
2.29 dd (12.1, 3.1)
1.16 br d (12.1)
2.24 dd (12.2, 3.1) 2.29 dd (4.2, 11.8)
1
.25 o
1.26 - 1.29 m
4.19 - 4.22 m
4.28 d (9.5)
1.16 d (12.2)
4.00 - 4.05 m
3.30 d (9.0)
1.31 t (11.8)
4.23 - 4.29 m
4.20 d (10.5)
1
1
.83 - 1.86 m
.75 - 1.80 m
2
4.17-4.23 m
4.10 - 4.15 m
3
5
4.24 d (9.3)
4.20 dd (9.6, 3.9)
3.41 d (9.4)
1.73 dd (11.3, 2.0)
1.83 br d (12.4)
1.68 - 1.74 m
1.29 - 1.32 m
1.57 - 1.62 m
1.23 - 1.26 m
1.86 d (9.2)
1.44 br d (12.6)
1.62 - 1.67 m
1.26 - 1.32 m
1.50 - 1.56 m
1.17 - 1.24 m
1.64 d (8.4)
0.92 o
0.91 - 0.93 m
1.47 - 1.53 m
1.29 - 1.32 m
1.44 - 1.46 m
1.25 - 1.28 m
1.71 - 1.76 m
1.98 - 2.02 m
1.92 - 1.95 m
5.47 s
1.80 - 1.84 m
1.75 - 1.79 m
1.48 - 1.52 m
1.71 - 1.76 m
1.48 - 1.53 m
1.84 - 1.86 m
2.18 - 2.22 m
1.84 - 1.87 m
5.48 t (3.4)
1
1
1
1
.72 - 1.76 m
.29 - 1.33 m
.61 - 1.64 m
.25 o
1.52 br d (11.7)
1.32 br d (12.8)
1.42 - 1.46 m
1.25 - 1.29 m
1.61- 1.69 m
6
7
9
1.77 br d (9.3)
1.95 - 1.99 m
5.45 t (3.6)
1
1
.82 - 1.91 m
.72 - 1.81 m
1
1
1
2
1.93 - 2.00 m
5.46 t (3.2)
1.85 - 1.92 m
5.46 t (3.5)
5.50 t (3.2)
7

2
1
.08 o
2.06 - 2.09 m
1.03 - 1.08 m
2.07 - 2.10 m
1.05 - 1.09 m
2.11 o
2.11 - 2.14 m
1.10 - 1.15 m
1.86 dd (6.0, 13.7)
1.14 d (15.2)
1
1
5
6
.06 - 1.11 m
1.08 - 1.12 m
2
2
.09 - 2.12 m
2.11 - 2.13 m
2.07 - 2.10 m
2.12 - 2.15 m
2.16 - 2.18 m
2.10 - 2.19 m
2.20 d (13.9)
1.14 d (13.9)
2.09 - 2.15 m
.08 o
2.11 o
1
1
8
9
3.38 dd (13.7, 3.75) 3.38 dd (14.1, 3.6)
3.6 dd (13.3, 4.4)
1.95 t (14.0)
1.38 - 1.42 m
-
3.40 dd (12.7, 3.7)
2.00 t (14.0)
1.42 - 1.46 m
-
3.40 br d (12.6)
1.98 - 2.02 m
1.42 - 1.44 m
-
2.70 br s
-
1
1
-
.94 d (13.9)
.31 - 1.34 m
1.94 t (14.0)
1.41 dd (14.0, 4.7)
-
2
2
0
1
1.93 d (7.6)
4.39 d (5.3)
3.87 - 3.91 m
3.87 - 3.90 m
2.64 dd (13.5, 4.3)
2.32 o
3.86 - 3.89 m
2.66 dd (13.3, 4.4)
2.32 t (12.6)
4.05 d (9.3)
4.00 d (9.3)
0.97 s
3.92 - 3.96 m
3.92 - 3.96 m
2
2
.65 dd, (13.2, 4.2)
.32 t (13.2)
2.68 dd (12.8, 2.6)
2.34 t (12.8)
2.67 dd (12.8, 2.8) 1.63 dd (5.3, 11.0)
2
2
2
3
2.34 t (12.8)
3.51 d (11.0)
4.20 d (9.7)
3.74 d (10.5)
1.09 s
4
3
.56 d (10.7)
.71 d (10.7)
4.05 br s
1.24 s
1.41 s
2
2
2
2
2
3
4
5
6
7
9
0
1.00 s
1.01 s
1.01 s
1.05 s
1.00 s
1.00 s
0.88 s
0.90 s
0.90 s
1.09 s
0.97 s
0.99 s
0.95 s
0.95 s
0.96 s
0.98 s
1.11 s
1.17 s
1.15 s
1.20 s
1.27 s
1.53 s
1.39 s
1.39 s
1.39 s
1.42 s
1.42 s
1.12 d (7.3)
1.36 s
1.36 s
1.36 s
1.36 s
1.38 s
1.26 s
Glc′-1
5.03 d (7.7)
4.03 - 4.08 m
4.43 - 4.46 m
4.21 - 4.26 m
3.90 - 3.96 m
5.04 d (7.6)
4.11 - 4.15 m
3.94 - 3.96 m
4.25 - 4.27 m
5.04 d (7.6)
4.02 - 4.08 m
4.33 - 4.37 m
4.18 - 4.23 m
5.05 d (7.6)
4.25 - 4.29 m
3.95 - 3.99 m
4.25 - 4.29 m
5.05 d (7.4)
4.21 - 4.25 m
4.25 - 4.28 m
4.33 - 4.36 m
-
-
-
-
-
2
3
4
5
3.92 - 3.94 m
4.49 - 4.52 m
4.42 - 4.46 m
5.19 d (7.2)
4.33 - 4.37 m
4.48 - 4.52 m
4.45 - 4.48 m
4.90 d (7.6)
4.33 - 4.37 m
4.47 - 4.50 m
4.33 - 4.37 m
5.39 d (7.6)
3.89 - 3.92 m
4.58 - 4.62 m
4.46 - 4.49 m
5.39 d (7.3)
4
4
.45 - 4.53 m
.31 - 4.37 m
6
-
Glc′′-1 4.93 d (7.7)
-
-
-
-
-
2
3
4
5
4.21 - 4.27 m
4.32 - 4.38 m
4.18 - 4.20 m
4.16 - 4.17m
4.22 - 4.24 m
4.25 - 4.27 m
4.22 - 4.24 m
4.20 - 4.22 m
3.91 - 3.92 m
4.33 - 4.37 m
4.12 - 4.15 m
4.23 - 4.25 m
4.34 - 4.38 m
4.14 - 4.18 m
4.08 - 4.13 m
4.21 - 4.25 m
4.11 – 4.13 m
4.22 - 4.24 m
4.31 - 4.34 m
5.38 d (7.6)
3.92 - 3.95 m
4.54 dd (11.8, 2.6)
4.35 - 4.39 m
5.40 d (7.4)
4.26 - 4.29 m
4.47 - 4.50 m
4.33 - 4.37 m
5.10 d (7.7)
3.89 - 3.92 m
4.46 - 4.49 m
4.33 - 4.37 m
4.97 d (7.7)
4
4
.57 dd (11.7, 2.4)
.38 - 4.42 m
6
-
Glc′′′-1 5.38 d (7.6)
-
-
-
-
-
2
3
4
5
4.12 - 4.18 m
4.20 - 4.29 m
4.33 - 4.39 m
3.90 - 3.96 m
4.15 - 4.18 m
3.87 - 3.94 m
4.35 - 4.38 m
4.13 - 4.18 m
4.23 - 4.27 m
4.18 - 4.23 m
4.03 - 4.06 m
3.93 – 3.95 m
4.21 - 4.23 m
4.08 - 4.13 m
4.33 - 4.37 m
4.21 - 4.25 m
3.87 - 3.94 m
4.49 - 4.52 m
4.34 - 4.37 m
3.91 - 3.97 m
4.48 - 4.52 m
4.38 - 4.41 m
4.01 - 4.03 m
4.51 - 4.53 m
4.33 - 4.37 m
4.26 - 4.28 m
4.46 - 4.49 m
4.33 - 4.37 m
4
4
.45 - 4.53 m
.31 - 4.37 m
6
-
a
“
o” represents the overlapped peak.
1
3
Table 2 C NMR Data of Compounds 1-6 in Pyridine-d (150 MHz)
5
pos.
1
1
2
3
4
5
6
47.7
69.1
47.7
67.4
38.8
27.8
45.6
80.8
47.5
67.0
48.4
69.3
2
8

3
4
5
6
7
8
9
78.1
43.9
48.6
18.9
33.2
40.1
48.4
38.7
24.3
123.1
144.5
42.5
28.6
25.2
48.9
41.7
47.8
6.4
88.9
45.1
47.7
18.6
33.1
40.1
48.5
38.2
24.3
123.2
144.3
42.6
28.7
25.3
48.9
41.7
47.8
6.4
72.6
43.2
49.0
18.9
33.3
40.0
48.4
6.5
81.5
40.2
55.7
18.9
33.5
40.0
48.3
38.7
24.2
123.0
144.3
42.6
28.6
25.3
49.0
41.8
47.9
6.4
95.8
41.0
56.0
18.9
33.4
40.0
48.4
38.2
24.3
123.1
144.2
42.6
28.6
25.2
48.9
41.7
47.9
6.4
78.4
44.0
48.6
19.0
33.4
41.7
47.8
38.7
24.7
130.7
137.3
42.2
29.1
29.9
45.1
56.8
73.9
45.0
82.5
33.8
66.6
14.9
18.3
17.6
26.2
183.7
14.3
28.5
-
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
24.1
123.2
144.4
42.5
28.6
25.2
48.9
41.7
47.9
6.4
83.6
40.7
74.3
14.7
17.8
17.9
26.5
179.5
29.7
18.8
105.1
75.7
78.2
72.0
78.7
63.1
105.7
83.9
78.7
72.2
79.1
63.4
106.5
77.6
78.3
72.0
78.9
63.0
83.6
40.7
64.3
15.2
17.7
17.8
26.4
179.5
29.7
18.8
105.2
75.9
78.3
71.8
78.7
63.1
106.2
83.9
78.3
72.0
78.7
62.7
106.5
77.6
78.8
72.0
78.9
62.9
83.6
40.7
75.6
13.6
16.3
17.8
26.5
179.5
29.7
18.8
105.1
75.7
78.7
72.1
78.9
63.0
105.9
83.9
78.2
72.0
78.8
63.3
106.5
77.6
78.3
72.0
79.1
63.1
83.6
40.7
29.7
18.1
16.9
17.7
26.4
179.5
29.8
18.8
105.2
84.0
78.3
72.0
78.8
63.0
106.6
77.6
78.7
72.0
78.9
62.8
104.4
75.6
78.3
71.9
78.7
63.1
83.6
40.7
28.9
18.5
17.0
17.8
26.4
179.5
29.8
18.7
105.1
83.9
79.1
72.0
78.7
62.9
106.5
77.6
78.9
71.9
78.3
62.9
106.9
76.0
78.7
71.9
78.2
63.1
Glc′-1
2
-
3
-
4
-
5
-
6
-
Glc′′-1
-
2
-
3
-
4
-
5
-
6
-
Glc′′′-1
-
2
3
4
5
6
-
-
-
-
-
9

H
COOH
OH
H
COOH
OH
HO
HO
OH
HO
O
H
H
O
O
O
OH
O
HO
O
HO
HO
OH
HO
HO
HO
H
O
H
OH
O
OH
HO
HO
OH
OH
O
HO
HO
O
O
OH
HO
HO
O
1
2
OH
OH
H
COOH
OH
O
H
COOH
OH
HO
HO
O
H
O
OH
OH
HO
O
H
HO
O
O
HO
O
H
HO
HO
OH
O
OH
HO
HO
HO
OH
H
O
O
HO
HO
O
HO
HO
O
OH
OH
3
4
H
COOH
HO
O
OH
HO
OH
O
OH
H
O
HO
HO
O
HO
HO
HO
HO
OH
O
O
H
H
O
H
HO
HO
O
OH
OH
6
5
Fig. 2. Key HMBC correlations of compounds 1-6.
1
2
3
4
5
6
Fig. 3. Key NOESY correlations of compounds 1-6.
1
0

We established an in vitro experimental model of neonatal rat cardiomyocytes
injury to evaluate the protective effects of all the isolated compounds 1-28. Neonatal
rat cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) insult for a
different time (3, 6, 9 h). Cell viability, LDH, and CK-MB levels were measured. As
shown in Fig. 4A-C, the viability of neonatal rat cardiomyocytes was decreased and
LDH, CK-MB levels were increased after subjected to H/R after 3 h. Therefore, the
condition of underwent H/R injury for 3/3 h was chosen for the further research.
Fig. 4. The ability of compounds 1-28 against H/R-induced neonatal rat cardiomyocytes injury.
(A-C) The neonatal rat cardiomyocytes were subjected to H/R in different time (3, 6, 9 h), and
cells viability, LDH and CK-MB levels were measured. (D) Neonatal rat cardiomyocytes were
pretreated with compounds 1-28 (100 µM) for 4 h and underwent H/R injury for 3/3 h.
Next, the potential protective effects of compounds 1-28 were evaluated. The
results showed that the poor survival of neonatal rat cardiomyocytes was significantly
ameliorated after treated with compounds 4, 6-17, 21-22 and 26-28 compared with the
H/R group (Fig. 4D), demonstrating their protective effects on neonatal rat
cardiomyocytes injured by H/R. The preliminary structure-activity relationship
indicated that triterpenoids (1-3, 23) with three glucose moieties in the structures
showed no protective activity, and the absence of 3-OH or 23-OH could obviously
1
1

decrease the protective effects of triterpenoids (18-20, 23-25). And these compounds
showed no effect on cell viability (IC >200 µM) in neonatal rat cardiomyocytes with
5
0
2
4 h incubation.
The protective mechanism of the representative new compound 6 on myocardial
ischemia/reperfusion injury was further studied. Compound 6 antagonized the
reduction of viability and the increase of apoptosis induced by H/R insult in primary
neonatal rat cardiomyocytes subjected to 3 h hypoxia followed by 3 h reoxygenation.
The reduction of cell viability mainly results from apoptosis in primary neonatal rat
cardiomyocytes when subjected to H/R insult. The results of Hoechst 33342 staining
showed that cells subjected to H/R significantly increased the number of cells with
condensed or blebbing nuclei. In contrast, when cells were pretreated with compound
6
, these phenomena were reduced (Fig. 5A). The mitochondrial membrane potential
(MMP) in primary neonatal rat cardiomyocytes was also evaluated to investigate the
effects of compound 6 on the mitochondrial function of neonatal rat cardiomyocytes
induced by H/R. The results demonstrated that the reduction in MMP was
significantly attenuated by pretreatment with compound 6 (Fig. 5B). In addition,
Western blot analysis indicated that H/R downregulated the expression of
apoptosis-associated protein Bcl-2 and upregulated Bax expression. Compound 6
rescued the down-regulated Bcl-2 and decreased the up-regulated Bax. These results
confirmed that compound 6 may antagonize the apoptosis induced by H/R insult.
Fig. 5. Effects of 6 on cell viability and apoptosis in neonatal rat cardiomyocytes and neonatal rat
cardiomyocytes subjected to H/R. (A-B) The cells were treated with compound 6 for 4 h before
1
2

subjected to H/R, stained with Hoechst 33342 and Rhodamine 123 solution, then observed using a
fluorescence microscope (200 × magnification). (C) Cells were treated with compound 6 for 4 h
before subjected to H/R followed by Western blot analysis for the detection of Bcl-2 and Bax.
Oxidative stress is widely reported to serve a critical role in mediating
myocardial H/R injury, thus the effect of compound 6 on oxidant stress in neonatal rat
cardiomyocytes in response to H/R injury was investigated. To examine whether the
protective effect of compound 6 was actually mediated by attenuating ROS generation,
the DCFH-DA fluorescent probe was used to evaluate the level of intracellular ROS
in neonatal rat cardiomyocytes subjected to H/R insult. DCFH-DA passes through the
cellular membrane and is hydrolyzed to DCFH by intracellular esterases. DCFH is
then unable to cross the membrane and is thus reserved in the cytosol. With the
excessive production of ROS, DCFH is quantitatively oxidized into the fluorescent
dichlorofluorescein (DCF). The results of the present experiments revealed that the
fluorescence in the H/R group was markedly higher than that in the control group.
Upon treatment with different concentrations of 6, the fluorescence intensity of DCF
was reduced (Fig. 6A), implying that compound 6 was able to alleviate the
overproduction of intracellular ROS in neonatal rat cardiomyocytes induced by H/R.
Under H/R, the activity of LDH in the culture medium of neonatal rat
cardiomyocytes significantly increased compared with the control group,
demonstrating that leakage of LDH from the cytosol occurred in the H/R group and
cell survival decreased. When treated with compound 6, the release of LDH in
neonatal rat cardiomyocytes was significantly decreased (Fig. 6B). These results
indicated the compound 6 affected the LDH release in neonatal rat cardiomyocytes
induced by H/R. As demonstrated in Fig. 6C, H/R treatment significantly increased
CK-MB level in neonatal rat cardiomyocytes compared with the normoxia group and
this parameter was significantly decreased by compound 6 pretreatment. By contrast,
pretreatment with compound 6 significantly increased GSH levels induced by H/R in
neonatal rat cardiomyocytes (Fig. 6D).
To determine the intracellular calcium level, the fluorescence probe Fluo-3 AM
was used. Following treatment of H/R, the fluorescence intensity of the intracellular
1
3

calcium in the H/R group was significantly elevated compared with the control group
Fig. 6E). However, when compared with the H/R group, compound 6 treatment
(
attenuated the overload of intracellular calcium as observed by the reduced
fluorescence intensity in neonatal rat cardiomyocytes induced by H/R.
Fig. 6. Compound 6 inhibits oxidant stress in H/R-induced neonatal rat cardiomyocytes. (A) Cells
were pretreated with 6 for 4 h before subjected to H/R insult. Cells were stained with DCFH-DA.
The DCF fluorescence intensity was measured by fluorescence microscope. (B-D) Cells were
pretreated compound 6. LDH, CK-MB and GSH were measured with microplate. (E) The content
and intracellular calcium concentration of primary culture cardiomyocytes subjected to H/R.
Activation of autophagy usually occurs in cardiomyocytes following H/R. To
identify the role of compound 6 in regulating H/R-mediated autophagy in
cardiomyocytes, we examined whether compound 6 could inhibit autophagy in
cardiomyocytes following H/R by MDC staining. The autofluorescent substance
MDC has been shown to be a specific marker for autophagic vacuoles (AVs). When
cells are viewed with a fluorescence microscope, AVs stained by MDC appear as
distinct dot-like structures distributed within the cytoplasm or localized to the
perinuclear regions. In the H/R group, an increase of MDC-labeled vesicles was
observed, as indicated by punctuate MDC fluorescence (Fig. 7A), suggesting an
induction of AV formation after H/R. In the compound 6-treated groups, the number
of MDC-labeled vesicles declined. LC3-II is an accepted marker for autophagosome
formation. Western blot analysis revealed that LC3-II was up-regulated in H9c2 cells
when exposed to H/R (Fig. 7B). And compound 6 could inhibit the expression of
1
4

LC3-II. Besides, the result of immunofluorescence experiments of LC3-II also
confirmed the Western blot result (Fig. 7C). To further investigate the effect of
compound 6 on autophagy, we used Western blot to determine the expression level of
autophagy-related gene, Beclin-1. Expression of Beclin-1 was increased in neonatal
rat cardiomyocytes subjected to H/R, and compound 6 reduced the expression of
Beclin-1 (Fig. 7B). The above data clearly indicated that compound 6 could inhibit
autophagy induced by H/R in neonatal rat cardiomyocytes.
The underlying molecular mechanism of compound 6 inhibiting H/R-induced
autophagy was next investigated. Since p38 MAPK is involved in myocardial injury
caused by H/R, Western blot analysis was performed to investigate whether
compound 6 inhibited the activation of p38 MAPK in neonatal rat cardiomyocytes. As
demonstrated Fig. 7D, H/R treatment significantly induced the phosphorylation of p38
MAPK compared with the normal group. However, compound 6 significantly
alleviated H/R-induced phosphorylation of p38 MAPK in neonatal rat
cardiomyocytes.
Fig. 7. Effect of compound 6 on H/R-induced autophagy in neonatal rat cardiomyocytes. (A)
Autophagic vacuoles were stained with MDC. (B) Protein expression of LC3II and Beclin-1. (C)
The expression of LC3II was detected by immunocytochemistry. (D) Protein expression of p-p38
and p38. Quantification of panel E with densitometry. β-actin was used as a loading control. The
data shown are represented as the means ± SD confirmed in three separate experiments.
1
5

3
. Conclusion
Herein, 28 triterpenoids including 6 new (1-6) and 22 known analogues (7-28)
were isolated from the aerial parts of A. italica. All these isolated compounds were
assayed for their possible effects on hypoxia/reoxygenation (H/R) induced
cardiomyocytes injury. In our study, representative compound 6 significantly
suppressed the levels of H/R-induced apoptosis and autophagy in neonatal rat
cardiomyocytes, confirmed tripterpenoid 6 and its analogues may be the
pharmacodyamic material of A. italica as cardiovascular and cerebrovascular agent in
Xinjiang region (China), and offered a promising therapeutic approach for treating
cardiomyocyte injury induced by H/R..
4
. Experimental section
4
.1. General Experimental Procedures
Optical rotations were acquired on a PerkinElmer 241 polarimeter. IR spectra
were recorded on a Bruker IFS 55 spectrometer. NMR spectra were measured on an
AV-600 spectrometer. Chemical shift values are described as δ (ppm) by using one of
the resonances of Pyridine-d (δ 7.58 and δ 135.9) as reference, and the coupling
5
H
C
constants are expressed as J in Hz. HRESIMS data were tested on an Agilent 6210
TOF mass-spectrometer. Silica gel (200−300 mesh, Qingdao Marine Chemical
Factory), octadecyl silica gel (Merck Chemical Company Ltd., Germany), and
Sephadex LH-20 (Pharmacia, USA) were utilized for column chromatography (CC).
Silica gel GF254 TLC plates were purchased from Qingdao Marine Chemical Factory
(Qingdao, China). RP-HPLC was performed on an LC-6AD liquid chromatography
system equipped with a SPD-20A UV detector (Shimadzu, Kyoto, Japan) and an ODS
column (C18, 250 × 20 mm, 120 Å, 5 μm, YMC Co. Ltd.).
4
.2. Plant Material
The aerial parts of A. italica Retz. were collected from Shihezi, Xinjiang
Province, China, in March 2015, and were authenticated by Dr. Jin-Hui Wang,
Shenyang Pharmaceutical University. A specimen (NSC. 20150018) was preserved in
the herbarium of Shenyang Pharmaceutical University.
1
6

4
.3. Extraction and Isolation
The aerial parts of A. italica (30.0 Kg) were extracted with 75% EtOH (2 × 300
L × 2 h) under refluxing, and the crude extraction (1750 g) was obtained after
removing solvent in vacuo. The total extraction was absorbed by AB-8 macroporous
resin, and then eluted with H O, 30% EtOH, 50% EtOH, 70% EtOH and 95% EtOH.
2
The 70% EtOH layer (250 g) was subjected to silica gel CC eluted by CH Cl -MeOH
2
2
to afford nine fractions (D -D ). Fraction D (14.5 g) was separated by a silica gel
1
9
4
column (150 g) using CH Cl -MeOH as eluent to obtain six fractions (D -D ).
2
2
41
46
Subfraction D was subjected to Sephadex LH-20 CC (MeOH) and followed by an
4
6
ODS column (MeOH-H O, 10:90, 30:70, 50:50, 70:30, 100:0) to yield three
2
subfractions (D4621-D4623). Separation of subfraction D4623 through preparative HPLC
(
MeOH-H O, 65:35) gave compounds 13 (13.6 mg, t = 27.0 min) and 15 (4.2 mg, t
2 R R
=
36.2 min). Totally ten subfractions (D -D6-10) from fraction D6 (23.9 g) were
61
obtained via silica gel CC (50 g) eluted by CH Cl -MeOH (100:1, 80:1, 70:1, 50:1,
2
2
3
0:1, 10:1 and 0:1). Subfraction D was separated by ODS CC, and then purified by
63
Sephadex LH-20 CC (MeOH) to get fraction D634-13. Compounds 7 (4.2 mg, t = 15.2
R
min), 8 (203.4 mg, t = 55.1 min), 10 (365.2 mg, t = 43.2 min), and 11 (90.2 mg, t =
R
R
R
3
8.5 min) were purified by preparative HPLC (MeOH-H O, 65:35) from D634-13.
2
Fraction D was isolated by ODS CC to afford 4 subfractions (D -D ). D was
6
4
641
644
644
subjected to Sephadex LH-20 (MeOH) CC and followed by separation of preparative
HPLC (MeOH-H O, 60:40) to yield compound 26 (48.4 mg, t = 37.4 min). Fraction
2
R
D65 was chromatographed on an ODS column, and then subjected to a Sephadex
LH-20 column (MeOH) to obtain three fractions (D6521-D6523). D6522 was purified by
preparative HPLC (MeOH-H O, 65:35) to provide compounds 9 (200.1 mg, t = 41.0
2
R
min) and 24 (11.5 mg, t = 37.4 min), the rest was purified by preparative HPLC
R
(MeOH-H O, 60:40) to afford compound 14 (8.0 mg, t = 19.2 min). Compound 27
2 R
(
3.2 mg, t = 25.0 min) was obtained by preparative HPLC (MeOH-H O, 65:35).
R
2
Subfraction D was seperated by an ODS column, followed by preparative HPLC
6
9
(
MeOH-H O, 55:45) to yield compounds 3 (66.2 mg, t = 18.5 min), 4 (45.1 mg, t =
2
R
R
3
0.8 min), and 5 (39.5 mg, t = 10.5 min). Fraction D
was purified over ODS CC,
R
6-10
1
7

and then on preparative HPLC (MeOH-H O, 55:45) to provide compounds 1 (36.7 mg,
2
t = 10.7 min) and 2 (34.0 mg, t = 12.8 min). Fraction D (25.7 g) was isolated by
R
R
7
silica gel CC (50 g), eluted with CH Cl -MeOH (100:1, 80:1, 70:1, 50:1, 30:1, 10:1
2
2
and 0:1) to afford 5 fractions (D -D ). D (2.5 g) was repeatedly purified by silica
7
1
75
71
gel CC (CH Cl -MeOH), followed by preparative HPLC (HCN-H O, 25:75) to give
2
2
2
compound 28 (53.2 mg, t = 15.2 min), and the rest fraction was purified by
R
preparative HPLC (HCN-H O, 20:80) to afford compounds 12 (5.1 mg, t = 42.0 min),
2
R
1
6 (5.6 mg, t = 42.2 min), and 17 (6.4 mg, t = 25.7 min). Fraction D (25.6 g) was
R R 9
subjected to silica gel CC (50 g) using CH Cl -MeOH (100:1, 80:1, 70:1, 50:1, 30:1,
2
2
1
0:1 and 0:1) to yield 7 subfractions (D -D ). Compounds 22 (40.1 mg) and 6 (22.5
91 97
mg) were yielded by seperation with ODS CC from subfraction D . Compound 21
9
3
(
80.0 mg, t = 9.4 min) was purified from the remaining fraction of D by preparative
R
9
3
HPLC (MeOH-H O, 50:50). Subfraction D was subjected to a Sephadex LH-20
2
94
column (MeOH) to provide three fractions (D -D ). Separation of D through
9
41
943
942
preparative HPLC (HCN-H O, 25:75) to yield compound 23 (3.1 mg, t = 10.2 min).
2
R
Subfraction D943 was applied to preparative HPLC (MeOH-H O, 50:50) to obtain
2
compound 25 (3.3 mg, t = 28.9 min). Fraction D was chromatographed on a silica
R
96
gel column eluting with CH Cl -MeOH (100:1, 80:1, 70:1, 50:1, 30:1, 10:1 and 0:1),
2
2
followed by repeated separation with Sephadex LH-20 CC with MeOH as eluent to
yield five subfractions (D9621-D9625). Subfraction D9622 was purified by preparative
HPLC (MeOH-H O, 45:55) to obtain compound 20 (4550.0 mg, t = 28.9 min).
2
R
Compounds 18 (16.9 mg, t = 22.2 min) and 19 (3.3 mg, t = 34.7 min) were
R
R
respectively isolated from D9623 and D9624 by preparative HPLC (MeOH-H O, 50:50).
2
(2α,3β,21β)-21-(β-D-glucopyranosyloxy)-23-{[β-D-glucopyranosyl(1→2)-β-D-
glucopyranosyl]oxy}-2,3-dihydroxyolean-12-en-28-oic acid (1): white amorphous
2
D
0
powder (MeOH);



: -117.0 (c = 0.20, MeOH); IR (KBr) νmax: 3423, 2925, 1702,
384, 1078, 634 cm ; H (600 MHz, Pyridine-d ) and 13C NMR (150 MHz,
-
1
1
1
5
+
Pyridine-d ) data (Tables 1 and 2); HRESIMS m/z 1013.4966 [M + Na] (calcd for
5
+
C H O Na , 1013.4927).
4
8
78 21
1
8

(2α,3β,21β)-3-{[β-D-glucopyranosyl(12)-β-D-glucopyranosyl]oxy}-21-(β-D-
glucopyranosyloxy)-2,23-dihydroxyolean-12-en-28-oic acid (2): white amorphous
2
D
0
powder (MeOH);



: -113.9 (c = 0.24, MeOH); IR (KBr) νmax: 3424, 2927, 1639,
384, 1077, 634 cm ; H (600 MHz, Pyridine-d ) and 13C NMR (150 MHz,
-
1
1
1
5
+
Pyridine-d ) data (Tables 1 and 2); HRESIMS m/z 1013.4963 [M + Na] (calcd for
5
+
C H O Na , 1013.4927).
4
8
78 21
(3β,21β)-21-(β-D-glucopyranosyloxy)-23-{[β-D-glucopyranosyl(12)-β-D-
glucopyranosyl]-oxy}-3-hydroxyolean-12-en-28-oic acid (3): white amorphous
2
D
0
powder (MeOH);



: -114.2 (c = 0.20, MeOH); IR (KBr) νmax: 3423, 2929, 1702,
385, 1077, 635 cm ; H (600 MHz, Pyridine-d ) and 13C NMR (150 MHz,
-
1
1
1
5
+
Pyridine-d ) data (Tables 1 and 2); HRESIMS m/z 997.5019 [M + Na] (calcd for
5
+
C H O Na , 997.4978).
4
8
78 20
(2α,3β,21β)-2-(β-D-glucopyranosyloxy)-21-{[β-D-glucopyranosyl(12)-β-D-
glucopyranosyl]oxy}-3-hydroxyolean-12-en-28-oic acid (4): white amorphous powder
2
D
0
(MeOH);



: -95.7 (c = 0.30, MeOH); IR (KBr) νmax: 3423, 2923, 1629, 1384, 1075,
-1
1
13
5
74 cm ; H (600 MHz, Pyridine-d ) and C NMR (150 MHz, Pyridine-d ) data
5
5
+
+
(
Tables 1 and 2) ; HRESIMS m/z 997.5018 [M + Na] (calcd for C H O Na ,
48 78 20
9
97.4978).
2α,3β,21β)-3-(β-D-glucopyranosyloxy)-21-{[β-D-glucopyranosyl(12)-β-D-
glucopyranosyl]oxy}-2-hydroxyolean-12-en-28-oic acid (5): white amorphous powder
(
2
D
5
(MeOH);



-40 (c = 0.5, MeOH); IR (KBr) νmax: 3396, 2922, 2850, 1740, 1646,
-1
1
13
1
261, 802 cm ; H (600 MHz, Pyridine-d ) and C NMR (150 MHz, Pyridine-d )
5
5
+
+
data (Tables 1 and 2); HRESIMS m/z 997.4986 [M + Na] (calcd for C H O Na ,
4
8
78 20
9
97.4978).
α,3β,19α,23-tetrahydroxyurs-12-en-28,21β-olide (6): white amorphous powder
2
2
D
5
(MeOH);



+17 (c = 0.4, MeOH); IR (KBr) νmax: 3423, 2924, 2659, 1776, 1752,
-1
1
13
1
649, 1623, 1404, 1385, 1271, 703 cm ; H (600 MHz, Pyridine-d ) and C NMR
5
+
(
150 MHz, Pyridine-d data (Tables 1 and 2); HRESIMS m/z 503.3383 [M + H]
5
1
9

+
(
calcd for C H O , 503.3367).
30 47 6
4
.4. Enzymatic hydrolysis of compounds 1-5
Compounds 1-5 (each 4.0 mg) were dissolved in 4.0 mL water and about 25.0
mg snail enzyme was added, respectively. Then, the mixture was shaken in a 37 °C
constant temperature shaker, and the reaction progress was monitored by TLC every 2
h until the reaction was finished (8 h). The reaction was finally quenched with ethyl
acetate. The hydrolysate was extracted with 5 mL ethyl acetate for five times. The
solvent of organic phase was evaporated to obtain the corresponding aglycone, and
the aqueous phase containing glucose was centrifuged to remove enzyme, and then
freeze-dried. The dried aqueous phase was dissolved in acetonitrile-water (75:25),
filtered through a 0.45 μm filter, and then analyzed by HPLC using a JASCO
LC-NetII/ADC system [with a JASCO OR-4090 detector and a Shodex Asahipak
NH2P-50 4E column (250 × 4.6 mm, 5 μm)]. The HPLC-OR spectrum of both
standard D-glucose and glucose from compounds 1-5 showed a positive peak at 8.60
min (Supporting information Fig. S45).
4
.5. Primary culture of neonatal rat cardiomyocytes
The hearts of 1- to 3-day-old neonatal Sprague-Dawley (SD) rats were purchased
from Beijing HFK Bioscience Co. Ltd. (HFK) and surgically removed under sterile
3
conditions, washed three times with cold PBS, minced into ∼1 mm tissue chunks,
and then digested with trypsin (0.1%) for 15–20 min at 37 °C. The supernatants were
transferred to a fresh sterile tube, the DMEM (containing 10% FBS) was added to
stop digestion. After each digestion, cells were collected in DMEM and kept on ice.
The isolated cells were filtered with a 100 μm cell strainer, seeded into culture dishes
and incubated in DMEM for 1 h. Unattached cardiomyocytes were collected and
seeded into 35 mm culture dishes. Cardiomyocytes were cultured in DMEM for at
least 3 days before H/R exposure.
4
.6. Cell Culture and hypoxia/reoxygenation
2
0

Primary cultured neonatal rat cardiomyocytes were cultured at 37 °C with 95%
air and 5% CO in DMEM supplemented with 10% FBS, 100 μg/mL streptomycin,
2
and 100 units/mL penicillin. When cells grew to 70–80% confluence, they were
pretreated with compounds or vehicle (DMSO) for 4 h (a time point with better
efficacy in cardiomyocytes protection). The cells were then exposed to an anaerobic
medium (serum and glucose free) in a hypoxia incubator chamber (STEMCELL
Technologies, Vancouver, BC, Canada) with an anoxic mixture gas (95% N and 5%
2
CO ) for 6 h at 37 °C followed by reoxygenation for 3 h with fresh culture medium
2
(
95% air and 5% CO ) to simulate H/R injury in isolated hearts[32].
2
4
.7. Cell viability assay
To detect the protective effects of compounds 1-28 on neonatal rat
cardiomyocytes exposed to H/R, an MTT assay was performed. Subsequent to the
aforementioned treatments, the cells were incubated with 0.2 mL MTT (0.5 mg/mL)
for 4 h at 37 °C. Next, 200 µL DMSO was added into each well in order to dissolve
the formazan crystals. The optical density (OD) was recorded on a BioTek ELx800
microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 490 nm. The
results are expressed as the relative percentage of the control group.
4
.8. Extracellular LDH activity
To further confirm the protective effects of compounds 1-28, the LDH activity in
the culture medium was measured by an LDH assay kit, according to the
manufacturer’s protocol. Briefly, following treatment, the culture medium was
centrifuged at 400 × g and room temperature for 5 min, and then 20 µL supernatant
was collected and mixed with 20 µL 2,4-dinitrophenylhydrazine. After incubation at
3
7 °C for 15 min, 250 µL NaOH (0.4 M) was added into the mixture and incubated
for a further 15 min at 37 °C. The mixture was maintained at room temperature for 5
min, and subsequently the OD was recorded on a microplate reader at 450 nm. The
activity of LDH was derived from the OD values and expressed as U/L.
4
.9. Intracellular calcium level.
2
1

To monitor the intracellular calcium in neonatal rat cardiomyocytes, the
fluorescence dye Fluo-3 AM was employed, following the manufacturer’s protocol.
Briefly, the pretreated neonatal rat cardiomyocytes were loaded with 5 µM Fluo-3
AM at 37 °C for 30 min in the dark, and then washed with PBS for three times to
remove any excessive dye. The fluorescence intensity of Fluo-3 chelated with calcium
was recorded on a PerkinElmer EnVision fluorescence microplate reader
(PerkinElmer, Llantrisant, UK) at excitation and emission wavelengths of 488 and
5
25 nm, respectively.
4
.10. Measurement of ROS production
The production of ROS was detected by a fluorescence method using a ROS assay
kit. Subsequent to treatment, the medium was replaced and the cells were rinsed with
PBS. Next, 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in DMEM
was added and incubated at 37 °C for 30 min. The cells were then washed again with
PBS to remove the excessive dye, and the fluorescence intensity was recorded on a
fluorescence microplate reader at 485 nm (excitation wavelength) and 520 nm
(emission wavelength).
4
.11. MMP assay
The MMP was also detected using an MMP assay kit with Rhodamine 123. The
cells were stained Rhodamine 123 at 37 °C for 30 min, and then the morphology was
observed by fluorescence microscopy (Zeiss, OBSERVER D1/AX10 cam HRC).
4
.12. Assays for LDH and CK-MB
To determine the extent of myocardial injury, the release of creatine kinase-MB
(CK-MB) and lactate dehydrogenase (LDH) in coronary effluents were measured
using commercially available kits (Jian Cheng Bioengineering Institute, Nanjing,
China). After H/R, 0.2 mL and 0.04 mL of culture medium was taken and assayed for
LDH activity and CK-MB with a Microplate reader (Sunrise RC, Tecan, Switzerland),
following the manufacturer’s instruction.
4
.13. Western blot analysis
2
2

Total proteins of the cell lysates were extracted by using protein extraction RIPA
buffer with protease inhibitor. Protein concentration was estimated by using the BCA
protein assay Kit (Solarbio, Beijing, China). Proteins samples were separated via
SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, United
States). The membranes were blocked and incubated with primary antibodies (1:1000)
at 4 °C overnight. After washing, membranes were incubated with secondary antibody
(
1:10000) at room temperature for 2 h. Blots were visualized with ECLTM reagents
Advansta, Menlo Park, CA, United States), and the signals were captured with
(
chemiluminescence detection system (FluoChem E, San Jose, CA, United States). The
density of the band was analyzed with image analysis software.
4
.14. Statistical analysis
Data analysis was performed with GraphPad Prism 5.0 (GraphPad Software, Inc.,
San Diego, CA, United States). Results are presented as mean ± SEM. One-way
ANOVA followed by Bonferroni-test was used for comparisons of multiple groups. A
value of P < 0.05 was considered as statistically significant.
Acknowledgements
This study was financially supported by the National Natural Science Foundation
of China (Nos. U1803122, U1703111, 81473254, 81773594 and 81773637), Liaoning
Province Natural Science Foundation (No. 2019-MS-299), Liaoning Revitalization
Talents Program (No. XLYC1807182), and Shenyang Planning Project of Science
and Technology (No. 18-013-0-46).
Notes
The authors declare no competing financial interest.
Appendix A. Supplementary material
The material (1D and 2D NMR, HRESIMS, and IR spectra for new compounds
(
1-6)) is available on the website.
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2
6

Graphical abstract
2
7

Highlights
1
2
3
.
.
.
Six new triterpenoids (1-6) and 22 known analogues (7-28), were separated
from the aerial parts of Anchusa italica Retz.
Compounds 4, 6-17, 21-22 and 26-28 showed significant protective effects on
cardiomyocytes injury.
Tripterpenoid 6 and its analogues may be offer a promising therapeutic approach
for treating cardiomyocyte injury inducing by H/R.
2
8