Seven new triterpenoids from the aerial parts of Ilex cornuta and protective effects against H2O2-induced myocardial cell injury

Article abstract of DOI:10.1016/j.phytol.2015.10.010

Seven new triterpenoids (1-7), together with two known ones (8-9), were isolated from the aerial parts ofIlex cornuta. The leaves of I. cornuta are the major source of "Kudingcha", a popular herbal tea consumed in China and other countries. The structures of compounds 1-7 were determined as 20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (1), 20-epi-urs-12,18-dien-28-oic acid 2′-O-acetyl-3β-O-α-l-arabinopyranoside (2), 20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (3), 3β,23-dihydroxy-20-epi-urs-12,18-dien-28-oic acid (4), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (5), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronic acid (6), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (7), on the basis of spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR) and chemical reactions. Protective effects against H₂O₂-induced H9c2 cardiomyocyte injury were tested in vitro for compounds 1-9, and the data showed that compound 4 had significant cell-protective effect. Compounds 1-9 did not show significant DPPH radical scavenging activity.

Full text of DOI:10.1016/j.phytol.2015.10.010

Phytochemistry Letters 14 (2015) 178–184
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Seven new triterpenoids from the aerial parts of Ilex cornuta and
protective effects against H2O2-induced myocardial cell injury
Shanshan Lia,b, Jianping Zhaoc, Wenlian Wangb, Yuchen Lub, Qiongming Xub,
,
*
b
Yanli Liub, , Xiaoran Li , Ikhlas A. Khanb,c, Shilin Yangb
*
a
Children’s Hospital of Soochow University, Suzhou 215003, China
College of Pharmaceutical Science, Soochow University, Suzhou 215123, China
b
c
National Center for Natural Products Research, and Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy,
University of Mississippi, Mississippi 38677, United States
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 29 June 2015
Received in revised form 3 October 2015
Accepted 10 October 2015
Available online 10 November 2015
Seven new triterpenoids (1–7), together with two known ones (8–9), were isolated from the aerial parts
ofIlex cornuta. The leaves of I. cornuta are the major source of “Kudingcha”, a popular herbal tea consumed
in China and other countries. The structures of compounds 1–7 were determined as 20-epi-urs-12,18-
dien-28-oic acid 3
-arabinopyranoside (2), 20-epi-urs-12,18-dien-28-oic acid
methyl ester (3), ,23-dihydroxy-20-epi-urs-12,18-dien-28-oic acid (4), 23-hydroxy-20-epi-urs-
12,18-dien-28-oic acid 3 -O- -arabinopyranoside (5), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid
-O- -glucuronic acid (6), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3 -O- -glucuronopyr-
b
-O-a
-L
-arabinopyranoside (1), 20-epi-urs-12,18-dien-28-oic acid 20-O-acetyl-3
-O-
b
-O-
a
-L
3b
b-D-glucuronopyranoside-6-O-
3b
Keywords:
Ilex cornuta
Aquifoliaceae
b
a-L
3b
b
-D
b
b-D
anoside-6-O-methyl ester (7), on the basis of spectroscopic analyses (IR, ESI–MS, HR-ESI–MS, 1D and 2D
NMR) and chemical reactions. Protective effects against H2O2-induced H9c2 cardiomyocyte injury were
tested in vitro for compounds 1–9, and the data showed that compound 4 had significant cell-protective
effect. Compounds 1-9 did not show significant DPPH radical scavenging activity.
Triterpenoids
Structure identification
Cardiomyocytes oxidative injury
DPPH radical scavenging activity
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
Studies showed that Kudingcha possesses antioxidant, antidiabet-
ic, hepatoprotective, neuroprotective, anti-inflammatory, and
Ilex cornuta Lindl. et Paxt. (Aquifoliaceae family), a slow-
growing evergreen shrub or small tree, is cultivated widely in
South China. The leaves of I. cornuta are used to make the popular
herbal tea, called as “Kudingcha” or bitter tea (Chau and Wu, 2006)
in China. Commercially, Kudingcha is sold in the form of tea-bags
packed with ground leaves (1 to 2 g). The leaf extracts are also used
as ingredients in food or dietary supplement industries. Tradition-
ally, Kudingcha has been used for treatment of sore throat, as an
agent for weight loss (Ahai,1985), and for the relief of hypertension
(Jiangsu Medical College, 1975). It is believed that Kudingcha has
potential benefits for reducing cardiovascular and oxidative stress-
related diseases (Thuong et al., 2009). Recently, I. cornuta has been
gaining research attention because of its potential health benefits.
diuretic effects (Jiangsu New Medical College, 1986; Qin et al.,
1988; Puangpraphant and De Mejia, 2009). Previous phytochemi-
cal investigations showed that the whole plant of I. cornuta is a rich
source of triterpenoids and flavonoids as well as their correspond-
ing glycosides (Wang et al., 2014; Liao et al., 2013; Li et al., 2006).
Previous studies on the aerial parts of I. cornuta have resulted in the
isolation of a series of new triterpenoidal saponins, and some
triterpenoidal saponins showed potential benefit for inhibition of
peroxidative damage associated with ischemia and reperfusion (Li
et al., 2014). As part of a continuous search for potentially active
substances for the prevention of coronary artery disease, seven
new triterpenoids (1-7) (Fig. 1), along with two known ones were
obtained from the aerial parts of I. Cornuta. The isolation and
structural elucidation of the new compounds, as well as the
protective effects of the isolates against cardiomyocytes injury
induced by H2O2 are reported herein. In addition, their radical
scavenging activity was evaluated by DPPH assay.
* Corresponding authors. Fax: +86 512 65882089.
E-mail addresses: xuqiongming@suda.edu.cn (Q. Xu), liuyanli@suda.edu.cn
(Y. Liu).
http://dx.doi.org/10.1016/j.phytol.2015.10.010
1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

S. Li et al. / Phytochemistry Letters 14 (2015) 178–184
179
Thus, compound 1 was established as 20-epi-urs-12,18-dien-28-
oic acid 3 -O- -arabinopyranoside.
b
a-L
Compound 2 was obtained as white powder. Its molecular
formula was determined to be C37H56O8 according to the [M-H]ꢀ
ion peak at m/z627.3891(calc’d. 627.3897). An anomeric proton
observed at dH 4.83 (1H, d, J = 7.5 Hz) showed HSQC correlation
with the anomeric carbon at dC 104.8 (C-1 of Ara), Which indicated
the presence of one sugar unit in the structure of compound 2. The
structure of the sugar residue yielded from acid hydrolysis of 2 was
identified as L-arabinose by GC analysis. Comparing the NMR
spectrum of compound 2 with that of compound 1, it was
suggested that 2 was derived from the acetylation of 1, The
downfield shift of C-2 (+1.4 ppm) of Ara at dC 74.5 suggested that
the acetyl (Ac) group was located at C-2 of arabinose, and the
assignment was supported by the HMBC correlation between dH
5.98 (H-2 of Ara) and dC 170.1 (C-1 of Ac) (Fig. 2). Thus, compound 2
was elucidated as 20-epi-urs-12,18-dien-28-oic acid 3
acetyl)- -arabinopyranoside.
b
-O-(20-O-
a-L
Compound 3 was obtained as white powder. Its molecular
formula was determined to be C37H56O9 based on the positive HR-
ESI–MS. An anomeric proton observed at dH 5.13 (1H, d, J = 14.0 Hz,
H-1 of GlcA) showed HSQC correlation to the anomeric carbon at dC
107.5 (C-1 of GlcA), indicating the presence of a sugar unit in the
structure of compound 3. The sugar residue obtained from the acid
hydrolysis of 3 was identified as
analysis. The 13C NMR spectrum of 3 was very similar to that of urs-
12,18-dien-28-oic acid -O- -glucuronopyranoside-6-O-
methyl ester, and the main differences arising from the significant
downfield shifts of C-18 (+11.4) at 134.8 and C-22 (+1.1) at
35.9 due to the -effect of the 30- (axial) methyl group that were
observed when compared with urs-12,18-dien-28-oic acid 3 -O-
-glucuronopyranoside-6-O-methyl ester which possesses a 30-
a (equatorial) methyl group instead (Hidaka et al.,1987). Moreover,
the orientation assignment of Me-30 was also confirmed by
D-glucuronic acid by the GC
3
b
b-D
Fig. 1. Structures of the compounds 1–9.
d
d
2. Results and discussion
g
b
b
2.1. Structure Identification of the isolates
b-D
A 50% EtOH extract of the aerial parts of I. cornuta was subjected
to column chromatographic separations on macroporous resin
D101, silica gel and octadecylsilane (ODS) silica gel, yielding seven
new triterpenoids (1–7) and two known ones which were
b
comparing its spectroscopic data with those of compound 1.
Therefore, the structure of 3 was elucidated as 20-epi-urs-12,18-
dien-28-oic acid 3
ter.
b-O-b-D-glucuronopyranoside-6-O-methyl es-
identified as 3
b
-hydroxy-20
a
b
(H)-urs-12,18-dien-28-oic acid (8)
-O- -arabinopyranosyl-20 (H)-
-glucopyranoside (9) (Che
(Ali and Srivastava, 1990), 3
urs-12,18-dien-28-oic acid 28-O-b-D
a
-L
a
Compound 4 was obtained as white powder. Its molecular
formula was assigned as C30H46O4 on the basis of its positive HR-
ESI–MS [M + Na]+ ion peak at m/z493.3318 (calc’d. 493.3294). The
1H NMR spectrum showed the singlet resonances of five tertiary
et al., 2012) (Fig. 1).
Compound 1 was isolated as white powder, and its negative-ion
HR-ESI–MS showed a quasimolecular [M + Na]+ ion peak at m/z
609.3796, attributed to the molecular formula of C35H54O7Na
(calc’d. 609.3767). The 1H NMR spectrum showed the singlet
methyl groups at
s, Me-26), 1.07 (3H, s, Me-24), 1.01 (3H, s, Me-25), one methyl
doublet at 1.12 (3H, d, J = 10.0 Me-30), an olefinic proton signal at
5.69 (1H, m, H-12), and the signals of a hydroxymethylene group at
4.23 (1H, m, H-23), 3.76 (1H, d, J = 15.0 Hz, H-23), as well as the
d 1.08(3H, s, Me-27), 1.89 (3H, s, Me-29), 1.09 (3H,
d
resonances of six tertiary methyl groups at
d 0.97 (3H, s, Me-25),
1.06 (3H, s, Me-24),1.12 (3H, s, Me-26),1.23 (3H, s, Me-27),1.38 (3H,
s, Me-23), 1.97 (3H, s, Me-29), one methyl doublet at 1.21 (3H, d,
d
signal of a oxygen-bearing methine at d4.28 (1H, dd, J = 5.0, 10.0 Hz,
H-3). The 13C NMR showed resonances for 30 carbon atoms, whose
multiplicity patterns were revealed from the DEPT and HSQC
experiments as six methyls, ten methylenes, five methines, and
nine quaternary carbons. It showed four olefinic C-atoms at dC
126.4, 134.5, 135.6, 139.6, one oxymethylene group at dC 67.6,
J = 6.5 Hz Me-30), an olefinic proton signal at
d
5.73 (1H, m, H-12),
and the signal of a oxygen-bearing methine at
d
3.49 (1H, dd, J = 4.0,
12.0 Hz, H-3). The NMR spectra indicated that the aglycone of 1 was
a 3 -hydroxy-20-epi-urs-12,18-dien-28-oic acid by comparing its
b
spectroscopic data with those of compound 8 (Ali and Srivastava,
1990). In the 1H NMR spectrum, an anomeric proton was observed
at dH 4.90 (1H, d, J = 7.0 Hz), which showed HSQC correlation with
the anomeric carbon at dC 107.7 (C-1 of arabinose (Ara)), indicating
the presence of one sugar unit in the structure of compound 1. The
sugar residue yielded from acid hydrolysis of 1 was identified as L-
arabinose by GC analysis. The location of arabinopyranosyl group
was assigned at C-3 of the aglycone on the basis of the observations
corresponding to C-23, and one oxymethine carbon at
d 73.1,
corresponding to C-3, as well as one carboxyl group at dC 178.8,
corresponding to C-28. The 13C NMR spectrum of compound 4 was
similar to that of compound 8 except for the significant chemical
shift change of C-23 (+38.5 ppm) at dC 67.6, suggesting the methyl
group in compound 8 was replaced by the hydroxymethylene
group in compound 4. The assignment was confirmed by the
observation of HMBC correlations between C-23 at dC 67.6 and Me-
24 at dH 1.07, and between C-24 at dC 13.2 and Me-23 at dH 3.76.
of the down field shift of H-3 at d3.49, the correlation between dH
3.49 (H-3) and dC 107.7 (C-1 of Ara), as well as the correlation
between dH 4.90 (H-1 of Ara) and dC 89.0 (C-3 of the aglycone) in
Thus, compound 4 was identified as 3b,23-dihydroxy-20-epi-urs-
the HMBC spectrum (Fig. 2). The
anosyl unit was inferred from the NOESY correlations between
4.90 (H-1 of Ara) and 4.26 (H-3 of Ara), 4.42 (H-4 of Ara) (Fig. 2).
a-configuration of arabinopyr-
12,18-dien-28-oic acid.
d
Compound 5 was isolated as white powder. Its HR-ESI–MS
[M + HCOOH-H]ꢀ ion peak at m/z 647.3801 (calc’d. 647.3795)
d

180
S. Li et al. / Phytochemistry Letters 14 (2015) 178–184
Fig. 2. Key HMBC and NOESY correlations for 1–7.
suggested the molecular formula C35H54O8. An anomeric proton
observed at dH 5.09 (1H, d, J = 10.0 Hz) showed the HSQC correlation
with the anomeric carbon at dC 106.8 (C-1 of Ara), indicating the
presence of one sugar unit in the structure of compound 5. The
(H-1 of arabinose) and
of arabinose) (Fig. 2). Thus, compound 5 was established as
23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3b-O-a-L-arabino-
pyranoside.
d 4.15 (H-3 of arabinose), 4.33 (H-4
sugar residue yielded from acid hydrolysis of 5 was identified as
L
-
Compound 6 was obtained as white powder. The positive HR-
ESI–MS showed a quasi-molecular ion peak at m/z 645.3694,
indicating the molecular formula of C36H53O10 ([M-H]ꢀ, calc’d.
645.3639). The presence of a sugar unit in the structure of
compound 6 was confirmed by the observation of an anomeric
proton at dH 5.31 (1H, d, J = 10.0 Hz, H-1 of GlcA), which showed
HSQC correlation to the anomeric carbon at dC 106.4 (C-1 of GlcA).
The sugar residue obtained from the acid hydrolysis of 6 was
arabinose by GC analysis. Comparing the NMR spectral data
between compound 5 and compound 4, it was suggested that 5
was derived from the glycosylation of compound 4. The downfield
shift of C-3 (+8.8 ppm) of aglycone at dC 81.9 suggested that the
arabinopyranosyl group was located at C-3 of aglycone, and the
assignment was confirmed by the observation of the HMBC
correlations between dH 5.09 (H-1 of Ara) and dC 81.9 (C-3 of
aglycone), and between dH 4.38 (H-3 of aglycone) and dC 106.8 (C-1
identified as -glucuronic acid by the GC analysis. Compound 6 had
D
of arabinose) (Fig. 2). The
was assigned according to the NOESY correlations between
a
-configuration of arabinopyranosyl unit
5.09
a similar structure as compound 5 with comparison of the NMR
spectral data between them, and the only difference was that
d

S. Li et al. / Phytochemistry Letters 14 (2015) 178–184
181
compound 5 had an arabinopyranosyl group while compound 6
had a GlcA group. The configuration of GlcA group was confirmed
on the basis of the large 3JH-1,H-2 coupling constant. Therefore, the
structure of compound 6 was elucidated as 23-hydroxy-20-epi-
suggested that protective effects on H2O2 induced myocardial cell
injury were not directly associated with their radical scavenging
capacity.
b
Previous studies on the cardioprotective potential of natural
products or herbal medicines have revealed that except for radical
scavenging, other cellular and molecular anti-oxidation mecha-
nisms may be involved in their protective effects against
myocardial ischemia/reperfusion (I/R) injury (Liu et al., 2013).
For instance, it was found that senticoside B, a triterpenoidal
saponin exhibiting antioxidant action and cardiac protection, could
attenuate oxidative stress and protect cardiomyocytes from I/R
injury by enhancing the actions of catalase (CAT), glutathione
peroxidase (GSHPx), and superoxide dismutase (SOD) (Shi et al.,
2014; Liang et al., 2010; Luo et al., 2011). Since the compounds
isolated in this study belong to triterpenoidal saponins, we
speculated that these active compounds might exert their
protection on myocardial cells through enhancing the level of
antioxidant enzymes (CAT, GSHPx, and SOD) in cardiomyocyte,
thereby increasing the contents of endogenous antioxidants.
Nevertheless, further investigations are needed to explore the
mechanisms of protective effects of the saponins from I. cornuta on
myocardial cell injury.
urs-12,18-dien-28-oic acid 3b-O-b-D-glucuronic acid.
Compound 7 was obtained as white powder. The positive HR-
ESI–MS showed a quasi-molecular ion peak at m/z 683.3771,
indicating the molecular formula of C37H56O10Na ([M + Na]+, calcd.
683.3771). In the 1H NMR spectrum, an anomeric proton was
observed at dH 5.28 (1H, d, J = 10.0 Hz, H-1 of GlcA), which showed
the correlation to the anomeric carbon at dC 106.9 (C-1 of GlcA) in
the HSQC spectrum, indicating the presence of a sugar unit in the
structure of compound 7. The sugar residue obtained from the acid
hydrolysis of 7 was identified as D-glucuronic acid by the GC
analysis. The 13C NMR data of 7 were similar to those of 6 except for
the additional signal at dC 52.5 corresponding to one OCH3 group.
The OCH3 group was determined to attach at C-6 of GlcA group
according to the HMBC correlation between dC 171.3 (C-6 of GlcA)
and dH 3.74 (OCH3). Therefore, the structure of 7 was elucidated as
23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3b-O-b-D-glucuro-
nopyranoside-6-O-methyl ester.
2.2. Protective effects of the isolates against H2O2-induced myocardial
cell injury
Our previous investigation of the structure-activity relationship
of these natural triterpenoidal saponins revealed that the ursane-
Table 1
Many chronic diseases such as cardiovascular diseases, may be
caused by intracellular oxidative damage of biomolecules via ROS
(Fearon and Faux, 2009). Oxidative stress plays an important role
in the pathogenesis of cardiomyocytes ischemic injury. A number
of studies showed that antioxidants reduced oxidative stress-
mediated cellular injury (Yousuf et al., 2009). Among ROS, H2O2
readily crosses cellular membranes and gives rise to the highly
reactive hydroxyl radical, which is the most reactive and induces
severe damage to adjacent biomolecules(Valko et al., 2007). H2O2,
superoxide and hydroxyl radicals, have been used frequently in
models of oxidative stress in H9c2 cardiomyocytes to investigate
heart dysfunction(Hescheler et al., 1991; Yasuoka et al., 2004). The
previous investigation suggested that I. cornuta extract might have
a protective effect on myocardial ischemia (Liu et al., 2009; Li et al.,
2014). For the investigation of the protective effects against H2O2-
induced myocardial cell injury, compounds 1–9 were tested.
13C NMR data (
d) of compounds 1–7 (125 MHz in pyridine-d5).
No.
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
39.5
39.2
39.4
39.2
39.4
39.7
39.6
27.0
89.0
39.8
56.3
18.7
35.1
39.5
48.5
37.1
23.8
126.7
139.8
45.1
29.5
35.1
50.5
134.9
136.1
37.7
29.1
35.9
28.5
17.3
26.7
89.0
39.4
56.1
18.6
35.0
39.4
48.4
36.9
23.7
126.6
139.7
45.0
29.1
35.0
50.5
134.6
135.7
37.7
29.4
35.8
28.2
17.0
16.2
18.2
22.4
179.2
20.4
20.7
26.9
89.3
39.7
56.2
18.6
35.1
39.4
48.4
36.9
23.7
126.6
139.8
45.0
29.5
35.1
50.5
134.8
135.7
37.7
29.1
35.9
28.4
17.2
16.3
18.3
22.5
179.0
20.5
20.8
27.7
73.1
42.9
48.2
18.4
34.6
39.2
48.5
37.0
23.5
126.4
139.6
44.8
29.2
34.8
50.2
134.5
135.6
37.4
28.9
35.6
67.6
13.2
16.6
18.2
22.2
178.8
20.2
20.5
26.4
81.9
43.7
47.8
26.6
82.1
44.0
48.1
18.7
35.2
39.9
48.8
37.2
24.1
127.1
140.1
45.4
29.8
35.5
50.8
135.6
135.7
38.0
29.5
36.2
64.9
14.2
17.4
26.6
82.5
44.0
48.1
18.6
35.1
39.8
48.8
37.2
24.0
127.0
140.1
45.3
29.8
35.4
50.8
135.0
136.2
38.0
29.5
36.2
64.7
14.2
17.3
18.3
34.8
39.5
48.5
36.9
23.7
126.7
139.8
45.0
29.4
35.1
50.5
134.7
135.8
37.6
29.1
35.9
64.6
13.9
17.0
18.4
22.5
179.0
20.4
20.7
Among them compound 4 (12.5–200
the viability of H9c2 induced by H2O2 in a dose-dependent manner
with concentrations ranging from 12.5 to 200 M (Table 3).
According to the results, Compound exhibited significant
mM) significantly increased
m
4
protective effects on H2O2 induced myocardial cell injury. Vitamin
E, which was reported to have effects on inhibiting peroxidative
damage associated with ischemia and reperfusion, was used as a
positive control (Yamamoto et al., 1983; Fujimoto et al., 1984).
Vitamin E can incorporate into the membrane lipids of endothelial
cells to protect against membrane lipid peroxidation by quenching
cytotoxic ROS (Martin et al., 1996). We speculated that the active
16.5
18.3
22.5
179.1
20.5
20.8
18.7
22.9
179.3
20.8
21.1
18.7
22.8
179.3
20.7
21.1
29
compounds isolated from I. cornuta might play
a similar
30
C3-O-
GlcA-1
2
antioxidant role in the prevention of peroxidative damage.
Therefore, the radical scavenging capacities of the isolates were
evaluated in the present study.
107.5
75.6
78.1
73.3
77.4
171
106.4
75.7
78.7
74.0
78.0
174.0
106.9
75.9
78.3
73.7
77.7
3
4
5
6
2.3. DPPH radical scavenging capacity assay
171.3
52.5
Methyl-1
Ara-1
2
3
4
5
52.2
Compounds 1–9 were evaluated for antioxidant activity by
DPPH radical scavenging capacity assay. As shown in Table 4, the
maximal scavenging rates of compounds 1–9 were less than 20% at
107.7
73.1
74.8
69.7
66.9
104.8
74.5
72.6
69.9
67.3
21.4
106.8
73.3
74.9
69.8
67.1
the concentration of 200 mM. The test results showed that
compounds 1–9 were found to have no significant DPPH radical
scavenging capacity. Combined with H2O2-induced myocardial cell
injury experiment, the DPPH radical scavenging capacity assay
COOCH3
COOCH3
170.1

182
S. Li et al. / Phytochemistry Letters 14 (2015) 178–184
type triterpenoidal saponins with one double bond located at C-
12 is essential for the protective activity, and the glucuronic acid
methyl ester at C-3 of aglycone were active in protecting
myocardial cell injury (Li et al., 2014). In this paper, we found
that the ursane type triterpenoidal saponins with two conjugated
double bonds located at C-12 and C-18 also showed protective
activity, suggesting that the two conjugated double bonds did not
significantly alter the activity. Compound 4 with significant
activity was just a triterpene with no glycolic chain, suggesting
the glucosylation might diminish the activity.
purchased from Qingdao Marine Chemical Factory (Qingdao,
China). Macroporous resin (D101, Xi’an Sunresin New Materials
Co., Ltd., Xi’an, China) was used for column chromatography. TLC
was colored on 10% sulfuric acid alcohol solution. GC was carried
out on a GC-14C (Shimadzu Corp., Kyoto, Japan) with a flame
ionization detector (FID).
3.2. Plant material
The aerial parts of I. cornutawere collected from Liuan City,
Anhui province of China in April 2011, and authenticated by
Professor Xiao-Ran Li at Soochow University. A voucher sample
(No.11-11-06-01) was kept in the herbarium of the College of
Pharmaceutical Science, Soochow University.
3. Experimental
3.1. General experimental procedures
1H, 13C NMR and 2D NMR spectra were measured in C5D5N with
a Varian Inova 500 spectrometer (Varian Inc., Palo Alto, CA, USA)
using tetramethylsilane (TMS) as the internal standard. HR-ESI–MS
spectra were recorded on a Micromass Q-TOF2 spectrometer
(Micromass Corp., London, UK).
HPLC analysis and purification were performed on a Shimadzu
HPLC system equipped with a LC-20AT pump, a SPD-20A detector
(Shimadzu Corp., Kyoto, Japan), and the wavelength for detection
3.3. Extraction and isolation
Dried aerial parts (10 kg) of I. cornuta were extracted with 50%
EtOH. Evaporation of the solvent under reduced pressure yielded a
residue (325 g), which was dissolved in distilled water and
subjected to a D101 macroporous resin column (200 ꢁ 30 cm i.
d.), then eluted with 0%, 30%, 60% and 90% EtOH. The 90% EtOH
eluate was dried in vacuo to yield a fraction (30 g), which was
separated by a silica gel column (60-100 mesh) using CHCl3-MeOH
(90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 0:100, each 6.0 L)
for elution. The CHCl3-MeOH (80:20) eluate (4.9 g) was further
separated by MPLC over an ODS column to yield 5 fractions.
Fraction 1 (225 mg) was separated by semi-preparative HPLC,
eluted with MeOH-H2O (75:25) to yield compounds 1 (31 mg, tR
37.24 min), 7 (7 mg, tR 39.10 min). In the same way, compound 8
(25 mg, tR 49.21 min) was obtained from fraction 2 (170 mg). The
was 203 nm. The HPLC column (250 ꢁ 9.4 mm i.d., 5
mm, Agilent
Zorbax SB-C18 semipreparative) was purchased from Agilent Corp.
(Palo Alto, CA, USA). MPLC purification was taken on a Büchi Flash
Chromatography system composed of a C-650 pump and a flash
column (460 mm ꢁ 26 mm i.d., Büchi Corp., Flawil, Switzerland).
The ODS column for MPLC was purchased from Merck KGaA
(Darmstadt, Germany). Silica gel (60–100 mesh) used for open
column chromatography and precoated silica gel TLC plates were
Table 2
1H NMR data of compounds 1–7 (500 MHz in pyridine-d5).
No.
1
2
3
4
5
6
7
1
2
3
5
6
7
9
11
12
15
16
20
21
22
23
24
1.03m, 1.70m
1.98m, 2.28m
3.49dd (4.0,12.0)
0..94m
1.35m, 1.57m
1.55m, 1.72m
1.60m
0.98m, 1.65m
1.91m, 2.17m
3.35dd (4.5,11.8)
0.90m
1.36m, 1.58m
1.55m, 1.70m
1.59m
0.90m, 1.56m
1.95m, 2.24m
3.51dd (4.0,12.0)
1.10m
1.35m, 1.56m
1.56m, 1.74m
1.56m
1.12m, 1.69m
1.97m, 2.02m
4.28dd (5.0,10.0)
1.65m
1.43m, 1.71m
1.50m, 1.75m
1.58m
1.13m, 1.75m
2.11m, 2.36m
4.38m
0.99m
2.41m
4.44m
1.75m
1.39m
1.52m
1.66m
2.01m
5.72m
1.73m
1.68m
2.26m
1.31m
1.67m
3.79m
1.03s
0.94m, 1.57m
2.02m, 2.26m
4.35m
1.79m
1.70m
1.39m, 1.78m
1.51m, 1.81m
1.71m
2.03m
5.74m
1.70m, 1.79m
1.67m, 2.53m
2.24m
1.29m, 2.29m
1.60m, 2.44m
3.79m, 4.38m
1.01s
1.28m, 1.67m
1.45m, 1.74m
1.58m
1.95m
5.66m
1.65m, 1.72m
1.62m, 2.47m
2.20m
1.24m, 2.22m
1.60m, 2.37m
3.71m, 4.37m
0.95s
2.04m
5.73m
1.96m
5.73m
2.00m
5.73m
2.03m
5.69m
1.70m, 1.79m
1.65m, 2.56m
2.27m
1.35m, 2.31m
1.68m, 2.45m
1.38s
1.35m, 2.31m
1.61m, 2.58m
2.26m
1.73m, 1.80m
1.67m, 2.46m
1.21s
1.73m, 1.81m
1.67m, 2.59m
2.27m
1.35m, 2.30m
1.71m, 2.45m
1.41s
1.63m, 1.72m
1.62m, 2.51m
2.18m
1.27m, 2.26m
1.57m, 2.38m
3.76d(15.0), 4.23m
1.07s
1.06s
0.98s
1.06s
25
0.97s
0.92s
0.92s
1.01s
1.05s
1.02s
0.93s
26
1.12s
1.11s
1.10s
1.09s
1.13s
1.12s
1.05s
27
1.23s
1.22s
1.23s
1.08s
1.15s
1.18s
1.09s
29
1.97s
1.95s
1.98s
1.89s
1.96s
1.96s
1.89s
30
1.21d(6.5)
1.19d(7.0)
1.21d(7.0)
1.12d(10.0)
1.18d(10.0)
1.20d(10.0)
1.12d(8.5)
C3-O-
GlcA-1
2
3
4
5.13d(14.0)
4.18t(8.0)
4.36t(9.0)
4.57t(9.5)
4.69d(10.0)
3.85s
5.31d(10.0)
4.21m
4.29 m
4.60 m
4.62 m
5.28d(10.0)
4.13t(10.0)
4.20t(10.0)
4.48m
4.51m
3.74s
5
Methyl-1
Ara-1
4.90d(7.0)
4.52m
4.26m
4.42m
3.92m, 4.41m
4.83d(7.5)
5.98t(8.8)
4.26dd (3.0.9.5)
4.37m
4.39mchar_dot
3.88d(11.0)
2.20s
5.09d(10.0)
4.52t(10.0)
4.15m
4.33m
3.81m, 4.37m
2
3
4
5
COOCH3
*Data in parentheses are J values (in Hz). Ara referred to arabinose, GlcA referred to gluconic acid.

S. Li et al. / Phytochemistry Letters 14 (2015) 178–184
183
Table 4
CHCl3-MeOH (50:50) eluate (3.7 g) was separated into 4 fractions
by MPLC over the ODS column in a similar way described above,
and offered compounds 2 (12 mg), 3 (10 mg) and 6 (10 mg) after
Maximal scavenging rate of 1–9 against DPPH in vitro.
Compounda
Scavenging rate (200 mM)
semi-preparative HPLC purification. Moreover, compounds
4
1
2
3
4
5
6
7
8
9
VE
7.06 ꢂ 5.08
5.48 ꢂ 1.26
9.35 ꢂ 1.21
3.85 ꢂ 2.03
11.03 ꢂ 6.69
6.78 ꢂ 3.45
4.73ꢂ 2.63
8.28 ꢂ 2.11
3.52 ꢂ 1.90
92.74 ꢂ 1.09
(18 mg), 5(11 mg) and 9 (15 mg) were obtained from the CHCl3-
MeOH (40:60) eluate (2.9 g) after undertaking the similar steps of
MPLC and semi-preparative HPLC separations as described above.
3.4. Acid hydrolysis and sugar analysis of compounds 1-3 and 5-7
The acid hydrolysis and sugar analysis were carried out
according to the method described previously (Gao et al., 2011).
Using the method, the following sugar units in compounds 1–3 and
a
For 1–9, the test concentration range from 5 to 200
M.
mM; for VE,
the test concentration was 200
m
5–7 were identified by comparison with authentic samples:
glucose (tR 10.49 min), -glucose (tR 11.10 min), -glucuronic acid
(tR 9.21 min), -glucuronic acid (tR 9.66 min), -arabinose (tR
8.52 min), -arabinose (tR 8.33 min).
D-
L
D
radical scavenging rate in percent (I%) was calculated in the
following way:
L
D
L
ꢂ
ꢀ
ꢁꢃ
Asample ꢀ Ablank
I% ¼ 100 ꢁ 1 ꢀ
3.5. Assay for testing the protective effects against myocardial cell
injury induced by H2O2
AControl
AControl is the absorbance of the control reaction (containing all
reagents except the test compound), and Asample is the absorbance
of the test compound, Ablank is the absorbance of a blank sample.
H9c2 cells (the Cell Bank of the Chinese Academy of Sciences
Shanghai, China) were seeded into 96-well flat microtiter plates at
a density of 1 ꢁ105 per well and allowed 24 h to adhere before
drugs were introduced. After incubation with different concen-
3.7. Statistical analysis
trations of compounds 1–9 (12.5–200
mM) for 12 h, the cells were
treated with 200 M H2O2 for 24 h. (Konorev et al., 1999; Xu et al.,
m
The results were expressed as the mean ꢂ SD. Differences were
evaluated with one way ANOVA. The post hoc test was done with
student’s Dunnett test. P Values less than 0.05 were considered
statistically significant. Statistical calculations were performed
using the SPSS 16.0 for Windows software package.
2005). The cells in the control groups were treated with the same
volume of phosphate-buffered saline (PBS). Cell viability was
evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) assay, which is based on the reduction of
MTT by the mitochondrial dehydrogenase of intact cells to a purple
formazan product. Absorbance was read on an ELISA plate reader at
540 nm as a measure of quantity of formazan. The percentage cell
viability was calculated as a ratio of optical density (OD) value of
sample to the OD value of control (Shi et al., 2013). All experiments
were done for three times.
3.7.1. Compound 1
25
white amorphous powder; [
a
]
+17.3 (c 0.11, MeOH); UV
D
(MeOH)lmax (loge
) 223 (2.76) nm; IR nmax (KBr) cmꢀ1 3429, 2934,
2787, 1756, 1458, 1348, 1071; 1H NMR and 13C NMR, see
Tables 1 and 2; HR-ESI–MS m/z 609.3796 ([M + Na]+, calc’d. for
C35H54O7Na, 609.3767).
3.6. DPPH radical scavenging capacity assay
3.7.2. Compound 2
white amorphous powder; [a D
25
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging
activity of compounds 1–9 were measured in triplicate according
to a described method (Hatano et al., 1988). Briefly, a 0.5 mM of
]
+14.2 (c 0.12, MeOH); UV
(MeOH)lmax (loge
) 226 (2.78); IR nmax (KBr) cmꢀ1 3425, 2961, 2707,
1742, 1435, 1325, 1060; 1H NMR and 13C NMR, see Tables 1 and 2;
methylated DPPH solution was prepared. An aliquot (100
tested compound at final concentrations (5–200 M) in methanol
was mixed with 100 L of methylated DPPH radical solution and
mL) of
HR-ESI–MS m/z 627.3891 ([M-H]ꢀ, calc’d. for C37H55O8, 627.3897).
m
m
3.7.3. Compound 3
25
incubated for 30 min in the dark. Vitamin E served as a positive
control. A freshly prepared DPPH solution exhibits a deep purple
color with an absorption maximum at 517 nm. The DPPH free
white amorphous powder; [
(MeOH)lmax (log
2734,1797,1472,1363,1083; 1H NMR and 13C NMR, see Tables 1 and
a
]
+15.0 (c 0.10, MeOH); UV
D
e
) 229 (2.87); IR nmax (KBr) cmꢀ1 3345, 2972,
Table 3
Protective Effects of Triterpenoidal Saponins from Ilex cornuta on H2O2-induced H9c2Cell Injury.
Compound
Concentration(mM)
0
12.5
25
50
100
200
1
2
3
4
5
6
7
8
53.52 ꢂ 7.22
55.37 ꢂ 3.26
61.81 ꢂ 1.18
61.81 ꢂ 1.18
63.86 ꢂ 1.67
55.37 ꢂ 3.26
55.37 ꢂ 3.26
53.52 ꢂ 7.22
56.68 ꢂ 0.77
62.56 ꢂ 1.14
41.04 ꢂ 1.21
58.25 ꢂ 6.54
53.62 ꢂ 1.42
61.94 ꢂ 1.24
59.02 ꢂ 1.69
49.25 ꢂ 3.45
37.10 ꢂ 2.73
44.00 ꢂ 0.88
49.20 ꢂ 2.71
64.99 ꢂ 2.50
50.61 ꢂ 4.31
59.38 ꢂ 4.20
53.51 ꢂ 0.14
65.37 ꢂ 1.24
61.73 ꢂ 0.70
53.08 ꢂ 0.77
36.06 ꢂ 2.73
47.23 ꢂ 2.99
51.74 ꢂ 2.71
68.28 ꢂ 3.24
53.58 ꢂ 9.86
59.46 ꢂ 2.12
43.60 ꢂ 1.34
71.75 ꢂ 2.22*
64.09 ꢂ 0.43
52.62 ꢂ 0.77
38.83 ꢂ 5.40
50.44 ꢂ 2.99
51.21 ꢂ 4.52
70.27 ꢂ 1.86
15.35 ꢂ 0.66
16.05 ꢂ 0.60
42.77 ꢂ 4.27
25.27 ꢂ 0.14
76.76 ꢂ 2.79**
12.10 ꢂ 0.69
26.22 ꢂ 0.55
82.00 ꢂ 2.10**
66.11 ꢂ 1.26
72.06 ꢂ 0.81
11.06 ꢂ 0.54
37.36 ꢂ 5.64
57.30 ꢂ 6.17
51.43 ꢂ 4.12
75.27 ꢂ 1.09*
11.28 ꢂ 0.11
41.08 ꢂ 4.65
49.42 ꢂ 7.34
48.12 ꢂ 2.63
82.49 ꢂ 0.96**
9
Vitamin Ea

184
S. Li et al. / Phytochemistry Letters 14 (2015) 178–184
2; HR-ESI–MS m/z 679.3643 ([M + Cl]ꢀ, calc’d. for C37H56O9Cl,
679.3612).
Wang, W.L., Zhou, X., Liu, Y.L., Xu, Q.M., Li, X.R., Yang, S.L., 2014. Two new 20a (H)-
ursane-type triterpenoids from Ilex cornuta and their cytotoxic activities. J.
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saponins from Ilex cornuta and their cytotoxic activities. Phytochem. Lett. 6,
429–434.
Li, Y., Wu, T., Chen, Z.H., Wang, Z.T., 2006. New triterpene saponins and flavonol
glycosides from the leaves of Ilex cornuta. Chin. J. Chem. 24, 577–579.
Li, S.S., Zhao, J.P., Liu, Y.L., Chen, Z., Xu, Q.M., Khan, I., Yang, S.L., 2014. New
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Ali, M., Srivastava, T.N., 1990. Triterpenoids from Symplocos racemosa bark.
Phytochemistry 29, 3601–3604.
3.7.4. Compound 4
25
white amorphous powder; [
a
]
+23.4 (c 0.12, MeOH); UV
D
(MeOH)lmax (log
e
) 225 (2.78); IR nmax (KBr) cmꢀ1 3398, 2892,
2714,1674,1463,1327,1052; 1H NMR and 13C NMR, see Tables 1 and
2; HR-ESI–MS m/z 493.3318 ([M + Na]+, calc’d. for C30H46O4Na,
493.3294).
Che, Y.Y., Zhang, L., Li, N., Zeng, K.W., Tu, P.F., 2012. Triterpenoid saponins from Ilex
mamillata. Nat. Prod. Res. 26, 1991–1995.
Hidaka, K., Ito, M., Matsuda, Y., Kohda, H., Yamasaki, K., Yamahara, J., Chisaka, T.,
Kawakami, Y., Sato, T., Kagei, K., 1987. New triterpene saponins from Ilex
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3.7.5. Compound 5
25
white amorphous powder; [a D
]
+16.7 (c 0.15, MeOH); UV
(MeOH)lmax (log
e
) 226 (2.79); IR nmax (KBr) cmꢀ1 3413, 2969,
2760, 1772, 1446, 1369, 1049; 1H NMR and 13C NMR, see Tables 1
and 2; HR-ESI–MS m/z 647.3801 ([M + HCOOH-H]ꢀ, calc’d. for
647.3795).
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3.7.6. Compound 6
25
white amorphous powder; [a D
]
+15.2 (c 0.12, MeOH); UV
(MeOH)lmax (log
e
) 224 (2.77); IR nmax (KBr) cmꢀ1 3312, 2852,
2709,1789,1415,1374,1100; 1H NMR and 13C NMR, see Tables 1 and
2; HR-ESI–MS m/z 645.3694 ([M-H]ꢀ, calc’d. for C37H55O10
,
645.3639).
3.7.7. Compound 7
25
white amorphous powder; [a D
]
+11.8 (c 0.13, MeOH); UV
(MeOH)lmax (log
e
) 223 (2.77); IR nmax (KBr) cmꢀ1 3421, 2961, 2767,
1790, 1402, 1358, 1070; 1H NMR and 13C NMR, see Tables 1 and 2;
HR-ESI–MS m/z 683.3771 ([M + Na]+, calc’d. for C37H56O10Na,
683.3771).
Fujimoto, S., Mizoi, K., Yoshimoto, T., Suzuki, I.,1984. The protective effect of vitamin
Acknowledgement
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We sincerely thank Dr. Jon Parcher at the University of
Mississippi for helpful discussions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2015.
10.010.
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