Pentacyclic triterpenoids from Cyclocarya paliurus and their antioxidant activities in FFA-induced HepG2 steatosis cells

Article abstract of DOI:10.1016/j.phytochem.2018.03.010

Six undescribed pentacyclic triterpenoids including four triterpenoid aglycones, 1β,2a,3β,23-tetrahydroxyurs-12-en-28-ursolic acid, 2a,3a,6β,19α,23-pentahydroxyurs-12-en-28-ursolic acid, 2α,3α,20β,23-tetrahydroxyurs-12-en-28-ursolic acid and 1β,2a,3β,23-tetrahydroxyurs-12,20(30)-dien-28-ursolic acid, and two triterpenoid glucosides, 2a,3a,23-trihydroxy-12,20(30)-dien-28-ursolic acid 28-O-β-D-glucopyranoside and 1-oxo-3β,23-dihydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranoside, along with 5 known triterpenoids were isolated from a CH₃Cl-soluble extract of the leaves of Cyclocarya paliurus. Their structures were established on the basis of chemical and spectroscopic approaches. These compounds were assessed for their antioxidant effects on FFA-induced hepatic steatosis in HepG2 cells. The results revealed that three saponins and two aglycones markedly increased SOD activity and reduced MDA level.

Full text of DOI:10.1016/j.phytochem.2018.03.010

Phytochemistry 151 (2018) 119e127
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Pentacyclic triterpenoids from Cyclocarya paliurus and their
antioxidant activities in FFA-induced HepG2 steatosis cells
,
b,
c
,
,
,
, *
Hui-Min Yang a
1, Zhi-Qi Yin b 1, Meng-Ge Zhao b c, Cui-Hua Jiang c, Jian Zhang c
,
,
**
Ke Pan a
a Department of Natural Medicinal Chemistry, School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, PR China
b Department of TCMs Pharmaceuticals, School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, PR China
c Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Six undescribed pentacyclic triterpenoids including four triterpenoid aglycones,
1
b
,2a,3
b
,23-
acid,
,23-tetrahydroxyurs-12,20(30)-dien-
28-ursolic acid, and two triterpenoid glucosides, 2a,3a,23-trihydroxy-12,20(30)-dien-28-ursolic acid 28-
O- -glucopyranoside and 1-oxo-3 ,23-dihydroxyolean-12-en-28-oic acid 28-O- -xylopyranoside,
Received 29 December 2017
Received in revised form
27 March 2018
tetrahydroxyurs-12-en-28-ursolic
acid,
2a,3a,6
b
,19
a
,23-pentahydroxyurs-12-en-28-ursolic
,2a,3
2
a
,3 ,20 ,23-tetrahydroxyurs-12-en-28-ursolic acid and 1
a
b
b
b
Accepted 30 March 2018
b
-D
b
b-D
along with 5 known triterpenoids were isolated from a CH3Cl-soluble extract of the leaves of Cyclocarya
paliurus. Their structures were established on the basis of chemical and spectroscopic approaches. These
compounds were assessed for their antioxidant effects on FFA-induced hepatic steatosis in HepG2 cells.
The results revealed that three saponins and two aglycones markedly increased SOD activity and reduced
MDA level.
Keywords:
Cyclocarya paliurus
Juglandaceae
Pentacyclic triterpenoids
Antioxidant effects
FFA-induced hepatic steatosis
© 2018 Elsevier Ltd. All rights reserved.
1. Introduction
and tetracyclic type such as oleanane-, ursane- and dammarane-
type triterpenoids (Fu and Fang, 2009; Wang et al., 2016). Previ-
Cyclocarya paliurus (Batalin) Iljinskaja (Juglandaceae) is a tall
tree belonging to the Juglandaceae family and is an endemic species
distributed in south and southeast of China (Wu et al., 2014). The
leaves of C. paliurus have been used by indigenous people for the
prevention and treatment of hypertension, dyslipidemia and dia-
betes (Jiang et al., 2015a; Kurihara et al., 2003). Over the last several
decades, phytochemical studies of C. paliurus led to the identifica-
tion of nearly 100 compounds, containing polysaccharides, flavo-
noids, phenolic acids and triterpenoids (Wu et al., 2017; Xie and
Xie, 2008). Various triterpenoid aglycones and glycosides have
been identified from the leaves of C. paliurus, including pentacyclic
ous studies also reported that ethanol extract of C. paliurus
exhibited antioxidant effects, remarkably lowered the levels of
malondialdehyde, and elevated superoxide dismutase activity in
type 2 diabetic rats and high-fat diet mice (Ma et al., 2015; Wang
et al., 2013). Our findings have demonstrated that ethanol extract
of C. paliurus leaves was abundant in triterpenic acids and could
ameliorate high fat diet-induced hepatic oxidative stress and
inflammation, leading to block the development of nonalcoholic
fatty liver disease (NAFLD) (Lin et al., 2016). These results indicated
that triterpenoids might be the major functional components of
C. paliurus.
Currently NAFLD has become the most common liver disease
with about 20e30% of general population worldwide (Yopp and
Choti, 2015). NAFLD includes
a series of hepatic pathology
* Corresponding author. Laboratory of Translational Medicine, Jiangsu Province
Academy of Traditional Chinese Medicine, No.100, Shizi Street, Hongshan Road,
Qixia District, Nanjing, 210028 Jiangsu Province, PR China.
** Corresponding author. Department of Natural Medicinal Chemistry, School of
Traditional Chinese Medicine, China Pharmaceutical University, No.639, Longmian
Road, Nanjing, 211198 Jiangsu Province, PR China.
ranging from hepatic steatosis to nonalcoholic steatohepatitis,
which may trigger the development of fibrosis and cirrhosis,
eventually lead to hepatocarcinoma (Choi and Diehl, 2008). Though
the precise mechanism of NAFLD is still unknown, its pathogenesis
is often described as a “two-hits hypothesis” involving insulin
resistance and oxidative stress (Day and James, 1998). Oxidative
stress has also been verified to play an important role in the
E-mail addresses: zhangjian@jsatcm.com (J. Zhang), pan.ke.nj@gmail.com
(K. Pan).
1
These authors contributed equally.
https://doi.org/10.1016/j.phytochem.2018.03.010
0031-9422/© 2018 Elsevier Ltd. All rights reserved.

120
H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
pathogenesis of NAFLD in animal and human studies (De Almeida
et al., 2010; Madan et al., 2006). Furthermore, both malondialde-
hyde (MDA) and superoxide dismutase (SOD) are important indices
of oxidative stress. As a cytotoxic product of lipid peroxidation,
MDA impairs nucleotide and protein synthesis, plays a vital role in
hepatic fibrogenesis (Leonarduzzi et al., 1997). SOD, an enzyme that
catalyzes the dismutation of superoxide radicals into hydrogen
peroxide, prevents lipid peroxidation (Jiang et al., 2015b).
In a continuing search for the biological active compounds from
C. paliurus, six undescribed pentacyclic triterpenes (1e6) and five
known triterpenoids (7e11) were isolated. These compounds were
assessed for their antioxidant effects on FFA-induced hepatic
steatosis in HepG2 cells to validate whether they could block the
development of NAFLD via ameliorating hepatic oxidative stress.
methyl doublets at dH 0.94 (3H, d, J ¼ 6.0 Hz) and 0.99 (3H, d,
J ¼ 6.0 Hz), an oxygen-bearing methylene group at dH 4.23 (1H, d,
J ¼ 12.0 Hz) and 3.74 (1H, d, J ¼ 12.0 Hz), and three oxygenated
methine groups at dH 4.32 (1H, d, J ¼ 9.0 Hz), 4.16 (1H, dd, J ¼ 9.0,
9.0 Hz) and 3.71 (1H, d, J ¼ 9.0 Hz) were observed. Further, one
olefinic proton signal at dH 5.61 (1H, br s) was apparent. The 13C
NMR spectrum of 1 (Table 1) displayed 30 carbon resonances
including a carbonyl carbon (dC 180.0), two olefinic carbons (dC
138.3 and 127.2), and four oxygenated carbons (dC 66.3, 74.4, 74.8,
84.7). Therefore, 1 was assigned as an ursane-type triterpenoid
(Singab et al., 2000). The spectroscopic data above were similar to
those of a known compound, 1b,2a,3b,19a,23-pentahydroxyurs-12-
en-28-ursolic acid (Singab et al., 2000) except for the resonances
corresponding to a methyl doublet [H-29 (dH 0.94, d, J ¼ 6.0 Hz); dC
39.5 (C-19)] in 1 instead of signals for the H-29 (dH 1.44, 3H, s) and
C-19 (dC 72.5) groups. The 2D structure of 1 was established via
thorough analysis of the COSY, HSQC, and HMBC NMR spectra
2. Results and discussion
(Fig. 2). The
a
-orientation of 2-OH was confirmed by the strong
-ori-
Compound 1 was obtained as white and amorphous powder.
The molecular formula was determined to be C30H48O6 on the basis
of HRESIMS (m/z 527.3338 [MþNa]þ, calcd for C30H48NaO6,
527.3343) and 13C NMR data. In the 1H NMR spectrum (Table 1),
four methyl singlets at dH 1.11, 1.20, 1.22, 1.37 (each 3H, s), two
NOESY correlation from H-2 to H-25 and H-24 (Fig. 2).The
entations of 1-OH and 3-OH were confirmed by the NOESY corre-
lations from H-1 to H-9 and H-3 to H-5, respectively (Fig. 2). Thus,
the structure of 1 (Fig. 1) was elucidated as 1b, 2a, 3b, 23-
b
Table 1
1H [ppm, mult. (J in Hz)] and13C NMR data of compounds 1e3 (in C5D5N).
Position
1
1
2
3
a
b
c
d
c
d
dC
dH
dC
dH
dC
dH
84.7, CH
3.71, d (9.0)
45.4, CH2
1.98, m
1.88, m
4.45, m
4.15,d (4.5)
e
48.3, CH2
2.28, m
1.37, m
4.24,ddd (10.5,9.5,4.0)
4.19,d (9.5)
e
2
3
4
5
6
74.4, CH
74.8, CH
43.5, C
45.6, CH
18.1, CH2
4.16,dd (9.0,9.0)
4.32,d (9.0)
e
66.7, CH
81.1, CH
42.9, C
44.5, CH
68.4, CH
69.3, CH
78.8, CH
44.0, C
48.4, CH
18.9, CH2
1.84, m
1.83, m
1.61, m
1.74, m
1.40, m
e
2.30, m
4.97, m
1.77, m
1.71, m
1.43, m
1.67, m
1.32, m
e
7
33.5, CH2
41.7 CH2
2.14, m
1.89, m
e
33.6, CH2
8
40.7, C
40.3, C
40.4, C
9
10
11
49.1, CH
43.4, C
27.6, CH2
2.18, m
e
48.6, CH
38.4, C
24.5 CH2
2.30, m
e
48.5, CH
38.7, C
24.2 CH2
1.84, m
e
3.26, m
2.49, m
5.61, br s
e
2.30, m
2.04, m
12
13
14
15
127.2, CH
138.3, C
42.6, C
128.6, CH
139.7, C
43.0, C
5.66, br s
e
125.8, CH
139.1, C
43.0, C
5.54, br s
e
e
e
e
28.8, CH2
2.30, m
1.20, m
2.07, m
1.96, m
e
29.5, CH2
2.45, m
1.27, m
3.07, m
1.99, m
e
29.0, CH2
2.29, m
1.13, m
2.28, m
1.97, m
e
16
25.0, CH2
26.8, CH2
25.2, CH2
17
18
19
20
21
48.1, C
48.6, C
55.0, CH
73.1, C
42.7, CH
27.3, CH2
48.2, C
53.5, CH
39.5, CH
39.4, CH
31.1, CH2
2.64,d (12.0)
1.43, m
0.95, m
1.45, m
1.43, m
1.97, m
1.95, m
4.23,d (12.0)
3.74,d (12.0)
1.11, s
3.07, m
e
51.5, CH
43.2, CH
72.7, C
2.89,d (10.0)
2.24, m
e
1.50, m
2.06, m
1.33, m
2.06, m
38.7, CH2
2.15, m
1.88, m
2.10, m
1.96, m
4.18,d (10.0)
3.72,d (10.0)
1.06, s
1.07, s
1.07, s
1.14 s
22
23
37.5, CH2
66.3, CH2
38.8, CH2
72.2, CH2
35.5, CH2
67.1, CH2
4.16,d (10.0)
3.92,d (10.0)
1.50, s
1.72, s
1.68, s
24
25
26
27
28
29
30
14.4, CH3
14.0, CH3
18.1, CH3
23.9, CH3
180.0, C
20.0, CH3
18.9, CH3
18.7, CH3
25.1, CH3
181.1, C
14.7, CH3
17.9, CH3
18.0, CH3
23.9, CH3
180.2, C
1.37, s
1.20, s
1.22, s
e
1.68, s
e
e
21.4, CH3
17.5, CH3
0.94,d (6.0)
0.99,d (6.0)
27.4, CH3
17.1, CH3
1.44, s
1.11,d (5.0)
14.0, CH3
20.4, CH3
1.22,d (5.0)
1.38, s
a
Measured at 75 MHz.
Measured at 300 MHz.
Measured at 125 MHz.
Measured at 500 MHz.
b
c
d

H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
121
Fig. 1. Chemical structures of compounds 1e11.
Fig. 2. Key 1He1H COSY (
), HMBC (
), and NOE (
) correlations of 1.

122
H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
tetrahydroxyurs-12-en-28-ursolic acid.
HRESIMS (m/z 520.3629 [M
þ
NH4]þ, calcd for C30H50NO6,
Compound 2 was isolated as white and amorphous powder. The
molecular formula C30H48O7 was deduced from HRESIMS (m/z
538.3738 [M þ NH4]þ, calcd for C30H52NO7, 538.3738) and 13C NMR
data. The 1H NMR spectrum of 2 exhibited the typical spectroscopic
features of an ursane-type triterpene, including an olefinic proton
at dH 5.66 (1H, br s), three oxygenated methine protons at dH 4.97
(1H, m), 4.45 (1H, m) and 4.15 (1H, d, J ¼ 4.5 Hz), a pairs of
oxygenated methylene protons at dH 4.16 (1H, d, J ¼ 10.0 Hz) and
3.92 (1H, d, J ¼ 10.0 Hz), five methyl singlets at dH 1.44, 1.50, 1.68,
1.68,1.72 and a methyl doublet at dH 1.11 (3H, d, J ¼ 5.0 Hz) (Table 1).
In addition, the 13C NMR spectrum displayed one carboxylic carbon
at dC 181.1, five oxygenated carbons at dC 66.7, 68.4, 72.2, 73.1, 81.1 as
well as two olefinic carbons at dC 128.6 and 139.7 (Table 1). These
520.3633) and 13C NMR data. The 1H and 13C NMR spectra (Table 2)
were almost identical to those of 1 except for the replacement of a
methyl signal [dH 0.99 (3H, d, J ¼ 6.0 Hz)] by the NMR signals due to
an exomethylene [dH 4.76 (1H, br s), 4.81 (1H, br s); dC 154.3, 105.4]
in 4. The location of terminal double bond was deduced at C-20 and
C-30 from the HMBC correlations of dH 4.76 and 4.81 with C-19 (dC
38.1)/C-21 (dC 33.2), H-29 (dH 1.11) with C-19 (dC 38.1)/C-18 (dC
56.1)/C-20 (dC 154.3) (Fig. 5). The relative configuration of 4 was
consistent with that of 1 and confirmed by the NOE interactions in
the NOESY spectrum (Fig. 5). Hence, the structure of 4 (Fig. 1) was
elucidated as 1
ursolic acid.
b, 2a, 3b, 23-tetrahydroxyurs-12,20(30)-dien-28-
Compound 5 was obtained as white and amorphous powder. It
gave the molecular formula C36H56O10, according to its HRESIMS at
m/z 666.4210 [M þ NH4]þ (calcd for 666.4212) and 13C NMR data.
The 13C NMR spectrum of 5 showed 36 carbon signals due to the
aglycone and one sugar moiety (Table 2). The five sp3 carbons (dC
16.8, 17.6, 18.1, 18.1, 24.0), two sp2 carbons (dC 126.8, 138.6) and a
carboxylic carbon (dC 176.0) were coupled with the 1H NMR data
which were assigned as four methyl proton singlets (dH 1.17, 1.09,
1.05, 0.87), a methyl proton doublet (dH 1.04), a broad triplet-like
NMR data (Table 1) were almost identical to those of 2a, 3a,19a, 23-
tetrahydroxy-12-en-28-ursolic acid (Lee et al., 2005) except for the
replacement of one methylene by an additional oxymethine [dH
4.97 (1H, m); dC 68.4] in 2. The 1He1H COSY correlations of the
oxymethine proton between H-5 and H-7 (Fig. 3) revealed the
attachment of an OH group to C-6 in 2. The NOE interaction of H-5
with H-6 (Fig. 3) confirmed the
compound (Fig. 1) was elucidated as 2a,3a,6
pentahydroxyurs-12-en-28-ursolic acid.
b-orientation of 6-OH. Therefore,
2
b
,19 ,23-
a
vinyl proton signal
(dH 5.44). These data indicated that 5
Compound 3 appears as white and amorphous powder. Its
molecular formula was established as C30H48O6 by HRESIMS (m/z
522.3786 [M þ NH4]þ, calcd for C30H52NO6, 522.3789) and 13C NMR
data. The 1H NMR spectrum (Table 1) displayed resonances for five
methyl singlets (dH 1.06, 1.07, 1.07, 1.14, 1.38), a methyl doublet [dH
1.22 (3H, d, J ¼ 5.0 Hz)], two oxymethines [dH 4.24 (1H, ddd,
J ¼ 10.5,9.5,4.0 Hz), 4.19 (1H, d, J ¼ 9.5 Hz)], an oxygenated methy-
lene group [dH 4.18 (1H, d, J ¼ 10.0 Hz), 3.72 (1H, d, J ¼ 10.0 Hz)] and
an olefinic proton [dH 5.54 (1H, br s)]. The 13C NMR spectrum
(Table 1) showed 30 carbon resonances comprising four oxygen-
bearing carbons (dC 67.1, 69.3, 72.7, 78.8), two olefinic carbons (dC
139.1 and 125.8) and a carboxylic carbon (dC 180.2). These spec-
trometric data (Table 1) were comparable to those of 2a, 3a, 23-
trihydroxyurs-12-en-28-oic acid (Lee et al., 2008) except for
replacement of one methine by an additional oxygenated quater-
nary carbon (dC 72.7) in 3. Furthermore, the OH group was located
at C-20 based on the HMBC cross-peak from H-29 [dH 1.22 (3H, d,
J ¼ 5.0 Hz)] and H-30 [dH 1.38 (3H, s)] to C-20 (dC 72.7) (Fig. 4). The
possessed an urs-12-en-28-oic acid skeleton. Furthermore, the 1H
and 13C NMR spectrum (Table 2) exhibited an exomethylene signal
[
dH 4.71, 4.73 (each 1H, br s); dC 153.6, 105.6]. The comparison of
NMR data (Table 2) with those of 2 ,3 ,23-trihydroxy-12,20(30)-
dien-28-ursolic acid (Sashida et al., 1992) indicated the presence of
-glucopyranosyl moiety on the basis of the 13C NMR data as well
as the coupling constant of the anomeric proton (JH-1' ¼ 8.5 Hz)
(Yang et al., 2012). Acid hydrolysis of 5 afforded a -glucose from
a
a
a
b
D
HPLC analysis compared with an authentic sugar sample. The
linkage of the glucose was verified at C-28 by the HMBC correlation
from H-1' (dH 6.25) to dC 176.0 (Fig. 6). The relative configuration of
the aglycone was determined to be 2a, 3a, 23-trihydroxy-12,
20(30)-dien-28-ursolic acid by NOESY spectrum interpretation
(Fig. 6). Consequently, compound 5 was assigned as 2a, 3a, 23-
trihydroxy-12, 20(30)-dien-28-ursolic acid 28-O-b-D-glucopyrano-
side (Fig. 1).
Compound 6 was isolated as a white and amorphous powder. Its
molecular formula of C35H54O9 was determined from HRESIMS (m/z
641.3653 [MþNa]þ, calcd for C35H54NaO9, 641.3660) and 13C NMR
data. In the 1H NMR spectrum (Table 2), six methyl singlets (dH 1.40,
1.21, 1.19, 1.19, 0.93, 0.89), one olefinic proton signal at dH 5.50 (1H,
br s), together with one anomeric proton signal at dH 6.24 (1H, d,
J ¼ 7.0 Hz) were observed. The 13C NMR spectrum (Table 2) showed
signals at dC 213.5, 177.0 (for a keto carbonyl group and an ester
carbonyl group), two olefinic carbons signals (dC 143.8, 123.7), and
an anomeric carbon signal (dC 96.7). The analysis of NMR data and
comparison with the reference revealed that 6 shared the same
b-orientation of 20-OH was confirmed by the strong ROESY corre-
lations from H-19 [dH 2.24 (1H, m)] to H-30 [dH 1.38 (3H, s)]. The
configurations of the remaining stereogenic centers were identical
to those of 2a, 3a, 23-trihydroxyurs-12-en-28-oic acid based on the
ROESY correlations of 3 (Fig. 4). Accordingly, the structure of 3
(Fig. 1) was defined as 2a, 3a, 20b, 23-tetrahydroxyurs-12-en-28-
ursolic acid.
Compound 4 was purified as white and amorphous powder. It
showed a molecular formula of C30H46O6 on the basis of the
Fig. 3. Key 1He1H COSY (
), HMBC (
), and NOE (
) correlations of 2.

H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
123
Fig. 4. Key 1He1H COSY (
), HMBC (
), and ROE (
) correlations of 3.
Table 2
1H [ppm, mult. (J in Hz)] and13C NMR data of compounds 4e6 (in C5D5N).
Position
4
5
6
a
b
c
d
c
d
dC
dH
dC
dH
dC
dH
1
2
85.1, CH
74.8, CH
3.72, d (9.0)
43.3, CH2
66.6, CH
1.94, m
1.79, m
4.28, m
213.5, C
e
4.17,dd (9.0,9.0)
45.5, CH2
3.47,dd (12.0,12.0)
2.81,dd (12.5,5.0)
4.58,dd (12.0,5.0)
e
3
4
5
6
75.3, CH
43.9, C
46.0, CH
18.5, CH2
4.32,d (9.0)
e
79.4, CH
42.3, C
43.9, CH
18.7, CH2
4.14,d (2.0)
e
73.3, CH
44.2, C
47.8, CH
18.4, CH2
1.87, m
1.84, m
1.61, m
1.75, m
1.42, m
e
2.04, m
1.59, m
1.36, m
1.71, m
1.37, m
e
1.92, m
1.77, m
1.57, m
2.01, m
1.79, m
e
7
33.9, CH2
33.6, CH2
33.1, CH2
8
41.1, C
40.7, C
40.2, C
9
10
11
49.4, CH
43.8, C
28.0, CH2
2.17, m
e
48.4, CH
38.7, C
24.2, CH2
1.90, m
e
40.0, CH
52.8, C
26.2, CH2
2.66, m
e
3.25,dt (18.0,6.0)
2.50, m
5.61, br s
e
2.08, m
2.04, m
5.44,t (3.0)
e
2.73, m
2.02, m
5.50, br s
e
12
13
14
15
127.9, CH
138.4, C
43.0, C
126.8, CH
138.6, C
43.0, C
123.7, CH
143.8, C
42.8, C
e
e
e
29.1, CH2
2.33, m
1.21, m
2.30, m
2.08, m
e
28.9, CH2
2.42, m
1.17, m
2.22, m
2.04, m
e
28.6, CH2
2.25, m
1.11, m
2.02, m
1.93, m
e
16
25.4, CH2
25.0, CH2
23.8, CH2
17
18
19
48.8, C
56.1, CH
38.1, CH
48.9, C
55.8, CH
37.9, CH
47.7, C
42.4, CH
46.3, CH2
2.77,d (12.0)
2.44, m
2.64,d (12.0)
2.39, m
3.23, m
1.71, m
1.20, m
e
20
21
154.3, C
33.2, CH2
e
153.6, C
32.8, CH2
e
31.2, C
34.5, CH2
2.27, m
2.40, m
2.05, m
2.29, m
2.13, m
2.01, m
1.79, m
3.91,d (10.0)
3.76,d (10.0)
0.87, s
1.05, s
1.17, s
1.33, m
1.13, m
1.57, m
1.33, m
4.14,d (10.5)
3.73,d (10.5)
1.19, s
1.40, s
1.19, s
1.21, s
e
22
23
40.1, CH2
66.7, CH2
39.3, CH2
71.7, CH2
33.2, CH2
66.4, CH2
4.24,d (12.0)
3.76,d (12.0)
1.11, s
1.37, s
1.17, s
24
25
26
27
28
29
30
14.8, CH3
14.4, CH3
18.5, CH3
24.1, CH3
179.9, C
18.1, CH3
17.6, CH3
18.1, CH3
24.0, CH3
176.0, C
14.1, CH3
16.1, CH3
18.6, CH3
26.3, CH3
177.0, C
1.20, s
e
1.09, s
e
17.0, CH3
105.4, CH2
1.11,d (6.0)
4.81, br s
4.76, br s
16.8, CH3
105.6, CH2
1.04,d (7.5)
4.73, br s
4.71, br s
6.25,d (8.5)
4.17,dd (8.5,8.5)
4.27, m
33.5, CH3
24.0, CH3
0.89, s
0.93, s
10
20
30
40
50
96.2, CH
74.4, CH
79.3, CH
71.7, CH
79.6, CH
96.7, CH
74.1, CH
78.6, CH
71.3, CH
68.1, CH2
6.24,d (7.0)
4.20, m
4.23, m
4.25, m
4.39,dd (11.5,4.0)
3.84,dd (11.0,9.0)
4.30, m
4.01,ddd (9.0,4.8,2.8)
60
62.8, CH2
4.45,dd (12.0,2.6)
4.38,dd (12.0,4.6)
a
Measured at 75 MHz.
Measured at 300 MHz.
Measured at 125 MHz.
Measured at 500 MHz.
b
c
d

124
H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
Fig. 5. Key 1He1H COSY (
), HMBC (
), and NOE (
) correlations of 4.
Fig. 6. Key 1He1H COSY (
), HMBC (
), and NOE (
) correlations of 5.
aglycone structure with 1-oxo-3
b
, 23-dihydroxyolean-12-en-28-
cells. As shown in Fig. 8, compounds 1e8 and 10e11 had no
observable effect on cell viabilities at the concentration of 10 M,
while compound 9 exhibited no cytotoxicity at 1 M concentration.
The bioassay results showed that the SOD activity were signifi-
cantly decreased (p < 0.01) and the MDA levels were significantly
increased (p < 0.01) when cells were incubated with FFA mixture
compared with the blank group (Fig. 9). Interestingly, compounds
oic acid 28-O-
there was a xylopyranosyl unit instead of a
moiety (Yoshikawa et al., 2005). The configuration of
b-
D
-glucopyranoside (Yang et al., 2011). However,
-glucopyranosyl
-xylose
m
b
m
b-
D
was supported by the coupling constant (J ¼ 7.0 Hz) and acid hy-
drolysis. The sugar linkage at C-28 was deduced from the HMBC
correlations between H-10(dH 6.24) and C-28 (dC 177.0) (Fig. 7). The
relative configuration of 6 was also confirmed by the NOESY
spectrum (Fig. 7). Thus, the structure of 6 (Fig. 1) was characterized
4e7 (5
reduced levels of MDA in FFA-induced HepG2 steatosis cells
compared with the model group. Especially, compound 9 (1 M)
mM) and 9 (1 mM) markedly increased SOD activity and
as 1-oxo-3
xylopyranoside.
The five known compounds were identified as
trihydroxy-urs-12-en-28-oic acid 28-O- -glucopyranoside (7)
(Yang et al., 2012), 19 -hydroxyasiatic acid (8) (Lee et al., 2010), 2a-
b,
23-dihydroxyolean-12-en-28-oic acid 28-O-
b-D
-
m
exhibited better potential effect on the inhibition of MDA produc-
tion than the positive control (Vitamin E). These results implied
that compounds 4e7 and 9 might block the development of NAFLD
via ameliorating hepatic oxidative stress. However, compounds
1e3, 8, and 10e11 showed unfavourable effects on the antioxidant
2a,3a,23-
b-D
a
hydroxyursolic acid (9) (Cheng et al., 2010), 2a, 3a, 23-
trihydroxyurs-12, 20(30)-dien-28-oic acid (10) (Xiao et al., 2013),
activity at the concentration of 10 mM.
and 2a, 3
b
, 23-trihydroxyurs-12, 20(30)-dien-28-oic acid (11) (Xiao
Further analysis of their structures and bioactivities revealed
that 1 and 4 shared the same chemical skeleton only differing in the
C-30 functional group. However, their different activity suggested
that the terminal double bond may be crucial for the antioxidant
activity. Interestingly, compounds 10 and 11 with the terminal
double bonds had no antioxidant activity, indicating that 1-hydroxy
et al., 2013) by comparison of their spectroscopic data with those
reported in the literature.
All isolated compounds (1e11) were assayed for their antioxi-
dant effects on FFA-induced hepatic steatosis in HepG2 cells. Firstly,
the compounds were tested for cytotoxic activity against HepG2
Fig. 7. Key 1He1H COSY (
), HMBC (
), and NOE (
) correlations of 6.

H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
125
antioxidant activity than 10 without pyranose. It was proposed that
the glycosyl moiety had a significant contribution to the antioxi-
dant activity.
3. Conclusion
Phytochemical study of the leaves of C. paliurus generated six
undescribed pentacyclic triterpenoids (1e6) and five known tri-
terpenoids (7e11). All isolated compounds were assayed for their
antioxidant effects on Free Fatty Acid-induced hepatic steatosis in
HepG2 cells. Among them, three undescribed and two known tri-
terpenoids significantly increased superoxide dismutase activity
and reduced malondialdehyde level. Our findings enriched the di-
versity of pentacyclic triterpenoids for the genus Cyclocarya, and
suggested that pentacyclic triterpenoids may be major functional
constituents in C. paliurus contributing to ameliorate hepatic
oxidative stress.
4. Experimental section
4.1. General experimental procedures
Optical rotations were measured in methanol using a Jasco P-
1020 polarimeter. A Shimadzu UV-2500PC spectrophotometer and
Nicolet Impact 410 spectrophotometer were used for recording the
UV spectra (Methanol) and IR spectra (KBr pellets), respectively. 1D
and 2D NMR spectra were recorded on a Bruker Avance-300
(Bruker, Switzerland, 300 MHz for 1H NMR, 75 MHz for 13C NMR),
Bruker Avance-500 (Bruker, Switzerland, 500 MHz for 1H NMR,
125 MHz for 13C NMR) and Bruker Avance-600 (Bruker,
Switzerland, 600 MHz for 1H NMR, 150 MHz for 13C NMR) NMR
spectrometer with TMS as internal standard. Unless otherwise
Fig. 8. The cell viability assay of compounds 1e11 by the MTT method. (A) Cytotoxicity
of compounds 1e11 (10 M). (B) Cytotoxicity of compound 9 (1e9 M).
m
m
specified, chemical shifts (d) were expressed in ppm with reference
to the solvent signals, and coupling constants are expressed in
hertz. HRESIMS were performed on a HP-1100 LC/EST and Synapt™
Q-TOF mass spectrometer (Waters, Milford, America). Agilent 1260
Infinity equipped with UV and Alltech 3300 ELSD detector was used
to analyze the samples, and Alliance semi preparative HPLC with
COSMOSIL Phecda C18 (250 mm ꢀ 20 mm, 5
mm) was applied for
further purification. Column chromatography was performed with
silica gel (100e200 and 200e300 mesh, Qing-dao Marine Chemical
Group Co., Shandong, CN), ODS RP-18 gel (40e63 mm, Merck,
Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Swe-
den).
D
-Glucose,
D
-xylose, pyridine (Reagent Plus, ꢁ 99%),
L-cysteine
methyl ester hydrochloride, and isothiocyanate were purchased
from Sigma (St. Louis, MO, America). Methanol and acetonitrile
(HPLC-grade) was purchased from TEDIA. All other chemicals are of
analytical grade. The fractions were monitored by TLC and 1%
vanillin in H2SO4 was used as the spraying reagent to visualize
spots.
4.2. Plant material
The leaves of C. paliurus were collected from Nanjing Forestry
University, Nanjing, Jiangsu, People's Republic of China (GPS co-
ordinates: 118.822414, 32.085054), in October 2010 and authenti-
cated by Prof. Minjian Qin, China Pharmaceutical University,
People's Republic of China. A voucher herbarium specimen (No.
L20100033) has been deposited in the herbarium of the Depart-
ment of Natural Medicinal Chemistry, China Pharmaceutical Uni-
versity, Nanjing, Jiangsu, People's Republic of China.
Fig. 9. Effect of compounds 4e7 (5 mM) and 9 (1 mM) on the levels of SOD activity (A)
and MDA (B). The results include data from three experiments (mean SD, n ¼ 5).
#p < 0.05, ##p < 0.01 vs blank group; *p < 0.05, **p < 0.01 vs model group, Student's t-
test.
4.3. Extraction and isolation
group may have impact on their antioxidant activities. At a con-
centration of 5 mM, compound 5 with pyranose showed stronger
Air-dried leaves of C. paliurus (48.5 kg) were extracted three

126
H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
times with 80% ethanol under reflux and evaporated to afford a
crude extract (8.3 kg). The extract was suspended in water and
partitioned with chloroform to yield a chloroform-soluble extract
(3.6 kg). The chloroform extract (460 g) was subjected to a reduced
pressure chromatography and eluted with gradient mixtures of
CH3CleMeOH (100:0, 100:3, 100:5, 5:1 and 0:100) to yield five
fractions (Fr.1e5).
4.4. Acid hydrolysis of compounds 5e6
Compounds 5e6 (each 1 mg) were hydrolyzed in 2 M HCl under
reflux in a boiling water bath for 2 h. The reaction mixture was
neutralized by AgCO3 and extracted with CHCl3 (3 ꢀ 3 mL). The
aqueous layer was concentrated and dried to obtain the mono-
saccharide fraction. The residue was dissolved in pyridine (1 mL)
The isolation and purification of Fr.2 and Fr.3 have been finished
in our earlier research (Wu et al., 2017).
containing 2 mg of L-cysteine methyl ester hydrochloride and
heated at 60 ꢄC for 1 h. Then o-toryl isothiocyanate (2 mg) was
added and heated at 60 ꢄC for another 1 h. The reaction mixture was
analyzed by RP-HPLC under the following conditions: column,
Fr.4 (137.6 g) was chromatographed over a silica gel column and
eluted with gradient mixtures of CH3CleMeOH (35:1, 25:1, 15:1,
10:1, 1:1, 0:100) to obtain six subfractions (Fr.4a - 4f). Fr.4b was
subjected to a Sephadex LH-20 column eluted with CH3CleMeOH
(1:1) to yield two subfractions (Fr.4b1 - Fr.4b2). Fr.4b1 was frac-
tionated by silica gel column and eluted with CH3CleMeOH
(30:1 /1:1) to yield four subfractions (Fr.4b1a - Fr.4b1d). Fr.4b1a
was purified by semi-preparative HPLC (MeOHeH2O, 85:15) to give
1 (25 mg), 3 (9 mg), and 9 (20 mg). Fr.4b1b was purified by semi-
preparative HPLC (MeOHeH2O, 65:25) to obtain 2 (10 mg) and 8
(35 mg). Fr.4b1c was purified by semi-preparative HPLC (MeC-
NeH2O, 72:28) to yield 4 (10 mg), 10 (11 mg) and 11 (13 mg). Fr.4b2
was separated on ODS column chromatography with MeOHeH2O
(50:50 / 100:0) and purified by semi-preparative HPLC (MeCN
eH2O, 65:35) to afford 5 (8 mg), 6 (10 mg) and 7 (7 mg).
Agilent ZORBAX SB-C18 column (250 ꢀ 4.6 mm, 5
mm); solvent, 25%
CH3CN in 50 mM H3PO4 for 40 min; flow rate, 0.8 mL/min; detector,
UV; wavelength, 250 nm; temperature, 35 ꢄC; injection volume,
10 mL. The identification of D-glucose (tR 17.1 min) and D-xylose (tR
19.8 min) present in sugar fractions of compounds 5e6 were
confirmed by comparison with those of authentic standards
(Tanaka et al., 2007).
4.5. Cell culture, measurement of antioxidant enzymes and lipid
peroxide, and cell viability
Human hepatoma HepG2 cells were obtained from the Amer-
ican Type Culture Collection (ATCC) (Shanghai, China). The cells
were cultured in DMEM supplemented with 10% fetal bovine serum
penicillin (100 U/ml), streptomycin (100 m
g/mL) at 37 ꢄC in a hu-
4.3.1. 1b,2a,3b,23-tetrahydroxyurs-12-en-28-ursolic acid (1)
White amorphous powder; ½a D20
þ 26 (c 0.097, MeOH); UV
ꢂ
midified atmosphere containing 5% CO2. To induce hepatic stea-
tosis, FFA mixture (0.33 mM palmitate and 0.66 mM oleate)
(sodium salt of oleate and palmitate, Sigma, Malaysia) was added to
HepG2 cells with fatty acid free bovine serum albumin as supple-
ment in a final concentration of 1% in the culture medium as
described previously (Park et al., 2017; Wobser et al., 2009). HepG2
cells were seeded into 24-well plates with 3 ꢀ 105/well. After 24 h
of FFAs exposure with or without compounds (1e11), intracellular
SOD and MDA content were measured by an enzymatic kit ac-
cording to the manufacturer's instructions. Vitamin E (Sigma-
Aldrich, St. Louis, MO, USA) served as a positive control (Gayathri
et al., 2015). The effects of compounds (1e11) on cell viability
were evaluated using the MTT method (Lin et al., 2015). All values
were normalized by the total protein concentration of the same
sample. Measurements were performed in triplicate and are
representative of three independent experiments. Data analyses
were performed using the software SPSS 22.0. Data were expressed
as the mean SD. Statistical comparisons were made between
groups using a one-way ANOVA and the StudentꢃNewmanꢃKeuls
test. A value of p < 0.05 was considered as significant.
(MeOH) lmax (log ε) 210 (2.74) nm; IR (KBr) nmax 3441, 2946, 1639,
1050 cmꢃ1 1H and 13C NMR data, see Table 1; HRESIMS m/z
;
527.3338 [MþNa]þ (calcd for C30H48NaO6, 527.3343).
4.3.2. 2a,3a,6b,19a,23-pentahydroxyurs-12-en-28-ursolic acid (2)
White amorphous powder; ½a D20
þ 10 (c 0.110, MeOH); UV
ꢂ
(MeOH) lmax (log ε) 213 (4.06) nm; IR (KBr) nmax 3449, 2926, 1686,
1040 cmꢃ1 1H and 13C NMR data, see Table 1; HRESIMS m/z
;
538.3738 [M þ NH4]þ (calcd for C30H52NO7, 538.3738).
4.3.3. 2a, 3a, 20b, 23-tetrahydroxyurs-12-en-28-ursolic acid (3)
White amorphous powder; ½a D20
þ 46 (c 0.110, MeOH); UV
ꢂ
(MeOH) lmax (log ε) 210 (3.09) nm; IR (KBr) nmax 3455, 1640,
1049 cmꢃ1 1H and 13C NMR data, see Table 1; HRESIMS m/z
;
522.3786 [M þ NH4]þ (calcd for C30H52NO6, 522.3789).
4.3.4. 1b,2a,3b,23-tetrahydroxyurs-12,20(30)-dien-28-ursolic acid
(4)
White amorphous powder; ½a D20
þ 104 (c 0.097, MeOH); UV
ꢂ
Author contributions
(MeOH) lmax (log ε) 214 (3.64) nm; IR (KBr) nmax 3449, 1633,
1050 cmꢃ1 1H and 13C NMR data, see Table 2; HRESIMS m/z
;
520.3629 [M þ NH4]þ (calcd for C30H50NO6, 520.3633).
H.M. Yang and Z.Q. Yin contributed equally.
Notes
4.3.5. 2a, 3a, 23-trihydroxy-12, 20(30)-dien-28-ursolic acid 28-O-
b
-D-glucopyranoside (5)
The authors declare no competing financial interest.
Acknowledgments
White amorphous powder; ½a D20
þ 60 (c 0.095, MeOH); UV
ꢂ
(MeOH) lmax (log ε) 212 (4.30) nm; IR (KBr) nmax 3442, 2928, 1644,
1075 cmꢃ1 1H and 13C NMR data, see Table 2; HRESIMS m/z
;
666.4210 [M þ NH4]þ (calcd for C36H60NO10, 666.4212).
This research was supported by grants from the National Natural
Science Foundation of China (No. 81503316, 31270673) and the
Natural Science Foundation of Jiangsu Province (No. BK20161460,
BK20160926).
4.3.6. 1-oxo-3
xylopyranoside (6)
b, 23-dihydroxyolean-12-en-28-oic acid 28-O-
b-D-
White amorphous powder; ½a D20
ꢂ
þ 55 (c 0.099, MeOH); UV
(MeOH) lmax (log ε) 210 (3.45) nm; IR (KBr) nmax 3451, 2922, 1636,
Appendix A. Supplementary data
1057 cmꢃ1 1H and 13C NMR data, see Table 2; HRESIMS m/z
;
641.3653 [MþNa]þ (calcd for C35H54NaO9, 641.3660).
Supplementary data related to this article can be found at

H.-M. Yang et al. / Phytochemistry 151 (2018) 119e127
127
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