Triterpenoids from Cyclocarya paliurus and their inhibitory effect on the secretion of apoliprotein B48 in Caco-2 cells

Article abstract of DOI:10.1016/j.phytochem.2017.06.015

Five previously undescribed compounds including two triterpenoid aglycones, 3β,23-dihydroxy-1,12-dioxo-olean-28-oic acid and 3β,23,27-trihydroxy-1-oxo-olean-12-ene-28-oic acid, and three triterpenoid glucosides cyclocarioside L-N, along with 17 known compounds were isolated from a CH₃Cl-soluble extract of the leaves of Cyclocarya paliurus. Two 27-nor-triterpenoid glycosides were isolated from the genus for the first time. Furthermore, the characterized compounds were tested for the inhibitory effects on apoliprotein B48 secretion in Caco-2 cells. Seven triterpenoid aglycones together with four triterpenoid saponins significantly decreased the apoliprotein B48 oversecretion induced by oleic acid in Caco-2 cells.

Full text of DOI:10.1016/j.phytochem.2017.06.015

Phytochemistry 142 (2017) 76e84
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpenoids from Cyclocarya paliurus and their inhibitory effect on
the secretion of apoliprotein B48 in Caco-2 cells
,
b
,
,
c
,
,
Zheng-Feng Wu a 1, Fan-Cheng Meng a 1, Lan-Jie Cao a b, Cui-Hua Jiang b,
Meng-Ge Zhao a b, Xu-Lan Shang d, Sheng-Zuo Fang d, Wen-Cai Ye e, Qing-Wen Zhang c,
,
Jian Zhang b **, Zhi-Qi Yin a
,
, *
a Department of Natural Medicinal Chemistry & State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, PR China
b Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, 210028, PR China
c State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao SAR, PR China
d College of Forestry, Nanjing Forestry University, Nanjing, 210037, PR China
e Institute of Traditional Chinese Medicine and Natural Products & Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional
Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Five previously undescribed compounds including two triterpenoid aglycones, 3b,23-dihydroxy-1,12-
Received 3 April 2017
Received in revised form
19 June 2017
dioxo-olean-28-oic acid and 3b,23,27-trihydroxy-1-oxo-olean-12-ene-28-oic acid, and three triterpe-
noid glucosides cyclocarioside L-N, along with 17 known compounds were isolated from a CH3Cl-soluble
extract of the leaves of Cyclocarya paliurus. Two 27-nor-triterpenoid glycosides were isolated from the
genus for the first time. Furthermore, the characterized compounds were tested for the inhibitory effects
on apoliprotein B48 secretion in Caco-2 cells. Seven triterpenoid aglycones together with four tri-
terpenoid saponins significantly decreased the apoliprotein B48 oversecretion induced by oleic acid in
Caco-2 cells.
Accepted 25 June 2017
Keywords:
Cyclocarya paliurus
Juglandaceae
© 2017 Elsevier Ltd. All rights reserved.
Triterpenoids
Cyclocarioside
Apoliprotein B48
1. Introduction
observations and basic researches put insight that anomalous
elevated plasma concentration of apoB48, especially in the fasting
Digestion and absorption of excessive lipids is a well-known
contributor to the development of dyslipidemia, which is associ-
ated with metabolism syndrome including cardiovascular disease
and diabetes mellitus (Karpe, 1999). Apolipoprotein B48 (apoB48)
is critical for the intestinal absorption of dietary triglycerides and
cholesterol and has been shown to be a useful marker of intestinally
derived lipoprotein particles, especially chylomicron and chylomi-
cron remnants (Otokozawa et al., 2009; Young, 1990). Both clinical
state, was positive correlated with an elevated risk of coronary
artery disease (Mori et al., 2013; Valero et al., 2005).
Cyclocarya paliurus (Juglandaceae) (Batal) Iljinskaja (C. paliurus)
is an edible and medicinal plant distributed in the highland of
southern China (Shu et al., 1995b). Its leaves have been used to
prevent and ameliorate dyslipidemia and diabetes in Chinese
folkloric medicine. Previous studies reported that C. paliurus extract
suppressed an increase in plasma triacylglycerol levels in lipid-
loaded mice and ameliorate dyslipidemia in type 2 diabetic rats
(Kurihara et al., 2003; Wang et al., 2013).
Triterpenoids have been extensively investigated due to various
biological activities, such as anti-inflammatory, anti-tumor, anti-
hyperglycemic as well as anti-hyperlipidemic effects (Alqahtani
et al., 2013; Ghosh and Sil, 2013; Somova et al., 2003). For
example, ginsenoside Rg3 and ursolic acid, the main constituents of
red ginseng and Crataegii fructus, can inhibit the activities of
pancreatic lipase and 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase, and reduce the plasma levels of triglyceride
* Corresponding author. Department of Natural Medicinal Chemistry & State Key
Laboratory of Natural Medicines, China Pharmaceutical University, No. 24, Tong-
jiaxiang, Gulou District, Nanjing, 210009, Jiangsu Province, PR China.
** 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.
E-mail addresses: zhangjian@jsatcm.com (J. Zhang), cpu-yzq@cpu.edu.cn
(Z.-Q. Yin).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.phytochem.2017.06.015
0031-9422/© 2017 Elsevier Ltd. All rights reserved.

Z.-F. Wu et al. / Phytochemistry 142 (2017) 76e84
77
and cholesterol in hyperlipidemic mice (Min et al., 2008). Tri-
terpenic acids including oleanolic acid, ursolic acid, maslinic acid,
corosolic acid, 23-hydroxyursolic acid, arjunolic acid and echino-
cystic acid are all reported to have inhibitory effect on cholesterol
acyltransferase (ACAT) activity (Kim et al., 2005). Phytochemical
investigations of the leaves of C. paliurus have resulted in the
isolation of approximately 100 compounds, including poly-
saccharides, flavonoids, phenolic acids and triterpenoids (Shang
et al., 2015). Up to date, pentacyclic and tetracyclic triterpenoid
aglycones and glycosides, such as oleanane-, ursane- and
dammarane-type triterpenoids, have been identified from the
leaves of C. paliurus (Fu and Fang, 2009). Our previous findings
demonstrated that ethanol extract of C. paliurus leaves was abun-
dant in triterpenic acids and could mitigate dyslipidemia and
down-regulate intestinal apoB48 secretion, indicating that tri-
terpenoids might be the active components with biological activity
(Ma et al., 2015).
In the present study, 5 previously undescribed and 17 known
compounds were isolated from the chloroform extract of C. paliurus
using column chromatography and Pre-HPLC methods (Fig. 1).
Their structures were elucidated as 14 pentacyclic triterpenic acids
and 8 tetracyclic triterpenoid glycosides based on the detailed
spectroscopic analysis. Moreover, all the characterized compounds
were evaluated for the inhibitory effects on apoB48 secretion of
Caco-2 cells.
3441, 2945, 1696 and 1663 cmꢀ1 indicated the presence of hy-
droxyl, aliphatic CꢀH, carbonyl and olefinic groups. The 1H NMR
spectrum of 2 exhibited the spectral features of a typical oleanane
triterpene, including an olefinic proton at dH 5.86 (1H, t, J ¼ 3.6 Hz),
an oxygenated methine proton at dH 4.55 (1H, dd, J ¼ 4.9, 11.8 Hz),
two pairs of oxygenated methylene protons at dH 4.12 (1H, d,
J ¼ 10.7 Hz), 3.71 (1H, d, J ¼ 10.5 Hz), 4.04 (1H, d, J ¼ 12.0 Hz) and
3.93 (1H, d, J ¼ 11.9 Hz), and five methyl singlets at dH 1.38,1.20,1.13,
1.01 and 0.87 (Table 1). In addition, the 13C NMR spectrum dis-
played one keto carbonyl carbon at dC 213.5, one carboxylic carbon
at dC 180.7, three oxygenated carbons at dC 73.2, 66.6 and 64.8 as
well as two olefinic carbons at dC 128.4 and 139.8 (Table 1). These
NMR data were quite similar to those of 3b,23-dihydroxy-1-oxo-
olean-12-en-28-oic acid, except for an oxygenated methylene
instead of a methyl group at C-27 (Okada et al., 2003). The HMBC
correlations between H-27 (dH 4.04 and 3.93) and C-13 (dC 139.8), C-
14 (dC 48.5), C-8 (dC 40.8) and C-15 (dC24.1) confirmed that the
methylene was oxygenated and located at C-27 (Fig. 2). The relative
configuration of compound 2 was determined by the NOESY cor-
relations of H-3/H-5, H-5/H-9, H-9/H-27, H-18/H-30 and H-25/H-26
(Fig. 3). Therefore, compound 2 was elucidated as 3b,23,27-
trihydroxy-1-oxo-olean-12-ene-28-oic acid.
Compound 15 was obtained as a colorless powder. Its molecular
formula was established as C38H62O12 by HR-ESI-MS (m/z 728.4607
[MþNH4]þ, calcd for C38H66NO12, 728.4580), suggesting 8 degrees
of unsaturation. The UV spectrum of 15 exhibited absorption
maxima at 203 nm. The IR absorption bands at 3439, 2929 and
1640 cmꢀ1 indicated the presence of hydroxyl, aliphatic CꢀH and
carbonyl groups, respectively. In the 1H NMR spectrum, there were
two anomeric protons at dH 5.53 (1H, br s) and 4.98 (1H, d,
J ¼ 7.8 Hz), two oxymethine protons at dH 4.43 (1H, ddd, J ¼ 5.0,
10.5, 10.5 Hz) and 3.61 (1H, d, J ¼ 2.1 Hz), and six methyl protons at
dH 1.35, 1.27, 1.26, 1.04, 0.96 and 0.71 (Table 2). The 13C NMR of 38
carbons resonances confirmed the aforementioned moieties
(Table 2). Moreover, the 13C NMR showed a carbonyl carbon at
2. Results and discussion
2.1. Structural elucidation of the previously undescribed
compounds
Compound 1 was obtained as a colorless powder. The molecular
formula was determined to be C30H46O6 on the basis of HR-ESI-MS
(m/z 501.3218 [M-H]-, calcd for C30H45O6, 501.3216). The UV spec-
trum of 1 exhibited absorption maxima at 203 nm. The IR ab-
sorption bands at 3441, 2946 and 1696 cmꢀ1 indicated the presence
of hydroxyl groups, aliphatic CꢀH and carbonyl groups. In the 1H
NMR spectrum, six tertiary methyl groups at dH 1.26, 1.17, 1.10, 1.10,
1.01 and 0.93, an oxygen-bearing methylene group at dH 4.17 (1H, d,
J ¼ 10.7 Hz) and 3.73 (1H, d, J ¼ 10.6 Hz), and an oxygenated
methine group at dH 4.59 (1H, dd, J ¼ 4.6, 11.8 Hz) were observed
(Table 1). The 13C and DEPT NMR spectra displayed 30 carbon
resonances attributed to six methyl, ten methylene (one oxygen-
ated), five methine (one oxygenated) and nine quaternary carbons
(three carbonyls). Among them, two keto carbonyl groups were
apparent from the signals at dC 213.0 and 211.6 as well as one
carboxyl group from the signal at dC 181.1 (Table 1). Therefore,1 was
assigned as an olean-type triterpenoid. The spectroscopic data
d
176.9. These data suggested that 15 was a nor-triterpenoid
glycoside with a dammarane-type aglycone and two sugar moi-
eties. Comparing the NMR data of 15 with those of cyclocarioside I,
15 has a similar aglycone as those of cyclocarioside I, except for the
C-17 side chain (Shu et al., 1995a). Five-membered lactone ring
located at C-17 was proposed for the structure of compound 15
based on the HMBC correlations from H-23 (dH 2.64 and 2.50) to dC
176.9 (C-24), 89.7 (C-20), 32.5 (C-22) and H-22 (dH 1.97 and 1.82) to
dC 176.9 (C-24), 89.7 (C-20), 49.9 (C-17), 29.7 (C-23) and 24.8 (C-21)
(Fig. 2). Acid hydrolysis of 15 afforded L-arabinofuranose and D-
quinovopyranose on the basis of HPLC by comparing with an
authentic sugar sample and the attachments were confirmed by the
HMBC correlations from dH 5.53 (H-10, Ara) to dC 79.7 (C-3) and dH
above were closely related to those of a known compound, 3
b
,23-
4.98 (H-100, Qui) to dC 76.3 (C-12), respectively. The
of quinovopyranose was based on the coupling constant of the
anomeric proton (J ¼ 7.8 Hz), and configuration of arabinofur-
anose was determined by the NOESY correlations of H-1'/H-30 and
H-3'/H-5'. Additionally, the H-3 and H-12 were present in
orientation and -orientation according to the coupling constant of
the protons (J ¼ 2.1 Hz and J ¼ 10.5 Hz), respectively. The config-
uration at C-20 was determined to be S by a comparison of the 13
b configuration
dihydroxy-1-oxo-olean-12-en-28-oic acid (Okada et al., 2003).
Instead of a double bond between C-12 and C-13 in the above
known compound, compound 1 was established in the presence of
a carbonyl group at C-12 according to the observed HMBC corre-
lations from dC 211.6 (C-12) to dH 3.14 (H-11a), 2.25 (H-11b) and 3.22
(H-13) (Fig. 2). The configuration of the hydroxyl group at C-3 was
a
b
-
a
deduced to be
b
oriented on the basis of the coupling constant of
C
J ¼ 11.8 Hz and an observed NOE effect between H-3 and H-5. The
NOESY correlations of H-5/H-9, H-9/H-27, H-13/H-26 and H-18/H-
26 suggested that the A/B, B/C and C/D rings were all trans-fused
while the D/E ring was cis-fused (Fig. 3). Thus, the structure of 1
NMR chemical shift data of analogous dammaranes (Zhao et al.,
2015). Thus, compound 15 was deduced to be 20S,24-lactone-
dammar-(3a,12b)- 12-O-b-D-quinovopyranosyl-3-O-a-L-arabino-
furanoside, and named as cyclocarioside L.
was established as 3
b
, 23-dihydroxy-1,12-dioxo-olean-28-oic acid.
Compound 16 was obtained as a colorless powder. Its HR-ESI-
MS spectrum displayed an [MþNH4]þ molecular ion at m/z
775.4277 (calcd 770.4716), corresponding to a molecular formula of
Compound 2 was isolated as a colorless powder. The molecular
formula C30H46O6 was deduced from HR-ESI-MS (m/z 520.3625
[MþNH4]þ, calcd for C30H50NO6, 520.3633). The UV spectrum of 2
exhibited absorption maxima at 204 nm. The IR absorption bands at
C40H68NO13. The UV spectrum of 16 exhibited absorption maxima
at 224 and 203 nm. The IR absorption bands at 3442, 2929 and

78
Z.-F. Wu et al. / Phytochemistry 142 (2017) 76e84
Fig. 1. Chemical structures of compounds 1e22.
1643 cmꢀ1 assigned as hydroxyl, aliphatic CꢀH and carbonyl
groups, respectively. The structure of 16 was deduced as a 27-nor-
triterpenoid glycoside with a dammarane triterpenoid aglycone
and two sugars according to the NMR data (Table 2), which were in
good agreement with those of 15, except for the presence of the
acetyl group in 16. In the HMBC spectrum, the correlations from H-
5' (dH 4.80 and 4.60) to dC 171.1 indicated that the acetyl group was
linked at C-50 of the arabinofuranose. The linkage position of ara-
binofuranose at C-3 and the quinovopyranose at C-12 was
confirmed by the HMBC correlations from dH 5.50 (H-10) to dC 80.0
(C-3) and dH 4.97 (H-100) to dC 76.3 (C-12). Therefore, compound 16
was deduced as 20S,24- lactone-dammar-(3
novopyranosyl-3-O-
-(40-O-acetyl)-L-arabinofuranoside,
named as cyclocarioside M.
a
,12
b
)-12-O-
b
-D-qui-
a
and
Compound 17 was obtained as a colorless powder, having a
molecular formula of C44H74O13 as deduced from HR-ESI-MS (m/z
828.5473 [MþNH4]þ, calcd for C44H78NO13, 828.5468). The UV
spectrum of 17 exhibited absorption maxima at 225 and 203 nm.
The IR absorption bands at 3441 and 1642 cmꢀ1 assigned as hy-
droxyl and carbonyl groups, respectively. The NMR data of 17

Z.-F. Wu et al. / Phytochemistry 142 (2017) 76e84
79
Table 1
NMR data for compound 1 and 2 (d in ppm, all measured in C5D5N, J in Hz).
Position
1
2
a
dCb, type
dHa (J in Hz)
HMBC (H/C)
dCc, type
dH (J in Hz)
HMBC (H/C)
1
2
213.0, C
45.5, CH2
213.5, C
45.6, CH2
3.46, dd (11.8, 11.8)
2.80, dd (4.7, 11.4)
4.59, dd (4.6, 11.8)
1, 3, 4, 10
4, 23, 24
3.43, dd (11.9, 11.9)
2.81, dd (4.8, 11.8)
4.55, dd (4.9, 11.8)
1, 3, 4, 10
24
3
4
5
6
7
8
73.3, CH
44.4, C
73.2, CH
44.1, C
47.8, CH
18.5, CH2
33.5, CH2
40.8, C
47.7, CHd
18.4, CH2
31.8, CH2
41.9, C
1.91, d (12.0)
3, 4, 6, 7, 10, 23, 25
5, 7, 10
26
2.06, md
4, 6, 7, 9, 10, 24, 25
4, 6, 10, 26
1.87, d (13.0), 1.63, m
1.81, md, 1.59, md
2.07, md, 1.80, md
1.42, md, 1.20, md
9
10
11
42.4, CH
52.7, C
41.3, CH2
2.72, dd (3.8, 12.8)
10, 26
9, 12
40.9, CH
52.9, C
26.5, CH2
3.18, dd (5.9, 10.9)
2.86, dt (4.3, 18.7) 2.03, md
5.86, t (3.6)
1, 8, 10, 11, 25, 26
9, 12, 13
3.14, dd (3.8, 16.4)
2.25, dd (13.6, 5.7)
12
13
14
15
211.6, C
53.0, CH
43.1, C
128.4, CH
139.8, C
48.5, C
9, 11, 13
3.22, d (3.1)
12, 14, 18, 19, 27
14, 17
28.7, CH2
2.07, dt (5.0, 12.3),
1.06, br d (13.3)
1.96, m, 1.11, m
24.1, CH2
2.16, d (9.3)
1.61, md
8, 27
16
23.9, CH2
14, 15, 17, 18, 28
24.4, CH2
2.16, d (9.3)
27, 28
1.99, md
17
18
19
47.7, Cd
33.2, CH
37.2, CH2
47.1, C
42.5, CH
45.8, CH2
3.36, br d (13.3)
2.31, d (12.7)
1.42, md
13, 14, 17
17, 21, 29, 30
3.39, dd (4.1, 14.6)
1.80, md, 1.34, md
12, 13, 16, 17
18, 20, 30
20
21
22
31.4, C
35.2, CH2
34.1, CH2
31.3, C
34.5, CH2
33.9, CH2
1.42, md, 1.20, md
2.15, td (3.5,13.5)
1.75, d (13.4)
4.17, d (10.7)
3.72, d (10.6)
1.17, s
1.26, s
1.10, s
1.01, s
29, 30
17, 21, 28
1.42, md, 1.17, md
2.07, md, 1.37, md
20
20
23
65.9, CH2
3, 4, 5, 24
66.6, CH2
4.12, d (10.7), 3.71, d (10.5)
3, 4, 5, 24
24
25
26
27
13.9, CH3
15.4, CH3
16.9, CH3
21.1, CH3
3, 4, 5, 23
1, 5, 9, 10
9, 27
14.1, CH3
16.4, CH3
19.8, CH3
64.8, CH2
1.20, s
1.38, s
1.13, s
4.04, d (12.0)
3.93, d (11.9)
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
13, 14, 15
28
29
30
181.1, C
23.9, CH3
34.0, CH3
180.7, C
24.2, CH3
33.6, CH3
1.10, s
0.93, s
19, 20, 30
19, 20, 21, 30
1.01, s
0.87, s
19, 20, 30
19, 20, 21, 30
a
1H NMR spectra were measured at 600 MHz.
1H NMR spectra were measured at 500 MHz.
13C NMR spectra were measured at 125 MHz.
Overlapped signals.
b
c
d
(Table 2) revealed that it was a dammarane triterpenoid glycoside
with two sugars and were quite similar to cyclocarioside B, except
for the signals of the glycosyl unit (Jiang et al., 2006). The acetyl
group were observed in 17 and its linkage at C-40 of quinovopyr-
anose was determined by the correlations from dH 5.25 (H-40) to dC
170.8 (C¼O), 70.6 (C-50) and 18.5 (C-60) in the HMBC spectrum. The
long-range correlations from dH 4.71 (H-10) to dC 82.0 (C-3) and dH
5.00 (H-100) to dC 77.9 (C-12) implied the linkage of two quinovoses
at C-3 and C-12. Furthermore, the large coupling constant of the
2a b,23-trihydroxy-12-ene-28-ursolic acid (9) (Meng et al., 2009),
,3
2a,3a,23-trihydroxyurs-12-en-28-oic acid (10) (Xiao et al., 2013),
2a,3 ,23- trihydroxyoleana-11,13(18)-dien-28-oic acid (11)
b
(Mahato and Nandy, 1990), 2a,3a,23-trihydroxyurs- 12,20(30)-
dien-28-oic acid (12) (Xiao et al., 2013), actinidic acid (13) (Xiao
et al., 2013), 2a,3b,23-trihydroxyurs-11- oxo-12-ene-28-oic acid
(14) (Zhao et al., 2007), cylocarioside H (18) (Li et al., 2012),
cyclocarioside J (19) (Liu et al., 2014), cyclocarioside I (20) (Shu
et al., 1995a), pterocaryoside A (21) (Kennelly et al., 1995) and
pterocaryoside B (22) (Kennelly et al., 1995) by comparing their
spectroscopic data with those reported in the literature.
anomeric protons confirmed the
b relative configurations. The
configuration at C-20 and C-24 was determined to be S and R based
on comparison to the 13C NMR chemical shift data of analogous
expoxydammaranes (Jiang et al., 2006). The
a-orientation of H-3
2.2. The inhibitory effect on the secretion of apoB48 in Caco-2 cells
and H-12 was clarified by the NOESY correlations between dH 3.66
(H-3) and dH 1.03 (H-28) as well as dH 4.38 (H-12) and dH 0.61 (H-
30). Thereby, compound 17 was determined as (20S,24R)-epox-
Overconsumption of exogenous cholesterol and triglyceride was
proved to be associated with dyslipidemia. Apolipoprotein B48
(apoB48) is the constitutive protein involved in dietary lipid ab-
sorption, and a high level of apoB48 in plasma elevates cardiovas-
cular risk factors (Otokozawa et al., 2009). Caco-2, derived from a
human colorectal carcinoma, is an intestinal cell line as a common
research model for investigating intestinal-derived lipoprotein
synthesis and secretion (Levy et al., 1995). In the current study, the
isolated triterpenoids were tested for inhibitory effect on apoB48
oversecretion in Caco-2 cell model.
ydammarane (3
b
,12
b)-25-hydroxyl-12-O-b-D- quinovopyranosyl-
3-O-(40-O-acetyl)-
b-D-quinovopyranoside, and named as cyclo-
carioside N.
The 17 known triterpenoids were identified as cyclocaric acid B
(3) (Cui and Li, 2012), maslinic acid (4) (Zhang et al., 2006), arju-
nolic acid (5) (Cui and Li, 2012), hederagenin (6) (Kizu and
Tomimori, 1982), 2
a-hydroxyursolic acid (7) (Sashida et al., 1992),
3
b,23-dihydroxy-12-ene-28-ursolic acid (8) (Xie et al., 2007),

80
Z.-F. Wu et al. / Phytochemistry 142 (2017) 76e84
Fig. 2. Selected key COSY (bold lines) and HMBC (/) correlations of 1, 2, 15, 16 and 17.
Fig. 3. Selected key NOESY (4) correlations of 1, 2 and the aglycons of 15, 16 and 17.
Our results showed that the apoB48 levels in the medium were
replacement positions, while 8 and 9 with ursane-type showed
better inhibitory activity on apoB48 secretion than 5 and 6 with
oleanane-type. The above results suggested that the hydroxyl
group at C-23 might be essential for their activities. Under same
conditions, ursane-type may be more active skeleton than
significantly increased (p < 0.05) when cells were incubated with
oleic acid (Fig. 4). Compared with the control group, most of the
triterpenoids declined the apoB48 oversecretion induced by oleic
acid. Especially, the treatments of seven pentacyclic triterpenoids 1,
3, 5, 6, 8, 9 and 10, together with four triterpenoid saponins 17, 19,
21 and 22 significantly decreased the apoB48 oversecretion
(p < 0.05, Fig. 4). As a positive control, statin showed inhibitory
effect on the apoB48 secretion, in accordance with the previous
report (Pal et al., 2005).
Analysis of the structures and their activities of compounds
1e14 led to the following findings: (a) the compounds with
inhibitory effect on the apoB48 secretion all had a hydroxyl group
at C-23; (b) 4 and 7 with methyl groups at C-23 showed no
oleanane-type. Furthermore, double bonds and
a, b-unsaturated
ketone may be responsible for the activity decrease.
Among compounds 15e22, compounds 17 and 19 with pyra-
noses showed stronger inhibitory effects on the apoB48 over-
secretion than 18 and 20 with furanoses, indicating that the
glycosyl moiety may have different impact on their inhibitory
activity.
3. Conclusion
inhibitory activity; (c) 11e13 with two double bonds and 14 with
-unsaturated ketone had no effect on apoB48 secretion; (d)
compounds 5, 6, and had the same substituents and
a,
b
5 previously undescribed compounds and 17 known com-
pounds were isolated and identified from the chloroform extract of
8
9

Z.-F. Wu et al. / Phytochemistry 142 (2017) 76e84
81
Table 2
NMR data for compound 15e17 (
d
in ppm, all measured in C5D5N, J in Hz).
Position
15
16
17
a
a
a
dCb, type
dH
dCb, type
dH
dCb, type
dH
1
2
3
4
5
6
7
8
36.0, CH2
21.7, CH2
79.7, CH
38.4, C
51.3, CH
18.7, CH2
36.7, CH2
41.8, C
2.99, m, 1.81, mc
1.81, mc, 1.70, m
3.61, d (2.1)
36.0, CH2
21.1, CH2
80.0, CH
38.3, C
51.3, CH
18.6, CH2
36.7, CH2
41.7, C
2.99, m, 1.81, mc
1.81, mc, 1.66, mc
3.56, br s
36.0, CH2
22.5, CH2
82.0, CH
38.4, C
51.4, CH
18.7, CH2
36.8, CH2
41.9, C
3.14, br d (13.5), 2.10, m
1.95, mc, 1.92, mc
3.66, mc
1.56, mc
1.54, mc
1.65, mc
1.56, mc, 1.48, mc
1.52, mc, 1.16, m
1.51, mc, 1.50, mc
1.53, mc, 1.14, m
1.58, mc, 1.50, mc
1.50, mc, 1.20, m
9
10
11
54.3, CH
40.4, C
34.0, CH2
1.83, mc
54.3, CH
40.3, C
33.9, CH2
1.84, mc
54.3, CH
40.3, C
34.9, CH2
1.92, mc
2.64, m, 1.56, mc
2.64, mc, 1.55, mc
2.86, dd (3.7, 12.2)
1.50, mc
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
76.3, CH
42.1, CH
50.7, C
31.6, CH2
25.8, CH2
49.9, CH
17.4, CH3
17.0, CH3
89.7, C
24.8, CH3
32.5, CH2
29.7, CH2
176.9, C
23.3, CH3
30.3, CH3
16.8, CH3
4.43, ddd (5.0, 10.5, 10.5)
1.74, mc
76.3, CH
42.0, CH
50.6, C
31.6, CH2
25.8, CH2
49.9, CH
17.3, CH3
17.0, CH3
89.6, C
24.7, CH3
32.5, CH2
29.7, CH2
176.9, C
23.3, CH3
30.2, CH3
16.8, CH3
4.43, ddd, (5.2, 10.7, 10.7)
1.74, mc
77.9, CH
41.7, CH
50.5, C
31.9, CH2
27.1, CH2
49.6, CH
17.4, CH3
17.09, CH3
86.8, C
24.9, CH3
34.5, CH3
26.7, CH2
84.6, CH
71.6, C
26.5, CH3
28.0, CH3
23.6, CH3
30.2, CH3
17.11, CH3
102.0, CH
75.9, CH
76.1, CH
77.7, CH
70.6, CH
18.5, CH3
170.8, C
21.4, CH3
102.3, CH
75.9, CH
78.8, CH
77.2, CH
73.2, CH
18.9, CH3
4.38, ddd (4.7, 10.7, 10.7)
1.88, mc
1.40, m, 1.01, m
1.74, mc, 1.43, m
1.91, m
1.04, s
1.35, s
1.39, m, 1.00, mc
1.74, mc, 1.44, m
1.90, mc
1.03, s
1.33, s
1.43, mc, 0.99, m
1.84, mc, 1.75, mc
1.84, mc
1.10, s
1.43, s
1.26, s
1.25, s
1.17, s
1.97, m, 1.82, mc
2.64, mc, 2.50, m
1.95, m 1.82, mc
2.64, mc, 2.49, m
1.75, m, 1.55, mc
2.03, m, 1.98, mc
3.94, mc
0.96, s
1.27, s
0.71, s
0.95, s
1.25, s
0.70, s
1.44, s
1.38, s
1.03, s
1.27, s
0.61, s
106.8, CH
84.2, CH
79.8, CH
86.5, CH
63.4, CH2
5.53, br s
4.84, m
4.80, dd (6.3, 7.8)
4.72, m
107.0, CH
82.0, CH
80.2, CH
84.5, CH
65.5, CH2
5.50, d (1.4)
4.84, dd, (1.6, 4.0)
4.60, m
4.73, ddd (3.1, 7.0, 7.0)
4.80, dd (3.1, 11.7), 4.60, m
4.71, d (7.8)
3.99, t (8.4)
4.16, t (9.0)
5.25, t (9.5)
3.66, mc
1.34, d (6.2)
20
30
40
50
4.35, dd (3.0, 12.0), 4.25, dd (4.6, 11.8)
60
CH3C¼O
CH3C¼O
100
171.1, C
21.1, CH3
101.7, CH
75.9, CH
78.7, CH
77.3, CH
73.2, CH
19.0, CH3
1.97, s
2.04, s
101.7, CH
75.9, CH
78.7, CH
77.3, CH
73.2, CH
19.0, CH3
4.98, d (7.8)
3.95, dd (8.0, 8.5)
4.14, t (8.8)
3.68, t (9.0)
3.76, m
4.97, d (7.7)
3.94, dd (7.2, 8.8)
4.14, t (8.9)
3.68, t (9.0)
3.77, m
5.00, d (7.8)
200
3.93, mc
300
4.14, t (8.8)
3.66, mc
3.80, m
400
500
600
1.61, d (6.0)
1.60, d (6.0)
1.61, d (6.0)
a
1H NMR spectra were measured at 500 MHz.
13C NMR spectra were measured at 125 MHz.
Overlapped signals.
b
c
C. paliurus. 27-nor-triterpenoid glycosides (15 and 16) were first
isolated from the genus. All the characterized compounds were
evaluated for the inhibitory effects on apoB48 secretion of Caco-
2 cells. Among them, seven pentacyclic triterpenoids and four tri-
terpenoid saponins exerted to significantly decrease the apoB48
secretion. In conclusion, triterpenoids are major and functional
constituents in C. paliurus contributing to improve lipid disorders.
This present study is beneficial for understanding the chemical
composition and biological effects of C. paliurus as the popular and
functional tea in China.
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 specified, chemi-
cal shifts (d) were expressed in ppm with reference to the solvent
signals, and coupling constants are expressed in hertz. ESI-MS and
HR-ESI-MS were performed on a HP-1100 LC/EST and Synapt™ Q-
TOF mass spectrometer (Waters, Milford, America), respectively.
Agilent 1260 Infinity equipped with UV and Alltech 3300 ELSD
detector was used to analyse the samples and Alliance semi pre-
4. Experimental section
4.1. General experimental procedures
parative HPLC with COSMOSIL 5C18-AR-Ⅱ (250 mm ꢁ 20 mm, 5
mm)
used for further purification. Column chromatography were per-
formed 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
Melting points were obtained using a X-4 micro melting
point apparatus (Taike, Beijing, CN) and optical rotations were
measured in methanol using an JASCY-1020 polarimeter. A Shi-
madzu UV-2500PC spectrophotometer and Nicolet Impact 410

82
Z.-F. Wu et al. / Phytochemistry 142 (2017) 76e84
Fig. 4. Inhibitory effect of triterpenoids from C. paliurus on apoB48 secretion of Caco-2 cells. ApoB48 concentrations are expressed as a percentage of control, mean S.D. (n ¼ 3).
*p < 0.05, **p < 0.01 represents significant difference as compared with the data of blank group. #p < 0.05, ##p < 0.01 represents significant difference as compared with the data of
control group.
(Pharmacia, Uppsala, Sweden). D-Quinovose, L-Arabinose, pyridine
(Reagent Plus, ꢂ 99%), L-cysteine methyl ester hydrochloride, and
isothiocyanate were purchased from Sigma (St. Louis, MO, Amer-
ica). Methanol and acetonitrile (HPLC-grade) was purchased from
TEDIA. All other chemicals are analytically pure. The fractions were
monitored by TLC and 1% vanillin in H2SO4 was used as the spraying
reagent to visualize spots.
with CH3Cl-MeOH (100:3 / 1:1) to yield 5 subfractions (Fr.2b-1 -
Fr.2b-5). Fr.2b-2 was subjected to Sephadex LH-20 column and
eluted with CH3Cl-MeOH (1:1), followed by ODS column eluted
with MeOH-H2O (50:50 / 100:0) to obtain 5 (1000 mg), 9 (4 mg),
11 (15 mg) and 13 (30 mg). Fr.2b-3 subjected to column chroma-
tography on ODS with MeOH-H2O (50:50 / 100:0) and purified
by semi-preparative HPLC (MeOH-H2O, 73:27) to give 1 (5 mg)
and 2 (2 mg). 14 (3 mg) was obtained from Fr.2c using Sephadex
LH-20 column (CH3Cl-MeOH, 1:1), followed by ODS column
(MeOH-H2O, 50:50 / 100:0) and semi-preparative HPLC (MeOH-
H2O, 65:35).
Fr.3 (100 g) was chromatographed over a silica gel column and
eluted with CH3Cl-MeOH (100:5, 7:1, 5:1, 1:1, 0:100) to yield 5
subfractions (Fr.3a - Fr.3e). Fr.3b was fractioned by ODS column
(MeOH-H2O, 40:60 / 100:0) to obtain 4 subfractions (Fr.3b-1 -
Fr.3b-4). Fr.3b-1 was purified by a silica gel column (CH3Cl-MeOH,
20:1) to obtain 17 (18 mg). 18 (60 mg), 19 (25 mg) and 20 (35 mg)
were isolated from Fr.3b-2 by Sephadex LH-20 column
(CH3Cl-MeOH, 1:1) and a silica gel column (CH3Cl-MeOH, 10:1).
Fr.3b-3 was isolated using a silica gel column (CH3Cl-MeOH,
100:5 / 0:100) and semi-preparative HPLC (MeOH-H2O, 80:20)
to yield 15 (10 mg) and 16 (5 mg). Fr.3c was separated by ODS
column (MeOH-H2O, 60:40), silica gel column (CH3Cl-MeOH, 10:1)
and semi-preparative HPLC (CH3CN-H2O, 50:50) to yield 21
(60 mg) and 22 (40 mg).
4.2. Plant material
The leaves of Cyclocarya paliurus (Batal) Iljinskaja (Juglandaceae)
were collected in October 2010 in the campus of the Nanjing
Forestry University, China (GPS coordinates: 118.822414,
32.085054), and were authenticated by Prof. Minjian Qin (China
Pharmaceutical University). A voucher herbarium specimen (No.
L20100033) has been deposited in Department of Natural Medici-
nal Chemistry of the university.
4.3. Extraction and isolation
Air-dried leaves of C. paliurus (48.5 kg) were extracted three
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
CH3Cl-MeOH (100:0, 100:3, 100:5, 5:1 and 0:100) to yield 5 frac-
tions (Fr.1 - 5).
Fr.2 (210 g) was chromatographed over a silica gel column and
eluted with gradient mixtures of CH3Cl-MeOH (100:3 / 0:100) to
obtained 4 subfractions (Fr.2a - 2d). Fr.2a was subjected to a silica
gel column and eluted with gradient mixtures of CH3Cl-MeOH
(100:03 / 1:1), Sephadex LH-20 column eluted with CH3Cl-
MeOH (1:1) and followed by semi-preparative HPLC (MeOH-H2O,
80:20) together with recrystallization to obtain 3 (15 mg), 4
(10 mg), 6 (700 mg), 7 (35 mg), 8 (900 mg), 10 (600 mg) and 12
(20 mg). Fr.2b was fractionated by silica gel column and eluted
4.3.1. 3b,23-dihydroxy-1,12-dioxo-olean-28-oic acid (1)
Colorless amorphous powder; ½a D27
ꢃ
þ44ꢄ(c 0.095 MeOH); UV
(MeOH) lmax (log ε) 203 (3.40) nm; IR (KBr) nmax 3441, 2946, 1696,
1050 cmꢀ1
;
1H and 13C NMR data, see Table 1; HR-ESI-MS m/z
501.3218 [M-H]- (calcd for C30H45O6, 501.3216).
4.3.2. 3b,23,27-trihydroxy-1-oxo-olean-12-ene-28-oic acid (2)
Colorless amorphous powder; ½a D27
ꢃ
þ73.6ꢄ (c 0.063, MeOH); UV
(MeOH) lmax (log ε) 204 (3.70) nm; IR (KBr) nmax 3441, 2945, 1696,
1633 cmꢀ1 1H and 13C NMR data, see Table 1; HR-ESI-MS m/z
;
520.3625 [MþNH4]þ (calcd for C30H50NO6, 520.3633).

Z.-F. Wu et al. / Phytochemistry 142 (2017) 76e84
83
4.3.3. Cyclocarioside L (15)
University) for authenticating the plant material. This work was
partially sponsored by grants from the National Natural Science
Foundation of China (No. 81503316, 31270673), the Natural Science
Foundation of Jiangsu Province (No. BK20161460, BK20160926),
College Students Innovation Project for the R&D of Novel Drugs (No.
J1030830), the Priority Academic Program Development of Jiangsu
Higher Educational Institutions.
Colorless amorphous powder; ½a D27
ꢃ
ꢀ17.6ꢄ (c 0.081, MeOH); UV
(MeOH) lmax (log ε) 203 (3.77) nm; IR (KBr) nmax 3439, 2929, 1640,
1070 cmꢀ1 1H and 13C NMR data, see Table 2; HR-ESI-MS m/z
;
728.4607 [MþNH4]þ (calcd for C38H66NO12, 728.4580).
4.3.4. Cyclocarioside M (16)
Colorless amorphous powder; ½a D27
ꢃ
ꢀ12.3ꢄ (c 0.104, MeOH); UV
(MeOH) lmax (log ε) 224 (3.10), 203 (3.35) nm; IR (KBr) nmax 3442,
2929, 1643, 1068 cmꢀ1; 1H and 13C NMR data, see Table 2; HR-ESI-
MS m/z 775.4277 [MþNH4]þ (calcd for C40H68NO13, 770.4716).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2017.06.015.
4.3.5. Cyclocarioside N (17)
Colorless amorphous powder; ½a D27
ꢃ
ꢀ8.1ꢄ (c 0.086, MeOH); UV
References
(MeOH) lmax (log ε) 225 (3.17), 203 (3.35) nm; IR (KBr) nmax 3441,
1642, 1071 cmꢀ1; 1H and 13C NMR data, see Table 2; HR-ESI-MS m/z
828.5473 [MþNH4]þ (calcd for C44H78NO13, 828.5468).
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4.4. Acid hydrolysis of compounds 15e17
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Compounds 15e17 (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)
containing 2 mg of L-cysteine methyl ester hydrochloride and
heated at 60 ꢄC for 1 h. Then o-torylisothiocyanate (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,
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Grace Alltima C18 column (20 ꢁ 250 mm, 4.6
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 retention times of the sugar derivatives for 15e17 are
21.21 and 32.18 min, respectively, consistent with those of de-
rivatives of standard L-arabinose and D-quinovose (Huang et al.,
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polycarbonate microporous membranes (0.4 mm pore size, inserts
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The cultural condition was previously reported with a little modi-
fication (Pal et al., 2005). The medium in both apical and basolateral
wells of the blank group (Blank) contained DMEM with 0.1% bovine
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The medium were tested for apoB48 quantification by Elisa kit
(Sakai et al., 2003). Measurements were performed in triplicate and
are representative of three independent experiments. The data
were evaluated using the SPSS software package (Version 19.0, SPSS
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as significantly different at p < 0.05.
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The authors declare no competing financial interest.
Acknowledgments
The authors thank Prof. Minjian Qin (China Pharmaceutical

84
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