New dammarane triterpenoid saponins from the leaves of Cyclocarya paliurus

Article abstract of DOI:10.1080/10286020.2018.1457653

Three new dammarane triterpenoid saponins, cyclocariosides O-Q (1–3), were isolated from the ethanolic extracts of the leaves of Cyclocarya paliurus. The structures of these compounds were elucidated by spectroscopic methods.

Full text of DOI:10.1080/10286020.2018.1457653

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
New dammarane triterpenoid saponins from the
leaves of Cyclocarya paliurus
Yi-Rui Wang, Bao-Song Cui, Shao-Wei Han & Shuai Li
To cite this article: Yi-Rui Wang, Bao-Song Cui, Shao-Wei Han & Shuai Li (2018): New
dammarane triterpenoid saponins from the leaves of Cyclocarya paliurus, Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2018.1457653
To link to this article: https://doi.org/10.1080/10286020.2018.1457653
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Journal of asian natural Products research, 2018
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2018.1457653
ꢀ
New dammarane triterpenoid saponins from the leaves of
Cyclocarya paliurus
Yi-Rui Wang, Bao-Song Cui, Shao-Wei Han and Shuai Li
sꢁꢇꢁꢈ Kꢈy lꢇbꢄꢆꢇꢁꢄꢆy ꢄꢉ Bꢅꢄꢇꢊꢁꢅvꢈ sꢋbꢂꢁꢇꢌꢊꢈ ꢇꢌꢃ fꢋꢌꢊꢁꢅꢄꢌ ꢄꢉ nꢇꢁꢋꢆꢇꢍ Mꢈꢃꢅꢊꢅꢌꢈꢂ, iꢌꢂꢁꢅꢁꢋꢁꢈ ꢄꢉ Mꢇꢁꢈꢆꢅꢇ Mꢈꢃꢅꢊꢇ,
cꢀꢅꢌꢈꢂꢈ aꢊꢇꢃꢈmy ꢄꢉ Mꢈꢃꢅꢊꢇꢍ sꢊꢅꢈꢌꢊꢈꢂ ꢇꢌꢃ Pꢈkꢅꢌg uꢌꢅꢄꢌ Mꢈꢃꢅꢊꢇꢍ cꢄꢍꢍꢈgꢈ, Bꢈꢅjꢅꢌg 100050, cꢀꢅꢌꢇ
ABSTRACT
ARTICLE HISTORY
Three new dammarane triterpenoid saponins, cyclocariosides O-Q (1–
rꢈꢊꢈꢅvꢈꢃ 12 dꢈꢊꢈmbꢈꢆ 2017
aꢊꢊꢈpꢁꢈꢃ 23 Mꢇꢆꢊꢀ 2018
3
), were isolated from the ethanolic extracts of the leaves of Cyclocarya
paliurus. The structures of these compounds were elucidated by
spectroscopic methods.
KEYWORDS
Cyclocarya paliurus;
Jꢋgꢍꢇꢌꢃꢇꢊꢈꢇꢈ; ꢃꢇmmꢇꢆꢇꢌꢈ
ꢁ
ꢆꢅꢁꢈꢆpꢈꢌꢄꢅꢃ ꢂꢇpꢄꢌꢅꢌꢂ;
ꢊyꢊꢍꢄꢊꢇꢆꢅꢄꢂꢅꢃꢈ o;
ꢊyꢊꢍꢄꢊꢇꢆꢅꢄꢂꢅꢃꢈ Q
1
. Introduction
Cyclocarya paliurus (Batal.) Iljinsk is a Chinese endemic plant, which belongs to the genus
Cyclocarya of Juglandaceae. It is widely distributed in the south of Yangtze region [1]. e
leaves of C. paliurus are used as sweet tea for the treatment of hypertension and diabetes
[
[
[
2]. In recent years, the constitutents of triterpenoids [3–6], flavonoids [7], polysaccharides
8], and the pharmacological activities such as hypoglycemic and hypolipidemic activity
9–11], antioxidant activity [7], immunomodulatory effect [12] had been reported. In our
previous study, 3,4-secodammarane, epoxydammarane triterpenoid saponins, oleanane-
and ursane-type triterpenoids, terpenoids, flavonoid glycosides had been isolated from the
CONTACT sꢀꢋꢇꢅ lꢅ
ꢍꢅꢂꢀꢋꢇꢅ@ꢅmm.ꢇꢊ.ꢊꢌ
tꢀꢈ ꢂꢋppꢍꢈmꢈꢌꢁꢇꢆy mꢇꢁꢈꢆꢅꢇꢍ ꢉꢄꢆ ꢁꢀꢅꢂ ꢇꢆꢁꢅꢊꢍꢈ ꢅꢂ ꢇvꢇꢅꢍꢇbꢍꢈ ꢄꢌꢍꢅꢌꢈ ꢇꢁ ꢀꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2018.1457653
©
2018 iꢌꢉꢄꢆmꢇ uK lꢅmꢅꢁꢈꢃ, ꢁꢆꢇꢃꢅꢌg ꢇꢂ tꢇyꢍꢄꢆ & fꢆꢇꢌꢊꢅꢂ Gꢆꢄꢋp

2
Y.ꢀR. WANG ET AL.
ethyl acetate soluble fraction of ethanolic extract of the leaves [13–15]. By further chemical
investigation of this fraction, three new dammarane triterpenoid saponins (1–3, Figure 1)
were isolated and elucidated. is report describes the isolation and characterization of
three new triterpenoid saponins from the leaves of C. paliurus. Chemical and spectroscopic
methods were used to elucidate the structures of the compounds.
2
. Results and discussion
Cyclocarioside O (1) was obtained as white amorphous powder. e molecular formula
+
of 1 was determined to be C H O by HR-ESI-MS (m/z 745.4516 [M + Na] ), indicated
4
0
66 11
1
eight degrees of unsaturation. e H NMR spectrum of 1 exhibited seven methyl singlets
at δ 0.89 (3H, s, H-29), 0.92 (6H, s, H-30), 0.99 (3H, s, H-18), 1.10 (3H, s, H-19), 1.15 (3H,
H
s, H-21), and 1.82 (3H, s, H-27); two pairs of olefinic protons: one pair of coupled olefinic
protons at δ 5.82 (1H, dt, J = 16.0, 8.0 Hz, H-23) and 6.18 (1H, d, J = 16.0 Hz, H-24), the
H
coupling constant (J = 16.0 Hz) showed that the configuration of the double bond was E, the
other pair of terminal methylene olefinic protons at δ 4.85 (1H, br s, H-26) and 4.86 (1H,
H
br s, H-26); two oxygenated methine protons at δ 3.29 (1H, t, J = 2.0 Hz, H-3), 4.09 (1H,
H
dt, J = 11.0, 5.0 Hz, H-12), the coupling constant showed that the relative configuration of
H-3 was β-orientation, and H-12 was α-orientation; in addition, partially overlapped multi
-
ples of methylene and methine protons were between δ 1.03 and δ 2.52. According to these
data, 1 was a triterpenoid. Two anomeric proton signals at δ 4.28 (1H, d, J = 7.0 Hz, H-1″)
H
and 4.95 (1H, br s, H-1′), as well as other oxygenated methine protons and two oxygenated
methylene of glycosyls suggested that 1 possesed two glycosyls and was a triterpenoid
1
3
saponin. e C NMR spectrum of 1 revealed 40 carbon resonances, 30 of the 40 carbons
were assigned to the triterpenoid skeleton and 10 to 2 pentoses. ey consisted of 7 methyls,
1
1 methylenes (one olefinic), 16 methines (two olefinic and ten oxygenated) and 6 quater-
nary carbons (one olefinic, one oxygenated). According to the molecular formula of 1, the
unsaturation degrees of 1 were 8, including two double bonds, two rings of glycosyls, four
13
rings of triterpenoid aglycone. e C NMR spectrum of 1 showed two signals of anomeric
carbons at δ 106.6 (C-1′) and 101.4 (C-1″) which supported the presence of two glycosyl
C
moieties in 1. Additionally, one set of carbon signals for ꢀ-arabinofuranose and another
1
3
set of carbon signals for ꢀ-arabinopyranose were observed in the C NMR spectrum of
1
[15–17]. Acid hydrolysis of 1 furnished arabinose, which was identified by comparison
with the authentic sample on TLC and HPLC chromatography. e HMBC correlations
from the anomeric proton at δ 4.95 (H-1′) to δ 79.5 (C-3) suggested the arabinofuranose
linked to C-3, the correlations from the anomeric proton at δ 4.28 (H-1″) to δ 76.3 (C-12)
H
C
H
C
Figure 1. tꢀꢈ ꢂꢁꢆꢋꢊꢁꢋꢆꢈꢂ ꢄꢉ ꢊꢄmpꢄꢋꢌꢃꢂ 1–3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
indicated the arabinopyranose linked to C-12 (Figure 2). e absolute configuration of C-20
was determined to be S on the basis of the same carbon chemical shif (C-17, 20, 21, 22)
comparison to cyclocarioside I [15]. e other HMBC correlations from δ 1.15 (H-21)
H
to δ 50.6 (C-17) and δ 45.1 (C-22), δ 2.30 (H-22) to δ 74.9 (C-20), δ 1.82 (H-27) to
C
C
H
C
H
δ 135.8 (C-24), δ 143.0 (C-25) and δ 114.7 (C-26) (Figure 2) confirmed the structure
C
C
C
of 1. Ultimately, the structure of 1 was deduced as (20S,23E)-dammarane-(3α,12β)-20-
trihydroxy-23, 25-dien-12-O-α-ꢀ-arabinopyranosyl-3-O-α-ꢀ-arabinofuranoside, and 1 was
named cyclocarioside O (Figure 3).
Cyclocarioside P (2) was obtained as white amorphous powder, and it had the molecular
+
formula C H O as determined by HR-ESI-MS analysis (m/z 759.4670 [M + Na] ). e
4
1
68 11
NMR spectrum of 2 showed that it was a triterpenoid saponin with a dammarane aglycone
skeleton and two glycosyl moieties (Table 1). Detailed analysis of the NMR spectra revealed
that the signals of 2 and 1 were nearly identical, except that the NMR signals due to the
arabinopyranosyl of 1 were replaced by signals contributable to the quinovopyranosyl of 2
[
15,17]. e HMBC correlation between the anomeric proton of quinovopyranosyl at δ 4.36
H
(
H-1″) and δ 76.4 (C-12) indicated the quinovopyranose linkage site at C-12. e arabinose
C
and quinovose were identified by acid hydrolysis analysis of 2. erefore, the structure of 2
was deduced as (20S,23E)-dammarane-(3α, 12β)-20-trihydroxy-23,25-dien-12-O-β-ꢁ-qui-
novopyranosyl-3-O-α-ꢀ-arabinofuranoside, and was named cyclocarioside P (Figure 4).
Cyclocarioside Q (3) was obtained as white amorphous powder, and it had the molecular
+
formula C H O , as determined by HR-ESI-MS analysis (m/z 791.4927 [M + Na] ). e
4
2
72 12
NMR spectra of 3 showed that it was a triterpenoid glycoside with a dammarane triterpenoid
aglycone and two glycosyl moieties (Table 1). Detailed analysis of the NMR data revealed
that the aglycone signals of 3 and 2 were nearly identical, except for the signals of the side-
chain of the dammarane triterpenoid skeleton. In the NMR spectra of 3, there were one
Figure 2. tꢀꢈ kꢈy hMBc ꢊꢄꢆꢆꢈꢍꢇꢁꢅꢄꢌꢂ ꢄꢉ ꢊꢄmpꢄꢋꢌꢃꢂ 1 ꢇꢌꢃ 3.
Figure 3. tꢀꢈ kꢈy noe ꢊꢄꢆꢆꢈꢍꢇꢁꢅꢄꢌꢂ ꢄꢉ ꢊꢄmpꢄꢋꢌꢃ 1.

4
Y.ꢀR. WANG ET AL.
1
13
Table 1. h ꢇꢌꢃ c nMr ꢂpꢈꢊꢁꢆꢄꢂꢊꢄpꢅꢊ ꢃꢇꢁꢇ (500 Mhz, ꢇꢊꢈꢁꢄꢌꢈ-d ) ꢉꢄꢆ ꢊyꢊꢍꢄꢊꢇꢆꢅꢄꢂꢅꢃꢈꢂ o (1), P (2) ꢇꢌꢃ
6
Q (3).
Cyclocarioside O (1)
Cyclocarioside P (2)
Cyclocarioside Q (3)
δ ,
C
No
δC, type
δH (J in Hz)
δC δH
(J in Hz)
type
δH (J in Hz)
1
35.8, ch2
1.30, ꢄvꢈꢆꢍꢇp
35.7 1.30, ꢄvꢈꢆꢍꢇp
35.6,
ch2
1.36–1.39, m
2
.52, ꢃꢁ (14.0,2.5)
1.54, ꢄvꢈꢆꢍꢇp
.69, ꢄvꢈꢆꢍꢇp
2.52, ꢃꢁ (14.0,3.5)
21.1 1.56, ꢄvꢈꢆꢍꢇp
2.50, ꢃꢁ(14.0,
3.5)
2
21.2, ch2
21.5,
ch2
1.61, ꢄvꢈꢆꢍꢇp
1
1.70, ꢄvꢈꢆꢍꢇp
79.5 3.28, bꢆ ꢂ
1.72, ꢄvꢈꢆꢍꢇp
3.32, ꢁ (2.5)
3
4
5
6
79.5, ch
38.2, c
3.29, ꢁ (2.0)
81.2,
ch
38.2
38.3,
c
51.0,
ch
18.7,
ch2
51.3, ch
18.6, ch2
1.24, ꢄvꢈꢆꢍꢇp
1.42, ꢄvꢈꢆꢍꢇp
51.3 1.23, ꢄvꢈꢆꢍꢇp
18.6 1.42, ꢄvꢈꢆꢍꢇp
1.31, ꢄvꢈꢆꢍꢇp
1.42–1.45, m
1
.83, bꢆ ꢂ
1.83, bꢆ ꢂ
36.8 1.19, ꢄvꢈꢆꢍꢇp
1.83 ꢄvꢈꢆꢍꢇp
1.20, ꢄvꢈꢆꢍꢇp
7
36.8, ch2
1.19, ꢄvꢈꢆꢍꢇp
36.8,
ch2
1
.55, ꢄvꢈꢆꢍꢇp
1.56, ꢄvꢈꢆꢍꢇp
41.9
1.56, ꢄvꢈꢆꢍꢇp
8
9
41.9, c
41.9,
c
54.3,
ch
40.1,
c
34.7,
ch2
54.5, ch
40.2, c
1.65, bꢆ ꢃ (11.0)
54.5 1.65, bꢆ ꢃ (10.5)
40.2
1.68, bꢆ ꢃ
(10.5)
1
0
1
1
34.3, ch2
1.33, ꢄvꢈꢆꢍꢇp
34.6 1.33, ꢄvꢈꢆꢍꢇp
2.41, ꢃꢁ(13.0, 4.5)
76.4 4.08, ꢃꢁ (10.5,5.0)
41.0 1.86–1.87, m
51.0
1.33, ꢄvꢈꢆꢍꢇp
2.40, ꢃꢁ
2
.44, ꢃꢁ (12.5,4.5)
(
13.0,4.0)
12
13
14
15
76.3, ch
40.9, ch
51.0, c
4.09,ꢃꢁ(11.0, 5.0)
1.86–1.88, m
76.6,
ch
40.9,
ch
51.2,
c
31.6,
ch2
4.09, ꢃꢁ (10.5,
5.0)
1.84–1.86, m
31.5,ch2
1.03–1.05, m
31.5 1.02, ꢃꢃ(11.0,8.5)
1.05–1.06, m
1
.41, ꢄvꢈꢆꢍꢇp
1.41, ꢄvꢈꢆꢍꢇp
25.7 1.66, ꢄvꢈꢆꢍꢇp
1.41, ꢄvꢈꢆꢍꢇp
1.69, ꢄvꢈꢆꢍꢇp
16
25.7,ch2
1.65, ꢄvꢈꢆꢍꢇp
25.6,
ch2
1
.72–1.78, m
1.75, ꢄvꢈꢆꢍꢇp
50.6 1.77, ꢄvꢈꢆꢍꢇp
1.75, ꢄvꢈꢆꢍꢇp
1.78, ꢄvꢈꢆꢍꢇp
17
18
19
20
21
22
23
24
25
50.6, ch
17.4, ch3
16.9, ch3
74.9, c
1.76–1.78, m
50.2,
ch
0.99, ꢂ
17.3 0.99, ꢂ
16.8 1.10, ꢂ
17.3,
ch3
16.8,
ch3
74.6,
c
27.0,
ch3
45.0,
ch2
1.00, ꢂ
1.12, ꢂ
1.10, ꢂ
74.9
27.1, ch3
45.1, ch2
1.15, ꢂ
27.2 1.15, ꢂ
1.16, ꢂ
2.30,
45.1 2.30,ꢃꢃꢃ(14.0,8.0,2.0)
128.2 5.82, ꢃꢁ(15.5,7.5)
135.8 6.18, ꢃ (15.5)
143.1
2.21–2.24, m
ꢃꢃꢃ(14.0,8.0,2.0)
128.2, ch 5.82, ꢃꢁ (16.0,8.0)
135.8, ch 6.18, ꢃ (16.0)
143.0, c
127.3, 5.70,
ꢃꢁ(16.0,7.5)
138.9, 5.45, ꢃ(16.0)
ch
ch
75.1,
c
(
Continued)

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. (Continued).
Cyclocarioside O (1)
Cyclocarioside P (2)
Cyclocarioside Q (3)
δ ,
C
No
δC, type
δH (J in Hz)
δC δH
(J in Hz)
type
δH (J in Hz)
26
114.7,ch2 4.86, bꢆ ꢂ
114.7 4.86, bꢆ ꢂ
26.1,
ch3
1.21, ꢂ
4
.85, bꢆ ꢂ
4.85, bꢆ ꢂ
18.9 1.83, ꢂ
27
28
29
30
18.9, ch3
29.9, ch3
23.0, ch3
17.0,ch3
1.82, ꢂ
0.92, ꢂ
0.89, ꢂ
0.92, ꢂ
26.6,
ch3
29.9,
ch3
23.1,
ch3
17.0,
1.21, ꢂ
0.96, ꢂ
0.89, ꢂ
0.93, ꢂ
3.09, ꢂ
29.8 0.92, ꢂ
23.0 0.88, ꢂ
17.0 0.92, ꢂ
ch3
och3 50.3,
ch3
-
ꢇꢆꢇ(ꢉ)
ꢇꢆꢇ(ꢉ)
ꢇꢆꢇ(p)
1′
2′
3′
4′
5′
106.0, ch 4.95, bꢆ ꢂ
106.0 4.94, bꢆ ꢂ
100.2, 4.36, ꢃ (8.0)
ch
82.9, ch
79.3, ch
86.4, ch
62.9, ch2
3.99, ꢄvꢈꢆꢍꢇp
82.9 3.99, ꢄvꢈꢆꢍꢇp
79.3 3.89–3.91, m
86.4 3.99, ꢄvꢈꢆꢍꢇp
62.9 3.65, bꢆ ꢃ (12.5)
3.72, bꢆ ꢃ (12.0)
72.1,
ch
73.6,
ch
67.9,
ch
3.64, ꢄvꢈꢆꢍꢇp
3.63–3.64, m
3.81, ꢄvꢈꢆꢍꢇp
3.90–3.92, m
3.99, ꢄvꢈꢆꢍꢇp
3.65, ꢃꢃ(11.5,4.0)
64.8,
ch2
3.48, bꢆ ꢃ
(10.0)
3.80, ꢄvꢈꢆꢍꢇp
3
.71,ꢃꢃ (11.5,3.0)
ꢇꢆꢇ(p)
qꢋꢅ
1″
qꢋꢅ
1″
2″
3″
4″
5″
101.4, ch 4.28, ꢃ (7.0)
100.8 4.36, ꢃ (7.5)
75.4 3.11, ꢃꢃ (9.0,7.5)
77.9 3.29, ꢃꢃ (9.0,9.0)
76.8 2.99, ꢃꢃ (9.0,9.0)
72.5 3.25, ꢃq (9.0,6.0)
100.9, 4.38, ꢃ (7.5)
ch
72.7, ch
74.3, ch
69.5, ch
3.46, ꢃꢃ (9.0,7.0)
2″
3″
4″
5″
75.3,
ch
3.12, ꢃꢃ
(9.0,7.5)
3.31, ꢃꢃ
(9.0,9.0)
3.00, ꢃꢃ
(9.0,9.0)
3.26, ꢃq
(9.0,6.0)
3.51, ꢄvꢈꢆꢍꢇp
3.75–3.76, m
77.9,
ch
76.8,
ch
67.1, ch2 3.49, ꢄvꢈꢆꢍꢇp
.86, ꢃꢃ(12.5,1.5)
72.4,
ch
3
6
″
18.2 1.23, ꢃ (6.0)
18.2, 1.23, ꢃ (6.0)
ch3
Figure 4. tꢀꢈ kꢈy noe ꢊꢄꢆꢆꢈꢍꢇꢁꢅꢄꢌꢂ ꢄꢉ ꢊꢄmpꢄꢋꢌꢃ 2.
methoxyl at δ 3.09 (3H, s, OCH -25), two methyl singlets at δ 1.16 (3H, s, H-21), 1.21
H
3
H
(
(
3H, s, H-27) and two olefinic protons at δ 5.70 (1H, dt, J = 16.0, 7.5 Hz, H-23) and 5.45
H
1H, d, J = 16.0 Hz, H-24) in the side-chain of the dammarane triterpenoid skeleton, instead
of two methyl singlets at δ 1.15 (3H, s, H-21), 1.83 (3H, s, H-27) and four olefinic protons
H

6
Y.ꢀR. WANG ET AL.
at δ 5.82 (1H, dt, J = 15.5, 7.5 Hz, H-23), 6.18 (1H, d, J = 15.5 Hz, H-24), and 4.85, 4.86
H
(
2H, br s, H-26) in 2. is suggested that the side-chain of the aglycone of 3 was an addition
2
5
product of 2, and 3 was obtained by adding a molecule CH OH on the double bond (Δ )
3
of 2. e HMBC correlation from the methoxyl at δ 3.09 to C-25 at δ 75.1 suggested the
methoxy group was linked to C-25. e presence of two anomeric protons of glycosyls at δ
H
C
H
4
.38 (1H, d, J = 7.5 Hz, H-1″) and 4.36 (1H, d, J = 8.0 Hz, H-1′), and two signals of anomeric
carbons at δ 100.9 (C-1″) and 100.2 (C-1′) supported the presence of two glycosyl moieties
C
in the molecule. Additionally, one set of carbon signals for ꢀ-arabinopyranose and one set of
1
3
carbon signals for ꢁ-quinovopyranose were observed in the C NMR spectrum of 3 [15].
Acid hydrolysis of 3 furnished arabinose and quinovose. e HMBC correlation from δ
H
4
2
.38 (H-1″) and δ 76.6 (C-12) indicated the quinovopyranose was linked at C-12 (Figure
C
), and the HMBC correlation from δ 4.36 (H-1′) and δ 81.2 (C-3) was not observed, but
H
C
the arabinopyranose was linked to C-3 on the basis of the chemical shif at δ 81.2 (C-3).
C
Ultimately, the structure of 3 was deduced as (20S, 23E)-dammarane-25-methoxy-3α,12β-
2
0-trihydroxy-23-en-12-O-β-ꢁ-quinovopyranosyl-3-O-α-ꢀ-arabinopyranoside, and 3 was
named cyclocarioside Q.
Compounds 1–3 exhibited no cytotoxicity against five human cancer cell lines, TNF-α
inhibitory activity observed in LPS-induced murine peritoneal macrophages (RAW264.7)
and hepatoprotective effect via inhibiting the proliferation of HepG2.
3
. Experimental
3
.1. General experimental procedures
Melting points were recorded on an XT5B micromelting point apparatus (Tianjin Optical
Instrument Factory, Tianjin, China). Optical rotations were measured on PE Model 343
polarimeter (PerkinElmer Enterprise Management Co., Ltd, Shanghai, China). UV spec-
trum was measured with a JASCO V-650 spectrophotometer (JASCO Corporation, Tokyo,
Japan). IR spectrum was recorded on a Nicolet 5700 spectrometer using an FT-IR micro-
scope transmission method (ermo Electron Corporation, Madison, WI, U.S.A). NMR
spectra were obtained on an Varian INOVA-500 MHz spectrometer (Varian, Inc., Palo Alto,
CA, U.S.A) in acetone-d . Mass spectrum was measured on a Mass Agilent 1100 Series
6
LC-MSD-Trap-SL spectrometer (ESI-MS) and 6520 ESI-TOF spectrometer (HR-ESI-MS)
(Agilent Technologies Ltd., Santa Clara, CA, U.S.A). High-performance liquid chromatog-
raphy (HPLC) was carried out on a Shimadzu instrument: pump LC-6AD, UV detector:
SPD-20A (Shimadzu Corporation, Tokyo, Japan), and a semipreparative reversed-phase col-
umn: Grace, Allsphere ODS-25 μm, 250×10 mm (Grace Corporation,Columbia, Maryland,
U.S.A.). Column chromatography (CC) was performed on silica gel (Qingdao Marine
Chemical Factory, Qingdao, China), RP-18 (45–75 μm, YMC CO., Ltd, Kyoto, Japan), and
Sephadex LH-20 (GE Healthcare Corporation, Uppsala, Sweden). in layer chromatogra-
phy (TLC) was performed on silica gel plates (GF254, Yantai Chemical Industry Research
Insititute, Yantai, China) and RP-18F254 plates (Merck Corporation, New Jersey, U.S.A).

e analytical and chromatographic grade solvents were purchased from Beijing Chemical
Corporation (Beijing, China). Arabinose (A0515) was purchased from J&K CHEMICA Co.,
Ltd. (Beijing, China), and quinovose (D9761-100 mg) was purchased from Sigma-Aldrich

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
company (Beijing, China). Fractions were monitored by TLC, and spots were visualized on
precoated silica gel plates by spraying 10 % H SO in EtOH followed by heating.
2
4
3
.2. Plant material

e leaves of Cyclocarya paliurus were collected at Qimen, Anhui Province, China, and iden-
tified by Mr Ma Lin (Institute of Materia Medica, Chinese Academy of Medical Sciences).
A voucher specimen (No. ZH02001) had been deposited at the sample library, Institute of
Materia Medica, Chinese Academy of Medical Sciences.
3
.3. Extraction and isolation
e air-dried and powdered leaves of C. paliurus (1 kg) were extracted with 95% ethanol
6 L × 1 h × 3) by ultrasonication (230 W, 35 kHz) at room temperature. e extracts were

(
combined and concentrated under reduced pressure (at <60 °C) to give a dark brown
residue (51 g). e residue was suspended in water (3 L) and then partitioned with ethyl
acetate (3 L × 3). e ethyl acetate fraction (28 g) was subjected to column chromatography
(
(
CC) [silica gel, (500 g), column, 6 × 70 cm] eluted with a gradient of increasing methanol
0–20%) of the mixture of chloroform and methanol to yield 14 fractions (A–N) on the
basis of TLC analysis. Fraction M (3.9 g) was purified by CC [Sephadex LH-20 (100 g),
column, 2.5 × 75 cm] eluted with petroleum ether-CHCl -MeOH (5: 5: 1, 700 ml) to yield
3
six fractions (M1–M6) on the basis of TLC analysis. e subfraction M2 (1.27 g) was
purified by CC [silica gel, (50 g), column, 2.5 × 20 cm] eluted with a gradient of increasing
methanol (10–15%) of the mixture of chloroform and methanol to yield four fractions
(
M2A–M2D). e subfraction M2C (200 mg) was purified by preparative HPLC [column,
RP-18 (250 × 10 mm, 5 μm); MeOH-H O (80: 20), 2 ml/min, 210 nm] to obtain 3 (5 mg,
2
1
3.682 min). e subfraction M3 (415 mg) was purified by CC [Sephadex LH-20 (50 g),
column, 1.5 × 75 cm] eluted with petroleum ether-CHCl -MeOH (5: 5: 1, 300 ml) and then
3
applied to preparative HPLC [column, RP-18 (250 × 10 mm, 5 μm); MeOH-H O (85: 15),
2
2
ml/min, 210 nm] to obtain 1 (5 mg, 12.198 min) and 2 (7 mg, 15.465 min).
3
.3.1. Cyclocarioside O (1)
20
White amorphous powder (MeOH); m.p. 165.0–168.5 °C;[ꢀ] −13.39 (0.03, MeOH); UV
D
(
MeOH) λmax(log ε): 199(2.14), 226 (1.57), 277 (0.78) nm; IR: ν 3360.2, 2921.5, 1727.0,
max
−1 1
13
1
659.1, 1633.6, 1468.5, 1066.2 cm ; H and C NMR spectral data, see Table 1; ESI-MS: m/z
+
+
7
45 [M + Na] ; HR-ESI-MS: m/z 745.4516 [M + Na] (calcd for C H O Na, 745.4497).
4
0
66 11
3
.3.2. Cyclocarioside P (2)
20
White amorphous powder (MeOH); m.p. 164.2–166.5 °C;[ꢀ] −30.16 (0.09, MeOH); UV
D
(
MeOH) λmax(log ε): 199 (2.17), 227 (1.93), 277 (sh) nm; IR: ν 3359.8, 2920.9, 1727.5,
max
−1 1
13
1
659.1, 1633.4, 1468.5, 1065.9 cm ; H and C NMR spectral data, see Table 1; ESI-MS: m/z
+
+
7
59 [M + Na] ; HR-ESI-MS: m/z 759.4670 [M + Na] (calcd for C H O Na, 759.4654).
4
1
68 11
3
.3.3. Cyclocarioside Q (3)
20
White amorphous powder (MeOH); m.p. 167.0–169.3 °C;[ꢀ] −17.62 (0.06, MeOH); UV
D
(MeOH) λmax(log ε): 199 (2.12), 227 (sh) nm; IR: νmax 3362.8, 2922.4, 1724.6, 1657.8, 1634.4,

8
Y.ꢀR. WANG ET AL.
−
1 1
13
+
1
468.5, 1066.0 cm ; H and C NMR spectral data, see Table 1; ESI-MS: m/z 791 [M + Na] ;
+
HR-ESI-MS: m/z 791.4927 [M + Na] (calcd for C H O Na, 791.4916).
4
2
72 12
3
.4. Acid hydrolysis of compounds 1–3 and determination of the absolute
configuration of the sugars
Solutions of 1, 2, and 3 were mixed with 0.5 M HCl and refluxed for 1 h. Each reaction
mixture was diluted with water and extracted exhaustively with CHCl . e aqueous layer
3
was neutralized with sodium bicarbonate and allowed to dry at room temperature. TLC
analysis of the residues using CHCl -MeOH-H O (6: 4: 1) showed the presence of arabinose
3
2
as the only sugar for 1, and both arabinose and quinovose for 2 and 3 by comparison of
their R values with arabinose and quinovose. e sugar components in the aqueous layer
f
obtained af er acid hydrolysis were evaporated and dissolved in anhydrous pyridine (1 ml).
0
6
.5 mg ꢀ-cysteine methyl ester hydrochloride was added, and the mixture was warmed at
0 °C for 1 h, o-tolyl isothiocyanate was added to the mixture, which was warmed at 60 °C
for 1 h. e reaction mixture was directly analyzed by HPLC on a Cosmosil-Packed 5C -
1
8
AR-II column using an elution of 25% CH CN in 50 mM H PO at a flow rate of 0.8 ml/
3
3
4
min. Retention time for the authentic derivatives: ꢁ-arabinose (15.305 min), ꢀ-arabinose
14.601 min), ꢁ-quinovose (33.474 min) was used for comparison with retention times from
reaction mixtures for the saponin. e absolute configuration of arabinose of compounds
(
1
2
–3 was identified as ꢀ by the retention time of the reaction products of 1 (14.595 min),
(14.603 min), 3 (14.599 min), quinovose of compounds 2–3 was identified as ꢁ by the
retention times of the reaction mixtures of 2 (33.476 min), 3 (33.471 min).
3
.5. Pharmacological activity experiment
3
.5.1. Cytotoxic assay
Compounds 1–3 were tested for cytotoxicity against five human cancer cell lines (HCT116,
HepG2, BGC-823, A549, and MCF-7) using the MTT assay.
3
.5.2. TNF-α inhibitory activity assay
Compounds 1–3 were evaluated for their TNF-α inhibitory factor expression via the model
of murine peritoneal macrophages (RAW264.7) induced by LPS in vitro.
3
.5.3. Hepatoprotective effects assay
Compounds 1–3 were evaluated for their hepatoprotective activities against paracetamol-in-
duced HepG2 cell damage, and bicyclol, a hepatoprotective activity drug, was used as the
positive control.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is study was supported financially by CAMS Initiative for Innovative Medicine [grant number
CAMS-I2M-1-010].

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
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