Two new triterpenoid saponins from the leaves of Ilex kudingcha

Article abstract of DOI:10.1080/10286020.2011.653347

Two new triterpene saponins, ilekudinchosides F (1) and G (2), along with three known saponins were isolated from the leaves of Ilex kudingcha C. J. Tseng. The new compounds were characterized as 3β,19α-dihydroxy- 12α-ethoxy-urs-13(18)-ene-28,20β-lactone-

Full text of DOI:10.1080/10286020.2011.653347

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Two new triterpenoid saponins from the
leaves of Ilex kudingcha
a b
b
b
b
Wen-Jian Zuo
, Hao-Fu Dai , Yan-Bo Zeng , Hui Wang , Hui-
a b
a
Qin Chen
a
& Jin-Hui Wang
School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang, 110016, China
b
Hainan Key Laboratory for Research and Development of Natural
Products from Li Folk Medicine, Institute of Tropical Bioscience
and Biotechnology, Chinese Academy of Tropical Agricultural
Sciences, Haikou, 571101, China
Published online: 01 Mar 2012.
To cite this article: Wen-Jian Zuo , Hao-Fu Dai , Yan-Bo Zeng , Hui Wang , Hui-Qin Chen & Jin-Hui
Wang (2012): Two new triterpenoid saponins from the leaves of Ilex kudingcha , Journal of Asian
Natural Products Research, 14:4, 308-313
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Journal of Asian Natural Products Research
Vol. 14, No. 4, April 2012, 308–313
Two new triterpenoid saponins from the leaves of Ilex kudingcha
ab
Wen-Jian Zuo , Hao-Fu Dai , Yan-Bo Zeng , Hui Wang ,
b
b
b
ab
Hui-Qin Chen and Jin-Hui Wang *
a
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
b
Shenyang 110016, China; Hainan Key Laboratory for Research and Development of Natural
Products from Li Folk Medicine, Institute of Tropical Bioscience and Biotechnology,
Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
(Received 9 August 2011; final version received 22 December 2011)
Two new triterpene saponins, ilekudinchosides F (1) and G (2), along with three
known saponins were isolated from the leaves of Ilex kudingcha C. J. Tseng. The new
compounds were characterized as 3b,19a-dihydroxy-12a-ethoxy-urs-13(18)-ene-
2
8,20b-lactone-3-O-[b-D-glucopyranosyl(1 ! 3)]-[a-L-rhamnopyranosyl(1 ! 2)]-a-
L-arabinopyranoside (1) and 3b,19a-dihydroxy-12a-methoxy-urs-13(18)-ene-28,20b-
lactone-3-O-[a-L-rhamnopyranosyl(1 ! 2)]-a-L-arabinopyranoside (2). The new
structures were elucidated by spectroscopic methods including 1D and 2D NMR,
HR-TOF-MS, and CD spectrometry, and the known compounds were identified by the
comparison of their NMR and HR-TOF-MS data with those reported in the literature.
Keywords: Ilex kudingcha; triterpene saponins; ilekudinchosides F and G
1
.
Introduction
2. Results and discussion
Ilex kudingcha C. J. Tseng is an
economically very important plant and is
also widely distributed in the southern
region of China. The leaf of this plant is a
traditional tea product, known as ‘Ku-
Ding-Cha’, which is also used in the form
Compound 1 was obtained as white
powder. Its quasi-molecular ion peak at
þ
m/z 977.5078 [M þ Na] by HR-TOF-MS
1
3
and C NMR spectral data suggested the
molecular formula was C H O . The
4
9 78 18
UV spectrum showed an absorption
maximum at 233 nm. The IR spectrum
of
a
commercial herbal product,
2
1
especially for treating hypertension, obes-
ity, and sore throats [1]. Previous studies
indicated that saponins are the major
constituents of I. kudingcha [2–7]. Some
authors had reported inhibitory activity of
acyl-CoA cholesteryl acyl transferase of
triterpenoid saponins isolated from this
plant [8,9]. This paper reported the
isolation and the structure elucidation of
two new triterpene saponins from the
leaves of I. kudingcha, along with three
known saponins (Figure 1).
exhibited absorptions at 3369 cm
2
1
(hydroxyls), 1730 cm
(g-lactone), and
(C ¼ C). The spectroscopic
features suggested that 1 was a triterpene
saponin. The C NMR spectrum showed
49 carbons, of which 32 were assigned to
triterpene moiety and 17 to saccharide
2
1
1655 cm
1
3
1
portion. The H NMR spectrum (Table 1)
showed seven tertiary methyls at d 0.84,
0.88, 1.12, 1.21, 1.47, 1.49, and 1.58,
a triplet methyl signal at d 1.09
(t, J ¼ 6.6 Hz), and two multiple signals
*Corresponding author. Email: wjh1972@yahoo.com.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.653347
http://www.tandfonline.com

Journal of Asian Natural Products Research
309
3
0
1
2α
29
2
0
1
2β
19
O
O
21
22
O
HO
O
O
HO
O
1
2
18
28
OH
O
1
1
HO
HO
OH
25
26 13
17
OH
1
4
9
14
1
6
2
3
O
O
10
8
15
HO
O
OH
H
6
27
7
O
O
5
O
O
H
2
4
23
H C
O
H C
3
O
3
HO
HO
HO
HO
OH
OH
1
2
Figure 1. Structures of compounds 1 and 2.
at d 3.61 (1H, m) and 3.69 (1H, m)
ascribable to ethoxy protons; a typical
signal at d 3.27 (dd, J ¼ 12.0 and 4.5 Hz)
ascribable to an axial H-3; and a low-field
H-12 signal at d 5.42 (br s). Further
low-field ethoxy carbon signal (d 62.7),
and two olefinic carbons (d 139.5 and
142.5). The signals at d 74.0 (C-19), 85.7
(C-20), and 175.1 (C-28) and the carbon
resonances of the E ring indicated the
presence of a 19-O-substituent and a
1
3
features in C NMR spectrum (Table 2)
3
were eight sp carbon signals (d 15.9, 16.9,
1
hexacyclic lactone ring [2,3]. The H and
1
C NMR spectra of 1 were very similar
3
1
6.9, 18.2, 19.5, 22.3, 25.6, and 28.0), a
1
Table 1. H NMR spectral data of 1 and 2 (600 MHz in pyridine-d5).
Aglycon
1
1
2
3-O-sugar
1
2
1.65, 1.05
1.63, 0.99 td-like
(6.0, 0.8)
1.80, 1.73
Ara
0
0
2
3
1.84, 1.78
3.27 dd (12.0, 4.5) 3.25 dd (12.0, 4.2)
1
2
4.86 d (5.7)
4.67 dd
4.91 d (6.0)
4.58 t (6.0)
(
6.0, 7.2)
0
5
6
7
9
0.81 d (12.6)
1.48, 1.24
1.53, 1.38
1.97
0.79 d (12.0)
1.47, 1.21
1.46, 1.38
1.91
3
4
5
4.32
4.53
4.22
4.31
4.34
4.28
0
0
3.76 d (10.2) 3.85 d (9.6)
1
1
2.47 dd (12.0, 3.0) 2.48 dd (10.8, 3.0)
1.61
Rha
1.54
5.42 br s
0
0
0
0
0
0
12
15
16
5.32 br s
1.94, 1.18
2.08 dd (12.6, 3.6)
1.36
2.54 td (13.2, 3.0)
1.63
1
2
3
6.16 br s
4.60
4.75 d (1.8)
6.20 br s
4.64
4.76 br s
1.95, 1.20
2.06 br d (9.6)
1
.36
2.55 td (13.2, 3.0)
.62
00
21
4
4.30
4.32
1
0
0
0
0
2
2
2
2
2
2
2
3
1
1
2
3
4
5
6
7
9
0
1.82, 1.74
1.21 s
1.12 s
1.78, 1.70
1.18 s
1.07 s
5
6
4.62
1.65 d (6.0)
4.66
1.63 d (6.0)
Glc
0
0
0
0
0
0
00
00
00
00
00
00
0.84 s
0.88 s
0.84 s
0.88 s
1
2
3
4
5
6
5.11 d (7.8)
3.95
4.19
4.18
3.93
1.47 s
1.58 s
1.44 s
1.58 s
1.49 s
3.69 m, 3.61 m
1.09 t (6.6)
1.50 s
3.35 s
2a
2b
4.52, 4.35
Note: Overlapped proton NMR signals are reported without designated multiplicity.

3
10
W.-J. Zuo et al.
1
3
Table 2.
Aglycon
C NMR spectral data of 1 and 2 (150 MHz in pyridine-d5).
1
2
3-O-sugar
1
2
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
39.3
26.7
88.1
39.6
56.1
18.4
35.5
42.4
45.2
37.0
26.8
73.4
142.5
44.8
29.4
26.6
44.9
139.5
74.0
85.7
28.6
33.1
28.0
16.9
16.9
18.2
22.3
175.1
25.6
19.5
62.7
15.9
39.1
26.5
88.7
39.5
56.1
18.6
35.4
42.2
45.1
36.9
26.5
75.2
142.4
44.8
29.3
25.5
44.8
139.9
74.0
85.6
28.5
33.1
28.0
16.9
16.7
18.1
22.1
175.1
25.5
19.5
55.3
Ara
0
1
104.9
74.8
83.1
68.4
65.1
104.9
75.9
74.0
68.8
64.8
0
2
3
4
5
0
0
0
Rha
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
102.0
72.5
72.5
73.9
70.0
18.6
101.8
72.4
72.6
74.0
69.8
18.6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
1
Glc
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
104.8
75.0
78.3
71.4
78.6
62.5
2
3
4
5
6
7
8
9
0
2a
2b
to those of kudinoside A [3]. The only
difference was the ethoxy group substi-
tuted at C-12 position in 1, which was
revealed from the HMBC correlations
between H-12a (3.61, 1H, m; 3.69, 1H,
m) and C-12 (d 73.4). The a-orientation of
the ethoxy group was deduced from the
three anomeric proton signals at d 4.86
(d, J ¼ 5.7 Hz), 6.16 (br s), and 5.11 (d,
J ¼ 7.8 Hz), and of three corresponding
carbon signals at d 104.9, 102.0, and 104.8
also indicated that 1 contained a trisac-
charide of L-arabinose, L-rhamnose, and
3
D-glucose. The J
coupling constant
(5.7 Hz) indicated the a-anomeric con-
H1–H2
key correlations of H-12 with CH -29 and
3
H-11b in NOESY spectrum. Thus, the
aglycon of 1 was determined to be 3b,19a-
dihydroxy-12a-ethoxy-urs-13(18)-ene-28,
20b-lactone. C NMR chemical shift
of C-3 (d 88.1) suggested that 1 has a
figuration of the arabinose unit. The large
3
JH1 – H2 coupling constant (7.8 Hz)
suggested that anomeric configuration of
the glucose unit was b [6]. The sequence
of the trisaccharide moiety was determined
by the long-range correlations of H-1
(d 4.86) of the inner a-arabinose and C-3
(d 88.1) of the aglycon, H-1 (d 5.11) of the
terminal b-glucose with C-3 (d 83.1) of the
inner a-arabinose, and H-1 (d 6.16) of the
13
glycosyl linkage at C-3. Acid hydrolysis of
afforded sugar components identified as
1
L-arabinose, L-rhamnose, and D-glucose
by GC analysis of their trimethylsilyl-
imidazole derivatives. The presence of

Journal of Asian Natural Products Research
311
terminal a-rhamnose with C-2 (d 74.8) of
the inner a-arabinose in HMBC spectrum.
The same conclusion was supported by
NOESY experiment. Thus, the structure of
2 was determined to be 3b,19a-dihydroxy-
12a-methoxy-urs-13(18)-ene-28,20b-lac-
tone-3-O-[a-L-rhamnopyranosyl(1 ! 2)]-
a-L-arabinopyranoside and named as ile-
kudinchoside G.
1
1
was determined to be 3b,19a-dihydroxy-
2a-ethoxy-urs-13(18)-ene-28,20b-lac-
Additionally, the known compounds
ilekudinoside L [4], ilekudinoside R [4],
and kudinoside A [3] were also isolated
and identified by comparing NMR and
HR-TOF-MS data with those in literature.
tone-3-O-[b-D-glucopyranosyl(1 ! 3)]-
[
a-L-rhamnopyranosyl(1 ! 2)]-a-L-arabi-
nopyranoside, and named as ilekudincho-
side F.
Compound 2 was assigned a molecular
formula of C H O based on an ion
42 66 13
þ
3. Experimental
peak at m/z 801.4424 [M þ Na] in HR-
1
TOF-MS and C NMR spectral data. The
3
3
.1 General experimental procedures
1
H NMR spectrum (Table 1) showed seven
tertiary methyls at d 0.84, 0.88, 1.07, 1.18,
Optical rotations were determined on a
Beckman DU800 spectrometer (Beckman
Coulter Corporation, Danvers, MA, USA).
IR spectra (KBr) were recorded on a Bruker
IFS-55 spectrometer (Bruck Corporation,
Ettlingen, Germany). UV spectra were
measured with a Shimadzu UV-2200
spectrophotometer (Shimadzu Corporation,
Kyoto, Japan). 1D and 2D NMR spectra
were obtained on a Bruker ARX-600
spectrometer with tetramethylsilane as the
internal standard (Bruck Corporation).
HR-TOF-MS were measured on a LCT
Premier XE mass spectrometer (Waters
Corporation, Milford, MA, USA). CD
spectra were recorded with a Jasco CD-
2095 Plus circular dichroism detector
(JASCO Corporation, Tokyo, Japan). For
column chromatography (CC), silica gel
(Qingdao Marine Chemical Industry, Qing-
dao, China, 60–80 mesh and 200–300
mesh), D101 macroporous resin (Cangzhou
Bon Adsorber Technology Co., Ltd., Cangz-
hou, China, 20–40 mesh), and Sephadex
LH-20 (Pharmacia Fine Chemical Co., Ltd.,
Uppsala, Sweden) were used. Thin layer
chromatography was carried out with silica
gel GF254 (Qingdao Marine Chemical
Industry). Semi-preparative HPLC was
carried out using a system composed of
a Hitachi Pump L-7110, with a Hitachi
L-7420 UV spectrophotometric detector and
a TEDAchrom YWG C18 reversed phase
column (10 £ 250 mm, detected at UV 260
and 210 nm). GC analysis was carried out on
1
.44, 1.50, and 1.58, a typical signal at d
.25 (dd, J ¼ 12.0 and 4.2 Hz) ascribable
3
to an axial H-3 in the aglycon moiety, a
signal at d 3.35 (3H, s) ascribable to
methoxy protons, and a low-field H-12
1
3
signal at d 5.32 (br s). The C NMR
3
spectrum (Table 2) showed seven sp
carbon signals (d 16.7, 16.9, 18.1, 19.5,
2
2.1, 25.5, and 28.0), a low-field methoxy
carbon signal (d 55.3), and two olefinic
carbons (d 139.9 and 142.4). The NMR
spectral data of 2 were very similar to
those of ilekudinoside R [4] except for a
methoxy group substituted at C-12 pos-
ition, which was confirmed by the HMBC
correlation from H-12 (d 5.32) to the
methoxy carbon signal (d 55.3). The a-
orientation of methoxy was deduced from
the key NOESY correlations of H-12 with
CH -29 and H-11b. Thus, the aglycon of 2
3
was determined to be 3b,19a-dihydroxy-
1
2a-methoxy-urs-13(18)-ene-28,20b-lac-
1
3
tone. C NMR chemical shift of C-3 (d
8.7) suggested that 2 has a glycosyl
8
linkage at C-3. Acid hydrolysis of 2
yielded L-arabinose and L-rhamnose by
GC analysis. The linkage position and the
sequence of the disaccharide moiety were
determined by HMBC correlations of H-1
(
d 4.91) of the inner a-arabinose and C-3 (d
8.7) of the aglycon, and H-1 (d 6.20) of the
8
terminal a-rhamnose with C-2 (d 75.9) of
the inner a-arabinose. Thus, the structure of

3
12
W.-J. Zuo et al.
927, and 632 cm
2
1
an FL-9790 apparatus using an OV-17
column (30 m £ 0.32 mm), with flame
ionization detector (detector temperature,
;
CD (MeOH)
D1236.0 nm 2 98.0; for H NMR spectral
1
data (C D N, 600 MHz), see Table 1; for
5
5
1
3
2808C; column temperature, 2208C; carrier
gas, N2).
C
150 MHz), see Table 2; HR-TOF-MS:
NMR spectral data (C D N,
5 5
þ
m/z 977.5078 [M þ Na] (calculated for
C H O Na, 977.5086).
49 78 18
3
.2 Plant material
The leaves of I. kudingcha were purchased
from the Weikang Pharmaceutical Com-
pany, Shenyang city, Liaoning Province,
China, in July 2007 and identified by
Professor Jin-Cai Lu of Shenyang Phar-
maceutical University, China. A voucher
specimen (No. 20070715) has been
deposited in the Research Department of
Natural Medicine, Shenyang Pharmaceu-
tical University.
3.3.2 Ilekudinchoside G
20
White powder; ½aꢀ 2 23:1 (c 0.1,
D
MeOH); UV (MeOH) l
(
(log e) 230
3.56) nm; IR (KBr) nmax 3421, 2939, 1734,
max
1
6
650, 1458, 1369, 1250, 1131, 1073, and
21
12 cm ; CD (MeOH) D1234.0 nm 2 85.0;
1
for H NMR spectral data (C D N,
5
5
13
00 MHz), see Table 1; for C NMR
6
spectral data (C D N, 150 MHz), see Table
5
5
þ
2
(calculated for C42 13Na, 801.4401).
; HR-TOF-MS: m/z 801.4424 [M þ Na]
3
.3 Extraction and isolation
H O
66
The air-dried and powdered leaves of I.
kudingcha (6.0 kg) were extracted three
times (each for 2 h) with 95% EtOH under
reflux. After filtration, the extracts were
combined and concentrated to dryness
underreduced pressure, andthen the residue
3
.4 Sugar analysis
Acid hydrolysis and determination of
absolute configuration of sugar were carried
out according to the method described
previously [10]. The absolute configuration
of sugar was confirmed by the same
retention times of their acetylated thiazoli-
dine derivatives by comparing with those of
standard monosaccharides prepared in a
similar manner. The retention times of L-
arabinose, L-rhamnose, and D-glucose were
(1130g) was suspended in H O (2.4 liters)
2
and extracted with petroleum ether, CHCl3,
EtOAc, and n-BuOH, successively. The n-
BuOH layer (640 g) was passed through a
macroporous resin D101 CC and succes-
sively eluted with H O and 30%, 70%, and
2
9
5% EtOH (each 50 liters) to yield four
3
.75, 4.65, and 9.51 min, and the corre-
fractions. Fr. C (180 g) was chromato-
graphed on silica gel CC eluted with
CHCl –MeOH to obtain 196 subfractions
sponding retention times of standard sugars
were 3.81, 4.62, and 9.45 min, respectively.
3
(Fr. C –Fr. C196). Fr. C38 (1.2 g) was
purified onSephadex LH-20 column eluting
1
Acknowledgments
with CH OH, and then separated by semi-
3
This work was financially supported by
National Non-profit Institute Research Grant
of CATAS-ITBB110403.
preparative HPLC using MeOH–H O (v/v,
2
72.8:27.2) at 2.5ml/min to yield 1 (15.6mg,
t 28.40 min) and 2 (21.0mg, t 17.30min).
R
R
References
3
.3.1 Ilekudinchoside F
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20
White powder; ½aꢀ 2 45:8 (c 0.1,
D
MeOH); UV (MeOH) l
(
(log e) 233
3.84) nm; IR (KBr) nmax 3369, 2939,
max
1
730, 1655, 1461, 1368, 1255, 1133, 1075,

Journal of Asian Natural Products Research
313
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