全 文 ： 22 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 2011 年 1 月 第 9 卷 第 1 期

Chinese Journal of Natural Medicines 2011, 9(1): 0022−0025
doi: 10.3724/SP.J.1009.2011.00022
Chinese
Journal of
Natural
Medicines







Triterpenoid Saponins from the Leaves of Ilex kudingcha
CHE Yan-Yun1, LI Ning2, ZHANG Liang2, TU Peng-Fei1, 2*
1Key Laboratory of Modern Chinese Medicines, Ministry of Education, Department of Pharmacognosy, China Pharmaceutical Univer-
sity, Nanjing 210009, China;
2State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center,
Beijing 100191, China
Available online 20 Jan. 2011
[ABSTRACT] AIM: To study the chemical constituents of the leaves of Ilex kudingcha C. J. Tseng. METHODS: Compounds were
isolated by column chromatography with silica gel and preparative HPLC. Their structures were identified on the basis of physico-
chemical properties and spectroscopic analysis. RESULTS: Three triterpenoid saponins were isolated and identified as 3-O-α-L-
rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl pomolic acid 28-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester (1),
3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-α-kudinlactone (2) and 3-O-β-D-glucopyranosyl-β-kudinlactone (3).
CONCLUSION: Compound 1 is a new triterpenoid saponin.
[KEY WORDS] Aquifoliaceae; Ilex kudingcha; Triterpenoid saponin
[CLC Number] R284.1 [Document code] A [Article ID] 1672-3651(2011)01-0022-04

1 Introduction
The leaves of Ilex kudingcha C.J. Tseng (Aquifoliaceae)
has been employed traditionally in China as an herbal tea
known as “Ku-Ding-Cha” exhibiting hypotensive and
anti-obesity activities [1]. It has been reported that the triter-
penoid saponins from Ku-Ding-Cha have inhibitory effects
on acyl-CoA cholesteryl acyl transferase (ACTA) and aggre-
gate LDL-induced lipid deposition in macrophages [2-3]. Also,
some triterpenoid glycosides from Ku-Ding-Cha show in-
hibitory activity against tobacco mosaic virus [4]. As part of
our search for bioactive saponins from Ilex kudingcha, this
paper presents the isolation and structural elucidation of a
new triterpene saponin (1) from this plant.
2 Experimental
2.1 General experimental procedures
IR spectra were determined using a Nicolet Avatar 360
FT-IR spectrometer. UV spectra were recorded by a Shima-
dzu spectrometer. Mass spectra were measured with a MS

[Received on] 19-Oct-2010
[Research Funding] This project was supported by the National
Natural Science Foundation of China (No.30672608).
[*Corresponding author] TU Peng-Fei: Prof., E-mail: pengfeitu@
vip.163.com
These authors have no any conflict of interest to declare.
Agilent 1100 Series LC/MSD Trap mass spectrometer
(ESI-MS) and an AutoSpec Ultimate-TOF MS (HR-ESI-MS),
respectively. NMR spectra were achieved in C5D5N at 300 K
by Bruker ACF-500 NMR spectrometer. Preparative HPLC
was performed on a Waters model 2487 instrument (All-
sphere ODS, 10 id × 250 mm, detected at UV 226 nm and
210 nm). GC analysis was equipped with an Agilent 6890N
gas chromatographer using a HP-5 capillary column (28 m ×
0.32 mm, id.); detection, FID; detector temperature, 260 °C;
column temperature, 180 °C; carrier gas, N2; flow rate, 1
mL·min−1. D-101 porous polymer resin (Hebei Bonherb
Technology Corporation, Hebei Province, China), silica gel
(Qingdao Ocean Chemical Corporation, Qingdao, Peoples’
Republic of China), and ODS (50 µm, Fuji Sylisia Chemical,
Ltd., Aichi, Japan) were used for column chromatography.
2.2 Plant material
Leaves of Ilex kudingcha C.J. Tseng were collected in
June 2003 from Daxin Prefecture, Guangxi Zhuang Autono-
mous Region, China, and were identified by one of the au-
thors (Prof. TU Peng-Fei). A voucher specimen (No.
20030601) is maintained at the Department of Natural Medi-
cine, Peking University.
2.3 Extraction and isolation
Air-dried leaves (5 kg) were extracted with 70% aqueous
ethanol (V/V) three times (15 L, 3 h each) under reflux. The
extract was concentrated to yield a dark green residue which
was suspended in cool water and partitioned between petro-
CHE Yan-Yun, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 22−25
2011 年 1 月 第 9 卷 第 1 期 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 23

leum ester and H2O. Then the water layer was chroma-
tographied on a macroporous resin D101 column eluted with
20%, 70% and 95% EtOH, respectively. The 70% EtOH frac-
tion was subjected to a silica gel column eluted with
CHCl3-MeOH-H2O (9:1:0.1→8:2:0.1→7:3:1→6.5:3.5:1) to
get four fractions (Fractions 1-4). Fraction 2 was further sub-
jected to repeated Rp-C18 column with a gradient of
60%-100% MeOH in H2O and then the eluent of fraction 2.2
(MeOH-H2O, 7:3) were separated by preparative HPLC
(MeOH in H2O, 60:40, UV detection at 226 nm and 210 nm)
to afford 2 (13 mg) and 3 (8 mg) respectively. Fraction 3 was
eluted with MeOH-H2O (55:45→80:20) to give four fractions
and compound 1 (15 mg) was obtained by further HPLC
purification (MeOH in H2O, 50:50, UV detection at 226 nm
and 210 nm) from fraction 4.1.
3 Results and Discussion
Compound 1 was obtained as a white amorphous pow-
der. The high-resolution electrospray ionization mass spec-
trometry (HR-ESI-MS) (positive-ion mode) gave a pseudo-
molecular ion peak at m/z 1097.528 77 [M + K]+ (Calcd.
1097.528 77), consistent with a molecular formula of
C53H86O21. The UV spectra showed an absorption maximum
at 210 nm. Its IR spectrum revealed absorption bands at
3 419 cm−1 (hydroxyl) and 1 729 cm−1 (carbonyl). The 1H
NMR spectrum of 1 (Table 1) showed characteristic proton
signals of seven tertiary methyl groups at δH 1.69 (3H, s),
1.39 (3H, s), 1.12 (3H, s), 1.07 (3H, s), 1.05 (3H, d, J = 6.5
Hz), 1.00 (3H, s) and 0.88 (3H, s) of the triterpenoid moiety,
and four anomeric protons at δH 4.85 (1H, d, J = 5.5 Hz),
6.11 (1H, br.s), 6.14 (1H, d, J = 8 Hz) and 6.67 (1H, br s). In

Table 1 1H and 13C NMR Data (δ) of compound 1 (C5D5N, 500 MHz, 125 MHz)
Position δH J in Hz δC Position δH J in Hz δC
aglycon 1 0.89 , 1.50 39.4 Suger C-3
2 2.19 , 3.12 26.0 Ara 1 4.85 d, 5.5 104.8
3 3.17 88.9 2 4.46 75.1
4 39.0 3 4.26 73.8
5 0.72 d, 11.5 56.0 4 4.24 68.7
6 1.30 , 1.39 18.7 5 4.28 , 3.79 d, 10.5 64.7
7 1.52 , 1.71 33.6 6
8 1.78 40.4 Rha 1 6.67 br s 101.3
9 47.8 2 4.78 72.3
10 36.9 3 4.71 72.3
11 2.03 24.0 4 4.23 73.8
12 5.56 br s 128.2 5 4.58 69.6
13 139.2 6 1.60 d, 6.0 18.5
14 41.8 28-C
15 1.53 , 2.23 29.6 Glc 1 6.13 d, 8.0 94.9
16 1.28 , 1.81 26.6 2 4.49 75.8
17 2.87 br s 48.7 3 4.35 80.0
18 54.6 4 4.24 71.5
19 72.6 5 4.00 78.9
20 1.42 42.2 6 4.34,4.43 62.3
21 2.05 26.5 Rha 1 6.11 br s 101.7
22 1.92 , 2.21 37.5 2 4.56 72.5
23 1.07 s 28.0 3 4.58 72.6
24 1.00 s 16.9 4 4.28 73.9
25 0.88 s 15.6 5 4.52 69.8
26 1.12 s 17.4 6 1.74 d, 6.0 18.6
27 1.69 s 24.3
28 176.8
29 1.39 s 27.0
30 1.05 d, 6.5 16.6
CHE Yan-Yun, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 22−25
24 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 2011 年 1 月 第 9 卷 第 1 期


Fig. 1 Structures of compounds 1-3

Fig. 2 Selected ROESY and HMBC correlations of com-
pound 1

turn, the 13C NMR and DEPT NMR spectra of 1 indicated the
presence of 53 signals including those of four anomeric car-
bons at δC 94.9, 101.3, 101.6, and 104.8 suggesting 1 to be a
triterpenoid saponin with four sugar units. The low-field sig-
nal for olefinic proton δH 5.56 (1H, br s) and two coupled
olefinic carbons at δC 128.2 (CH), 139.2 (C) confirmed the
presence of the 12, 13-ene ursane skeleton. In the HMBC
spectrum, cross-peak observed for H3-29/C-19 (C) showed
the location of hydroxyl groups at C-19. A typical H-3ax
proton δH 3.17 (1H, m) in the 1H NMR spectrum revealed the
presence of a β-O-function at C-3. Thus, the aglycon of 1 was
identified as pomolic acid [5]. On acidic hydrolysis with
2mol·L−1 HCl, 1 afforded sugar moieties identified as
L-arabinose, D-glucose and L-rhamnose in the ratio of 1:1:2
based on the GC-MS analysis of their chiral derivatives [6-7].
Direct evidence for the sugar sequence and their linkage sites
at C-3 was derived from the results of the HMBC experiment
that showed unequivocal correlation peaks between reso-
nances at δ 4.85 and 88.9 (H-1Ara−C-3), δ 6.67 and 75.2
(H-1Rha−C-2Ara). On irradiation of the anomeric proton signal
δ 4.85 of arabinose, ROE was observed for H-3 of aglycone
moiety. The anomeric proton signal δH 6.13 (1H, d, J = 8 Hz)
of the D-glucosyl moiety was correlated to the carbon signal
at δC 94.9 in the HSQC spectrum and to the C-28 carbon
signal at δC 176.8 in the HMBC NMR spectrum. Long-range
coupling was observed between the proton signal at δH 6.11
(Rha-H-1) and δC 75.8 (Glc-C-2) in the HMBC spectrum.
ROE was observed at H-2 of glucose on irradiation of the
anomeric proton signal of δH 6.11 (1H, br s) of rhamnose. The
anomeric configuration of the L-arabinosyl and D-glucosyl
moiety was α and β from the 3JH1-H2 coupling constants (5.5
and 8.0 Hz). And the anomeric pronton of the rhamnose unit
was observed as a broad singlet. Thus, Ilekudinoside W (1)
was concluded to be 3-O-α-L-rhamnopyranosyl-(1 →
2)-α-L-arabinopyranosyl pomolic acid 28-O-α-L- rhamno-
pyranosyl-(1→2)-β-D-glucopyranosyl ester.
Compound 2 White powder. HR-ESI-MS m/z 1
515.842 35 [2M + Na]+, C41H62O12. 1H NMR (C5D5N, 500
MHz) δ: 0.82 (3H, s, H-26), 0.87 (3H, s, H-25), 1.03 (3H, s,
H-24), 1.03 (3H, s, H-27), 1.16 (3H, s, H-23), 1.51 (3H, s,
H-30), 1.68 (3H, s, H-29), 3.24 (1H, dd, J = 4.5, 10.5 Hz,
H-3), 7.50 (1H, d, J = 10.5 Hz, H-11), 5.75 (1H, d, J = 10.5
Hz, H-12), 4.90 (1H, d, J = 5.0 Hz, H-1 of Ara), 6.15 (1H, br
s, H-1 of Rha); 13C NMR (C5D5N, 125 MHz) δ: 38.2 (C-1),
26.3 (C-2), 88.6 (C-3), 39.5 (C-4), 55.2 (C-5), 18.4 (C-6),
32.9 (C-7), 42.2 (C-8), 54.5 (C-9), 36.6 (C-10), 127.2 (C-11),
128.4 (C-12), 140.7 (C-13), 42.2 (C-14), 25.8 (C-15), 26.3
(C-16), 43.8 (C-17), 135.0 (C-18), 74.1 (C-19), 85.9 (C-20),
28.5 (C-21), 32.9 (C-22), 27.7 (C-23), 16.4 (C-24), 18.4
(C-25), 16.5 (C-26), 18.6 (C-27), 175.2 (C-28), 23.7 (C-29),
19.5 (C-30), 104.9 (C-1), 76.0 (C-2), 74.0 (C-3), 68.7 (C-4),
64.8 (C-5), 101.8 (C-1), 72.6 (C-2), 72.4 (C-3), 74.0
(C-4), 69.8 (C-5), 18.6 (C-6). Compound 2 was deter-
mined as 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopy-
ranosyl-α-kudinlactone [8].
Compound 3 White powder. ESI-MS m/z, 647 [M −
H]−. 1H NMR (C5D5N, 500 MHz) δ: 0.84 (3H, s, H-24), 0.89
(3H, s, H-26), 0.99 (3H, s, H-25), 1.32(3H, s, H-23), 1.49
(3H, s, H-30), 1.61 (3H, s, H-27), 1.64 (3H, s, H-29), 3.39
(1H, dd, J = 4.5, 10.5 Hz, H-3), 4.92 (1H, d, J = 8 Hz, H-1);
13C NMR (C5D5N, 125 MHz) δ: 38.9 (C-1), 28.2 (C-2), 88.9
(C-3), 39.6 (C-4), 56.0 (C-5), 18.5 (C-6), 35.5 (C-7), 41.7
(C-8), 44.1 (C-9), 36.9 (C-10), 28.8 (C-11), 66.1 (C-12),
146.4 (C-13), 43.9 (C-14), 28.4 (C-15), 26.7 (C-16), 44.8
(C-17), 137.5 (C-18), 74.3 (C-19), 85.7 (C-20), 26.2 (C-21),
32.8 (C-22), 28.4 (C-23), 16.9 (C-24), 16.6 (C-25), 18.2
(C-26), 23.4 (C-27), 175.4 (C-28), 25.2 (C-29), 19.5 (C-30),
107.0 (C-1), 75.8 (C-2), 78.7 (C-3), 71.8 (C-4), 78.3 (C-5),
CHE Yan-Yun, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 22−25
2011 年 1 月 第 9 卷 第 1 期 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 25

63.0 (C-6). Compound 3 was characterized as 3-O-β-D-
glucopyranosyl- β-kudinlactone [8].
Acid hydrolysis of compound 1 and determination of
absolute configuration of monosaccharides
Ilekudinoside W (0.5 mg) was heated in 2 mol·L−1 HCl
(0.4 mL) at 90 °C for 2 h, and then the solvent was evapo-
rated with a stream of N2. The residue was dissolved in tri-
methylchlorosilane and pyridine (0.2 mL), and the solution
was stirred at 60 °C for 5 min. The reaction mixture was par-
titioned with CHCl3. The CHCl3 layer was subjected to
GC-MS analysis. The absolute configurations of the mono-
saccharides were confirmed to be L-rhamnose (5.37 min),
L-arabinose (5.30 min) and D-glucose (12.46 min) by com-
parison of the retention times of monosaccharide derivatives
with those of standard samples.
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苦丁茶冬青叶的三萜皂苷类成分
车彦云 1, 李 宁 2, 张 梁 2, 屠鹏飞 1, 2*
1中国药科大学现代中药教育部重点实验室、生药学教研室, 南京 210009;
2北京大学药学院天然药物及仿生药物国家重点实验室, 北京 100191
【摘 要】 目的：研究苦丁茶冬青 Ilex kudingcha C. J. Tseng 叶的三萜皂苷类成分。方法：利用硅胶柱色谱和半制备高效液
相色谱对苦丁茶冬青的叶进行分离纯化, 利用光谱学手段结合理化性质鉴定其结构。结果：分离鉴定了 3 个化合物, 结构分别为：
3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl pomolic acid 28-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester (1),
3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-α-kudinlactone (2) and 3-O-β-D-glucopyranosyl-β-kudinlactone (3). 结论：化
合物 1 为新的三萜皂苷类成分。
【关键词】 冬青属; 苦丁茶冬青; 三萜皂苷

【基金项目】 国家自然科学基金资助项目(No. 30672608)