全 文 ： 2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 415

Chinese Journal of Natural Medicines 2013, 11(4): 04150417
doi: 10.3724/SP.J.1009.2013.00415
Chinese
Journal of
Natural
Medicines







A new triterpenoid glycoside from the roots of Ilex asprella
ZHAO Zhong-Xiang1, LIN Chao-Zhan2*, ZHU Chen-Chen2*, HE Wen-Jiang1
1 School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China;
2 Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine, Guangzhou, 510405, China
Available online 20 July 2013
[ABSTRACT] AIM: To study the chemical constituents of the roots of Ilex asprella Champ. ex Benth. METHODS: Compounds were
isolated by silica gel, ODS, and Sephadex LH-20 column chromatography, and their structures were elucidated on the basis of phys-
icochemical properties and spectroscopic analysis. RESULTS: Four triterpenoid glycosides were isolated and identified as
(3β)-19-hydroxy-28-oxours-12-en-3-yl β-D-glucopyranosiduronic acid n-butyl ester (1), ilexasoside A (2), monepaloside F (3), and
ilexoside A (4). CONCLUSION: Compound 1 is a new triterpenoid glycoside, and compounds 3 and 4 were isolated from this plant
for the first time.
[KEY WORDS] Ilex asprella; Triterpenoid glycosides; Treterpenoids
[CLC Number] R284.1 [Document code] A [Article ID] 1672-3651(2013)04-0415-04

1 Introduction
Ilex asprella Champ. ex Benth. (Aquifoliaceae), a de-
ciduous shrub, is mainly distributed in the south of China,
including Guangdong Province, Hunan Province and the
Guangxi Zhuang Autonomous Region. The roots of Ilex
asprella, called “Gangmei” in China, are a well-known tradi-
tional Chinese medicine, and have been used singly, or
combined with other plants, to treat laryngopharyngitis,
amygdalitis, tracheitis, and pertussis for a long time. Previous
phytochemical investigations on the roots and leaves of I.
asprella have led to the isolation of triterpenoids and flavon-
oids [1-5]. In an effort to search for novel bioactive constituents
from natural sources, the chemical constituents of I. asprella
were investigated. In this paper, the isolation and structural
e l u c i d a t i o n o f a n e w t r i t e r p e n o i d g l y c o s i d e ,
(3β)-19-hydroxy 28-oxours-12- en-3-yl β-D-glucopyranosidu-
ronic acid n-butyl ester (1), together with three known triterpe-

[Received on] 20-Aug.-2012
[Research funding] This project was supported by the National
Natural Science Foundation of China (Nos. 81270054, 30901954),
Specialized Research Fund for the Doctoral Program of Higher Edu-
cation (No.20094425110008) and the project of Zhu Jiang New Star
of Science and Technology in Guangzhou city (No. 2011J2200047)
[*Corresponding author] ZHU Chen-Chen: Prof., Tel: 86-20-36585303,
E-mail: zhuchenchen@vip.sina.com; LIN Chao-Zhan: Associate Prof.,
Tel: 86-20-36585303, E-mail: linchaozhan@sina.com
These authors have no conflict of interest to declare.
noid glycosides 2−4 (Fig. 1) are reported.
2 Results and Discussion
Compound 1 (Fig. 1) was isolated as colorless needle
crystals, and was considered to be a triterpenoid glycoside
due to the positive results for both Libermann-Burchard and
Molish reactions. Its molecular formula was established as
C40H64O10 from HR-ESI-MS for the pseudomolecular ion
peak at m/z 703.439 2 ([M − H]−, Calcd. 703.442 1). The IR
spectrum of 1 indicated the presence of hydroxyl (3433cm−1)
carboxyl (1 735 cm−1), and carbonyl (1 696 cm−1) groups.
The 1H NMR spectrum of 1 (Table 1) indicated the
presence of six tertiary methyl signals at δ 0.86 (3H, s), 0.99
(3H, s), 1.09 (3H, s), 1.31 (3H, s), 1.47 (3H, s), and 1.77 (3H,
s), one secondary methyl signal at δ 1.14 (3H, d, J = 6.8 Hz),
one primary methyl signal at δ 0.78 (3H, t, J = 7.6 Hz), a
typical signal at δ 3.37 (dd, J = 11.6, 4.4 Hz, H-3) ascribable
to an axial H-3, an anomeric sugar proton at δ 5.01 (1H, d, J
= 7.6 Hz), and one olefinic proton at δ 5.61 (1H, br s).
The 13C NMR spectrum of 1, analyzed with the aid of
the DEPT and HSQC spectra, revealed the existence of a
carboxylic carbon at δ 181.3 (s), an ester carbonyl carbon at δ
170.9 (s), one double bond at δ 128.5 (d) and 140.5 (s), which
were typical signals of C-12 and C-13 of a Δ12-ursane skele-
ton, and an anomeric carbon at δ 107.9 (d), which correlated
with the protons at δ 5.01 (1H, d, J = 7.6 Hz) in the HSQC
spectrum. In addition, an oxygenated methine carbon at δ
89.7 (d), and an oxygenated quaternary carbon signal at δ
73.2 (s) could be observed in the 13C NMR spectrum. Acidic
ZHAO Zhong-Xiang, et al. /Chinese Journal of Natural Medicines 2013, 11(4): 415418
416 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期



Fig. 1 Structures of compounds 1−4

Table 1 1H and 13C NMR data of 1 at 400 MHz for protons and 100 MHz for carbons (in C5D5N)
No. C H, mult, J (in Hz) No. C H, mult, J (in Hz)
1 39.3t 1.50 (1H, m), 0.89 (1H, m) 22 39.0t 2.17 (1H, m), 2.08 (1H, m)
2 27.2t 2.17 (1H, m), 1.88 (1H, m) 23 28.7q 1.31 (3H, s)
3 89.7d 3.37 (1H, dd, 4.4, 11.6) 24 17.4q 0.99 (3H, s)
4 40.0s 25 16.0 q 0.86 (3H, s)
5 56.4d 0.85 (1H, m ) 26 17.7q 1.09 (3H, s)
6 19.1t 1.56 (1H, m), 1.34 (1H,m) 27 25.2q 1.77 (3H, s)
7 34.0t 1.63 (1H, m), 1.39 (1H, m) 28 181.3s
8 40.9s 29 27.7q 1.47 (3H, s)
9 48.2d 1.81 (1H,m) 30 17.3q 1.14 (3H, d, 6.8)
10 37.5s 3-O-GlcA
11 24.5t 2.04 (2H, m) 1′ 107.9d 5.01 (1H, d, 7.6)
12 128.5d 5.61 (1H, br s) 2′ 75.9d 4.11 (t, 8.0)
13 140.5s 3′ 78.5d 4.30 (t, 8.8)
14 42.6s 4′ 73.6d 4.52 (1H, t, 9.2)
15 29.8t 2.33 (1H, m), 1.31 (1H, m) 5′ 77.9d 4.62 (1H, d, 10.0)
16 26.9t 3.16 (1H, m), 2.06 (1H, m) 6′ 170.9s
17 48.8s 6′-O-n-butyl
18 55.1d 3.07 (1H, br s) 1″ 65.5t 4.31 (2H, t, 6.4)
19 73.2s 2″ 31.4t 1.62 (2H, m)
20 42.9d 1.52 (1H, m) 3″ 19.8t 1.35 (2H, m)
21 27.5t 2.08 (1H, m), 1.37 (1H, m) 4″ 14.3q 0.78 (3H, t, 7.6)

hydrolysis of 1 gave D-glucuronic acid, which was confirmed
by high-performance thin-layer chromatography (HPTLC)
comparison with an authentic sample. The β-configuration of
the glycosidic bond was deduced from the coupling constant
of the anomeric proton and the 13C NMR data of the sugar
units (Table 1). The above spectroscopic features and phys-
icochemical properties suggested that 1 is a triterpenoid
saponin consisting of a triterpenoid aglycone and a glu-
curonic acid moiety.
The 1H and 13C NMR signals of 1 were assigned by a
combination of HSQC, 1H-1H COSY, and HMBC experi-
ments (Table 1). The presence of a hydroxyl group at C-19
was deduced from the 13C NMR resonance at δ 73.2 (s), as
well as the long-range correlations of CH3-29 (δ 1.47) with
C-19 (δ 73.2), C-18 (δ 55.1), and C-20 (δ 42.9) in the HMBC
experiment (Fig. 2). Moreover, a typical H-3ax proton at δ
3.37 (dd, J = 11.6, 4.4 Hz, H-3) in the 1H NMR spectrum
revealed the presence of a β-O-function at the C-3 position.
These observations further confirmed the structure of the
aglycone of 1 to be pomolic acid.
A detailed comparison between the 1H and 13C NMR
spectral data (Table 1) of 1 and the co-occurring known ilex-
asoside A (2) [5] revealed that structure of 1 was very similar
to that of ilexasoside A (2), except for the disappearance of a
methoxy group in 1, which was replaced by a O-n-butyl
group [δ 4.31 (2H, t, J = 6.4 Hz), 1.62 (2H, m), 1.35 (2H, m),
and 0.78 (3H, t, J = 7.6 Hz); δC 65.5 (t), 31.4 (t), 19.8 (t), and
14.3 (q)] (Table 1). The presence of the O-n-butyl group was
ZHAO Zhong-Xiang, et al. /Chinese Journal of Natural Medicines 2013, 11(4): 415418
2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Nov. 2013 Vol. 11 No. 4 417

also confirmed through a 1H-1H COSY experiment from the
correlations of H2-1″ (δ 4.31) to H2-2″ (δ 1.62), H2-2″(δ 1.62)
to H2-3″ (δ 1.35), and H2-3″(δ 1.35) to H3-4″ (δ 0.78) (Fig. 2).
The location of the O-n-butyl group and the site of glycosyla-
tion were confirmed by HMBC experiments showing long
range correlations of H-1″ (δ 4.31) of the n-butyl with C-6′ (δ
170.9) of the D-glucuronyl moiety, and of the anomeric pro-
ton H-1′ (δ 5.01) with C-3 (δ 89.7) of the aglycone (Fig. 2).
Comparison of chemical shifts and coupling constants of 1
with those of triterpenoids with similar structure [5-7] estab-
lished the relative configuration of 1 as shown in Fig. 1. In
addition, the correlations observed in the NOESY experiment
(Fig. 2) also supported the proposed relative configuration of
1, in which NOESY correlations of H-3 (δ 3.37) with Me-23
(δ 1.31) and H-5 (δ 0.85) proved the α-orientation of these
protons, and a NOESY correlation of H-18 (δ 3.07) with
Me-29 (δ 1.47) indicated the β-orientation of Me-29. Based
on the above results, the structure of 1 was identified as
(3β)-19-hydroxy-28-oxours-12-en-3-yl β-D-glucopyranosid-
uronic acid n-butyl ester.



Fig. 2 Selected HMBC, 1H -1H COSY and NOESY cor-
relations of compound 1

The known compounds 2−4 were purified as white pow-
ders, and characterized as ilexasoside A (2) [5], monepaloside F
(3) [8], and ilexoside A (4) [9] by comparison of their 1H and 13C
NMR data with those reported, respectively. Compounds 3 and
4 were isolated from this plant for the first time.
3 Experimental
3.1 General procedures
Melting points were determined by an X-6 melting-point
apparatus with a microscope and uncorrected. Optical rota-
tion was measured on a Polaptronic-HNQW5 high-resolution
polarimeter. The IR spectrum was recorded by a Bruker
Equinox 55 FTIR spectrometer. NMR spectra were run on a
Bruker AVANCE AV 400 spectrophotometer. Precoated silica
gel GF254 plates (Qingdao Marine Chemical Co.) were em-
ployed for TLC. For column chromatography, silica gel
(Qingdao Marine Chemical Co.), reversed-phase C18 silica
gel (Merck) and Sephadex LH-20 (Pharmacia) were used. All
the reagents were of analytical grade (Guangzhou Reagent
Factory).
3.2 Plant material
Roots of Ilex asprella Champ. ex Benth were purchased
from Zhixin Medicine Health Co., Ltd., Guangzhou, and
authenticated by Prof. ZHU Chen-Chen. A voucher specimen
(No. GM2007-1) was deposited in the School of Chinese
Materia Medica, Guangzhou University of Chinese Medicine.
3.3 Extraction and isolation
The dried roots (20 kg) of I. asprella were ground and
extracted consecutively with methanol (90 L) at room tem-
perature. After removal of solvent under vacuum, the residue
was suspended in water and partitioned successively with
CHCl3, EtOAc, and n-BuOH to afford the CHCl3 extract (430
g), EtOAc extract (80 g), and n-BuOH extract (900 g). A part
of the n-BuOH extract (240 g) was chromatographed over a
silica gel column eluting with CHCl3−MeOH (10 : 1→1 : 1)
and monitored by TLC analysis to yield five combined frac-
tions (Fr. A1−Fr. A5). Fr. A1 was separated on a silica gel
column eluted with petroleum ether-acetone (5 : 1→1 : 1),
and then subjected repeatedly to a RP-C18 column
(MeOH-H2O, 2 : 1→4 : 1) to give compounds 1 (120 mg)
and 2 (200 mg). Fr. A3 was applied to silica gel column
chromatography (CHCl3-MeOH, 5 : 1→2 : 1), followed by
Sephadex LH-20 chromatography (MeOH), and finally puri-
fied on RP-C18 column chromatography (MeOH : H2O, 1 :
1→3 : 1) to yield 3 (40 mg) and 4 (15 mg).
(3β)-19-Hydroxy-28-oxours-12-en-3-yl β-D-glucopy-
ranosiduronic acid n-butyl ester (1, Fig 1), Colorless needle
crystals (MeOH), mp 205−207 ºC, 25
D]α[ +16.2 (c 1.36,
MeOH). IR max (KBr) cm−1: 3 433, 2 936, 2 876, 1 735,
1 696, 1 649, 1 462, 1 374, 1 065. HR-ESI-MS m/z 703.439 2
[M − H]−, Calcd. for C40H63O10 703.442 1. 1H and 13C NMR,
see Table 1.
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岗梅根中一个新的三萜皂苷
赵钟祥 1，林朝展 2*，祝晨蔯 2*，何文江 1
1广州中医药大学中药学院, 广州 510006；
2广州中医药大学临床药理研究所，广州 510405
【摘 要】 目的：研究岗梅根的化学成分。方法：采用硅胶、反相硅胶、Sephadex LH-20 等柱色谱法对岗梅根的化学成分
进行分离纯化，应用谱学技术和化学方法鉴定化合物的结构。结果：从岗梅根中分离并鉴定了 4 个三萜苷类化合物：
(3β)-19-hydroxy-28-oxours-12-en-3-yl β-D-glucopyranosiduronic acid n-butyl ester (1), ilexasoside A (2), Monepaloside F(3) 和
ilexoside A(4)。结论：化合物 1 为新化合物, 化合物 3 和 4 为首次从该植物中分离得到。
【关键词】 岗梅; 三萜皂苷; 五环三萜

【基金项目】 国家自然科学基金（Nos. 81270054, 30901954）；高等学校博士学科点专项科研基金（No. 20094425110008）；广州
市珠江科技新星专项（No. 2011J2200047）资助