Fitoterapia 97 (2014) 98–104

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Triterpene saponins from the roots of Ilex pubescens

Yuan Zhou a, Kewu Zeng a, Jiayu Zhang c, Ning Li a, Xingyun Chai b, Yong Jiang a, Pengfei Tu a,b,⁎
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
b
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
c
Center of Scientific Experiment, Beijing University of Chinese Medicine, Beijing 100029, China

a r t i c l e i n f o a b s t r a c t

Article history: Five new triterpene saponins, Ilexpublesnins N–R (1–5), along with seven known analogs
Received 14 March 2014 were isolated from the root of Ilex pubescens. Their structures were elucidated on the basis of
Accepted in revised form 24 May 2014 extensive spectroscopic analysis, including 1D and 2D NMR experiments. Ilexpublesnin N (1)
Available online 6 June 2014 possessed a rare 20-hydroxyursolic acid scaffold from natural resource. These compounds
were evaluated in vitro for their cytotoxic effects on human cancer cell lines HepG2, HLE,
Keywords: BEL7402, BEL7403, BEL7405, MCF-7, HeLa. Among them, only compounds 5 and 10 showed
Ilex pubescens cytotoxic potentiality against BEl-7403 and HEL cell lines [inhibition (%): 35.38 and 45.12,
Aquifoliaceae respectively].
Ilexpublesnins N–R
© 2014 Elsevier B.V. All rights reserved.
Triterpene saponin
20-hydroxyursolic acid
Cytotoxic

1. Introduction previously, which led to a revelation of the absolute structure

Ilex pubescens Hook. et Arn., under the Chinese name
‘Mao-dong-qing’, is an evergreen shrub belonging to
Aquifoliaceae. Its roots are commonly used as Chinese
herbal medicine for treatment of cardiovascular disease
and hypercholestaemia in south China [1]. Previous phyto-
chemical investigation on its roots and leaves led to the
isolation of triterpene saponins [2–7], lignan glycosides [8],
phenylethanols [9,10], and other minor compounds [11–13].
Pharmacological evaluation proved that I. pubescens extracts
exhibited a variety of bioactivities, i.e., to enlarge blood vessels,
improve microcirculation, ease the blood pressure, inhibit
platelet aggregation, prevent thrombosis, and reduce cardiac
ischemia, as well as radiosensitization effect and so on [14,15].
As part of systematic research on seeking the bioactive
constituents from Ilex species [16–24], a phytochemical
investigation on the roots of I. pubescens was undertaken
⁎ Corresponding author at: State Key Laboratory of Natural and Biomimetic
Drugs, School of Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, China. Tel./fax: +86 10 8280 2750.
E-mail address: pengfeitu@vip.163.com (P. Tu).
of Ilexpublesnins C–M [25]. As an ongoing program toward
the discovery of novel bioactive constituents, five new
triterpene saponins, namely Ilexpublesnins N–R (1–5), and
seven known ones (6–12) were isolated. This paper describes
the isolation and structural elucidation of these compounds
(Fig. 1). In addition, the cytotoxic evaluation for these
compounds against the human cancer cell lines HepG2, HLE,
BEL-7402, BEL-7403, BEL-7405, MCF-7, and HeLa is included.
2. Experimental
2.1. General procedures
HRESIMS were measured on a Bruker APEX IV FT_MS
(7.0T) spectrometer in positive ion mode. Optical rotations
were measured on an Autopol-IV polarimeter (RudoPH
Research analytical). IR spectra were obtained using a
NEXUS-470 FTIR (Nicolet) spectrometer. 1D and 2D NMR
spectra were recorded on Bruker Avance DRX-400 and
Vnmrs-500 spectrometers. Semi-preparative HPLC was per-
formed on a Waters model 2487 (Agilent ODS column,
250 × 10 mm i.d., 5 μm) with an Alltech evaporative light

http://dx.doi.org/10.1016/j.fitote.2014.05.020
0367-326X/© 2014 Elsevier B.V. All rights reserved.
 Y. Zhou et al. / Fitoterapia 97 (2014) 98–104
Fig. 1. Structures of compounds 1–5.
scattering detector (ELSD). GC analysis was carried out on an
Agilent 6890N gas chromatograph, with an HP-5 capillary
column (28 m × 0.32 mm) and an FID detector operated at
260 °C (column temp. 180 °C), 1.0 mL/min N2 as carrier gas.
Macroporous resin (HPD100) was purchased from Hebei
Bao-En Biotech Ltd. Thin-layer chromatography and column
chromatography were performed using silica gel (Qingdao
Haiyang Chemical Co. Ltd., GF254 or 200–300 mesh) and
Sephadex LH-20 (Pharmacia Biotech Ltd.) and ODS (Merck &
Co., Inc. USA). All the solvents were of analytical grade and
were purchased from Beijing Chemical Company Ltd.
2.2. Plant material
The roots of I. pubescens were purchased from Guilin
San-Jin Pharmaceutical Co. Ltd, and were originally collected
from Guangxi province, China. The plants were identified by
Prof. Peng-Fei Tu (one of the authors in this paper). A
voucher specimen (No. 091005) is deposited in the Herbar-
ium of Modern Research Center for Traditional Chinese
Medicine (TCM), Peking University, Beijing.
2.3. Extraction and isolation
Dry crude materials (18 kg) were grinded and extracted
with 70% EtOH at the temperature of 70 °C. After the retrieval
of the ethanol, the residue suspended in water (50 L) was
subjected to column chromatography (CC) on macroporous
resin with an EtOH–H2O gradient (30:70, 70:30, 90:10)
to yield three fractions (Frs. 1–3). Fr. 2 (160 g) was
chromatographed on silica gel (2.5 kg, 100 × 10 cm) with a
gradient of CHCl3–MeOH (40:1–1:1) elution to yield thirteen
fractions (Frs. A–M).
Fr. A (11 g) was recrystallized with mixed solution of
MeOH–CHCl3 (1:1) yields compound 6 (8 g). Fr. C (1.5 g) was
99
subjected to CC on silica gel (50 g, 15 × 2 cm) eluted with
CHCl3–MeOH (20:1) yields compound 10 (500 mg). Fr. G
(25 g) was subjected to CC on silica gel (500 g, 50 × 4 cm)
eluted with CHCl3–MeOH (10:1, 5:1, 2:1) yields seven fractions
(Fr. G-1–Fr. G-7). Fr. G-4 (1.2 g) was chromatographed on
Sephadex LH-20 (50 × 3 cm) in MeOH afforded three
fractions (Fr. G-4-1–Fr. G-4-3). Fr. G-4-3 (660 mg) was
separated by semi-preparative HPLC (MeOH/H2O 80:20,
2.0 mL/min) yields compounds 7 (5 mg), 8 (180 mg) and 11
(15 mg). Fr. I (3.6 g) was subjected to CC on silica gel (300 g,
50 × 3 cm) eluted with CHCl3–MeOH (5:1, 2:1) yields two
fractions (Fr. I-1–Fr. I-2). Fr. I-1 (2.5 g) was chromatographed
on Sephadex LH-20 (50 × 3 cm) in MeOH, then subjected to
RP-C18 CC (40 × 3 cm) eluted with MeOH–H2O (70:30) and
further purified by semi-preparative HPLC (MeOH/H2O 56:44,
2.0 mL/min) yields compound 2 (10 mg). Fr. I-2 (400 mg) was
chromatographed on Sephadex LH-20 (100 × 2 cm) in MeOH
and then separated by semi-preparative HPLC (MeOH/H2O
65:35, 2.0 mL/min) yields compound 3 (15 mg). Fr. J (18 g)
was subjected to CC on silica gel (400 g, 50 × 4 cm) eluted
with CHCl3–MeOH–H2O (60:30:10, low layer) yields three
fractions (Fr. J-1–Fr. J-3). Fr. J-3 (2.8 g) was chromatographed
on Sephadex LH-20 (50 × 3 cm) in MeOH afforded three
fractions (Fr. J-3-1–Fr. J-3-3). Fr. J-3-3 (1.7 g) was subjected to
RP-C18 CC (40 × 3 cm) eluted with MeOH–H2O (70:30) and
further purified by semi-preparative HPLC (MeOH/H2O 41:59,
2.0 mL/min) yields compounds 4 (4 mg), 5 (5 mg) and 9
(60 mg). Fr. L (9 g) was chromatographed on RP-C18 CC
(50 × 4 cm) using a gradient of MeOH–H2O (20:80, 40:60,
60:40, 80:20) afforded five fractions (Fr. L-1–Fr. L-4). Compound
1 (10 mg) was obtained from Fr. L-3 (650 mg) by semi-
preparative HPLC (MeOH/H2O 53:47). Compound 12 (17 mg)
was obtained from Fr. L-4 (1.6 g) by Sephadex LH-20
(50 × 3 cm) and then semi-preparative HPLC (MeOH/H2O
65:35).
 100
Table 1
1
H NMR data (in pyridine-d5) of 1–2 (400 MHz) and 3–5 (500 MHz), J in Hz and δ in ppm.

Position 1 2 3 4 5 Position 1 2 3 4 5

1 0.91 m; 1.52 m 0.88 m; 1.52 m 0.90 m; 1.53 m 0.87 m; 1.45 m 0.87 m; 1.44 m 3-O- Xyl Xyl Xyl Ara Ara
2 1.85 m; 2.07 m 2.07 m 1.99 m; 2.23 m 1.92 m 1.92 m 1 4.91 d (6.8) 4.95 d (6.4) 4.90 d (8.0) 4.87 d (7.6) 4.88 d (7.6)
3 3.31 d (11.6) 3.24 (11.6) 3.51 m 4.04 m 4.06 m 2 4.02 m 4.10 m 3.96 t (8.0) 5.34 me 5.34 mf
5 0.79 d (11.6) 0.77 d (11.6) 0.93 m 0.76 d (11.0) 0.76 d (11.0) 3 3.84 m 4.27 m 4.15 t (8.0) 4.04 m 4.04 m
6 1.28 m; 1.47 m 1.33 m; 1.47 m 1.33 m; 1.62 m 1.29 m; 1.49 m 1.29 m; 1.49 m 4 4.26 m 4.21 m 4.04 m 4.23 m 4.23 m
7 1.31 m; 1.44 m 1.37 m; 1.55 m 1.35 m; 1.50 m 1.43 m; 1.58 m 1.43 m; 1.58 m 5 3.69 m; 4.26 mb 3.74 m; 4.33 m 3.18 t (10.5); 3.71 m; 4.28 m 3.71 m; 4.28 m
4.37 dd (10.5)

Y. Zhou et al. / Fitoterapia 97 (2014) 98–104

9 1.54 m 1.73 m 1.78 m 1.78 m 1.80 m Intermediate Glc Glc
sugar
11 1.99 m; 2.24 m 1.29 m 1.79 m 2.04 m 2.04 m 1 5.83 d (7.6) 5.43 d (7.6)
12 5.49 br s 5.53 br s 5.52 br s 5.55 br s 5.50 br s 2 4.02 m 4.17 m
15 1.13 ma; 2.43 m 1.43 m 1.25 m; 2.26 m 2.47 m 2.47 m 3 4.04 m 3.83 m
16 1.85 m; 2.07 m 1.83 m; 2.07 m 2.04 m; 1.84 m; 2.05 m 2.22 m 4 4.27 m 4.13 m
3.20 t (13.5)
18 2.80 d (12.4) 3.28 br s 3.26 br s 2.93 br s 3.59 br s 5 4.07 m 3.85 m
19 2.22 m 3.53 br s 6 4.29 m; 4.46 m 4.36 m; 4.54 m
20 2.04 m 2.00 m 1.30 m Terminal Rha Glc
21 1.74 m; 2.11 m 1.74 m 1.28 m; 2.68 m 1.84 m; 2.05 m 1.46 m 1 6.41 br s 5.37d (7.8)
22 1.88 m 1.94 m; 2.28 m 1.92 m; 2.20 m 1.94 d (7.0); 2.04 m 2 4.70 m 4.42 m
2.14 m
23 1.34 s 1.22 s 1.50 s 1.23 s 1.23 s 3 4.09 m 3.93 m
24 1.06 s 1.05 s 2.58 d (12.0); 1.15 s 1.18 s 4 4.25 m 4.19 m
4.34 d (12.0)
25 0.84 s 0.84 s 0.78 s 0.89 s 0.87 s 5 5.02 m 4.42 m
26 1.12 s 1.06 s 1.02 s 0.96 s 0.98 s 6 1.79 d (6.4) 4.45 m; 4.54 m
27 1.16 s 1.73 s 1.74 s 1.72 s 1.66 s 28-O- Glc Glc Glc
29 1.18 d (7.2) 1.42 s 1.41 s 1.39 s 1.14 s 1 6.32 d (8.0) 6.29 d (8.0) 6.37 d (8.0)
30 1.24 s 1.12 d (6.8) 1.10 d (7.0) 1.05 d (7.5) 0.96 s 2 4.22 m 4.24 m 4.24 m
3 4.30 m 4.06 m 4.06 m
4 4.32 m 4.37 m 4.37 m
5 4.02 m 4.30 m 4.30 m
6 4.29 mc; 4.46 md 4.39 m; 4.45 m 4.39 m; 4.45 m
a, b, c, d, e, f
These signals were overlapped.
 Y. Zhou et al. / Fitoterapia 97 (2014) 98–104 101

Ilexpublesnin N (1): white, amorphous powder; [α] 20

D −
9.47 (c 0.10, MeOH); IR (KBr) νmax 3425, 2930, 1726, 1632,
1071 cm−1; 1H and 13C NMR data (pyridine-d5, 400/
100 MHz), see Tables 1 and 2; HRESIMS m/z 1092.59461
[M + NH4]+ (calcd. for C53H86O22NH4, 1092.59490).
Ilexpublesnin O (2): white, amorphous powder; [α] 20
D +
10.1 (c 0.12, MeOH); IR (KBr) νmax 3424, 2927, 1695, 1635,
1074 cm−1; 1H and 13C NMR data (pyridine-d5, 400/
100 MHz), see Tables 1 and 2; HRESIMS m/z 951.49384
[M + Na]+ (calcd. for C47H76O18Na, 951.49238).
Ilexpublesnin P (3): white, amorphous powder; [α] 20
D +
1.45 (c 0.12, MeOH); IR (KBr) νmax 3410, 2927, 1697,
1071 cm−1; 1H and 13C NMR data (pyridine-d5, 500/
125 MHz), see Tables 1 and 2; HRESIMS m/z 1241.78632
[2 M + H]+ (calcd. for C70H113O18, 1241.78632).
Ilexpublesnin Q (4): white, amorphous powder; [α] 20
D +
9.6 (c 0.15, MeOH); IR (KBr) νmax 3433, 2941, 1732, 1636,
1073 cm−1; 1H and 13C NMR data (pyridine-d5, 500/
125 MHz), see Tables 1 and 2; HRESIMS m/z 845.39938
[M − H]− (calcd. for C41H65O16S, 845.39988).
Ilexpublesnin R (5): white, amorphous powder; [α] 20
D +
8.3 (c 0.12, MeOH); IR (KBr) νmax 3433, 2943, 1731, 1636,
1073 cm−1; 1H and 13C NMR data (pyridine-d5, 500/
125 MHz), see Tables 1 and 2; HRESIMS m/z 845.39933
[M − H]− (calcd. for C41H65O16S, 845.39988).
dioxane was removed, the solution was extracted with EtOAc
(3 mL × 3) to yield the aglycon and the sugar, respectively. The
sugar components in the aqueous layer left after acid hydrolysis
of 1–5 were analyzed by silica gel TLC by comparison with
standard sugars. The solvent system was n-BuOH–TEA–H2O
(60:0.7:30), and spots were visualized by spraying with
Phenylamine–Diphenylamine, and then heated at 200 °C for
1 min. For sugars of 1–5, the Rf of glucose, arabinose, xylose, and
rhamnose by TLC was 0.57, 0.62, 0.66, 0.71 respectively. The
results were confirmed by GC analysis of the methyl sugar
peracetates. The aqueous layer was evaporated and dissolved in
anhydrous pyridine (100 μL), 0.1 mL cysteine methyl ester
hydrochloride (200 μL) was added, and the mixture was warmed
at 60 °C for 1 h. The trimethysilylation reagent HMDS–TMCS
(hexamethyldisilazane–trimethylchlorosilanepyridine, 2:1:10)
(Acros Organics, Geel, Belgium) was added and warmed
at 60 °C for 30 min. The thiazolidine derivatives were
analyzed by GC for sugar identification. The retention times
of L-arabinose (tR, 5.21 min), L-rhamnose (tR, 5.35 min),
D-xylose (tR, 5.49 min) and D-glucose (tR, 11.72 min) were
confirmed by comparison with those of authentic standards
[26].
2.5. Cytotoxicity assay

2.4. Acid hydrolysis The method described in a literature [27] was used for
the measurement of cytotoxic activities against human
Compounds 1–5 (3 mg) were heated in 3 mL of 10% cancer cell lines. For detailed protocols, see Supporting
HCl-dioxane (1:1) at 90 °C for 4 h in a sealed amp. After the information.

Table 2
13
C NMR Data (in pyridine-d5) of 1–2 (100 MHz) and 3–5 (125 MHz), δ in ppm.

Position 1 2 3 4 5 Position 1 2 3 4 5

1 38.8 38.7 38.5 38.7 38.5 3-O- Xyl Xyl Xyl Ara Ara
2 26.6 27.1 27.0 26.7 26.1 1 105.8 104.9 106.7 106.9 106.9
3 89.6 88.7 88.8 88.6 88.6 2 79.4a 82.5 75.3 84.0 84.0
4 39.6 39.5 44.3 39.5 55.9 3 77.8 77.8 78.4 73.8c 73.8d
5 56.0 55.8 56.2 55.9 48.2 4 71.2 71.6 71.2 70.0 70.0
6 18.5 18.6 18.9 18.6 18.6 5 66.6 65.9 67.1 66.4 66.4
7 33.5 33.5 33.7 33.1 33.0 Intermediate Glc Glc
8 40.1 40.2 40.1 40.5 40.2 1 102.2 103.2
9 48.0 47.7 47.5 47.6 48.2 2 79.2a 84.4
10 36.9 37.0 36.7 37.1 36.9 3 79.1b 77.8
11 24.6 24.9 24.8 24.1 24.0 4 72.7 70.1
12 125.8 127.2 127.1 128.2 123.5 5 79.0b 77.8
13 137.9 139.5 139.4 139.2 144.2 6 63.2 62.6
14 42.5 42.1 42.1 42.1 42.0 Terminal Rha Glc
15 28.6 29.3 29.2 29.2 29.0 1 102.0 106.3
16 26.6 26.5 26.9 26.5 28.2 2 72.3 76.6
17 48.1 47.9 47.9 48.6 46.4 3 72.7 79.1
18 50.7 47.4 47.4 54.4 44.6 4 74.3 71.0
19 42.6 73.4 73.4 72.6 81.0 5 69.5 79.1
20 72.0 43.0 43.0 42.1 35.5 6 18.9 62.5
21 37.9 24.0 24.3 26.5 29.2 28-O- Glc Glc Glc
22 34.5 32.5 32.4 37.7 33.4 1 95.7 95.8 95.8
23 28.4 28.1 23.3 28.2 28.2 2 74.0 74.0c 74.1d
24 16.8 16.8 63.2 16.9 16.8 3 78.5 78.9 79.2
25 15.6 15.5 15.3 15.6 15.4 4 71.1 71.2 71.0
26 17.6 17.3 17.1 17.4 17.5 5 78.9 79.3 79.3
27 23.4 24.4 24.3 24.1 24.6 6 62.2 62.1 62.3
28 176.2 180.6 180.7 177.0 177.3
29 13.6 29.8 29.8 27.0 28.7
30 19.9 16.1 16.1 16.6 24.6
a, b, c, d
These signals under the same superscript may be interchanged.
102 Y. Zhou et al. / Fitoterapia 97 (2014) 98–104
3. Results and discussion
Ilexpublesnin N (1) was assigned a molecular formula of
C53H86O22 determined on the basis of its positive HRESIMS
[M + NH4]+ ion peak at m/z 1092.59461 (calcd. 1092.59490).
The IR spectrum showed absorption bands for hydroxyl
(3425 cm−1), methyl (2930 cm−1), carbonyl (1726 cm−1),
and olefinic (1632 cm−1) groups. Acid hydrolysis of 1 afforded
sugar components as D-glucose, L-rhamnose and D-xylose
identified by TLC and GC analyses, which was also verified by
comparing 13C NMR data of Ilexpublesnin E [25]. The 1H and
13
C NMR data (Tables 1 and 2) assigned by HSQC and HMBC
experiments, revealed six singlets for tertiary methyl at δH 0.84,
1.06, 1.12, 1.24, 1.34, 1.65, two doublets at δH 1.18 (d, J =
7.2 Hz), 1.79 (d, J = 7.2 Hz, Rha-CH3), one olefinic linkage (δH
5.49, δC 125.8, C-12; δC 137.9, C-13), one carboxyl (δC 176.2,
COOR-28), and four sugar components (δH 4.91, δC 105.8,
CH-Xyl-1; δH 5.83, δC 102.2, CH-Glc-1; δH 6.41, δC 102.0,
CH-Rha-1; δH 6.32, δC 95.7, CH-Glc-1) in 1.
Analyzing above data suggested 1 to be a hydroxylated
ursane triterpene saponin. Doublet of H-18 (δH 2.80, J =
12.4 Hz) indicated the only proton on C-19. Detailed HMBC
correlations, including from H-12 to C-11, C-14 and C-18,
from H-30 to C-19, C-20 and C-21, from H-29 to C-18 and
C-20, supported this assignments, and this analysis further
indicated the structure of 1 closely resembles Ilexpublesnin E
with the main difference that hydroxyl group of 1 transferred
to C-20. A careful comparison of its 1H data with those of
3β-O-cis-p-coumaroyl-20β-hydroxy-12-ursen-28-oic acid [28]
revealed they possessed the same aglycone. HMBC correlations
(Fig. 2) from the inner-xyl-H-1 (δH 4.91, d, J = 6.8 Hz) to C-3
(δC 89.6), from Glc-H-1 (δH 6.32, d, J = 8.0 Hz) to C-28 (δC
176.2) determined its glycosylation sites at 3-O- and 28-O-.
Fig. 2. Key HMBC and NOESY correlations of 1.
Furthermore, HMBC correlations from the intermediate-glc-H-1
(δH 5.83, d, J = 7.6 Hz) to inner-xyl-C-2 (δC 79.4) and from
terminal-rha-H-1 (δH 6.41, br s) to intermediate-glc-C-2 (δC
79.2) established all the linkages of sugar moieties. NOESY
correlations of CH3-30/CH3-27 suggested the configuration of
20-OHβ (Fig. 2). On the basis of the above analysis, compound
1 was elucidated as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-
β-D-glucopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β, 20β-
dihydroxyurs-12-en-28-oic-O-β-D-glucopyranosyl ester.
Ilexpublesnin O (2) was assigned a molecular formula
C47H76O18 determined on the basis of its positive HRESIMS m/z
951.49384 [M + Na]+ (calcd. 951.49238). The IR spectrum
showed absorption bands for hydroxyl, methyl, carbonyl and
olefinic groups. Acid hydrolysis of 2 afforded sugar components
of D-glucose and D-xylose identified by TLC and GC analysis. 1H
and 13C NMR data (Tables 1 and 2) assigned by HSQC and HMBC
experiments, revealed six singlets for tertiary methyl at δH 0.85,
1.05, 1.06, 1.20, 1.41, 1.73 (each 3H, s), one doublet at δH 1.12 (d,
J = 6.8 Hz), one olefinic linkage (δH 5.53, δC 127.2, C-12; δC
139.5, C-13), one carboxyl (δC 180.6, COOH-28), and three sugar
components (δH 4.95, δC 104.9, CH-Xyl-1; δH 5.43, δC 103.2,
CH-Glc-1; δH 5.37, δC 106.3, CH-Glc-1) in 2. A careful
comparison of its 1H and 13C NMR data with those of
Ilexpublesnin J [25] revealed their structural similarity, except
the difference that no sugar moiety at its C-28 carboxyl in
2. Therefore, compound 2 was elucidated as (20 S)-3-O-
[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-
xylopyranosyl]-3β, 19α-dihydroxyurs-12-en-28-oic acid.
Analysis of the HRESIMS data ([2 M + H]+ at m/z
1241.78632, calcd. 1241.79268) of Ilexpublesnin P (3)
determined its molecular formula C35H56O9. Its IR spectrum
showed absorption bands for hydroxyl, methyl, carbonyl, and
olefinic functional groups. Acid hydrolysis of 3 afforded sugar
components of D-xylose by TLC and GC analysis. The signals
characteristic for an ursane triterpene saponin were observed
from its 1H and 13C NMR data (Tables 1 and 2), including five
singlets for tertiary methyls, an olefinic proton at δH 5.52
(br s), one anomeric carbon as well. A comprehensive
analysis and comparison of its 1H and 13C NMR data with
those of Ilexpublesnin G [25] implied that they are highly
similar in structure, except the difference that no sugar
moiety at its C-28 carboxyl in 3. Eventually, 3 was elucidated
as (20 S)-3-O-β-D-xylpyranosyl-3β, 19α, 24β-trihydroxyurs-
12-en-28-oic acid.
A molecular formula C41H66O16S was determined for
Ilexpublesnin Q (4) on the basis of its negative HRESIMS
([M–H]- at m/z 845.39938, calcd. 845.39988). Overall
appearance of its IR spectrum and 1H, 13C NMR data
(Tables 1 and 2) displayed a high resemblance with those of
ilexoside II [29]. Interpretation of its spectroscopic data
revealed the only difference between them in a sugar moiety.
Acid hydrolysis of 4 yielded pomolic acid [30], D-glucose and
L-arabinose identified by TLC and GC analyses. Comparison of
the 13C NMR data with those of Ilexpublesnin H [25] revealed
sulfonylation on the hydroxyl attaching to Ara-C-2, which
was supported by HMBC correlations from Ara-H-2 (δH 5.34)
to Ara-C-3 (δC 73.8) and Ara-C-1 (δC 106.9). Same analysis of
its HMBC correlations from Ara-H-1 (δH 4.87) to C-3 (δC
88.6), and from Glc-H-1 (δH 6.29) to C-28 (δC 177.0)
suggested 6 of 3-O-, 28-O-diglycosides of pomolic acid. By
the way, R-orientation of chiral C-20 was from the evidences
 Y. Zhou et al. / Fitoterapia 97 (2014) 98–104
of chemical shift at δC 54.4 (C-18) and δC 37.7 (C-22) [31].
Subsequently, 4 was elucidated as (20 R)-3-O-[α-L-(2′-
sulfonyl)-arabinpyranosyl]-3β,
19α-dihydroxyurs-12-en-28-oic-O-β-D-glucopyranosyl ester.
The molecular formula C41H66O16S for Ilexpublesnin R (5)
was given by HRESIMS data. The IR spectra showed absorption
bands for hydroxyl, methyl, carbonyl, and olefinic. The 1H and 13C
NMR data (Tables 1 and 2) displayed seven singlets for tertiary
methyls, an olefinic, sugar moiety, including glucose and
arabinose. These assignments were confirmed by acid hydro-
lysis and/or 1H and 13C NMR comparison with those reported
data. Analysis of 13C NMR data indicated 5 closely resembled
oblonganoside K [32] except the only difference that one more
sulfonylated arabinose unit linked to C-3-OH. Comparison of
the 13C NMR data with those of 4 revealed sulfonylation on the
hydroxyl attaching to Ara-C-2, which was supported by HMBC
correlations from Ara-H-2 (δH 5.34) to Ara-C-3 (δC 73.8) and
Ara-C-1 (δC 106.9). Eventually, an extensive analysis of its
HMBC correlations was applied to establish its structure as 3-
O-[α-L-(2′-sulfonyl)-arabinpyranosyl]-3β, 19α-dihydroxyolea-
12-en-28-oic-O-β-D-glucopyranosyl ester.
Additionally, seven known analogs (6–12) (see Supporting
information) were also isolated. By comparing its 1H, 13C NMR
and MS data with reported values, their structures were
identified as ilexgenin A (6) [4], pedunculoside (7) [33],
ilexsaponin A1 (8) [34], mussaendoside V (9) [35], ilexoside A
(10) [36], 3β, 19α-dihydroxyolea-12-en-24, 28-dioic-O-β-D-
glucopyranosyl ester (11) [21], oblonganoside K (12) [32].
Compound 1 showed an aglycone of 20-hydroxyl ursolic
acid which was rarely reported from natural resource [28,
37,38]. As was identified simply by IR, MS and 1H NMR
spectra in the former reference, we elucidated its structure
on the basis of extensive spectroscopic analysis in this
study.
Compounds 1–12 were evaluated for their cytotoxic
activities in vitro against the human hepatoma cell lines
HepG2, HLE, BEL-7402, BEL-7403, BEL-7405, human breast
cancer cell line MCF-7, and human cervical cancer cell line
HeLa, using MTT assay. Unfortunately, no significant cytotox-
ic results [inhibition (%) b50] were observed against all seven
cancer cell lines at the concentration of 40 μM. Among these
isolates, only 10 showed a cytotoxic potential against HepG2
and HEL cell lines [inhibition (%) = 34.38, 45.2, respectively],
and 5 exhibited a weak cytotoxic activities on HepG2 and
Bel-7403 cell lines [inhibition (%) = 30.31, 35.38, respec-
tively]. The cytotoxic effects of 5 and 10 may be due to their
aglycon oleanic acid. Meanwhile, 3-O-glycoside may display a
stronger biology activity than 28-O-glycoside.
Supporting information
Cytotoxicity assay and MTT data of 1–12, structures of
6–12, and the HRESIMS, IR, 1D and 2D NMR spectra of 1–5 are
available in Supporting information.
Conflict of interest
The authors of the present manuscript have declared that
no competing interests exist.
103
Acknowledgments
The authors express thanks to the analytical center of
Peking University Health Science Center for measurements of
MS and NMR. This work is financially supported by grants
from the National Natural Science Foundation of China
(No.30672608) and the Scientific and Technological Major
Special Project for ‘Significant Creation of New Drugs’
(2009ZX09311-004).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2014.05.020.
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