Triterpene saponins from the roots of ilex asprella

Article abstract of DOI:10.1002/cbdv.201300155

Six new triterpene saponins, ilexasprellanosides A-F (1-6, resp.), together with eleven known compounds were isolated from the roots of Ilex asprella. The new saponins were characterized as ursa-12,18-dien-28-oic acid 3-O-β-D-xylopyranoside (1), 19α-hydroxyursolic acid 3-O-β-D-(2′-O-acetylxylopyranoside) (2), 19α-hydroxyursolic acid 3-O-β-D-glucuronopyranoside (3), 3β,19α-dihydroxyolean-12- en-23,28-dioic acid 28-O-β-D-glucopyranoside (4), 19α- hydroxyoleanolic acid 3-O-β-D-(2′-O-acetylxylopyranoside) (5), 19α-hydroxyoleanolic acid 3-O-β-D-glucuronopyranoside (6). The structures of the new compounds were elucidated by analysis of their spectroscopic data and chemical degradation. Compounds 2, 4, oleanolic acid 3-O-β-D-glucuronopyranoside, 3-β-acetoxy-28-hydroxyurs-12-ene, and pomolic acid showed significant cytotoxic activities against human tumor cell line A549 (IC₅₀ values of 1.87, 2.51, 1.41, 3.24, and 5.63 μM, resp.).

Full text of DOI:10.1002/cbdv.201300155

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
767
Triterpene Saponins from the Roots of Ilex asprella
a
b
c
a
a
a
by Yu Lei ) ), She-Po Shi ), Yue-Lin Song ), Dan Bi ), and Peng-Fei Tu* )
a) State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking
University Health Science Center, No. 38 Xueyuan Road, Beijing 100191, P. R. China
(
phone: þ86-10-82802750; e-mail: pengfeitu@vip.163.com)
b) Laboratory Animal Center, Peking University, Beijing 100871, P. R. China
c) Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine,
Beijing 100029, P. R. China
Six new triterpene saponins, ilexasprellanosides A–F (1–6, resp.), together with eleven known
compounds were isolated from the roots of Ilex asprella. The new saponins were characterized as ursa-
1
2,18-dien-28-oic acid 3-O-b-d-xylopyranoside (1), 19a-hydroxyursolic acid 3-O-b-d-(2’-O-acetylxylo-
pyranoside) (2), 19a-hydroxyursolic acid 3-O-b-d-glucuronopyranoside (3), 3b,19a-dihydroxyolean-12-
en-23,28-dioic acid 28-O-b-d-glucopyranoside (4), 19a-hydroxyoleanolic acid 3-O-b-d-(2’-O-acetyl-
xylopyranoside) (5), 19a-hydroxyoleanolic acid 3-O-b-d-glucuronopyranoside (6). The structures of the
new compounds were elucidated by analysis of their spectroscopic data and chemical degradation.
Compounds 2, 4, oleanolic acid 3-O-b-d-glucuronopyranoside, 3-b-acetoxy-28-hydroxyurs-12-ene, and
pomolic acid showed significant cytotoxic activities against human tumor cell line A549 (IC50 values of
1.87, 2.51, 1.41, 3.24, and 5.63 mm, resp.).
Introduction. – Ilex asprella (Aquifoliaceae) is mainly distributed in the south and
east regions of the Peopleꢀs Republic of China. Its roots and leaves are widely used as a
Traditional Chinese Medicine (TCM) for the treatment of headache, tussis, febris,
dysentery, sore throat, etc. In addition, the leaves of I. asprella are also one of the raw
materials of Cantonese herbal tea [1]. Previous investigations have resulted in the
isolation of triterpenes and triterpene saponins from I. asprella [2–9]. Encouraged by
the notable pharmacological properties of I. asprella, we have re-investigated the
constituents of the roots and characterized six new triterpene saponins, 1–6, together
with eleven known compounds (Fig. 1). Herein, we report the isolation and structure
elucidation of the new compounds, as well as their cytotoxic activities against A549 cell
line.
Results and Discussion. – Compound 1 was obtained as amorphous powder, with a
þ
molecular formula of C H O deduced from the [MþNa] ion peak at m/z 609.3771
35
54
7
13
(
calc. 609.3767) in the HR-ESI-MS and supported by the C-NMR data (Table 1). The
ꢀ
1
IR spectrum of 1 exhibited absorptions at 3445 (OH), 1697 (C¼O), and 1647 cm
(
3
C¼C). The UV spectrum of 1 indicated the maximum absorption at 227 nm (log e
1
.79), attributed to a conjugated diene system. The H-NMR spectrum (Table 1) of 1
revealed the presence of six tertiary Me groups resonating at d(H) 1.84, 1.28, 1.13, 0.99,
.96, and 0.84, a secondary Me group resonating at d(H) 1.07 (d, J¼5.5), an olefinic H-
atom resonating at d(H) 5.60 (t, J¼3.5), and a H-atom due to an O-bearing CH group
0
ꢁ
2014 Verlag Helvetica Chimica Acta AG, Zꢂrich

7
68
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Fig. 1. Structures of compounds 1–6
13
at d(H) 3.36 (dd, J¼10.5, 5.5). The C-NMR spectrum (Table 2) of 1 exhibited signals
of four olefinic C-atoms at d(C) 139.4, 134.7, 126.3, and 123.6, of one C¼O group at
1
d(C) 178.7, and of one O-bearing CH group at d(C) 88.5. In addition, the H- and
13
C-NMR spectra of 1 indicated the presence of a pentose moiety with the anomeric H-
atom resonating at d(H) 4.80 (d, J¼7.0). Acid hydrolysis and GC analysis evidenced
the occurrence of a d-xylosyl moiety in 1. The coupling constant (J¼7.0) of the
anomeric H-atom suggested that the anomeric C-atom of the d-xylosyl moiety was b-
configured. Comparison of the NMR data of 1 with those of ursa-12,18-dien-28-oic acid
3-O-b-d-arabinopyranoside revealed that the structures of both compounds were very
similar except for the sugar moiety [10]. Thus, the structure of 1 (Fig. 1) was proposed
to be ursa-12,18-dien-28-oic acid 3-O-b-d-xylopyranoside, which was further confirmed
by the following HMBCs: HꢀC(12) (d(H) 5.60 (t, J¼3.5)) to C(13) (d(C) 139.4) and
C(18) (d(C) 123.6); Me(30) (d(H) 1.07 (d, J¼5.5)) to C(19) (d(C) 134.7); Me(29)
(
d(H) 1.84 (s)) to C(19); and the anomeric H-atom (d(H) 4.80 (d, J¼7.0 )) to C( 3 )
(d(C) 88.5) of the aglycone (Fig. 2). Accordingly, the structure of 1 was unambiguously
established as ursa-12,18-dien-28-oic acid 3-O-b-d-xylopyranoside, named ilexasprel-
lanoside A.
Fig. 2. Key HMBCs (H!C) of compound 1

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
769

7
70
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 2. 1C-NMR Data (125 MHz; (D )pyridine) of 1–6. d in ppm.
5
Position
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
39.1
26.7
88.5
39.4
55.9
18.3
34.8
39.1
48.1
36.7
23.4
126.3
139.4
44.7
29.1
26.6
50.1
123.6
134.7
34.8
29.1
35.5
28.1
16.9
16.1
18.0
22.2
178.7
20.2
20.4
107.5
75.3
78.4
71.0
38.4
26.4
88.9
40.2
55.7
18.6
33.5
40.3
47.9
37.0
24.0
128.0
139.9
42.3
29.3
26.4
48.2
54.6
72.7
42.1
26.9
38.4
28.3
17.4
15.4
16.7
24.8
180.6
26.5
16.6
104.8
75.6
76.2
71.3
67.1
38.4
26.9
89.1
40.3
54.7
18.5
33.5
39.9
47.6
36.8
23.9
128.0
139.8
42.3
29.1
26.3
48.2
54.6
72.6
42.1
26.9
38.4
28.3
17.4
15.4
16.7
24.8
180.9
27.1
16.8
107.3
75.5
78.1
73.4
77.8
172.9
–
38.6
27.6
75.3
51.8
54.2
21.6
32.7
40.3
48.3
36.7
23.9
123.3
144.2
41.8
28.5
28.6
46.2
44.3
80.8
35.3
28.7
32.7
180.6
11.9
15.7
17.3
24.3
177.2
28.7
24.6
95.6
73.8
79.0
70.8
78.6
61.9
–
38.4
26.5
88.9
39.9
55.8
18.6
33.2
40.0
48.2
37.1
24.1
123.3
144.8
42.1
28.8
29.1
46.0
44.8
81.2
35.7
29.2
33.2
28.3
16.7
15.3
17.4
24.8
180.9
28.8
24.9
104.8
75.6
76.2
71.3
67.1
38.4
26.3
89.1
39.9
55.8
18.5
33.2
40.2
48.1
37.0
24.0
123.3
144.8
42.1
28.8
29.2
46.0
44.7
81.1
35.7
29.2
33.2
28.1
16.7
15.3
17.1
24.7
180.6
28.9
24.8
107.3
75.5
78.1
73.4
77.8
172.9
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1
2
3
4
5
6
’
’
’
’
’
’
66.9
Ac
–
170.0, 21.2
170.0, 21.2
For compound 2, the molecular formula was determined as C H O based on the
3
7
58
9
þ
[
MþNa] ion peak at m/z 669.3967 (calc. 669.3979) in the HR-ESI-MS and supported
13
by the C-NMR data (Table 2). Acid hydrolysis of 2 gave a known compound, pomolic
acid [11] and d-xylose, suggesting 2 to be a pomolic acid xyloside. Comparison of the
13
C-NMR data of 2 with those of pomolic acid disclosed that the chemical shift of C(3)
of 2 was shifted downfield to d(C) 88.9, revealing the position of attachment of the
xylosyl residue. This was confirmed by the HMBC between the anomeric H-atom
resonating at d(H) 4.78 (d, J¼8.0) and C(3) (d(C) 88.9) of the aglycone. The coupling
constant (J¼8.0) of the anomeric H-atom indicated that the anomeric C-atom of the

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
771
d-xylosyl moiety was b-configured. Additionally, the H- and 13C-NMR spectra of 2
evidenced the presence of an Ac group (d(H) 2.12 (s), d(C) 21.2, 170.0). The HMBC
between HꢀC(2’) of the xylose (d(H) 4.16 (t, J¼7.0)) and the C¼O C-atom of the Ac
group indicated that the Ac group was located at C(2’) of the xylose [12]. Therefore, the
structure of 2 was established as 19a-hydroxyursolic acid-3-O-b-d-(2’-O-acetylxylopyr-
anoside), named ilexasprellanoside B.
1
Compound 3 was obtained as an amorphous powder, with a molecular formula
þ
C H O deduced from the [MþNa] ion peak at m/z 671.3765 (calc. 671.3771) in the
36
56 10
1
13
HR-ESI-MS. The H- and C-NMR spectra of 3 displayed many similarities with those
of 2 except for the sugar moiety, suggesting that 2 and 3 possessed the same aglycone.
The sugar moiety was elucidated as d-glucuronic acid (GluA) by comparison of the
NMR data of 3 with those of ilexpernoside D, a known saponin previously isolated from
I. asprella [13]. The b-linkage of the GluA moiety was determined by the J value
(
7.5 Hz) of the anomeric H-atom. In the HMBC spectrum of 3, the correlation between
the anomeric H-atom signal at d(H) 5.02 (d, J¼7.5) and C(3) (d(C) 89.1) indicated that
the GluA moiety was linked to C(3) of the agylcone. Accordingly, the structure of 3 was
established as 19a-hydroxyursolic acid 3-O-b-d-glucuronopyranoside, named ilexa-
sprellanoside C.
Compound 4 was obtained as an amorphous powder. The HR-ESI mass spectrum
þ
showed a [MþNa] ion peak at m/z 687.3713 corresponding to the molecular formula
1
C H O (calc. 687.3720). The H-NMR spectrum of 4 (Table 3) exhibited six Me
36
56 11
singlets at d(H) 1.59, 1.52, 1.11, 1.09, 0.96, and 0.93, and the signal of an olefinic H-atom
13
at d(H) 5.51 (br. s). The C-NMR spectrum of 4 displayed the signals attributed to two
C¼O groups (d(C) 180.6 and 177.2), two O-bearing CH groups (d(C) 75.3 and 80.8),
and two olefinic C-atoms (d(C) 123.3 and 144.2). The above evidences suggested that 4
possessed an olean-12-ene skeleton. The cross-peak Me(25) (d(H) 0.96 (s))/Me(24)
(
d(H) 1.59 (s)) in the NOESY spectrum and the HMBC between HꢀC(3) (d(H) 4.62
(dd, J¼10.0, 6.0)) and the C¼O C-atom (d(C) 180.6) suggested that the COOH group
was at C(23). Additionally, the NMR spectra of 4 revealed the presence of a glucosyl
moiety, which was confirmed by acid hydrolysis to yield glucose and ilexolic acid B [14].
The coupling constant of the anomeric H-atom (d(H) 6.28 (d, J¼7.5)) evidenced b-
configuration of the glucosyl moiety. In the HMBC spectrum, the correlation between
the anomeric H-atom and C(28) (d(C) 177.2) of the aglycone indicated that the glucosyl
moiety was attached to C(28). From the evidences mentioned above, the structure of
4
was established as 3b,19a-dihydroxyolean-12-en-23,28-dioic acid 28-O-b-d-gluco-
pyranoside, named ilexasprellanoside D.
Compound 5 was obtained as an amorphous powder. Its molecular formula was
þ
determined as C H O by the presence of [MþNa] ion peak at m/z 669.3957 (calc.
37
58
9
1
13
6
69.3979) in its HR-ESI-MS. The H- and C-NMR data of the aglycon moiety of 5
were superimposable with those of the aglycon part of the known compound
siaresinolic acid [15]. Comparison of the NMR data of 5 with those of 2 suggested
that a 2’-O-acetylxylosyl moiety was also present in 5. The linkage of the Ac group was
confirmed by the HMBC between HꢀC(2’) (d(H) 4.16 (t, J¼7.0)) and the C¼O C-atom
of the Ac group (d(C) 170.0). Acid hydrolysis yielded d-xylose and siaresinolic acid.
The HMBC between the anomeric H-atom at d(H) 4.78 (d, J¼8.0) and C(3) (d(C)
88.9) of the aglycone indicated that the xylosyl moiety was at C(3). The b-linkage of the

7
72
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 3. 1H-NMR Data (500 MHz; (D )pyridine) of 4–6. d in ppm, J in Hz.
5
Posi-
tion
4
5
6
Aglycone moiety
1
2
3
5
6
7
9
1
2
5
6
8
9
1
2
3
4
5
6
7
9
0
1.09–1.11 (m), 1.49–1.55 (m) 0.84–1.01 (m), 1.47–1.55 (m) 0.83–0.88 (m), 1.29–1.35 (m)
1.87–1.90(m), 2.02–2.08 (m) 1.98–2.07 (m), 2.05–2.09 (m) 1.85–1.91 (m), 2.01–2.07 (m)
4.62 (dd, J¼10.0, 6.0)
3.19 (dd, J¼12.0, 4.5)
3.37 (dd, J¼12.0, 4.5)
1.94–1.99 (m)
0.73–0.79 (m)
0.79–0.86 (m)
1.49–1.52 (m), 1.63–1.69 (m) 1.02–1.06 (m), 1.09–1.15 (m) 1.27–1.30 (m), 1.46–1.49 (m)
1.36–1.39 (m), 1.63–1.70 (m) 1.26–1.32 (m), 1.99–2.07 (m) 1.30–1.35 (m), 1.56–1.62 (m)
1.90–1.93 (m)
1.97–1.99 (m)
5.51 (br. s)
1.82–1.88 (m)
1.94–1.96 (m)
5.53 (t, J¼4.5)
1.74–1.80 (m)
1.90–1.94 (m)
5.51 (br. s)
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
1.08–1.11 (m), 2.24–2.29 (m) 1.18–1.23 (m), 2.09–2.11 (m) 1.10–1.13 (m), 2.02–2.07 (m)
1.95–1.98 (m), 2.25–2.29 (m) 1.29–1.32 (m), 1.47–1.55 (m) 2.12–2.17 (m), 2.27–2.33 (m)
3.47 (br. s)
3.54 (br. s)
3.61 (br. s)
3.58 (br. s)
3.59 (br. s)
3.58 (d, J¼4.5)
0.98–1.05 (m), 1.12–1.15 (m) 1.27–1.35 (m), 1.47–1.49 (m) 1.15–1.18 (m), 1.23–1.28 (m)
1.87–1.90 (m), 1.98–2.02 (m) 1.37–1.41 (m), 2.01–2.03 (m) 1.31–1.35 (m), 2.01–2.07 (m)
–
1.07 (s)
0.87 (s)
0.81 (s)
1.01 (s)
1.65 (s)
1.10 (s)
1.18 (s)
1.28 (s)
0.96 (s)
0.80 (s)
1.04 (s)
1.66 (s)
1.00 (s)
1.17 (s)
1.59 (s)
0.96 (s)
1.11 (s)
1.52 (s)
1.09 (s)
0.93(s)
3
-O-Sugar
1
2
3
4
5
’
’
’
’
’
–
–
–
–
–
4.78 (d, J¼8.0)
4.16 (t, J¼7.0)
5.01 (d, J¼7.0)
4.12 (t, J¼6.0)
4.32 (t, J¼8.5)
4.59 (t, J¼8.5)
4.68 (d, J¼7.5)
4.20 (t, J¼9.0)
4.19–4.23 (m)
4.28 (dd, J¼13.0, 5.5),
3
.73 (dd, 13.0, 5.5)
2
8-O-Sugar
1
2
3
4
5
6
’
’
’
’
’
’
6.28 (d, J¼7.5)
4.16 (t, J¼8.5)
3.98–4.01 (m)
4.27–4.28 (m)
4.32–4.39 (m)
–
–
–
–
–
–
4.33 (dd, J¼8.5, 4.5),
3
–
.40 (dd, 8.5, 4.5)
Ac
2.12 (s)
–
xylosyl moiety was established by the J value of the anomeric H-atom (J¼8.0). Thus,
the structure of 5 was established as 19a-hydroxyoleanolic acid 3-O-b-d-(2’-O-
acetylxylopyranoside), named ilexasprellanoside E.
The molecular formula of compound 6 was C H O as deduced from the [Mþ
3
6
56 10
þ
Na] ion peak at m/z 671.3765 (calc. 671.3771) in the HR-ESI-MS. Comparison of the
NMR data of 6 with those of 5 and 3 allowed elucidation of the structure of 6 as 19a-

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
773
hydroxyoleanolic acid 3-O-b-d-glucuronopyranoside, named ilexasprellanoside F. The
linkage between the GluA moiety and the aglycone was confirmed by the HMBC
between the anomeric H-atom at d(H) 5.01 (d, J¼7.0) and C(3) (d(C) 89.1) of the
aglycone.
The other eleven known compounds were identified as ursa-12,18-dien-28-oic acid
3
-O-b-d-arabinopyranoside [10], suavissimoside R1 [16], 3b-[(a-l-arabinopyranosyl)-
oxy]-19a-hydroxyurs-12-en-28-oic acid 28-b-d-glucopyranosyl ester [10], oleanolic acid
-O-b-d-glucuronopyranoside [17], 3-b-acetoxy-28-hydroxyurs-12-ene [18], pomolic
3
acid [11], 2b,3a-dihydroxyurs-12-en-28-oate [19], 3-O-[6’-O-palmitoyl-b-d-glucosyl]-
stigmasta-5,25(27)-diene [20], 3-O-[6’-O-stearoyl-b-d-glucosyl]stigmasta-5,25(27)-di-
ene [20], 3-O-b-d-glucosylstigmasta-5,25(27)-diene [8], and syringic acid [21], respec-
tively. They were isolated for the first time from this plant except 3-O-b-d-
glucosylstigmasta-5,25(27)-diene.
All the compounds obtained were evaluated for their inhibitory activities against
human tumor cell A549 with fluorouracil (5-FU) as a positive control. Compounds 2, 4,
oleanolic acid 3-O-b-d-glucuronopyranoside, 3-b-acetoxy-28-hydroxyurs-12-ene, po-
molic acid, and 5-Fu showed notable cytotoxic activities against human tumor cell line
A549 (IC50 values 1.87ꢁ0.13, 2.51ꢁ0.29, 1.41ꢁ0.16, 3.24ꢁ0.18, 5.63ꢁ0.76, and
28.96ꢁ1.13 mm, resp.).
The authors thank the National Natural Science Foundation of China (grant No. 30672608) for
financial support of this work, which was also supported by Program for the Significant New Formulation
of New Drugs (No. 2009ZX09311-004).
Experimental Part
General. Column chromatography (CC): silica gel H (SiO , 200–300 mesh; Qingdao Marine
2
Chemical Industry) and Sephadex LH-20 gel (Pharmacia). Semi-prep. HPLC: Waters 600 pump with 600
controller, Waters C18 Nova-Pak column (300ꢂ7.8 mm, 5 mm), with ELSD detector (Alltech; split ratio,
1
0%); flow rate, 2.5 ml/min. GC: Agilent 6890 N gas chromatograph; cap. column (28 mꢂ0.32 mm i.d.;
HP-5); FID detector, operated at 2608 (column temp. 1808); N as carrier gas (40 ml/min). Optical
2
rotations: Perkin-Elmer 243 B digital polarimeter. UV Spectra: TU-1901 spectrophotometer; l (log e)
max
in nm. IR Spectra: NEXUS-470 FTIR (Nicolet) spectrometer; KBr pellets; ˜n in cmꢀ1. NMR Spectra:
1
13
Varian Inova-500 and Varian Unity-500 instruments; at 500 ( H) or 125 MHz ( C) in C D N at r.t.; d in
5
5
ppm rel. to Me Si as internal standard, J in Hz. ESI-MS (pos.): QSTAR (ABI, USA) mass spectrometer;
4
in m/z. HR-ESI-MS (pos.): Bruker APEX II FT-ICRMS mass spectrometer; in m/z.
Plant Material. The roots of Ilex asprella (Hook et Arn.) Champ. ex Benth were collected in
August 2006 from Guangdong Province, P. R. China. The plant was identified by one of the authors (P.-
F. T. ). A voucher specimen (CM200608) was deposited with the Herbarium of Peking University,
Modern Research Center for Traditional Chinese Medicine.
Extraction and Isolation. The dried roots (20 kg) of I. asprella were extracted with 70% EtOH (160 l,
2
h, 788) for three times. After removal of the solvent under reduced pressure at 608, the residue (1.7 kg)
was suspended in H O, and defatted with petroleum ether (PE). The aq. layer was further extracted with
2
AcOEt (3ꢂ1 l) and BuOH (3ꢂ1 l), successively, to provide the AcOEt (170 g) and BuOH extract
(
700 g). BuOH extract (500 g) was subjected to CC (D101 porous polymer resin; H O and 10, 30, 50, 70,
2
9
5% aq. EtOH, resp.). The fractions eluted with 50% EtOH (90 g) and 70% EtOH (65 g) had similar
compositions. They were pooled, and a portion (100 g) of the combined fraction was subjected to CC
SiO ; CHCl /MeOH/H O 8 :1:0!1:1:0.1) to afford four subfractions, Frs. 1–4. Fr. 1 (8 g) was
(
2
3
2
subjected to CC (SiO ; CHCl /MeOH 10 :1) to afford 2b,3a-dihydroxyurs-12-en-28-oate (9 mg). Fr. 2
2
3

7
74
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
(
25 g) was separated by CC (SiO ; CHCl /MeOH 10 :1) to afford Frs. 2.1 and 2.2. Fr. 2.2 was purified by
2
3
CC (ODS; MeOH/H O 1:3!3 :1) to afford Frs. 2.2.1 and 2.2.2. Fr. 2.2.1 was subjected to semiprep.
2
HPLC (MeOH/H O 55 :45) to yield compound 1 (t 20.5 min, 9.0 mg) and ursa-12,18-dien-28-oic acid 3-
2
R
O-b-d-arabinopyranoside (t 18.5 min; 5.0 mg). Fr. 2.2.2 was subjected to semiprep. HPLC (MeOH/H O
R
2
4
5 :55) to yield compounds 2 (tR 11.5 min; 6.0 mg) and 5 (tR 16 min; 5 mg). Fr. 3 (10 g) was subjected to
CC (SiO ; CHCl /MeOH 100 :1!100 :5) to afford pomolic acid (23 mg). A portion of the AcOEt
2
3
extract (130 g) was subjected to CC (SiO ; CHCl /MeOH 100 :0!100 :15) to afford six subfractions,
2
3
Frs. 1–6. Fr. 1 (5 g) was submitted to CC (SiO ; CHCl /MeOH 100 :10!100 :20) to afford 3b-acetoxy-
2
3
2
8-hydroxyurs-12-ene (9 mg). Fr. 2 (5 g) was subjected to CC (SiO ; CHCl /MeOH 100 :1) and
2 3
semiprep. HPLC (MeOH/H O 1:1) to afford syringic acid (t 11.5 min; 7 mg). Fr. 3 (10 g) was subjected
2
R
to CC (SiO ; CHCl /MeOH 100 :10!100 :20) to afford Frs. 3.1 and 3.2. Fr. 3.2 was separated by CC
2
3
(
Sephadex LH-20; H O) and semiprep. HPLC (MeOH/H O 1:1) to afford 3-O-[6’-O-palmitoyl-b-d-
2 2
glucosyl]stigmasta-5,25(27)-diene (tR 15 min; 10.0 mg) and 3-O-[6’-O-stearoyl-b-d-glucosyl]stigmasta-
,25(27)-diene (tR 17 min; 10.0 mg). Fr. 4 (13 g) was subjected to CC (SiO ; CHCl /MeOH 100 :10) to
5
2
3
afford 3-O-b-d-glucosylstigmasta-5,25(27)-diene (32 mg). Fr. 5 (15 g) was subjected to CC (ODS;
MeOH/H O 2 :1) to afford oleanolic acid 3-O-b-d-glucuronopyranoside (5 mg), 3b-[(a-l-arabinopyr-
2
anosyl)oxy]-19a-hydroxyurs-12-en-28-oic acid 28-b-d-glucopyranosyl ester (8 mg) and Fr. 5.1. Fr. 5.1 was
purified by CC (Sephadex LH-20; MeOH/H O 1:1) and semiprep. HPLC (MeOH/H O 43 :57) to afford
2
2
compounds 3 (t 8 min; 6.0 mg) and 6 (t 13 min; 5.0 mg). Fr. 6 (18 g) was subjected to CC (SiO ; CHCl /
R
R
2
3
MeOH 100 :3!100 :15) to afford Frs. 6.1 – 6.3. Fr. 6.1 was separated by CC (SiO ; CHCl /MeOH 100 :8)
2
3
to afford compound 4 (15.0 mg) and suavissimoside R1 (18 mg).
Ilexasprellanoside A (¼ 3-(b-d-Xylopyranosyloxy)ursa-12,18-dien-28-oic Acid; 1). Amorphous
2
6
powder. [a] ¼ þ110.0 (c¼0.96, MeOH). UV (MeOH): 227 (3.79). IR: 3445, 2935, 2874, 1697,1647,
D
1
13
1
6
454, 1388, 1372, 1275, 1203, 1166, 1074, 1044. H- and C-NMR: see Tables 1 and 2, resp. HR-ESI-MS:
þ
þ
7
09.3771 ([MþNa] , C H NaO ; calc. 609.3762).
35
54
Ilexasprellanoside B (¼ 3-[(2-O-Acetyl-b-d-xylopyranosyl)oxy]-19-hydroxyurs-12-en-28-oic Acid;
2
6
2
1
). Amorphous powder. [a] ¼ þ1.67 (c¼0.12, MeOH). IR: 3419, 2927, 2874, 1697, 1730, 1631, 1453,
D
1
13
384, 1318, 1261, 1111, 1072. H- and C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 669.3967 ([Mþ
þ
þ
Na] , C37
H58NaO
9
; calc. 669.3979).
Ilexasprellanoside C (¼(3b)-19,28-Dihydroxy-28-oxours-12-en-3-yl b-d-Glucopyranosiduronic
2
6
Acid; 3). Amorphous powder. [a] ¼ þ4.1 (c¼0.48, MeOH). IR: 3444, 2937, 2877, 1693, 1455, 1388,
D
1
13
þ
1
205, 1165, 1090. H- and C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 671.3765 ([MþNa] ,
þ
C H NaO ; calc. 671.3771).
36
56
10
Ilexasprellanoside D (¼1-O-[(19a)-3,19,23-Trihydroxy-23,28-dioxoolean-12-en-28-yl]-b-d-gluco-
2
6
pyranose; 4). Amorphous powder. [a] ¼ þ45.0 (c¼0.24, MeOH). IR: 3426, 2933, 2877, 1709, 1622,
D
1
13
1
6
452, 1388, 1262, 1231, 1165, 1075, 1030. H- and C-NMR: see Tables 3 and 2, resp. HR-ESI-MS:
þ
þ
11
87.3713 ([MþNa] , C H NaO ; calc. 687.3720).
36
56
Ilexasprellanoside E (¼(19a)-3-[(2-O-Acetyl-b-d-xylopyranosyl)oxy]-19-hydroxyolean-12-en-28-
oic Acid; 5). Amorphous powder. [a]2 ¼ þ110.7 (c¼0.94, MeOH). IR: 3419, 2927, 2874, 1697,1730,
6
D
1
13
1
6
631, 1453, 1384, 1318, 1261, 1111, 1072. H- and C-NMR: see Tables 3 and 2, resp. HR-ESI-MS:
þ
þ
9
69.3967 ([MþNa] , C H NaO ; calc. 669.3979).
37
58
Ilexasprellanoside F (¼(3b,19a)-19,28-Dihydroxy-28-oxoolean-12-en-3-yl b-d-Glucopyranosidur-
2
6
onic Acid; 6). Amorphous powder. [a]
1
C H NaO10 ; calc. 671.3771).
D
¼ þ4.1 (c¼0.24, MeOH). IR: 3444, 2937, 2877, 1693, 1455, 1388,
1
13
þ
205, 1165, 1090. H- and C-NMR: see Tables 3 and 2, resp. HR-ESI-MS: 671.3765 ([MþNa] ,
þ
36
56
Acid Hydrolysis and Determination of Sugar Components. Compounds 1, 2, 4, and 5 (2 mg) were
hydrolyzed with 2n aq. CF COOH (10 ml) at 1108 for 8 h in a sealed tube. The mixture was diluted with
3
H O (20 ml) and extracted with AcOEt (3ꢂ10 ml).
2
The aq. layer was concentrated under reduced pressure and analyzed by TLC (CHCl /MeOH/H O
3
2
8
:5 :1). Spots were visualized by spraying with 95% EtOH/H SO /anisaldehyde 9 :0.5 :0.5, then heated
2 4
at 1208 for 10 min. The R values of glucose and xylose were 0.40 and 0.56, resp. For the determination of
f
the absolute configuration, the aq. layer was completely dried and dissolved in anh. pyridine (100 l), l-
cysteine methyl ester hydrochloride (0.1m, 200 ml) was added, and the mixture was warmed at 608 for 1 h.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
775
The trimethysilylation reagent HMDS (hexamethyldisilazane)/TMCS (Me SiCl)/pyridine 2 :1 :10
3
(Acros Organics, Belgium) was then added, and the soln. was incubated at 608 for further 30 min. The
thiazolidine derivatives were analyzed by GC for sugar identification. d-Glucose (tR 11.25 min) and d-
xylose (tR 5.32 min).
Cytotoxicity Assay. Human tumor cell line A549 was maintained in RPMI 1640 medium (Thermo
Fisher) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin.
ꢀ
2
2
Cells were cultured in 96-well microtiter plates for the assay. Appropriate dilutions (10 to 10 mm) of
the test compounds were added to the cultures. After 72 h incubation in humidified air containing 5%
CO at 378, growth inhibition of cancer cells was evaluated by the MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-
2
2
,5-diphenyl-2H-tetrazolium bromide) method [22]. Results are expressed as the mean value of triplicate
data points. 5-FU was used as the positive control.
REFERENCES
1] Compiler group on compilation of Chinese traditional and herbal drugs, compilation of Chinese
traditional and herbal drugs; Peopleꢀs health publisher. Beijing, 1994, p. 449.
2] L. Wang, Y. Cai, X.-Q. Zhang, C.-L. Fan, Q.-W. Zhang, X.-P. Lai, W.-C. Ye, Carbohydrate Res. 2012,
[
[
349, 39.
[
[
[
[
3] W.-J. He, Z.-X. Zhao, Z.-Z. Lin, C.-C. Zhu, Hua Xi Pharm. J. 2012, 27, 51.
4] J.-L. Wen, X.-L. Chen, R.-Y. Mo, L.-B. Guo, J. Guangdong Pharm. Univ. 2011, 27, 468.
5] J.-C. Huang, F.-L. Chen, Y.-E. Zeng, P. Cao, H.-H. Xu, Chin. J. Mag. Res. 2011, 28, 142.
6] Y. Cai, Q.-W. Zhang, Z.-J. Li, C.-L. Fan, L.Wang, X.-Q. Zhang, W.-C. Ye, Chin. Trad. Herb. Drugs
2010, 41, 1426.
[
[
[
7] H.-L. Wang, L.-J. Wu, Y. Lei, S.-X. Zhou, P.-F. Tu, J. Shenyang Pharm. Univ. 2009, 26, 279.
8] M.-H. Li, S.-J. Du, S.-J. Yu, Chin. Trad. Herb. Drugs 1997, 28, 454.
9] Y. Kashiwada, D.-C. Zhang, Y.-P. Chen, C.-M. Cheng, H.-T. Chen, H.-C. Chang, J. Nat. Prod. 1993,
5
6, 2077.
10] Y. Mimaki, M. Fukushima, A. Yokosuka, Y. Sashida, S. Furuya, H. Sakagami, Phytochemistry 2001,
7, 773.
[
5
[
[
[
[
[
[
[
[
[
[
[
[
11] P. Liu, H.-Q. Duan, Q. Pan, Y.-W. Zhang, Z. Yao, China J. Chin. Materia Med. 2006, 31, 1875.
12] N.-Q. Zhu, Y. Jiang, M.-F. Wang, C.-T. Ho, J. Nat. Prod. 2001, 64, 627.
13] G.-B. Xie, J. Zheng, P.-F. Tu, G.-Q. Liu, Helv. Chim. Acta. 2008, 91, 1630.
14] K. Amimoto, K. Yoshikawa, S. Arihara, Phytochemistry 1993, 33, 1475.
15] W.-J. He, Z.-X. Zhao, Z.-Z. Lin, C.-Y. Zhu, West Chin. J. Pharm. 2012, 27, 51.
16] S.-H. Du, W.-Y. Liu, J.-H. Rao, J. Bai, Chin. Trad. Herb. Drugs 2005, 36, 348.
17] J. Zhang, W. Li, H.-Y. Gao, B. Wu, L.-J. Wu, J. Shenyang Pharm. Univ. 2005, 22, 183.
18] M. Katai, T. Terai, H. Meguri, Chem. Pharm. Bull 1983, 31, 1567.
19] H. Kojima, H. Ogura, Phytochemistry 1989, 28, 1703.
20] A.-P. Guevara, C.-Y. Lim-Sylianco, F.-M. Dayrit, P. Finch, Phytochemistry 1989, 28, 1721.
21] S. Inoshiri, M. Sasaki, H. Kohda, H. Otsuka, K. Yamasaki, Phytochemistry 1987, 26, 2811.
22] J.-M. Sargent, C.-G. Taylor, Br. J. Cancer 1989, 60, 206.
Received May 13, 2013