Two antiproliferative triterpene saponins from Nematostylis anthophylla from the highlands of central madagascar

Article abstract of DOI:10.1002/cbdv.201200156

Investigation of the endemic Madagascan plant Nematostylis anthophylla (Rubiaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the known triterpene saponin randianin (1), and the two new bioactive trit

Full text of DOI:10.1002/cbdv.201200156

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
233
Two Antiproliferative Triterpene Saponins from Nematostylis anthophylla
from the Highlands of Central Madagascar1)
by Yumin Daia), Liva Harinantenainaa), Peggy J. Brodiea), Chris Birkinshawb),
Richard Randrianaivob), Wendy Applequistc), Michel Ratsimbasond), Vincent E. Rasamisond),
Yongchun Shene), Karen TenDykee), and David G. I. Kingston*a)
a) Department of Chemistry and Virginia Tech Center for Drug Discovery, M/C 0212, Virginia Tech,
Blacksburg, Virginia 24061, USA (phone: þ1-540-231-6570; e-mail: dkingston@vt.edu)
b) Missouri Botanical Garden, B.P. 3391, Antananarivo 101, Madagascar
c) Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166–0299, USA
d) Centre National dꢀApplication des Recherches Pharmaceutiques, B.P. 702, Antananarivo 101,
Madagascar
e) Eisai Inc., 4 Corporate Drive, Andover, Massachusetts 01810, USA
Investigation of the endemic Madagascan plant Nematostylis anthophylla (Rubiaceae) for
antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the known
triterpene saponin randianin (1), and the two new bioactive triterpene saponins 2’’-O-acetylrandianin (2)
and 6’’-O-acetylrandianin (3). The structures of the two new compounds were elucidated based on
analysis of their 1D- and 2D-NMR spectra, and mass spectrometric data. The three isolated triterpene
saponins displayed moderate but selective antiproliferative activities, with IC50 values of 1.2, 1.7, and
2.2 mm, respectively, against the A2780 ovarian cancer, but only weak inhibitions of the proliferation of
A2058 melanoma and the H522 lung cancer cell lines.
Introduction. – As part of our engagement in an International Cooperative
Biodiversity Group (ICBG) program, we are focusing on the search for antiprolifer-
ative natural products from a diversity of vegetation types in Madagascar [1–3]. The
A2780 human ovarian cancer cell line is used as the primary screen, because it is a
stable and yet relatively drug-sensitive cell line and gives reproducible results. As a part
of this research, an EtOH extract from the roots of Nematostylis anthophylla
(Rubiaceae) from the Highlands of Central Madagascar was investigated and found
to exhibit antiproliferative activity against the A2780 cell line, with an IC50 value of 6.9
mg/ml. The Rubiaceae is a large family of 630 genera and ca. 13,000 species found
worldwide [4]. This family is a rich source of indole alkaloids, terpenoids, and
anthraquinones, all of which are well-known for their broad range of bioactivity,
including antimicrobial, antimalarial, antidiabetic, vasorelaxant, cytotoxic, antioxidant,
and anti-inflammatory activities among others [5–9]. Since Nematostylis is one of the
many genera of the Rubiaceae that have not been systematically investigated for their
phytochemical composition, the EtOH extract of N. anthophylla was selected for
bioassay-guided fractionation to isolate its active components.
1
)
ꢃBiodiversity Conservation and Drug Discovery in Madagascarꢀ, Part 52. For Part 51, see [1].
ꢁ 2013 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Results and Discussion. – Isolation of Bioactive Compounds. An EtOH extract of
the roots of N. anthophylla was subjected to liquidꢀliquid partitioning to give an active
BuOH fraction with an IC50 value of 2.2 mg/ml. Bioassay-guided separation, including
LH-20 size-exclusion, HP-20 Diaion, and silica-gel normal-phase chromatography, was
used to obtain three bioactive compounds comprising the known triterpene saponin
randianin (1), and the two new related glycosides 2’’-O-acetylrandianin (2) and 6’’-O-
acetylrandianin (3). All three compounds had moderate antiproliferative activities
against A2780 ovarian cancer cells, with IC50 values of 2.2, 1.2, and 1.7 mm, respectively.
Herein, we report the structure elucidation and antiproliferative properties of the two
new isolates.
Identification of Compounds 1 and 2. Compound 1 was identified as randianin
(¼oleanolic acid 3-O-b-d-glucopyranosyl-(1!3)-b-d-glucopyranoside) by compari-
son of its chemical and spectroscopic data with those reported in the literature for the
aglycone [10] and the glycoside [11].
Compound 2, [a]2D1 ¼ þ12 (c¼1.2, MeOH), was isolated as a light-yellow solid. Its
positive-ion HR-ESI-MS exhibited cationized molecular-ion peaks at m/z 845.4692
([MþNa]þ ) and 861.4618 ([MþK]þ ), corresponding to the molecular formula
C44H70O14. The observation of a C¼O absorption at 1734 cmꢀ1 in the IR spectrum, a
13C-NMR resonance at d(C) 170.7 ppm, and a singlet signal at d(H) 1.98 ppm in the
1H-NMR spectrum (Table 1) suggested the presence of an Ac group. Meanwhile, its
glycosidic nature was corroborated by the presence of two anomeric H-atom signals at
d(H) 4.83 and 5.43 ppm. In addition to the Me and C¼O C-atoms of the Ac group, there
were 42 C-atom signals in the 13C-NMR spectrum, among which 30 C-atom signals were
assigned to a triterpenoid aglycone and the remaining 12 C-atoms to a disaccharide
moiety. The 1H-NMR spectrum of 2 indicated that the aglycone had seven Me groups
corresponding to 3-H singlets at d(H) 0.80, 0.89, 0.97, 1.00, 1.03, 1.27 and 1.33, and one
olefinic H-atom signal appeared at d(H) 5.49. Correspondingly, signals for seven Me C-
atoms at d(C) 15.8, 17.2, 17.8, 24.1, 26.6, 28.5 and 33.7 ppm, and for two olefinic C-atoms
at d(C) 122.9 and 145.2 ppm were observed in the 13C-NMR spectrum. The presence of
a C¼O absorption at 1689 and a broad OH absorption at 3453 cmꢀ1 in its IR spectrum,
together with a 13C-NMR resonance at d(C) 180.6 ppm, supported the presence of a
carboxylic acid group.
Inspection of the 1H- and 13C-NMR spectra of compound 2 indicated that it had the
same oleanolic acid aglycone as compound 1. The HMB correlation between HꢀC(18)
(dd, J¼4.1, 14.0) and C(28) confirmed that the carboxylic C-atom was connected to
C(17) [12]. HMBCs between the anomeric HꢀC(1’) and C(3), as well as between

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
235
Table 1. 1H- and 13C-NMR Data for 1–3. Recorded in (D5)pyridine at 500 and 125 MHz, resp.; d in ppm,
J in Hz.
Position
1
2
3
d(H)
d(C) d(H)
d(C) d(H)
d(C)
1
2
1.21–1.25 (m),
1.39–1.42 (m)
1.75–1.78 (m),
2.14–2.18 (m)
39.0 1.21–1.25 (m),
1.37–1.40 (m)
26.8 1.76–1.79 (m),
2.15–2.19 (m)
39.0 1.22–1.26 (m),
1.39–1.42 (m)
26.9 1.74–1.77 (m),
2.14–2.18 (m)
39.0
26.8
3
4
5
6
3.36 (dd, J¼4.4, 11.9) 89.3 3.36 (dd, J¼4.4, 11.9) 89.3 3.36 (dd, J¼4.4, 11.7)
89.4
40.1
56.1
18.8
–
40.1
–
40.1 –
0.76–0.80 (m)
1.21–1.25 (m),
1.45–1.49 (m)
1.78–1.82 (m),
1.85–1.87 (m)
–
56.1 0.76–0.79 (m)
18.8 1.22–1.25 (m),
1.46–1.50 (m)
33.6 1.78–1.82 (m),
1.85–1.87 (m)
56.1 0.78–0.82 (m)
18.8 1.23–1.26 (m),
1.46–1.50 (m)
33.6 1.78–1.82 (m),
1.85–1.87 (m)
7
33.6
8
9
39.8
48.3 1.64 (br. t, J¼8.9)
37.3
–
39.8
48.4 1.65 (br. t, J¼8.9)
37.3
–
39.8
48.3
37.3
1.65 (br. t, J¼8.9)
10
11
12
13
14
15
–
–
–
1.88–1.92 (m)
5.50 (t, J¼3.3)
–
24.1 1.88–1.92 (m)
122.8 5.49 (t, J¼3.3)
24.1 1.88–1.92 (m)
122.9 5.50 (t, J¼3.3)
24.1
122.8
145.3
42.5
145.3
42.5
–
–
145.2
42.6
–
–
–
1.18–1.21 (m),
2.02–2.05 (m)
1.76–1.79 (m),
2.18–2.21 (m)
–
28.7 1.18–1.21 (m),
2.02–2.05 (m)
24.1 1.75–1.78 (m),
2.17–2.20 (m)
28.7 1.18–1.21 (m),
2.02–2.05 (m)
24.1 1.76–1.79 (m),
2.18–2.21 (m)
28.7
16
24.1
17
18
19
47.0
–
47.1
–
47.0
42.4
46.9
3.32 (dd, J¼4.1, 14.0) 42.4 3.32 (dd, J¼4.1, 14.0) 42.4 3.32 (dd, J¼4.0, 13.9)
1.28–1.31 (m),
1.82–1.84 (m)
–
46.9 1.28–1.31 (m),
1.82–1.84 (m)
46.9 1.28–1.31 (m),
1.82–1.84 (m)
20
21
31.3
–
31.3
–
31.3
34.6
1.49–1.52 (m),
1.82–1.84 (m)
1.45–1.49 (m),
2.05–2.08 (m)
1.27 (s)
34.6 1.49–1.52 (m),
1.82–1.84 (m)
33.6 1.45–1.49 (m),
2.05–2.08 (m)
17.8 1.27 (s)
34.6 1.49–1.52 (m),
1.82–1.84 (m)
33.6 1.46–1.50 (m),
2.05–2.08 (m)
17.8 1.32 (s)
22
33.5
23
24
25
26
27
28
29
30
17.8
28.5
15.8
17.3
26.5
180.7
24.1
33.6
0.89 (s)
28.5 0.89 (s)
28.5 1.01 (s)
0.82 (s)
15.8 0.80 (s)
15.8 0.82 (s)
1.00 (s)
17.4 1.00 (s)
17.2 1.00 (s)
1.33 (s)
26.5 1.33 (s)
26.6 1.33 (s)
–
180.7
–
180.6 –
1.03 (s)
0.97 (s)
24.1 1.03 (s)
33.7 0.97 (s)
24.1 1.02 (s)
33.7 0.97 (s)
3-O-Glucosyl
1’
2’
3’
4’
5’
6’
4.91 (d, J¼7.8)
106.7 4.83 (d, J¼7.8)
74.8 3.96–4.02 (m)
89.3 4.15 (t, J¼8.8)
70.2 4.04–4.08 (m)
78.3 3.89–3.93 (m)
62.9 4.26 (d, J¼11.4),
4.48 (dd, J¼2.1, 11.5)
107.1 4.91 (d, J¼7.6)
74.4 4.05–4.08 (m)
89.3 4.18 (t, J¼8.9)
70.7 4.11 (t, J¼9.3)
78.1 3.92–3.98 (m)
106.7
74.7
89.7
70.0
78.3
4.09–4.11 (m)
4.23 (t, J¼8.8)
4.11–4.14 (m)
3.92–3.98 (m)
4.32 (d, J¼11.3),
4.51 (d, J¼11.0)
63.1 4.32 (dd, J¼6.2, 11.8), 62.9
4.52 (dd, J¼2.2, 11.8)

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Table 1 (cont.)
Position
1
2
3
d(H)
d(C) d(H)
d(C) d(H)
d(C)
3’-O-Glucosyl
1’’
2’’
3’’
4’’
5’’
6’’
5.32 (d, J¼7.8)
106.3 5.43 (d, J¼8.1)
75.9 5.66 (dd, J¼8.1, 9.1)
75.8 4.31 (t, J¼9.1)
72.0 4.20 (t, J¼9.2)
79.1 4.07–4.10 (m)
62.8 4.28 (d, J¼11.1),
4.58 (dd, J¼2.1, 11.5)
103.7 5.25 (d, J¼7.9)
75.7 4.03–4.05 (m)
76.6 4.22 (t, J¼9.1)
72.3 4.01 (t, J¼9.1)
79.1 4.23–4.26 (m)
62.7 4.67 (dd, J¼6.8, 11.7),
4.95 (dd, J¼2.2, 11.8)
106.3
75.7
75.7
71.9
78.3
64.9
4.02–4.05 (m)
4.26 (t, J¼9.1)
4.20 (t, J¼9.2)
4.07–4.09 (m)
4.34 (d, J¼11.1),
4.56 (d, J¼10.7)
2’’-AcO
CO
Me
170.7
21.5
1.98 (s)
6’’-AcO
CO
Me
171.2
21.0
2.00 (s)
Figure. HMBC (H!C), COSY (H$H), and NOESY (H ꢀ !H) correlations of 2 (a) and 3 (b)
HꢀC(3) and the anomeric C(1’), confirmed that the disaccharide moiety was connected
to C(3) (Fig., a).
Both sugar molecules, which were represented by the of two sets of anomeric atom
signals at d(H) 4.83/d(C) 107.1 ppm and d(H) 5.43/d(C) 103.7 ppm, respectively, were
identified as glucose, based on the similarity of their 13C-NMR chemical shifts with
those of the sugar moiety of 1. The linkage between the two glucopyranosyl units was
determined as 1!3 on the basis of HMBCs between HꢀC(3’) and the two anomeric C-
atoms C(1’) and C(1’’), as well as the cross-peak between HꢀC(3’) and HꢀC(2’) in the

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
237
COSY spectrum (Fig., a). The coupling constants between HꢀC(1’) and HꢀC(2’), and
HꢀC(1’’) and HꢀC(2’’) (J¼7.8 and 8.1, resp.) indicated their axialꢀaxial conformation,
and thus the b-configuration of the two sugar units. The AcO group was deduced to be
1
located at C(2’’) of a glucopyranosyl residue, based on the comparison of the H- and
13C-NMR spectra of 2 with those of 1. Due to this acetylation, the chemical shift of
HꢀC(2’’) of 2 was d(H) 5.66 as compared to d(H) 4.02–4.05 for 1, while other H-atoms
in the distal glucose had chemical shifts similar to those of compound 1. The position of
the Ac group was confirmed by the COSY cross-peak between the downfield HꢀC(2’’)
and the corresponding anomeric HꢀC(1’’), and a three-bond HMBC between HꢀC(2’’)
and the C¼O C-atom of the Ac group (Fig.,a).
To determine the absolute configuration of the two glucose moieties and to confirm
the overall structure assignment, compound 2 was hydrolyzed with 6m NH4OH to yield
a product identified as randianin (1) by its 1H- and 13C-NMR spectra. Further
hydrolysis of 1 with 3m HCl yielded oleanolic acid, identified by its 1H- and 13C-NMR
spectra, and a single monosaccharide, identified as d-glucose on the basis of a single
TLC spot observed with the same Rf value as a d-glucose standard. Its absolute
configuration was determined as d based on its positive optical rotation.
Based on these evidences, the structure of 2 was elucidated as oleanolic acid 3-O-b-
d-glucopyranosyl-(1!3)-(2’’-O-acetyl)-b-d-glucopyranoside, or 2’’-O-acetylrandianin.
Identification of Compound 3. Compound 3, isolated as light-yellow solid, [a]2D1 ¼
þ17 (c¼1.2, MeOH), had the same molecular formula as compound 2 as determined
by HR-ESI-MS (m/z 845.4643 ([MþNa]þ ) and 861.4569 ([MþK]þ )), corresponding
to the molecular formula of C44H70O14. Due to the similarity of its NMR data with those
of compounds 1 and 2, the aglycone portion of 3 was also assigned as oleanolic acid,
with the disaccharide moiety connected to C(3) of the aglycone.
As in compound 2, the presence of two sugar moieties was evidenced by the NMR
spectra, which showed two sets of anomeric-atom signals at d(H) 4.91/d(C) 106.7 and
d(H) 5.25/d(C) 106.3, respectively. The two sugar moieties were determined as
glucosyls, as corroborated by the similarity of the 13C-NMR chemical shifts of all C-
atoms compared to those of compound 1. The linkage between the two glucopyranosyl
units was determined as 1!3 on the basis of the HMBCs between HꢀC(3’) and two
anomeric C-atoms (C(1’) and C(1’’)), as well as the cross-peak between HꢀC(3’) and
HꢀC(2’) in the COSY spectrum (Fig.,b). The coupling constants between HꢀC(1’) and
HꢀC(2’), and HꢀC(1’’) and HꢀC(2’’) (J ¼ 7.8 and 7.8, resp.) indicated their axialꢀaxial
orientation and thus the b-configuration of the two sugar units. The presence of an Ac
group was evidenced by a C¼O absorption at 1727 cmꢀ1 in its IR spectrum, 13C-NMR
resonances at d(C) 171.2 ppm, and a singlet signal at 2.00 ppm in its 1H-NMR spectrum.
The 6’’-OH group of the outer glucose moiety of 3 was acetylated, insted of the 2’’-OH
group of 2. This was established by comparing the NMR data of the outer glucose
moiety of 3 with those of 1. The chemical shift of the two diastereotopic H-atoms,
CH2(6’’), of 1 were shifted from d(H) 4.34 and 4.56 ppm to d(H) 4.67 and 4.95 ppm in 3,
while the resonances of the other H-atoms of the outer glucose moiety were similar to
those of compound 1. Furthermore, the location of the AcO group at C(6’’) was
confirmed by the COSY cross-peak between CH2(6’’) and HꢀC(5’’), and a three-bond
HMBCs between CH2(6’’) and the Ac C¼O C-atom at 171.2 ppm, and between
HꢀC(5’’) and the anomeric C(1’’) (Fig.,b).

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
As with compound 2, the absolute configuration of the two glucose units and the
overall structure assignment were confirmed by successive basic hydrolysis of 3 to
randianin, followed by acidic hydrolysis to oleanolic acid and d-glucose. Based on this
evidence, the structure of 3 was elucidated as oleanolic acid 3-O-b-d-glucopyranosyl-
(1!3)-(6’’-O-acetyl)-b-d-glucopyranoside, or 6’’-O-acetylrandianin.
Biological Evaluation. Compounds 1–3 were tested for their antiproliferative
activities against the A2780 ovarian cancer, the A2058 melanoma, and the H522 lung
cancer cell lines. All three compounds showed modest inhibitions of the proliferation of
A2780 ovarian cancer cells, with IC50 values in the low micromolar range. However,
they showed only weak inhibition of the proliferation of A2058 melanoma and the
H522 lung cancer cell lines (Table 2). Several hundred cytotoxic triterpene saponins
have been identified from plants, but only a few of them showed selective
antiproliferative activity [13]. 2’’-O-Acetylrandianin (2) and 6’’-O-acetylrandianin
(3) are examples of compounds that selectively inhibit the proliferation of A2780
ovarian cancer cells. Furthermore, in the A2780 assay, the cytotoxicities of the two
acetylated saponins are stronger than that of randianin (1), which has no Ac group in its
structure. This suggests that the increase in activity on acetylation may be due to an
increase in lipophilicity, facilitating cellular uptake [14].
Table 2. Antiproliferative Activities (IC50 [mm]) of Compounds 1–3
Compound
A2780a)
A2058
H522
7.32
>10
>10
ND
1
2
3
2.2ꢁ0.2
1.7 ꢁ0.1
1.2 ꢁ0.3
0.028ꢁ0.003
ND
7.63
>3.3, <10
>3.3, <10
NDb)
Paclitaxel
Vinblastine
0.004
0.009
a) Mean of three replicates. b) ND, Not determined.
This project was supported by the Fogarty International Center, the National Cancer Institute, the
National Science Foundation, the National Heart, Lung and Blood Institute, the National Institute of
Mental Health, the Office of Dietary Supplements, and the Office of the Director of NIH, under
Cooperative Agreement U01 TW000313 with the International Cooperative Biodiversity Groups. This
project was also supported by the National Research Initiative of the Cooperative State Research,
Education and Extension Service, USDA, Grant #2008-35621-04732. Funds for the plant collection and
extraction were provided by Dow AgroSciences and Eisai Inc. This support is gratefully acknowledged.
This work was also supported by the National Science Foundation under Grant No. CHE-0619382 for
purchase of the Bruker-Avance 500 NMR spectrometer and Grant No. CHE-0722638 for the purchase of
the Agilent 6220 mass spectrometer. We thank Mr. Bill Bebout for recording the mass spectra, Dr. Hugo
Azurmendi for assistance with the NMR spectra, and Drs. Donald Hahn (Dow AgroSciences) and
Edward Suh (Eisai Inc.) for their encouragement and support. Field work essential for this project was
conducted under a collaborative agreement between the Missouri Botanical Garden and the Parc
Botanique et Zoologique de Tsimbazaza, and a multilateral agreement between the ICBG partners,
including the Centre National dꢀApplications des Recherches Pharmaceutiques. We gratefully acknowl-
`
ˆ
edge courtesies extended by the Government of Madagascar (Ministere des Eaux et Forets).

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
239
Experimental Part
General. Optical rotations: Jasco P-2000 polarimeter. IR Spectra: MIDAC M-series FT-IR
spectrophotometer as a film; ˜n in cmꢀ1. NMR Spectra: in (D5)pyridine on a Bruker Avance 500
spectrometer; chemical shifts d in ppm, and coupling constants J in Hz. MS: Agilent 6220 LC-TOF-MS in
the pos.-ion mode; m/z.
Antiproliferative Bioassays. Antiproliferative activities were evaluated at Virginia Polytechnic
Institute and State University against the drug-sensitive A2780 human ovarian cancer cell line as
described in [15]. The values reported are the mean of three replicates. Antiproliferative activities
against the A2058 melanoma and the H522 lung cancer cell lines were determined at Eisai Inc. by similar
procedures to those used for the H460 cell line [16].
Plant Materials. A sample of the roots of Nematostylis anthophylla (A.Rich.) Baill. was collected in
March 2011. The sample was a shrub of 60 cm with red flowers and succulent leaves, growing in rocky
habitat on Ibity Massif in the Vakinakaratra region of the Antsirabe II district, Madagascar at an
elevation of 1650 m, and coordinates 20803’59’’S 047800’01’’E (ꢀ20.0663889, 47.0002778). Duplicate
voucher specimens (Richard Randrianaivo et al. 1803) have been deposited with the Parc Botanique et
Zoologique de Tsimbazaza (TAN), the Centre National dꢀApplication des Recherches Pharmaceutiques
in Antananarivo, Madagascar (CNARP), the Missouri Botanical Garden in St. Louis, Missouri (MO),
´
and the Museum National dꢀ Histoire Naturelle in Paris, France (P).
Extraction and Isolation. Dried root parts of N. anthophylla (273 g) were ground in a hammer mill,
then extracted with EtOH by percolation for 24 h at r.t. to give the crude extract MG 4657 (12.4 g), of
which 3.2 g was shipped to Virginia Tech for bioassay-guided isolation. A 1.1-g sample of MG 4657 (IC50
6.9 mg/ml) was suspended in aq. MeOH (MeOH/H2O 9 :1; 100 ml), and extracted with hexane (3ꢂ
100 ml). The aq. layer was then diluted to 60% MeOH (v/v) with H2O and extracted with CH2Cl2 (3ꢂ
150 ml). The remaining aq. layer was further extracted with BuOH (3ꢂ100 ml). The hexane fraction was
evaporated in vacuo to leave 131.2 mg of material with IC50 >20 mg/ml. The residue from the CH2Cl2
fraction (166.1 mg) had an IC50 value of 7.7 mg/ml, the residue from the BuOH fraction (248.6 mg) had an
IC50 value of 2.5 mg/ml and the remaining aq. MeOH fraction had an IC50 value of 20 mg/ml.
Chromatography of the CH2Cl2 fraction over a Sephadexꢁ LH-20 size-exclusion column with CH2Cl2/
MeOH 1:1 was used to obtain six fractions, of which the most active fraction (40.3 mg) had an IC50 value
of 2.0 mg/ml. This fraction was then applied to a silica-gel column with CHCl3/MeOH 9 :1 to give fourteen
fractions, of which Fr. 11 (4.8 mg) was the most active (IC50 1.0 mg/ml) and yielded compound 3. The
BuOH fraction was applied to an open column of Diaion HP-20 resin and eluted with a step MeOH/H2O
gradient of 40, 70, and 100% MeOH. The 100% MeOH fraction was the most active fraction (100 mg)
with an IC50 value of 2.2 mg/ml. This fraction was applied to a silica-gel column and eluted with CHCl3/
MeOH 6 :1 to give thirteen fractions, of which Fr. 4 (1.8 mg) yielded compound 2, with an IC50 value of
1.5 mg/ml, and Fr. 7 (6.3 mg) yielded compound 1, with an IC50 value of 1.9 mg/ml.
2’’-O-Acetylrandianin (¼(3b)-3-{[3-O-(2-O-Acetyl-b-d-glucopyranosyl)-b-d-glucopyranosyl]oxy}-
olean-12-en-28-oic Acid; 2). Light-yellow solid. [a]2D1 ¼ þ12 (c¼1.2, MeOH). IR: 3453, 2935, 1734,
1689, 1027. 1H- and 13C-NMR: see Table 1. HR-ESI-MS: 845.4692 ([MþNa]þ , C44H70NaO1þ4 ; calc.
845.4663).
6’’-O-Acetylrandianin (¼(3b)-3-{[3-O-(6-O-Acetyl-b-d-glucopyranosyl)-b-d-glucopyranosyl]oxy}-
olean-12-en-28-oic Acid; 3). Light-yellow solid. [a]2D1 ¼ þ17 (c¼1.2, MeOH). IR: 3439, 2935, 1727,
1689, 1027. 1H- and 13C-NMR: see Table 1. HR-ESI-MS: 845.4643 ([MþNa]þ , C44H70NaO1þ4 ; calc.
845.4663).
Hydrolysis of Compounds 2 and 3. Compound 3 (3.0 mg) was hydrolyzed with 6m NH4OH for 2 h at
1108. The soln. was evaporated to dryness under reduced pressure, and then the residue was dissolved in
H2O and extracted with BuOH (3ꢂ ) [17][18]. The BuOH extract was evaporated to dryness and yielded
1
a light-yellow powder (2.6 mg) identified as compound 1 by its H- and 13C-NMR spectra. The light-
yellow powder was further hydrolyzed with 3m HCl for 4 h at 1008. The soln. was extracted with AcOEt
(3ꢂ ), and both the org. and the aq. layers were evaporated to dryness under reduced pressure. The
structure of the white powder (1.4 mg) obtained from the org. layer was determined to be oleanolic acid
by 1H- and 13C-NMR spectroscopy. The semisolid carbohydrate mixture from the aq. layer (0.9 mg) was

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
dissolved in 2 ml of H2O and kept overnight before TLC analysis and determination of its optical
rotation. The same procedure was also applied to compound 2. The sugars from both 2 and 3 had Rf
values identical to that of glucose by TLC on a silica-gel plate with CHCl3/MeOH/H2O 15 :6 :1, and had
[a]2D1 values of þ13.9 and þ14.2, resp. (c¼0.1, H2O).
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Received May 7, 2012