New cytotoxic oleanane saponins from the infructescences of Polyscias amplifolia from the Madagascar rainforest

Article abstract of DOI:10.1055/s-2003-39711

Bioassay-guided fractionation of an ethanolic extract of the infructescences of Polyscias amplifolia resulted in the isolation of two new oleanolic acid saponins, polyfoliolides A (1) and B (2), in addition to the two known saponins 3-O-β-D-galactopyrano-syloleanolic acid (3) and 3-O-β-D-galactopyranosyl-(1→4)-β-D-galactopyranosyloleanolic acid (4). The structures of the two new compounds were established as 3-O-β-D-galactopyranosyl-(1→4)-β-D-xylopyranosyloleanolic acid (1) and 3-O-β-D-galactopyranosyl-(1→4)-α-L-arabinopyranosyloleanolic acid (2) on the basis of extensive 1D and 2D NMR spectroscopic data interpretation and chemical conversions. All the isolated compounds showed weak cytotoxicity against A2780 human ovarian cancer cell line, with IC₅₀ values in the range 6.7 to 10.8 μg/mL.

Full text of DOI:10.1055/s-2003-39711

V. S. Prakash Chaturvedula1
Jennifer K. Schilling1
James S. Miller2
Rabodo Andriantsiferana3
Vincent E. Rasamison3
David G. I. Kingston1
New Cytotoxic Oleanane Saponins from the
Infructescences of Polyscias amplifolia from the
Madagascar Rainforest
Abstract
(1®4)-b-D-xylopyranosyloleanolic acid (1) and 3-O-b-D-galacto-
pyranosyl-(1®4)-a-L-arabinopyranosyloleanolic acid (2) on the
Bioassay-guided fractionation of an ethanolic extract of the in- basis of extensive 1D and 2D NMR spectroscopic data interpreta-
fructescences of Polyscias amplifolia resulted in the isolation of tion and chemical conversions. All the isolated compounds
two new oleanolic acid saponins, polyfoliolides A (1) and B (2), showed weak cytotoxicity against A2780 human ovarian cancer
in addition to the two known saponins 3-O-b-D-galactopyrano- cell line, with IC50 values in the range 6.7 to 10.8 mg/mL.
syloleanolic acid (3) and 3-O-b-D-galactopyranosyl-(1®4)-b-D-
galactopyranosyloleanolic acid (4). The structures of the two Keywords
new compounds were established as 3-O-b-D-galactopyranosyl- Polyscias amplifolia ´ Araliaceae ´ saponins ´ cytotoxicity
Introduction
galactopyranosyl-(1®4)-b-D-galactopyranosyloleanolic acid (4)
[11], [12] by comparison of their spectral data with the values
In our continuing search for bioactive constituents from the flora reported in the literature. In this paper we report the structure
of the Suriname and Madagascar rainforests as part of an Inter- elucidation of the two new saponins, polyfoliolides A (1) and B
national Cooperative Biodiversity Group (ICBG) program [1], [2], (2).
440
an ethanol extract of the infructescences of Polyscias amplifolia
(Araliaceae) collected in Madagascar was found to be weakly cy-
totoxic, with an IC50 of 25.5 mg/mL against the A2780 cell line.
Materials and Methods
The genus Polyscias is a rich source of triterpenoid glycosides [3], General experimental procedures
[4], [5], [6], [7], [8] but no phytochemical studies have been re- Optical rotations were recorded on a Perkin-Elmer 241 polarime-
ported on Polyscias amplifolia. This fact, together with the cytoxi- ter. IR and UV spectra were measured on MIDAC M-series FTIR
city of its ethanol extract, caused it to be selected for bioassay- and Shimadzu UV-1201 spectrophotometers, respectively. NMR
guided fractionation. The crude extract was purified by extensive spectra were obtained on a JEOL Eclipse 500 spectrometer, and
bioassay-directed chromatography over Sephadex LH-20 fol- chemical shifts are given in d (ppm) with TMS (tetramethylsi-
lowed by reversed-phase HPLC. This furnished the two new sa- lane) as internal reference; coupling constants are reported in
ponins 1 and 2, in addition to the two known glycosides 3 and 4. Hz. Mass spectra were obtained on a JEOL HX-110 instrument.
The structures of the two known compounds were identified as Sephadex LH-20 was used for column chromatography. HPLC
3-O-b-d-galactopyranosyloleanolic acid (3) [9], [10] and 3-O-b-D- was performed on a Shimadzu LC-10AT instrument with an ODS
Affiliation
1 Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
2 Missouri Botanical Garden, St. Louis, Missouri, USA
3 Centre National d'Application et des Recherches Pharmaceutiques, Antananarivo, Madagascar
Correspondence
Dr. David G. I. Kingston ´ Department of Chemistry ´ M/C 0212 ´
Virginia Polytechnic Institute and State University ´ Blacksburg ´ VA 24061 ´ USA ´ Phone: +1-540-231-6570 ´
Fax: +1-540-231-7702 ´ E-mail: dkingston@vt.edu
Received September 12, 2002 ´ Accepted January 25, 2003
Bibliography
Planta Med 2003; 69: 440±444 ´ ꢀ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943

A323, 50”10 mm column and a flow rate of 2 mL/min, and detec- min) and 2 (4.4 mg, tR 10.3 min) in addition to the two known
tion by UV absorption at 210 nm.
compounds 3-O-b-D-galactopyranosyloleanolic acid (3, 2.6 mg,
[a]D25: + 22.48, c 0.72, MeOH), and 3-O-b-D-galactopyranosyl-
(1®4)-b-D-galactopyranosyloleanolic acid (4, 5.1 mg, [a]D25
:
Plant material
441
Infructescences from P. amplifolia (Baker) Harms (Araliaceae) + 34.48, c 0.68, MeOH). The known compounds 3 and 4 were
were collected in the eastern region of Madagascar, near Anta- identified by comparison of their spectral data with literature
nandava, by Sennen Randrianasolo, Stephan Rakotonandrasa- values [9], [10], [11], [12].
na, Chris Birkinshaw, and Patrice Antilahimena in 1999. Vou-
cher specimens (SSR 115) are preserved at the Herbarium of Polyfoliolide A (1): colorless solid; [a]D25: + 12.48 (c 1.42, MeOH);
the Centre National d'Application des Recherches Pharmaceu- IR: nmax = 3400, 2960, 1685, 1545, 1423, 1150, 1050, 850 cm±1
;
tiques, Madagascar, at the Herbarium of the Direction des Re- 1H- and 13C-NMR, see Tables 1 and 2; FABMS (positive mode):
cherches Foresti›res et Piscicoles, Madagascar, at the Herbar- m/z (rel. int.) = 773 [M + Na]+ (6), 751 [M + H]+ (4.5), 570 (32),
ium of the MusØum National d'Histoire Naturelle de Paris, 525 (7), 440 (4), 439 (45), 438 (16), 396 (28), 393 (18), 369 (16),
France and at the Herbarium of the Missouri Botanical Garden, 297 (8), 249 (8), 218 (24), 204 (16), 203 (100), 189 (43), 161 (31),
St. Louis, USA.
107 (52); HRFABMS: m/z = 773.4444 [M + Na]+ (calcd. for
41H66O12Na: 773.4452).
C
Extraction and isolation of compounds
The infructescences (145 g) of P. amplifolia were extracted with Acid hydrolysis of polyfoliolide A (1): To a solution of 1 (1.5 mg)
ethanol for 24 hours at room temperature. Evaporation of the in MeOH (2 mL) was added 1 mL of 3% H2SO4 in MeOH and the
solvent under vacuum yielded 3.2 g of crude dried extract mixture was refluxed for 6 h. The mixture was then neutralized
MG053, which had an IC50 of 25.5 mg/mL in the A2780 bioassay. with saturated Na2CO3 solution and extracted with EtOAc
A portion (0.2 g) of the extract was fractionated over Sephadex (3”10 mL) to give an aqueous fraction containing sugars and
LH-20 (10 g) using n-hexane/CHCl3 (100:0 to 0:100, 150 mL an EtOAc fraction containing the aglycone part. Purification of
each) and CHCl3/MeOH (100:0 to 1:1, 100 mL each) to furnish the EtOAc-soluble fraction by normal phase PTLC with the sol-
eight 300 mL fractions (A±H), of which fractions E and F were vent system n-hexane/EtOAc (90:10) yielded a solid (0.7 mg)
found to be active; these fractions were combined on the basis whose spectral data (1H-, 13C-NMR and MS) was identical to
of their similar TLC and 1H-NMR spectroscopic patterns. The those of oleanolic acid [13]. The aqueous phase was concentrat-
combined fraction was separated on reversed-phase HPLC (ODS ed and compared with standard sugars using the TLC systems
A323, 250”10 mm; flow rate: 2 mL/min; detection, UV absorp- EtOAc/n-BuOH/H2O (20:70:10) [3], and CH2Cl2/MeOH/H2O
tion at 210 nm) with the mobile phase MeOH/H2O (70:30, 600 (10:6:1) [4]; the two sugars were identified as D-xylose and D-
mL) yielded the two new triterpene glycosides 1 (4.9 mg, tR 9.8 galactose.
Chaturvedula VSP et al. New Cytotoxic Oleanane¼ Planta Med 2003; 69: 440±444

Cytotoxicity bioassays
Table 1 Selected 1H-NMR Data for Compounds 1 and 2 (CD3OD)a
The A2780 ovarian cancer cell line assays were performed at Vir-
ginia Polytechnic Institute and State University as previously re-
ported using actinomycin D as the positive control [19].
Position
1
2
12
23
24
25
26
27
29
30
1¢
5.24 (1H, s)
5.23 (1H, s)
1.03 (3H, s)
1.04 (3H, s)
Results and Discussion
0.94 (3H, s)
0.95 (3H, s)
0.82 (3H, s)
0.84 (3H, s)
Polyfoliolide A (1) was obtained as an optically active amorphous
solid. The HRFABMS of 1 showed an accurate [M + Na]+ ion peak
at m/z = 773.4444 in accordance with an empirical molecular
formula C41H66O12, and this composition was supported by 13C-
NMR and DEPT spectral data. Compound 1 thus had nine degrees
of unsaturation. The IR spectrum of 1 showed the characteristic
absorption of a carbonyl group at 1685 cm±1, as well as a broad
absorption due to hydroxy groups at 3400 cm±1. The 1H-NMR
showed the presence of seven methyl singlets at d = 0.77, 0.82,
0.89, 0.94, 0.96, 1.03, and 1.14 (each 3H, s) and an olefinic proton
at d = 5.24 (s), characteristic for pentacyclic triterpenoids of the
oleanolic acid type [13]. Furthermore, the 1H-NMR spectrum
showed two anomeric protons at d = 4.49 (d, J = 6.0 Hz) and
4.57 (d, J = 7.5 Hz), indicating the possibility of two sugar moi-
eties in the structure. Hydrolysis of 1 with 3% H2SO4 in methanol
resulted in the formation of an aglycone which was identified as
oleanolic acid (5) through comparison of its 1H- and 13C-NMR
data with those reported in the literature [13]and xylose and ga-
lactose as the sugar moieties. The identification of the sugar mol-
ecules was carried out by comparison with standard sugars by
co-TLC as reported earlier [3], [4]. The C-28 carbonyl group was
0.77 (3H, s)
0.77 (3H, s)
1.14 (3H, s)
1.16 (3H, s)
0.89 (3H, s)
0.87 (3H, s)
0.96 (3H, s)
0.96 (3H, s)
4.49 (1H, d, 6.0)
3.63 (1H, dd 5.2, 6.8)
3.37 (1H, dd, 11.6, 4.6)
3.74 (1H, ddd 2.2, 6.5, 9.2)
4.38 (1H, d 7.8)
3.52 (1H, dd 5.4, 8.6)
3.42 (1H, dd 8.2, 4.2)
3.58 (1H, ddd 1.2, 3.5, 4.2)
2¢
3¢
4¢
5¢
3.82 (1H, dd 11.2, 9.8)
3.64 (1H, dd 11.0, 4.4)
3.64 (1H, dd 10.6, 4.2)
3.28 (1H, dd 10.4, 2.0)
1¢¢
2¢¢
3¢¢
4¢¢
5¢¢
6¢¢
4.57 (1H, d, 7.5)
4.64 (1H, d, 7.8)
3.52 (1H, dd 9.2, 7.6)
3.25 (1H, dd 9.8, 4.2)
3.33 (1H, dd 4.6, 3.5)
3.28 (1H, ddd 1.2, 5.5, 7.2)
3.82 (1H, dd, 11.6, 6.6)
3.64 (1H, dd, 11.2, 3.8)
3.40 (1H, dd 9.0, 7.2)
3.22 (1H, dd 9.6, 7.6)
3.31 (1H, dd 4.2, 3.6)
3.30 (1H, ddd 1.2, 5.1, 7.4)
3.84 (1H, dd, 11.6, 6.4)
3.60 (1H, dd, 11.4, 3.6)
a Assignments made on the basis of COSY, HMQC and HMBC correlations.
Partial hydrolysis of polyfoliolide A (1): Compound 1 (1.5 mg) observed at d = 180.7 in the 13C-NMR spectrum, indicating that
was treated with 5 mg of oxalic acid in 1 mL of MeOH-H2O no sugar linkage was present at C-28 [4], and in the absence of
(1:1) at 608C with reaction monitoring by TLC. After 30 h, the
reaction mixture was extracted with EtOAc (3”10 mL) and the
EtOAc-soluble fraction purified by normal phase PTLC with the
442
solvent system n-hexane/EtOAc (75:25) to yield a solid (0.6
mg), whose spectral data (1H-, 13C-NMR and MS) and [a]D
Table 2 13C-NMR Data for Compounds 1 and 2 (CD3OD)a
were identical to those of 3-O-b-D-xylopyranosyloleanolic acid
Position
1
2
position
1
2
(6) [14], [15]. The aqueous phase was neutralized with satura-
ted Na2CO3 solution and concentrated to yield a white solid
which on purification over normal phase PTLC using the sol-
vent system EtOAc/n-BuOH/H2O (20:70:10) furnished the su-
gar galactose (0.3 mg); its [a]D25 was similar to that of D-galac-
tose [16].
1
2
39.3
39.1
22
23
24
25
26
27
28
29
30
1¢
32.7
32.8
27.1
15.8
14.7
16.9
25.5
27.2
89.9
39.1
55.7
18.1
32.4
39.3
47.9
36.6
25.0
122.3
143.9
41.6
27.5
23.2
46.0
41.4
47.7
32.2
33.7
27.0
90.2
39.4
55.9
18.4
32.0
39.1
47.6
36.8
25.4
122.5
144.1
41.4
27.5
23.1
46.0
41.4
47.4
31.9
33.8
27.3
15.6
14.6
16.6
25.7
180.7
32.4
22.8
104.0
76.6
77.8
80.7
67.5
103.5
72.4
74.6
70.4
76.7
61.6
3
4
5
6
Polyfoliolide B (2): colorless solid; [a]D25: + 26.48 (c 0.96, MeOH);
7
180.6
32.6
22.6
103.8
72.7
74.9
78.0
64.2
103.6
72.6
74.8
70.6
76.9
61.8
IR: nmax = 3420, 2945, 1670, 1535, 1415, 1150, 1050, 855 cm±1
;
8
1H- and 13C-NMR, see Tables 1 and 2; FABMS (positive mode):
m/z (rel. int.) = 773 [M + Na]+ (6), 751 [MH+](2), 570 (32), 525
(12), 440 (6), 439 (38), 438 (28), 396 (16), 393 (14), 369 (8), 297
(16), 249 (12), 218 (36), 204 (21), 203 (100), 189 (28), 161 (23),
107 (36); HRFABMS: m/z = 773.4450 [M + Na]+ (calcd. for
9
10
11
12
13
14
15
16
17
18
19
20
21
2¢
3¢
4¢
5¢
C41H66O12Na: 773.4452).
1¢¢
2¢¢
3¢¢
4¢¢
5¢¢
6¢¢
Acid hydrolysis of polyfoliolide B (2): Hydrolysis of polyfoliolide B
(2, 1.3 mg) as described above furnished oleanolic acid, L-arabi-
nose and D-galactose.
Partial hydrolysis of polyfoliolide B (2): Partial hydrolysis of 2 (1.3
mg) as described above yielded 3-O-a-L-arabinopyranosylolea-
nolic acid (7, 0.6 mg) [17], [18].
a Assignments made on the basis of HMQC and HMBC correlations.
Chaturvedula VSP et al. New Cytotoxic Oleanane¼ Planta Med 2003; 69: 440±444

any other oxygenated carbon, the diglycoside had to be placed at
the C-3 position of oleanolic acid. The presence of a diglycoside
at C-3 was also supported by the presence of intense fragment
ions in the positive mode EI mass spectrum at m/z = 439 and
438 formed by the loss of C11H19O10 and C11H20O10 from the mole-
cular ion peak. This evidence further supported the presence of
one hexose and one pentose unit in 1.
Table 3 Cytotoxicities of Compounds 1±7a
Compound
IC50, mg/mL
1
2
3
4
5
6
7
6.7  0.4
9.2  0.3
10.8  0.5
9.6  0.3
20.4  0.4
8.9  0.6
8.6  0.3
The 13C-NMR values for all the carbons were assigned on the ba-
sis of HMQC and HMBC spectra and are given in Table 2. Compar-
ison of the carbon chemical shifts thus assigned with those of re-
ference methyl glycosides [20]taking into account the known ef-
fects of O-glycosylation, and the results of acid hydrolysis, indi-
cated that 1 contained a terminal b-galactopyranosyl and a 4-
substituted b-xylopyranosyl unit in its structure. Partial hydroly-
sis of 1 with oxalic acid resulted in the formation of the known
monoglycoside, 3-O-b-D-xylopyranosyloleanolic acid (6) [14] ,
a The concentration of compound that inhibited 50% (IC50) of the growth of the A2780 mam-
malian cell line according to the procedure described19, with actinomycin D (IC50 2±5 ng/
mL) as the positive control.
[15], supporting the presence of the terminal galactopyranosyl lysis by H2SO4, whereas on hydrolysis with oxalic acid it yielded
group and the D-absolute stereochemistry of xylose. The connec- 3-O-a-L-arabinopyranosyloleanolic acid (7) [17], [18]. These
tivity of the sugars was confirmed by cross-peaks due to long- experiments indicated the presence of D-galactose and L-arabi-
range correlations in an HMBC spectrum. Cross peaks were ob- nose in 2. The presence of the terminal galactopyranosyl group
served between the C-3 carbon of the aglycone (d = 89.9) and was supported by the prominent mass fragment ion observed at
the H-1¢ proton of the arabinose unit (d = 4.49), and between m/z = 570 in the FAB mass spectrum indicating the loss of a hex-
the C-4¢ carbon of the xylose unit (d = 80.7) and the H-1¢¢ proton ose unit. The 13C-NMR values were assigned on the basis of
of the galactose unit (d = 4.47). The fragment ion observed at m/ HMQC and HMBC spectra and by comparison with 1 and are giv-
z = 570 in the positive mode FAB mass spectrum, formed by the en in Table 2. The orientation at the two anomeric centers was as-
loss of C6H12O6 from the molecular ion, also supported the pro- signed as b by the relatively large coupling constants of their
posed structure. The sequence of the two sugars and their link- anomeric protons, as in 1, and the galactose was assigned as D-ga-
age to the aglycone was fully supported by the HMBC correla- lactose on the basis of its identical [a]D value with that of the prod-
tions as shown in Fig.1. The b-orientations at the two anomeric uct obtained by the partial hydrolysis of 1 and with the reported
centers and at the C-3 position of oleanolic acid were supported value [16]. Thus, the structure of 2 was established as 3-O-b-D-ga-
by the relatively large coupling constants of their anomeric pro- lactopyranosyl-(1®4)-a-L-arabinopyranosyloleanolic acid.
tons [21]. The D-absolute stereochemistry of the galactose unit
443
was assigned by comparison of its [a]D value with the value re- All the isolated compounds 1±4 were tested for cytotoxicity
ported in the literature [16].
against A2780 ovarian cancer cells along with oleanolic acid (5),
and the two monoglycosides 3-O-b-D-xylopyranosyloleanolic
On the basis of all the above spectroscopic and chemical evi- acid (6) and 3-O-a-L-arabinopyranosyloleanolic acid (7) formed
dence, the structure of 1 was assigned as 3-O-b-D-galactopyrano- by the partial hydrolysis of 1 and 2, respectively. As shown in
syl-(1®4)-b-D-xylopyranosyloleanolic acid.
Table 3, all the compounds except oleanolic acid (5) are moder-
ately active with IC50 values ranging between 6.7 to 10.8 mg/mL.
The molecular formula of polyfoliolide B (2) was also determined
as C41H66O12 by HRFABMS. Its IR spectrum also showed the pres-
ence of a carbonyl (1670 cm±1) and hydroxy (3420 cm±1) groups, Acknowledgements
1
similar to that of 1. The H-NMR spectrum of 2 also showed the
presence of seven methyl singlets, an olefinic proton, and two This work was supported by International Cooperative Biodiver-
anomeric protons similar to that of 1 (Table 1). The mass frag- sity Grant Number U01 TW/CA-00313 from the Fogarty Center,
ments observed at m/z = 439 and 438 in the positive mode of National Institutes of Health, and this support is gratefully ac-
EIMS showed the presence of a diglycoside unit (one each of pen- knowledged. We also thank Mr. Bill Bebout for obtaining the
tose and hexose) as in 1. To identify the two sugar units attached mass spectra.
at the C-3 position of oleanolic acid, compound 2 was hydrolyzed
completely with 3% H2SO4 and partially with oxalic acid. Com-
pound 2 gave oleanolic acid, galactose, and arabinose on hydro- References
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