Cytotoxic 16-β-[(d-xylopyranosyl)oxy]oxohexadecanyl triterpene glycosides from a Malagasy plant, Physena sessiliflora

Article abstract of DOI:10.1016/j.phytochem.2009.06.002

Brine shrimp lethality assay-guided separation of the MeOH extract of leaves of Physena sessiliflora, which is endemic to Madagascar, afforded eight triterpene glycosides, Physenoside S1-4 and 16-β-[(d-xylopyranosyl)oxy]oxohexadecanyl homologues, Physenos

Full text of DOI:10.1016/j.phytochem.2009.06.002

Phytochemistry 70 (2009) 1195–1202
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cytotoxic 16-b-[(D-xylopyranosyl)oxy]oxohexadecanyl triterpene glycosides
from a Malagasy plant, Physena sessiliflora
a
a
a
b
Masaki Inoue a, , Kazuhiro Ohtani , Ryoji Kasai , Mayu Okukubo , Marta Andriantsiferana ,
*
Kazuo Yamasaki a, Tohru Koike a
a Department of Medicinal Chemistry, Graduate School of Biomedical Sciences, Hiroshima University, 734-8551 Hiroshima, Japan
b Laboratoire de Chimie Organigue, Produits Naturels, Universite’ d’Antananarivo, Madagascar
a r t i c l e i n f o
a b s t r a c t
Article history:
Brine shrimp lethality assay-guided separation of the MeOH extract of leaves of Physena sessiliflora, which
is endemic to Madagascar, afforded eight triterpene glycosides, Physenoside S1–4 and 16-b-[(D-xylopyr-
anosyl)oxy]oxohexadecanyl homologues, Physenoside S5–8. Structural elucidation of these compounds
was based on both spectroscopic analyses and chemical properties. Physenoside S7 and S8 have signifi-
cant cytotoxic activities in the brine shrimp lethality assay.
Received 27 February 2009
Received in revised form 21 May 2009
Available online 13 July 2009
Keywords:
Physena sessiliflora
Capparidaceae
Ó 2009 Elsevier Ltd. All rights reserved.
Physenaceae
Physenoside
Triterpene glycoside
Cytotoxicity
16-Hydroxy-oxohexadecanoic acid
1. Introduction
2. Results and discussion
A bushy tree, Physena sessiliflora Tul. is endemic to Madagascar
and only two species, P. sessiliflora and Physena madagascariensis,
belong to this genus Physena. This genus generally classifies into
Capparidaceae, but some botanists classify it into the Physenaceae
(Dickison and Miller, 1993), which is an endemic family in Mada-
gascar and contains only this genus. In Madagascar, the decoction
of these leaves is used for rites by witch doctor (Samyn, 1999). Tri-
terpenes (Deng et al., 1999) and prenyl biflavones (Deng et al.,
2000; Cao et al., 2006) were isolated from P. madagascariensis,
but no phytochemical and biological investigations on P. sessiliflora
have been reported. As a part of our studies on the phytochemical
constituents of Malagasy plants, we have undertaken chemical
investigation of the leaves of this plant. Besides, the MeOH extract
of these leaves showed cytotoxic activity in the brine shrimp
lethality assay (Meyer et al., 1982; Anderson et al., 1991; Solis
et al., 1993; Carballo et al., 2002). Herein, we describe isolation
and structure elucidation of eight novel triterpene glycosides, Phy-
The methanolic extract of the leaves of P. sessiliflora showed
cytotoxic activity using the brine shrimp lethality assay at
100 lg/mL. This extract was partitioned into n-hexane, EtOAc, n-
BuOH, and water layers, successively. The n-BuOH extract exhib-
ited the strongest activity and its activity-guided separation ex-
tract afforded eight triterpene glycosides, Physenoside S1–8(1–8),
whose structures are shown in Fig. 1. An alkaline hydrolysis prod-
uct 9 was obtained from 7 and 8. The various NMR spectroscopic
analyses such as 1H, 13C, DEPT, COSY, HMQC, and HMBC, in con-
junction with FABMS spectroscopy, established that the com-
pounds 1–9 (Fig. 1) were olean-12-en-28-oic acid type triterpene
glycosides with oxygenated carbons (C-2, C-3, C-23, and/or C-16).
Acid hydrolysis of 1–6 afforded the same aglycone, bayogenin
(2b,3b,23-trihydroxyolean-12-en-28-oic acid) 1a (see Fig. 2),
which was identified on the basis of NMR spectroscopic data when
compared with literature data (Jurenitsch et al., 1986). On the
other hand, alkaline hydrolysis of 1–6 gave a glucoside 1b
senoside S1–4, and 16-b-[(D-xylosyl)oxy]oxohexadecanyl homo-
(Fig. 2), which was identified as 3-O-b-D-glucopyranoside of 1a
logues, Physenoside S5–8. In general, keto-fatty acids such as
oxohexadecanoic acid are rarely found as plant lipid constituents
(Deas et al., 1974).
based on the comparison of its spectroscopic data with reference
data (Kasai et al., 1986; Marston et al., 1988).
Compound 1, named Physenoside S1, was obtained as a white
amorphous powder. Its negative FABMS exhibited a quasi-molecu-
lar ion peak at m/z 1191 [MÀH]À, indicating a molecular weight of
1192. The molecular formula was established as C57H92O26 by a
* Corresponding author. Tel.: +81 82 257 5281; fax: +81 82 257 5336.
E-mail address: D066862@hiroshima-u.ac.jp (M. Inoue).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.06.002

1196
M. Inoue et al. / Phytochemistry 70 (2009) 1195–1202
Fig. 1. Structures of 1–15, Acyl A, and Acyl B.
positive-ion mode HRFABMS with a pseudo-molecular ion peak at
and dC 83.1 (C-3 of aglycone), dH 6.48 (H-1 of inner Ara) and dC
176.2 (C-28 of aglycone), dH 5.80 (H-1 of Rha) and dC 74.1 (C-2 of
inner Ara), dH 5.08 (H-1 of Xyl) and dC 84.0 (C-4 of Rha), and dH
5.11 (H-1 of outer Ara) and dC 74.9 (C-3 of inner Ara). Based on
the 13C chemical shifts and coupling constants of proton signals
of inner arabinosyl residue, the conformation of ester-linked arabi-
nosyl residue of 1 was 1C4 form, which is congruent with the data
reported by Nagao et al. (1989). The configuration of the rhamno-
syl residue was determined by the same method as reported
m/z 1215.5790 [M+Na]+ (calcd for 1215.5775 C57H92O26Na). Acid
hydrolysis of 1 gave
D-glucose (Glc), L-arabinose (Ara), L-rhamnose
(Rha), and
D
-xylose (Xyl) as sugar components. The 1H and 13C
NMR spectra of 1 displayed five anomeric protons at d 6.48 (d,
J = 3.0 Hz), 5.80 (brs), 5.12 (d, J = 7.7 Hz), 5.11 (d, J = 6.2 Hz), and
5.08 (d, J = 7.3 Hz), and five anomeric carbons at d 107.1, 105.5,
103.4, 101.7, and 92.7 (see Tables 1 and 2). Each proton and carbon
signal of 1 was assigned to individual functional groups using 1D-
TOCSY, COSY, and HMQC spectra. An HMBC experiment was car-
ried out for determination of the glycosyl linkages. The HMBC
spectrum showed cross-peaks between each anomeric proton
and the corresponding carbon resonance; dH 5.12 (H-1 of Glc)
Table 1
Selected 1H NMR spectroscopic data (500 MHz, in pyridine-d5) of the aglycone and
anomeric protons of Physenoside S1 (1), S7 (7), and S8 (8).
1
7
8
Aglycone
16
18
23
24
25
26
27
29
30
5.50 (m)
5.14 (brs)
5.14 (brs)
3.20 (dd, 13.7, 4.3)
3.68 (d, 10.6)
1.30 (s)
1.52 (s)
1.12 (s)
1.23 (s)
0.88 (s)
0.95 (s)
1.85 (s)
1.46 (s)
1.03 (s)
1.67 (s)
0.88 (s)
1.00 (s)
1.86 (s)
1.46 (s)
1.03 (s)
1.66 (s)
0.88 (s)
1.00 (s)
C-3
Glc-
1
5.11 (d, 6.2)
4.99 (d, 7.8)
4.99 (d, 7.8)
C-28
inner-Ara-
outer-Ara-
Rha-
1
1
1
1
6.48 (d, 3.0)
5.12 (d, 7.7)
5.80 (brs)
6.17 (d, 4.6)
4.92 (d, 6.4)
5.90 (brs)
6.17 (d, 3.7)
4.91 (d, 6.4)
5.90 (brs)
Xyl-
5.08 (d, 7.3)
5.04 (d, 7.6)
5.04 (d, 7.6)
Acyl group
Xyl-
1
4.64 (d, 7.6)
4.64 (d, 7.6)
Fig. 2. Structures of the hydrolysis products 1a, 1b, 4b, 9a, and 9b.

M. Inoue et al. / Phytochemistry 70 (2009) 1195–1202
1197
Table 2
13C NMR spectroscopic data (125 MHz, in pyridine-d5) of the glycosidic parts of 1–9.
1
2
3
4
5
6
7
8
9
C-3
Glc-
1
2
3
4
5
6
105.5
75.5
78.6
71.6
78.2
62.6
105.2
75.2
78.2
71.3
78.0
62.3
105.6
75.5
78.6
71.7
78.3
62.7
105.0
75.2
78.5
71.6
76.6
70.1
105.6
75.6
78.6
71.6
78.2
62.7
105.6
75.6
78.6
71.6
78.2
62.7
104.7
74.7
77.9
71.0
77.8
62.1
104.7
74.7
77.9
71.0
77.8
62.1
105.7
75.5
78.6
71.7
78.3
62.7
Glc-
1
2
3
4
5
6
104.9
75.3
78.2
71.7
78.4
62.7
C-28
inner-Ara-
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
92.7
74.1
74.9
65.3
63.5
103.4
71.1
74.1
68.4
66.2
92.9
73.3
77.0
65.5
63.6
92.4
74.1
73.2
64.7
63.0
103.0
71.1
74.2
68.4
66.3
92.5
74.1
74.2
64.7
63.0
103.0
70.8
74.2
68.3
66.2
93.2
73.4
73.3
69.9
62.6
93.2
73.4
73.3
69.9
62.6
93.8
73.5
73.3
69.9
62.2
104.9
71.6
73.7
68.2
66.0
93.8
73.5
73.3
69.9
62.3
104.9
71.6
73.7
68.2
66.0
92.7
74.1
74.9
65.3
63.5
103.4
71.1
74.1
68.4
66.2
outer-Ara-
Api-
110.0
77.9
80.1
75.2
64.9
110.3
78.2
80.5
75.5
65.8
110.3
78.2
80.5
75.5
65.8
Rha-
Rha-
Xyl-
1
2
3
4
5
6
101.7
71.9
72.5
84.0
68.7
18.4
101.5
71.5
72.2
83.5
68.7
18.3
101.6
72.3
80.7
77.8
69.4
18.7
101.5
71.6
80.7
77.8
69.3
18.6
101.9
71.7
72.5
84.1
68.9
18.6
101.9
71.7
72.5
84.1
68.9
18.4
101.3
71.4
71.9
82.8
68.5
18.2
101.3
71.4
71.9
82.8
68.6
18.2
101.6
71.9
72.5
84.0
68.7
18.4
1
2
3
4
5
6
103.9
71.7
72.7
74.4
70.1
18.7
104.0
72.2
72.7
74.1
70.0
18.6
1
2
3
4
5
107.1
76.0
78.6
70.9
67.4
106.7
75.7
78.2
70.7
67.1
105.5
75.5
78.3
70.9
67.1
105.4
75.2
78.2
71.1
67.1
107.2
76.0
78.6
71.0
67.5
107.2
76.0
78.6
71.0
67.5
106.2
75.5
77.8
70.7
66.9
106.2
75.5
77.8
70.7
66.9
107.1
76.0
78.6
70.9
67.4
Acyl group
Xyl-
1
2
3
4
5
105.3
75.0
78.4
71.2
67.2
105.3
75.0
78.4
71.2
67.2
104.8
74.4
77.6
70.5
66.7
104.8
74.5
77.6
70.5
66.7
previously (Kasai et al., 1979). From these experimental facts, we
confirmed the structure of compound 1 to be b- -xylopyranosyl-
(1-4)- -rhamnopyranosyl-(1-2)-[ -arabinopyranosyl-(1-3)]-
-arabinopyranosyl 3-O-b- -glucopyranosyl-2b,3b,23-trihydroxy-
olean-12-en-28-oate.
Physenoside S2 (2) was obtained a white powder, and its posi-
tive HRFABMS spectrum exhibited a quasi-molecular ion peak at
m/z 1215.5801 [M+Na]+ (calcd. for 1215.5775 C57H92O26Na) as for
as 1. The 13C NMR spectrum of 2 was similar to that of 1 except
tion by comparing the 13C NMR spectroscopic data with the
reported values for - and b- -apiofranosides (Kitagawa et al.,
1993). Consequently, the structure of 2 was assigned to b- -xylo-
pyranosyl-(1-4)- -rhamnopyranosyl-(1-2)-[b- -apio- -furanosyl-
(1-3)]- -arabinopyranosyl 3-O-b- -glucopyranosyl-2b,3b,23-tri-
hydroxyolean-12-en-28-oate.
The FABMS spectrum of Physenoside S3 (3) showed a quasi-
molecular ion peak at 1337 [MÀH]À, which is 146 mass units
greater than 1. Its positive HRFABMS spectrum also exhibited a
quasi-molecular ion peak at m/z 1361.6371 [M+Na]+ (calcd for
D
a
D
a-
L
a-
L
a-
D
L
D
a-L
D
D
a-
L
D
for a terminal
cose, -arabinose,
detected. HMBC spectrum coupled with 1D-TOCSY and HMQC sug-
gested that compound 2 had a structure in which the terminal
arabinose of 1 was replaced by b- -apio- -furanose. The anomeric
center of the apio- -furanose was determined to be b-configura-
L-arabinosyl residue. On acid hydrolysis of 2,
D-glu-
L
L
-rhamnose, -xylose, and -apiose (Api) were
D
D
1361.6354 C63H102O30Na). Acid hydrolysis of 3 gave
D-glucose, L-
arabinose, -rhamnose, and -xylose. Two methyl proton signals
L
D
a-L
-
at d 1.74 (d, J = 6.7 Hz) and 1.64 (d, J = 6.7 Hz) indicated the
presence of two rhamnosyl residues, and fragment ion peak at m/
z 1191 [MÀ146]À suggesting that one of rhamnosyl residues is
D
D
D

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M. Inoue et al. / Phytochemistry 70 (2009) 1195–1202
the terminal residue. A comparison of the 13C NMR spectrum of 3
with that of 1 showed glycosylation shifts for C-3 of Rha (
D
d
+8.2 ppm) on going from 1 to 3. The HMBC spectrum of 3, followed
by assignments of protons and carbons of sugar moieties, showed a
cross-peak between dH 5.78 (H-1 of terminal Rha) and dC 80.7 (C-3
of inner Rha). These data of 3 were consistent with the structure of
b-
pyranosyl-(1-2)-[
3-O-b- -glucopyranosyl-2b,3b,23-trihydroxyolean-12-en-28-oate.
Physenoside S4 (4), C69H112O35, was isolated as a white powder
having a molecular weight of 1500. Acid hydrolysis of 4 gave -glu-
cose, -arabinose, -rhamnose, and -xylose. On the other hand,
D-xylopyranosyl-(1-4)-[a-L-rhamnopyranosyl-(1-3)]-a-L-rhamno-
a-L-arabinopyranosyl-(1-3)]-a-L-arabinopyranosyl
D
D
L
L
D
alkaline hydrolysis of 4 yielded compound 4b. Two anomeric pro-
tons at d 4.89 (d, J = 7.8 Hz) and 4.98 (d, J = 7.8 Hz) in 1H NMR, and
two anomeric carbons at d 104.9 and 105.0 in 13C NMR were ob-
served. The carbon signals indicated the presence of gentiobiosyl
unit in 4b. These experimental results established that compound
Fig. 3. Fragmentation points observed in EIMS of 12 and 15.
The chromatographic behavior of Physenoside S6 (6) was al-
most the same as 5. The 1H and 13C NMR spectroscopic data of 6
were also quite similar to those of 5. Alkaline hydrolysis of 6 with
2% KOHaq at room temperature yielded 1 and 13. The NMR spec-
troscopic data suggested that compound 13 is the isomer of 10,
and that the difference between 10 and 13 is the position of the
carbonyl group. In the EIMS of the trimethylsililated methyl ester
15, a critical fragment ion at m/z 185 (Fig. 3) was observed (Deas
et al., 1974; Ray et al., 1995). Therefore, the structure of 13 was as-
4b is 3-O-b-D-glucopyranosyl-(1-6)-b-D-glucopyranosyl-bayoge-
nin. The 1H and 13C NMR spectra of 4 showed seven anomeric sig-
nals. In the 13C NMR spectrum of 4, carbon resonances of sugar
chain at C-28 were essentially the same as those of 3. These NMR
spectroscopic data suggested that compound 4 is b-
syl-(1-4)-[ -rhamnopyranosyl-(1-3)]- -rhamnopyranosyl-(1-2)-
-arabinopyranosyl-(1-3)]- -arabinopyranosyl 3-O-b- -gluco-
pyranosyl-(1-6)-b- -glucopyranosyl-2b,3b,23-trihydroxyolean-
D-xylopyrano-
a-L
a-L
[a-L
a-L
D
D
12-en-28-oate. This formulation was confirmed by HMBC
experiment coupled with COSY, 1D-TOCSY, and HMQC.
signed to 9-oxo-16-b-[(D-xylopyranosyl)oxy]hexadecanoic acid. In
the similar manner, the position of ester linkage between 1 and
13 in 6 was determined as O-4 of 28-O-linked arabinose. The above
Physenoside S5 (5) showed a quasi-molecular ion peak [M+Na]+
at m/z 1615.8251 in the positive HRFABMS, in accordance with an
empirical molecular formula of C78H128O33Na. The 1H and 13C NMR
spectra of 5 displayed six anomeric protons at d 6.33 (brs), 5.78
(brs), 5.76 (d, J = 1.6 Hz), 5.12 (d, J = 7.8 Hz), 5.07 (d, J = 7.3 Hz),
and 4.71 (d, J = 7.6 Hz), and six anomeric carbons at d 110.3,
107.2, 105.6, 105.3, 101.9, and 93.2. Acid hydrolysis of 5 afforded
evidence showed that compound 6 is b-
rhamnopyranosyl-(1-2)-{[b- -apio- -furanosyl-(1-3)]-4-O-(9-oxo-
16-b-[( -xylopyranosyl)oxy]hexadecanoyl)}- -arabinopyranosyl
3-O-b- -glucopyranosyl-2b,3b,23-trihydroxyolean-12-en-28-oate.
D-xylopyranosyl-(1-4)-a-L-
D
D
D
a-L
D
Alkaline treatment of Physenoside S7 (7) and S8 (8) with 2.5%
NaOHaq gave the same compound 9. On the other hand, acid
hydrolysis of physenoside S7 and S8 afforded the same aglycone,
D-glucose, L-arabinose, D-apiose, L-rhamnose, and D-xylose as sugar
components. Alkaline hydrolysis of 5 gave compound 10 as well as
1a. Alkaline treatment of 5 with 2% KOHaq also yielded 10 in addi-
tion to compound 1. These results indicated that compound 5 is es-
ter of 1 and 10. In the 13C NMR spectrum of 10, one carbonyl (dC
210.3), one carboxyl (dC 172.9), 14 methylene carbon signals to-
gether with signals assigned as xylosyl residue were observed.
The HRFABMS of 10 showed a quassi-molecular ion peak at m/z
441.2450 [M+Na]+, suggesting the molecular formula C21H38O8
zanhic acid (Dimbi et al., 1984) (2b,3b,16a-trihydroxyolean-12-
en-23,28-dioic acid) 9a, which was identified on the basis of the
NMR spectroscopic data compared with the reported values (La-
vaud et al., 1998; Sakai et al., 1999). Furthermore, alkaline hydro-
lysis of 7 and 8 yielded the same compound (9b), which was
identified as 3-O-b-
The 1H and 13C NMR spectroscopic data of the sugar moieties
including the b-[( -xylopyranosyl)oxy]acyl group of compounds 7
D-glucopyranoside of 9a.
D
(calcd. 441.2464 for C21H38O8Na). Acid hydrolysis of 10 gave
lose and compound 11, and the HMBC spectrum displayed a cross-
peak between dH 4.71 (d, J = 7.6 Hz, H-1 of Xyl) and dC 69.7 (CH2).
D
-xy-
and 8 were essentially identical to those of corresponding com-
pounds 1 and each acyl component (Tables 1 and 2). Therefore,
the structures of 7 and 8 were identified as 28-O-b-
syl-(1-4)- -rhamnopyranosyl-(1-2)-{[ -arabinopyranosyl-(1-
3)]-4-O-(8-oxo-16-b-[( -xylopyranosyl)oxy]hexadecanoyl)}-
arabinopyranosyl 3-O-b- -glucopyranosyl-2b,3b,16 -trihydroxy-
olean-12-en-23,28-dioic acid and 28- O-b- -xylopyranosyl-(1-4)-
-rhamnopyranosyl-(1-2)-{[ -arabinopyranosyl-(1-3)]-4-O-(9-
oxo-16-b-[( -xylopyranosyl)oxy]hexadecanoyl)}- -arabinopyr-
anosyl 3-O-b- -glucopyranosyl-2b,3b,16 -trihydroxyolean-12-en-
23,28-dioic acid, respectively.
D-xylopyrano-
These results showed that compound 10 is 16-b-[(
D
-xylopyrano-
a
-
L
a-L
syl)oxy]- -oxohexadecanoic acid. To determine the position of
v
D
a-L-
the carbonyl group, compound 11 was converted to 12 by methyl-
ation followed by trimethylsilylation. The ELMS of 12 showed a typ-
ical fragment ion peak at m/z 171 (Fig. 3), indicating that the
carbonyl group exist at C-8 position. This result was accorded with
the data in the literature (Deas et al., 1974; Ray et al., 1995). Thus,
D
a
D
a-L
a-L
D
a-L
D
a
compound 10 was elucidated as 8-oxo-16-b-[(D-xylopyrano-
syl)oxy]hexadecanoic acid. The ester substituent in 5 was placed
at C-4 of ester-linked arabinosyl residue as a result of downfield
shifts observed for H-4 and C-4 of arabinosyl unit in the 1H and
13C NMR spectra, respectively, compared to those of 1; This was
also confirmed by observation of correlations between dH 5.62
(ddd, J = 3.2, 3.2, 8.0 Hz, H-4 of inner Ara) and dC 173.3 (C-1 of Acyl
A) in the HMBC spectrum of compound 5. Consequently, the
Finally, we conducted the brine shrimp lethality assay for deter-
mination of the toxicity of the isolated compounds 1–8. The assay
is proven to be an effective and rapid screening method to deter-
mine cytotoxicity of water-soluble compounds (Meyer et al.,
1982; Anderson et al., 1991; Solis et al., 1993; Carballo et al.,
2002). Table 3 shows the assay results as LD50 values with compar-
ison of a positive control, Etoposide phosphate. Among the subfrac-
tions prepared from the MeOH extract by solvent partition, only
structure of 5 was determined to be b-
rhamnopyranosyl-(1-2)-{[b- -apio- -furanosyl-(1-3)]-4-O-(8-oxo-
16-b-[( -xylopyranosyl)oxy]hexadecanoyl)}- -arabinopyranosyl
3-O-b- -glucopyranosyl-2b,3b,23-trihydroxyolean-12-en-28-oate.
D-xylopyranosyl-(1-4)-a-L-
D
D
n-BuOH extract exhibited the lethality at 32
indicated that toxic compounds of this plant are less-lipophilic
substances. Compounds 7 (8.5 g/mL) and 8 (22 g/mL) exhibited
high degrees of toxicity in isolated compounds.
lg/mL. This result
D
a-L
D
l
l

M. Inoue et al. / Phytochemistry 70 (2009) 1195–1202
1199
Table 3
crude residue (55 g). The MeOH extract was partitioned between
equal portions of n-hexane/MeOH–H2O (7:3), then the solvent
was removed from the n-hexane layer to yield the n-hexane ex-
tract (3.2 g). After removing MeOH from the aqueous layer, H2O
then added until one liter, the latter was partitioned with EtOAc
(1 L) and then n-BuOH (1 L) successively. From each layer, the sol-
vents were removed in vacuo to afford residues of 9.1 g (EtOAc),
21.4 g (n-BuOH), and 21.5 g (H2O), respectively. The active n-BuOH
soluble fraction was subjected to silica-gel column chromatogra-
phy (CC) with a step gradient solvent system of CH2Cl2–MeOH–
H2O (30:10:1, 20:10:1, 10:5:1, and finally 7:5:1), to give six frac-
tions (Fr. I to VI). Active fraction IV was applied to a silica-gel
eluted with CH2Cl2–MeOH–H2O (30:10:1) to give 5 fractions (Fr.
IVa to IVe). Fraction IVb was further separated by HPLC on an
ODS column with MeCN–H2O (3:7) to give compound 1–3 (176,
Brine shrimp lethality assay results of the extracts of P. sessiliflora, the isolated
compounds 1–9, and Etoposide phosphate (a positive control).
Brine shrimp lethality
(LD50 in lg/mL)
MeOH extract
50.4
MeOH ? n-hexane
MeOH ? EtOAc
MeOH ? n-BuOH
>500.0
>500.0
32.0
1
2
3
4
5
6
7
8
>500.0
>500.0
>500.0
>500.0
394.0
>500.0
8.5
32, and 128 mg, respectively). Compound
7 (15 mg) and 8
22.1
9
420.0
3.5
(22 mg) was purified by HPLC on an ODS column with MeCN–
H2O (37:63) and further with MeOH/0.05% TFAaq (65:35) from
Fr. IVc. Fr. III was subjected to an ODS CC using a MeOH–H2O
(4:6 to 9:1) gradient solvent system give four fractions and then
Fr. IIIc was purified by HPLC on a Polyamine-II column with
MeCN–H2O 82.5: 17.5) to afford compounds 5 (14 mg) and 6
(16 mg), respectively. Fr. V was separated by MPLC on an ODS
using a MeOH–H2O gradient (4:6 to 100:0) to give eight fractions.
Fr. Vc was purified by HPLC on a Polyamine-II column with MeCN–
H2O (77.5:22.5) afforded compound 4 (18 mg). All separations was
guided by brine shrimp lethality testing.
Etoposide phosphate
3. Conclusions
In this study, we isolated eight new triterpene glycosides (Pyse-
noside S1–8) in the methanolic extract of the leaves of P. sessiliflora.
For four of the eight compounds, novel acyl moieties were found as
16-b-[(D-xylopyranosyl)oxy]-8-oxohexadecanyl and 16-b-[(D-xylo-
pyranosyl)oxy]-9-oxohexadecanyl groups. Pysenoside S7 and S8
gave significant lethal activities for the brine shrimp, suggesting
that the b-[(D-xylopyranosyl)oxy]acyl moiety and the aglycone
4.4. Identification of the component monosaccharides
having 23-carboxylic and 16a-hydroxyl groups play important role
for the cytotoxicity.
Compounds (2 mg) were heated with 1 mol/L H2SO4 (1 mL) at
80 °C for 2 h with reaction mixtures washed with Et2O, and aque-
ous layers neutralized with amberlite MB-3 resin and dried. Each
4. Experimental
residue dissolved in H2O (100 lL) and subjected to HPLC analysis
4.1. General procedure
equipped with an optical rotation detector. HPLC conditions: col-
umn, Polyamine-II (4.6 mm i.d. Â 15 cm, YMC); column temp.,
40 °C; mobile phase, MeCN–H2O (4:1); flow rate, 1.0 mL/min;
detection, OR-2090 detector (JASCO). Retention time and a symbol
Optical rotations were measured on a JASCO DIP-1030 auto-
matic polarimeter in MeOH at 30 °C. NMR spectra were recorded
on a JEOL JNM ECP-500 with a field gradient unit (500 MHz for
1H and 125 MHz for 13C) in pyridine-d5, unless otherwise stated,
using TMS as an internal standard at 35.0 0.1 °C. FABMS in glyc-
erol matrix in the negative-ion mode and EIMS were recorded on a
JEOL SX-102A instrument. FL-100D (Fuji silysia, Tokyo, Japan) and
ODS-A-120 (YMC, Kyoto, Japan) were used for column chromatog-
of optical rotation:
D
-Glc, 12.5 min, +;
L-Ara, 8.3 min, +; L-Rha,
6.0 min, À; -Api, 5.7 min, À;
D
D-Xyl, 9.1 min, +.
4.5. Physenoside S1 (1)
D +17.8° (c 1.0, MeOH); for 1H NMR spectrum
White powder; [a]
raphy. HPLC were carried out using
a
D-ODS-5 (20 mm
(pyridine-d5) assignments, see Table 1; and for 13C NMR (pyridine-
d5) assignments of the glycosidic parts, see Table 2; 13C NMR data
of aglycone d 15.0 (C-24), 17.3 (C-25), 17.6 (C-26), 18.1 (C-6), 23.2
(C-16), 23.7 (C-30), 24.0 (C-11), 26.1 (C-27), 28.3 (C-15), 30.8 (C-
20), 32.7 (C-22), 33.1 (C-7), 33.1 (C-29), 34.1 (C-21), 37.0 (C-10),
40.0 (C-8), 41.9 (C-18), 42.4 (C-14), 42.7 (C-4), 44.1 (C-1), 46.2
(C-19), 47.3 (C-17), 47.8 (C-9), 48.5 (C-5), 65.6 (C-23), 70.4 (C-2),
83.1 (C-3), 122.9 (C-12), 144.1 (C-13), 176.2 (C-28); FABMS (nega-
tive ion mode) m/z 1191 [MÀH]À, 1059 [MÀ132ÀH]À, 1029
[MÀ162ÀH]À, 913 [MÀ132À146ÀH]À, 649 [MÀ132Â3À146ÀH]À;
HRFABMS (positive-ion mode) m/z 1215.5790 [M+Na]+ (calcd. for
1215.5775 C57H92O26Na).
i.d. Â 15 cm, YMC) column or Polyamine-II (20 mm i.d. Â 15 cm,
YMC) with two JASCO PU-1500 pumps, and a JASCO UV-1500 UV
detector, and/or a JASCO RI-1500 differential refractometer as a
detector. TLC was performed using a Merck Art. 5554 (silica-gel)
TLC plate. All reagents and solvents were of the highest commercial
quality and were used without further purification.
4.2. Plant material
The leaves of P. sessiliflora were collected in the Berenty Reserve
in March 2000. The identity of the plant was confirmed by Dr. Ar-
mand Rakotozafy from the Institut Malgache de Recherches Appli-
quées. A voucher specimen (PHYS200012) is on deposit at ESSD,
Antananarivo, Madagascar.
4.6. Acid hydrolysis of 1
Compound 1 (10 mg) was dissolved in 1 mol/L H2SO4 (2 mL)
and heated at 80 °C for 2 h. The reaction mixture was extracted
with Et2O, and the organic layer washed with saturated NaHCO3,
and solvent then removed. The residue was crystallized from
4.3. Extraction and isolation
Air-dried leaves (200 g) were extracted with MeOH (2 L Â 3)
under conditions where the suspension was heated until reflux be-
gan, thus being maintained for 3 h. The suspensions were filtered
with the solvent removed from the filtrate in vacuo to yield the
MeOH to give 1a as colorless needles. Bayogenin (1a): [
+17.8° (c 1.0, MeOH); 1H NMR (pyridine-d5)
5.49 (1H, t,
J = 3.6 Hz, H-12), 4.80 (1H, ddd, J = 3.9, 3.6, 3.6 Hz, H-2), 4.35, 3.68
a]
D
d

1200
M. Inoue et al. / Phytochemistry 70 (2009) 1195–1202
(2H, each d, J = 10.8 Hz, H-23), 4.31 (1H, d, J = 3.9 Hz, H-3), 3.29
(1H, dd, J = 4.4, 13.4 Hz, H-18), 1.54 (3H, s, H-25), 1.33 (3H, s, H-
24), 1.27 (3H, s, H-26), 1.07 (3H, s, H-27), 1.00 (3H, s, H-30), 0.93
(3H, s, H-29); 13C NMR (pyridine-d5) d 15.0 (C-24), 17.2 (C-25),
17.5 (C-26), 18.4 (C-6), 23.7 (C-16), 23.8 (C-30), 24.0 (C-11), 26.3
(C-27), 28.3 (C-15), 31.0 (C-20), 33.0 (C-22), 32.8 (C-7), 33.3 (C-
29), 34.3 (C-21), 37.2 (C-10), 49.9 (C-8), 42.1 (C-18), 42.4 (C-14),
42.2 (C-4), 44.1 (C-1), 46.5 (C-19), 46.7 (C-17), 48.3 (C-9), 48.5
(C-5), 65.6 (C-23), 71.6 (C-2), 73.3 (C-3), 123.0 (C-12), 144.2 (C-
13), 180.2 (C-28).
NMR data of the aglycone were almost the same (within
0.2 ppm) as those for compound 1; FABMS (negative ion mode)
m/z 1499 [MÀH]À, 1367 [MÀ132ÀH]À, 1353 [MÀ146ÀH]À, 1337
[MÀ162ÀH]À, 1175 [MÀ162Â2ÀH]À, 811 [MÀ132Â3À146ÀH]À;
HRFABMS (positive-ion mode) m/z 1523.6901 [M+Na]+ (calcd. for
1523.6882 C69H112O35Na); Acid hydrolysis in the same manner as
described above gave 1a. Alkaline hydrolysis gave 1b.
4.11. Physenoside S5 (5)
White powder; [a]
D +1.3ꢀ(c 1.0, MeOH); for 13C NMR (pyridine-
4.7. Alkaline hydrolysis of 1
d5) assignments of the glycosidic parts: see Table 2. 13C NMR data
of the aglycone were almost the same (within 0.2 ppm) as those
for compound 1; FABMS (negative ion mode) m/z 1621 [MÀH]À,
1489 [MÀ132ÀH]À, 1459 [MÀ162ÀH]À, 1343 [MÀ146À132ÀH]À,
649 [MÀ132Â3À146À430ÀH]À, 441 [Acyl]À; HRFABMS (positive-
ion mode) m/z 1615.8252 [M+Na]+ (calcd. for 1615.8235
C78H128O33Na); Acid hydrolysis in the same manner as described
above gave 1a. Alkaline hydrolysis gave 1b.
Compound 1 (20 mg) was treated with 1 mol/L NaOHaq at 80 °C
for 30 min. The reaction mixture was acidified with 2 mol/L HCl,
and then extracted with n-BuOH. The n-BuOH layer was washed
with saturated NaHCO3 and evaporated to dryness. The residue
was chromatographed on a silica-gel with CH2Cl2–MeOH–H2O
(30:10:1) to give 1b (10 mg). 3-O-b-D-glucopyranosyl bayogenin
(1b): 1H NMR (pyridine-d5) d 5.49 (1H, t, J = 3.6 Hz, H-12), 4.80
(1H, ddd, J = 3.9, 3.6, 3.6 Hz, H-2), 4.35, 3.68 (2H, each d,
J = 10.8 Hz, H-23), 4.31 (1H, d, J = 3.9 Hz, H-3), 3.29 (1H, dd,
J = 4.4, 13.4 Hz, H-18), 1.54 (3H, s, H-25), 1.33 (3H, s, H-24), 1.27
(3H, s, H-26), 1.07 (3H, s, H-27), 1.00 (3H, s, H-30), 0.93 (3H, s, H-
29), 5.15 (1H, d, J = 7.8 Hz, H-1 of Glc), 4.17 (1H, dd, J = 7.8,
8.2 Hz, H-2 of Glc), 4.20 (1H, dd, J = 8.2, 8.7 Hz, H-3 of Glc), 4.16
(1H, dd, J = 8.7, 8.9 Hz, H-4 of Glc), 3.90 (1H, ddd, J = 8.9, 2.7,
5.0 Hz, H-5 of Glc), 4.45 (1H, dd, J = 2.7, 11.8 Hz, H-6a of Glc),
4.31 (1H, dd, J = 5.0, 11.8 Hz, H-6b of Glc); 13C NMR (pyridine-d5)
d 15.0 (C-24), 17.2 (C-25), 17.5 (C-26),18.0 (C-6), 23.7 (C-16),
23.8 (C-30), 24.0 (C-11), 26.3 (C-27), 28.3 (C-15), 31.0 (C-20),
33.0 (C-22), 33.3 (C-7), 33.3 (C-29), 34.3 (C-21), 37.0 (C-10), 40.0
(C-8), 42.1 (C-18), 42.4 (C-14), 42.8 (C-4), 44.1 (C-1), 46.5 (C-19),
46.7 (C-17), 47.8 (C-9), 48.6 (C-5), 62.7 (Glc-1), 65.6 (C-23), 70.4
(C-2), 71.7 (Glc-4), 75.5 (Glc-2), 78.3 (Glc-5), 78.6 (Glc-3), 83.1
(C-3), 105.7 (Glc-1), 123.3 (C-12), 144.9 (C-13), 180.2 (C-28).
4.12. Physenoside S6 (6)
White powder;
[
a]
+10.2° (c 1.0, MeOH); for 13C NMR
D
(pyridine-d5) assignments of the glycosidic parts: see Table 2. 13C
NMR data of the aglycone were almost the same (within
0.2 ppm) as those for compound 1; FABMS (negative ion mode)
m/z 1621 [MÀH]À, 1489 [MÀ132ÀH]À, 1459 [MÀ162ÀH]À, 1343
[MÀ146À132ÀH]À, 649 [MÀ132Â3À146À430ÀH]À, 441 [Acyl]À;
HRFABMS (positive-ion mode) m/z 1615.8239 [M+Na]+ (calcd. for
1615.8235 C78H128O33Na); Acid hydrolysis in the same manner as
described above gave 1a. Alkaline hydrolysis gave 1b.
4.13. Mild alkaline hydrolysis of 5 and 6
Each of the saponins (10 mg) was dissolved into 0.5 (w/v)% KO-
Haq and left at 20 °C for 15 min. The reaction mixture was acidified
by 1 mol/L HCl and extracted with EtOAc, and then the organic
layer was washed with saturated NaHCO3. The solvent was re-
moved in vacuo from the EtOAc layer to afford corresponding 8/
9-oxo-16-b-[(D-xylopyranosyl)oxy]hexadecanoic acid (10 and 13,
respectively). The aqueous layer was extracted n-BuOH to give
the corresponding saponin.
4.8. Physenoside S2 (2)
White powder; [a]
+18.8° (c 0.6, MeOH); for 13C NMR (pyri-
D
dine-d5) assignments of the glycosidic parts: see Table 2. 13C
NMR data of the aglycone were almost the same (within
0.2 ppm) as those for compound 1; FABMS (negative ion mode)
m/z 1191 [MÀH]À, 1159 [MÀ132ÀH]À, 1029 [MÀ162ÀH]À, 913
[MÀ132À146ÀH]À, 649 [MÀ132Â3À146ÀH]À; HRFABMS (posi-
tive-ion mode) m/z 1215.5801 [M+Na]+ (calcd. for 1215.5775
C57H92O26Na); Acid hydrolysis in the same manner as described
above gave 1a. Alkaline hydrolysis gave 1b.
4.14. 8-oxo-16-b-[(
D
-xylopyranosyl)oxy]hexadecanoic acid (10)
D +52.1° (c 0.3, MeOH); 1H NMR (pyridine-d5)
White powder; [
a
]
d 1.26–1.41 (12H, m, H-4, H-5, H-11, H-12, H-13 and H-14), 1.58
(8H, m, H-3, H-6, H-10 and H-15), 2.15 (2H, t, J = 7.6 Hz, H-2),
2.44 (4H, m, H-7 and H-9), 3.15 (1H, dd, J = 7.5, 9.2 Hz, H-2 of
Xyl), 3.18 (1H, dd, J = 10.3, 11.4 Hz, H-5a of Xyl), 3.31 (1H, dd,
J = 8.7, 9.2 Hz, H-3 of Xyl), 3.47 (1H, d, J = 5.3, 8.7, 10.3 Hz, H-4 of
Xyl), 3.53 (1H, dt, J = 9.6, 6.7 Hz, H-16a), 3.79 (1H, dt, J = 9.6,
6.7 Hz, H-16b), 3.84 (1H, dd, J = 5.2, 11.4 Hz, H-5b of Xyl), 4.18
(1H, d, J = 7.5 Hz, H-1 of Xyl); 13C NMR (pyridine-d5) d 24.5 (C-6),
24.8 (C-10), 25.8 (C-3), 26.9 (C-14), 29.7 (C-4), 30.2 (C-5), 30.4
(C-11, C-12 and C-13), 30.7 (C-15), 34.0 (C-2), 43.3 (C-7), 43.5 (C-
9), 66.9 (Xyl-5), 70.9 (C-16), 71.2(Xyl-4), 74.9(Xyl-2), 77.9 (Xyl-
3), 104.8(Xyl-1), 178.4 (C-1), 214.3 (C-8); HRFABMS (positive-ion
mode) m/z 441.2450 [M+Na]+ (calcd. 441.2464 for C21H38O8Na);
Compound 10 (10 mg) was dissolved in 1 mol/L H2SO4 (2 ml) and
heated at 80 °C for 2 h. The reaction mixture was extracted with
Et2O, and the organic layer washed with saturated NaHCO3, and
the solvent was removed to give 11 (5 mg).
4.9. Physenoside S3 (3)
White powder; [a]
+12.7° (c 0.9, MeOH); for 13C NMR (pyri-
D
dine-d5) assignments of the glycosidic parts: see Table 2. 13C
NMR data of the aglycone were almost the same (within
0.2 ppm) as those for compound 1; FABMS (negative ion mode)
m/z 1337 [MÀH]À, 1205 [MÀ132ÀH]À, 1191 [MÀ146ÀH]À, 1175
[MÀ162ÀH]À, 649 [MÀ132Â3À146Â12ÀH]À; HRFABMS (posi-
tive-ion mode) m/z 1361.6371 [M+Na]+ (calcd. for 1361.6354
C63H102O30Na); Acid hydrolysis in the same manner as described
above gave 1a. Alkaline hydrolysis gave 1b.
4.10. Physenoside S4 (4)
White powder; [
dine-d5) assignments of the glycosidic parts: see Table 2. 13C
a]
+16.6° (c 0.5, MeOH); for 13C NMR (pyri-
D

M. Inoue et al. / Phytochemistry 70 (2009) 1195–1202
1201
4.15. 9-oxo-16-b-[(
D
-xylopyranosyl)oxy]hexadecanoic acid (13)
m/z 1621 [MÀH]À, 1489 [MÀ132ÀH]À, 1459 [MÀ162ÀH]À, 1343
[MÀ132À146ÀH]À; Acid hydrolysis in the same manner as
described above gave 9a. Alkaline hydrolysis of 8 gave 9b. Mild
alkaline hydrolysis with 0.5 (w/v)% KOHaq also gave 9-oxo-16-b-
White powder; [
a
]
D +48.5° (c 0.3, MeOH); 1H NMR (pyridine-d5)
d 1.26–1.41 (12H, m, H-4, H-5, H-6, H-12, H-13 and H-14), 1.58
(8H, m, H-3, H-7, H-11 and H-15), 2.15 (2H, t, J = 7.6 Hz, H-2),
2.43 (4H, m, H-8 and H-10), 3.15 (1H, dd, J = 7.5, 9.2 Hz, H-2 of
Xyl), 3.18 (1H, dd, J = 10.3, 11.4 Hz, H-5a of Xyl), 3.31 (1H, dd,
J = 8.7, 9.2 Hz, H-3 of Xyl), 3.48 (1H, d, J = 5.3, 8.7, 10.3 Hz, H-4 of
Xyl), 3.51 (1H, dt, J = 9.6, 6.7 Hz, H-16a), 3.80 (1H, dt, J = 9.6,
6.7 Hz, H-16b), 3.84 (1H, dd, J = 5.2, 11.4 Hz, H-5b of Xyl), 4.19
(1H, d, J = 7.5 Hz, H-1 of Xyl); 13C NMR (pyridine-d5) d 24.8 (C-7),
24.9 (C-11), 25.9 (C-3), 29.8 (C-4), 30.1 (C-5), 30.2 (C-6, C-12 and
C-13), 30.7 (C-14 and C-15), 34.0 (C-2), 43.3 (C-8 and C-10), 66.9
(Xyl-5), 70.9 (C-16), 71.2(Xyl-4), 74.9(Xyl-2), 77.9 (Xyl-3), 105.1
(Xyl-1), 178.4 (C-1), 214.5 (C-9); HRFABMS (positive-ion mode)
m/z 441.2488 [M+Na]+ (calcd. 441.2464 for C21H38O8Na); Acid
hydrolysis of 13 (10 mg) gave 14 (5 mg).
[(D-xylopyranosyl)oxy]hexadecanoic acid (13).
4.20. Brine shrimp lethality assay
The bioassay was performed in the similar way described in ref-
erence (Meyer et al., 1982; Anderson et al., 1991; Solis et al., 1993;
Carballo et al., 2002). Brine shrimp eggs (Japan Pet Drugs Co., To-
kyo, Japan) were hatched in artificial sea water prepared with com-
mercial salt mixture (Senju Phamaceutical Co., Osaka, Japan) and
oxygenated with an aquarium pump. After 48 h incubation at
28 °C, nauplii were collected with pasteur pipette after attracting
the organisms to one side of vessel with a light source. Ten shrimps
were transferred to each sample vial, and artificial sea water was
added to make 2.5 mL. Samples for testing were made up to
1 mg/mL in artificial sea water (2.5 mL) except for water insoluble
4.16. Preparation of methyl 16-trimethylsilyloxy-8/9-
oxohexadecanoate for EIMS
samples which were dissolved in 50 lL DMSO prior to adding sea
water. Sample solutions (2.5 mL) were added to each test vial (fi-
nally, total 5 mL). The vials were maintained under illumination.
Survivors were counted after 2 and 24 h, and the percent deaths
at each dose and control were determined. DMSO in this concen-
tration did not affect this bioassay. The LC50 values were deter-
mined from 24 h counts using the probit analysis. The results are
summarized in Table 3.
Trimethylsilyldiazomethane (20
solution (100 L) of compounds 11 or 14 (1 mg). After 1 h, the sol-
vent was removed from the reaction mixture, and then added
TMSI-H (100 L) to the residue. After 15 min, H2O (1 drop) was
added to stop the reaction, and the reaction mixture was extracted
with n-hexane. The organic layer furnished methyl 16-trimethylsi-
lyloxy-8/9-oxohexadecanoate (12 and 15, respectively).
lL) was added to an EtOH
l
l
Acknowledgements
4.17. Compound 9 (alkaline treatment product of Physenoside S7 (7)
and S8 (8) with 2.5% NaOHaq)
This work was supported by Grants-in-Aid for Scientific Re-
search (B) (14380289 and 15390013) from the Japan Society for
the Promotion of Science.
White powder; [a]
D +6.9° (c 1.0, MeOH); for 13C NMR (pyridine-
d5) assignments of the glycosidic parts, see Table 2. 13C NMR
(pyridine-d5) data of the aglycone: d 13.9 (C-24), 16.8 (C-25),
17.2 (C-26), 20.9 (C-6), 23.7 (C-11), 24.4 (C-30), 26.8 (C-27), 30.5
(C-20), 31.6 (C-22), 32.8 (C-29), 33.0 (C-7), 35.6 (C-15), 35.7 (C-
21), 36.5 (C-10), 40.2 (C-8), 41.3 (C-18), 42.0 (C-14), 44.1 (C-1),
47.0 (C-19), 47.5 (C-9), 49.3 (C-17), 52.3 (C-5), 52.6 (C-4), 69.8
(C-2), 73.4 (C-16), 85.6 (C-3), 122.3 (C-12), 144.2 (C-13), 175.7
(C-28), 180.9 (C-23); FABMS (negative ion mode) m/z 1221
[MÀH]À, 1089 [MÀ132ÀH]À, 1059 [MÀ162ÀH]À, 943
[MÀ132À146ÀH]À, 649 [MÀ132Â3À146ÀH]À; HRFABMS (posi-
tive-ion mode) m/z 1245.5562 [M+Na]+ (calcd. for 1245.5516
C57H90O28Na).
References
Anderson, J.E., Goetz, C.M., McLaughlin, J.L., Suffness, M., 1991. A blind comparison
of simple bench-top bioassays and human tumour cell cytotoxicities as
antitumor prescreens. Phytochem. Anal. 2, 107–111.
Cao, S., Norris, A., Miller, J.S., Ratovoson, F., Birkinshaw, C., Andriantsiferana, R.,
Rasamison, V.E., Rakotonandrasana, S., Kingston, D.G., 2006. Cytotoxic
compounds of Physena madagascariensis from the Madagascar rain forest
paragraph sign. Nat Prod. Res. 20, 1157–1163.
Carballo, J.L., Hernández-Inda, Z.L., Pérez, P., García-Grávalos, M.D., 2002.
A
comparison between two brine shrimp assays to detect in vitro cytotoxicity in
marine natural products. BMC Biotechnol. 2, 17.
Deas, A.H.B., Baker, E.A., Holloway, P.J., 1974. Identification of 16-
hydroxyoxohexadecanoic acid monomers in plant cutins. Phytochemistry 13,
1901–1905.
Deng, Y., Jiang, T.Y., Sheng, S., Tianasoa-Ramamonjy, M., Snyder, J.K., 1999.
Remangilones A–C, new cytotoxic triterpenes from Physena madagascariensis.
J. Nat. Prod. 62, 471–476.
Deng, Y., Lee, J.P., Tianasoa-Ramamonjy, M., Snyder, J.K., Des Etages, S.A., Kanada, D.,
Snyder, M.P., Turner, C.J., 2000. New antimicrobial flavanones from Physena
madagascariensis. J. Nat. Prod. 63, 1082–1089.
4.18. Physenoside S7 (7)
White powder; [a]
+5.2° (c 1.0, MeOH); for 1H NMR spectro-
D
scopic (pyridine-d5) assignments, see Table 1, and for 13C NMR
(pyridine-d5) assignments of the glycosidic parts: see Table 2; 13C
NMR data of the aglycone were almost the same (within
0.2 ppm) as those for compound 9; FABMS (negative ion mode)
m/z 1621 [MÀH]À, 1489 [MÀ132ÀH]À, 1459 [MÀ162ÀH]À, 1343
[MÀ132À146ÀH]À; Acid hydrolysis in the same manner as
described above gave 9a. Alkaline hydrolysis of 7 gave 9b. Mild
alkaline hydrolysis with 0.5 (w/v)% KOHaq also gave 8-oxo-16-b-
Dickison, W.C., Miller, R.B., 1993. Morphology and anatomy of the malagasy genus
Physena (Physenaceae), with a discussion of the relationships of the genus.
Adansonia 1–4, 85–106.
Dimbi, M.Z., Warin, R., Delaude, C., Huls, R., Kapundu, M., 1984. Structure of
zanhic acid and zanhic acid gamma-lactone,
Zanha golungensis and Ganophyllum giganteum. Bull. Soc. Chim. Belg. 93, 323–
328.
2 novel triterpenoids from
Jurenitsch, J., Lichtenberger, E., Robien, W., Jentzsch, K., 1986. About the
constituents of Giant Golden Rod. Planta Med. 52, 236–238.
Kasai, R., Okihara, M., Asakawa, J., Mizutani, K., Tanaka, O., 1979. 13C NMR study of
[(
4.19. Physenoside S8 (8)
White powder. [ ]
+1.2° (c 1.5, MeOH); for 1H NMR spectro-
D
D-xylopyranosyl)oxy]hexadecanoic acid (10).
a- and b-anomeric pairs of
D-mannopyranosides and L-rhamnopyranosides.
Tetrahedron 35, 1427–1432.
Kasai, R., Miyakoshi, M., Matsumoto, K., Nie, R.L., Zhou, J., Morita, T., Tanaka, O.,
1986. Tubeimoside I, a new cyclic bisdesmoside from Chinese cucurbitaceous
folk medicine ‘‘tu bei mu”, a tuber of Bolbostemma paniculatum. Chem. Pharm.
Bull. (Tokyo) 34, 3974–3977.
Kitagawa, I., Hori, K., Sakagami, M., Hashiuchi, F., Yoshikawa, M., Ren, J., 1993.
Saponin and sapogenol. XLIX. On the constituents of the roots of Glycyrrhiza
inflata Batalin from Xinjiang, China. Characterization of two sweet oleanane-
a
scopic (pyridine-d5) assignments, see Table 1, and for 13C NMR
(pyridine-d5) assignments of the glycosidic parts, see Table 2. 13C
NMR data of the aglycone were almost the same (within
0.2 ppm) as those for compound 9; FABMS (negative ion mode)

1202
M. Inoue et al. / Phytochemistry 70 (2009) 1195–1202
type triterpene oligoglycosides, apioglycyrrhizin and araboglycyrrhizin. Chem.
Pharm. Bull. 41, 1350–1357.
Ray, A.K., Lin, Y.Y., Gerard, H.C., Chen, Z.J., Osman, S.F., Fett, W.F., Moreau, R.A., Stark,
R.E., 1995. Separation and identification of lime cutin monomers by high
performance liquid chromatography and mass spectrometry. Phytochemistry
38, 1361–1369.
Sakai, K., Nagao, T., Okabe, H., 1999. Triterpenoid saponins from the ground part of
Aster ageratoides var. Ovatus. Phytochemistry 51, 309–318.
Samyn, J.M., 1999. Plantes Utiles des Hautes Terres de Madagascar. Graphoprint,
Madagascar.
Lavaud, C., Voutquenne, L., Massiot, G., le Men-Olivier, L., Das, B.C., Laprevote, O.,
Serani, L., Delaude, C., Becchi, M., 1998. Saponins from the stem bark of Filicium
decipiens. Phytochemistry 47, 441–449.
Marston, A., Gafner, F., Dossaji, S.F., Hostettmann, K., 1988. Fungicidal and
molluscicidal saponins from Dolichos kilimandscharicus. Phytochemistry 27,
1325–1326.
Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., McLaughlin, J.L.,
1982. Brine shrimp: a convenient general bioassay for active plant constituents.
Planta Med. 45, 31–34.
Solis, P.N., Wright, C.W., Anderson, M.M., Gupta, M.P., Phillipson, J.D., 1993. A
microwell cytotoxicity assay using Artemia salina (brine shrimp). Planta Med.
59, 250–252.
Nagao, T., Hachiyama, S., Okabe, H., Yamauchi, T., 1989. Studies on the constituents
of Aster tataricus L.f. II. Structures of Aster saponins isolated from the root..
Chem. Pharm. Bull. (Tokyo) 37, 1977–1983.