Triterpenoid saponins from Albizia boromoensis Aubrv. & Pellegr

Article abstract of DOI:10.1016/j.phytol.2014.11.004

As part of our search of new bioactive saponins from Cameroonian medicinal plants, phytochemical investigation of the roots of Albizia boromoensis led to the isolation of four new oleanane-type saponins, named boromoenosides A-D (1-4). Their structures were established by direct interpretation of their spectral data, mainly HRESIMS, 1D NMR (¹H, ¹³C NMR, and DEPT) and 2D NMR (COSY, NOESY, HSQC and HMBC), and by comparison with the literature data. All isolated saponins were assayed for their cytotoxicity against U-87 MG human glioblastoma cell lines and TG1 glioblastoma stem-like cells with no positive activity detected.

Full text of DOI:10.1016/j.phytol.2014.11.004

Phytochemistry Letters 11 (2015) 37–42
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Triterpenoid saponins from Albizia boromoensis Aubr e´ v. & Pellegr
a,b,
c
d
e
Olivier Placide Not e´ *, Dong Jihu , Cyril Antheaume , Dominique Guillaume ,
b
c
a
Dieudonn e´ Emmanuel Pegnyemb , Marie Claude Kilhoffer , Annelise Lobstein
a
Pharmacognosie et Mol e´ cules Naturelles Bioactives, Laboratoire d’Innovation Th e´ rapeutique, UMR 7200, CNRS-Universit e´ de Strasbourg,
Facult e´ de Pharmacie, 74 route du Rhin, F-67401 Illkirch Cedex, France
b
Laboratoire de Pharmacochimie des Substances Naturelles, D e´ partement de Chimie Organique, Facult e´ de Sciences, Universit e´ de Yaound e´ , BP 812, Yaound e´ ,
Cameroon
c
Chimie-Biologie Int e´ grative, Laboratoire d’Innovation Th e´ rapeutique, UMR 7200, CNRS-Universit e´ de Strasbourg, Facult e´ de Pharmacie, 74 route du Rhin,
F-67401 Illkirch Cedex, France
d
Service Commun d’Analyse, UMR 7200, CNRS-Universit e´ de Strasbourg, Facult e´ de Pharmacie, 74 route du Rhin, F-67401 Illkirch Cedex, France
e
Laboratoire de Chimie Th e´ rapeutique, UMR 6229, 51 rue Congnacq-Jay, 51100 Reims, France
A R T I C L E I N F O
A B S T R A C T
Article history:
As part of our search of new bioactive saponins from Cameroonian medicinal plants, phytochemical
investigation of the roots of Albizia boromoensis led to the isolation of four new oleanane-type saponins,
named boromoenosides A–D (1–4). Their structures were established by direct interpretation of their
spectral data, mainly HRESIMS, 1D NMR ( H, C NMR, and DEPT) and 2D NMR (COSY, NOESY, HSQC and
HMBC), and by comparison with the literature data. All isolated saponins were assayed for their
cytotoxicity against U-87 MG human glioblastoma cell lines and TG1 glioblastoma stem-like cells with
no positive activity detected.
Received 23 August 2014
Received in revised form 5 November 2014
Accepted 14 November 2014
Available online 26 November 2014
1
13
Keywords:
Mimosaceae
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Albizia boromoensis
Triterpenoid saponins
Boromoenosides
NMR
1
. Introduction
Albizia boromoensis Aubr e´ v. & Pellegr. (Mimosaceae) is a large
In the present paper, we report the isolation and structure
characterization of four new triterpenoid saponins, named
boromoenosides A–D (1–4) having oleanolic acid as aglycone.
emergent tree of the legume family. It is found growing naturally in
Senegal, Soudan, and Cameroon where it is harvested for timber
2. Results and discussion
(
Arbonnier, 2009). No ethnomedicinal use of this plant is
documented. Its chemical constituents have not been previously
investigated but phytochemical studies on related species of
Albizia genus revealed the presence of triterpenoid saponins
The air-dried powdered roots of A. boromoensis (200 g) were
extracted with 70% EtOH in soxhlet and after evaporation of the
solvent, the hydroalcoholic extract was partitioned against n-BuOH
saturated with water. The n-BuOH fraction was then submitted to
vacuum–liquid chromatography (VLC) on reversed phase silica gel
yielding a methanol fraction that was subjected to a VLC on silica gel.
Purification of the eluted fractions by semipreparative high pressure
liquid chromatography (HPLC) afforded compounds 1 (7.9 mg), 2
(
Abdel-Kader et al., 2001; Haddad et al., 2003; Liu et al., 2009; Krief
et al., 2005; Zheng et al., 2006; Melek et al., 2007; Cao et al., 2007;
Not e´ et al., 2009; Miyase et al., 2010). As a part of our continuing
studies on saponins from Cameroonian medicinal plants (Note´
et al., 2013), we have investigated the saponins content of the roots
of A. boromoensis.
(
8.5 mg), 3 (7.2 mg), and 4 (5.3 mg). Their structures (Fig. 1) were
established by extensive analysis of NMR techniques, mainly 1D
1
13
NMR ( H, C NMR, and DEPT) and 2D NMR (COSY, NOESY, HSQC and
HMBC) experiments, and HRESIMS and by comparison with the
literature data.
*
Corresponding author at: Laboratoire de Pharmacochimie des Substances
Boromoenoside A (1), obtained as a white amorphous powder,
Naturelles, D e´ partement de Chimie Organique, Facult e´ de Sciences, Universit e´ de
Yaound e´ , BP 812, Yaound e´ , Cameroon. Tel.: +237 72700015; fax: +237 22239588.
E-mail address: oliviernote1@yahoo.fr (O.P. Not e´ ).
was assigned a C64
H
104
O
31 molecular formula, as deduced from the
+
[M+NH
4
] ion at m/z 1386.6789 in the positive ion mode HRESIMS.
http://dx.doi.org/10.1016/j.phytol.2014.11.004
1
874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

3
8
O.P. Not e´ et al. / Phytochemistry Letters 11 (2015) 37–42
Ara
Glc'
Glc
Xyl
R
Glc
H
H
Glc
1
R
2
Glc
(1)
(2)
(3)
(4)
Glc(1→6)Glc-
Glc
H
Fig. 1. Structures of isolated compounds 1–4.
1
13
The H NMR and C NMR data of boromoenoside A displayed
resonances due to the aglycone part characteristic for oleanolic
acid (Table 1), a common triterpene of triterpene glycosides, which
were in full agreement with literature data (Nigam et al., 1997;
Mimaki et al., 2004). The sugar moieties of 1 were determined to be
D
-glucose (Glc),
L
-arabinose (Ara) and D-xylose (Xyl) by acid
hydrolysis followed GC analysis of their trimethylsilylated
derivatives by comparison with authentic samples (Section 3).
The sugar portion of compound 1 exhibited in the H NMR
1
spectrum, six anomeric proton signals at
d 4.84 [d, J = 7.6 Hz,
Table 1
1
13
a
NMR spectroscopic data (500 MHz for H and 125 MHz for C) for the aglycone moieties of compounds 1–4 (d in ppm and J in Hz).
No.
1
2
3
4
d
C
d
H
d
C
d
H
d
C
d
H
d
C
d
H
1
2
3
4
5
6
7
8
9
38.9
27.0
89.5
39.5
56.1
18.8
33.4
42.2
48.3
37.2
24.0
0.98; 1.51
1.88; 2.33
3.16 (brd, 11.8)
–
39.0
27.0
89.7
39.5
56.1
18.7
33.2
42.4
48.2
37.1
24.1
0.97; 1.50
1.88; 2.32
3.15
38.9
27.0
89.4
39.5
56.1
18.9
33.5
42.4
48.2
37.2
24.1
0.97; nf
38.9
27.0
89.7
39.8
56.1
18.7
33.4
42.3
48.2
37.1
24.0
0.97; 1.49
1.87; 2.33
1.92; 2.34
3.17 (brd, 12.6)
3.20 (d, 11.5)
–
–
–
0.62 (brd, 11.5)
1.26; 1.46
1.44; 1.60
–
0.63 (brd, 11.8)
1.21; 1.44
1.41; 1.53
–
0.63 (brd, 11.8)
0.64 (brd, 9.8)
1.21; 1.45
1.22; 1.40
1.44; 1.56
1.37; 1.53
–
–
1.59 (d-like, 9.3)
–
1.58 (d-like, 8.8)
–
1.58 (t-like, 9.8; 8.2)
1.57 (t-like, 9.8; 8.2)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
–
–
1.89
1.87
0.94; 1.90
5.41 (brs 5.7)
–
1.87
122.9
144.7
42.4
29.4
23.3
47.3
42.0
46.6
31.0
34.4
32.6
28.5
17.1
15.8
17.7
26.4
176.9
33.4
24.1
5.41 (brs)
–
129.9
144.7
42.4
29.4
23.3
47.3
42.0
46.7
31.0
34.3
32.5
28.4
17.1
15.8
17.7
26.4
176.9
33.4
24.0
5.40 (brs)
–
122.6
144.7
42.4
29.4
23.3
47.3
42.1
46.6
31.0
34.4
32.5
28.5
17.1
15.8
17.7
26.4
176.8
33.5
24.1
123.3
144.6
42.3
29.4
23.3
47.3
41.9
46.6
31.0
34.2
32.8
28.4
17.1
15.9
17.8
26.4
176.8
33.4
24.0
5.39 (brs)
–
–
–
–
–
1.39; 2.27
2.12; 2.33
–
1.37; 2.26
2.12; 2.33
–
1.40; nf
nf; nf
–
nf; nf
nf; nf
–
3.19 (d, 11.5)
1.26; 1.77
–
3.17
3.19
3.20
1.26; 1.79
–
1.28; nf
–
1.27; 1.76
–
1.07; 1.33
1.74; 1.86
1.18 (s)
1.04 (s)
0.80 (s)
1.01 (s)
1.22 (s)
–
1.08; 1.33
1.75; 1.86
1.27 (s)
1.12 (s)
0.75 (s)
0.99 (s)
1.21(s)
–
1.07; 1.39
1.79; nf
1.20 (s)
1.02 (s)
0.79 (s)
1.03 (s)
1.23 (s)
–
1.12; 1.36
1.78; 1.92
1.28 (s)
1.11 (s)
0.74 (s)
1.01 (s)
1.21 (s)
–
0.89 (s)
0.88 (s)
0.89 (s)
0.88 (s)
0.92 (s)
0.90 (s)
0.92 (s)
0.91 (s)
Assignments were based on the HMBC, HSQC, COSY and DEPT experiments.
a
1
Overlapped H NMR signals are reported without designated multiplicity; nf: not found.

O.P. Not e´ et al. / Phytochemistry Letters 11 (2015) 37–42
39
glucose (Glc)], 4.93 [d, J = 6.5 Hz, arabinose (Ara)], 5.38 [d,
J = 7.4 Hz, xylose (Xyl)], 6.16 [d, J = 8.2 Hz, glucose (Glc )],
aglycone, respectively),
and 5.38 (H-1 of Xyl) with
sugar arrangement in the 3-O-saccharide chain was established
as b-D-
glucopyranosyl (Fig. 2). The sequence and binding sites of the
sugar units at C-28 were determined by following HMBC
d
4.24 (H-6a of Glc) with
d
4.93 (H-1of Ara)
0
d
d 4.19 (H-2 of Glc). Accordingly, the
00
5
(
.71 [d, J = 7.7 Hz, glucose (Glc )], and 5.02 [d, J = 7.7 Hz, glucose
000
13
Glc )]. Their associated C resonances assigned from HSQC and
HMBC experiments were at 104.9, 105.6, 106.3, 93.8, 104.9, and
05.6, respectively. All proton signals due to the sugar moieties
a-
L
-arabinopyranosyl-(1!6)-[
b
-D
-xylopyranosyl-(1!2)]-
d
1
0
were identified by careful analysis of COSY, TOCSY and NOESY
spectra, and the carbon signals were assigned based on HSQC and
further confirmed by HMBC spectrum (Table 2). Data from the
correlations:
aglycone),
(H-1 of Glc ) with
d
6.16 (H-1 of Glc ) with
d
176.9 (C-28 of the
00
0
d
5.71 (H-1 of Glc ) with
d
69.3 (C-6 of Glc ) and
d
5.02
000
0
d
78.5 (C-2 of Glc ). Therefore, the sugar
above experiments indicated that these six sugar residues were in
sequence at C-28 of the aglycone was determined as
glucopyranosyl-(1!6)-[ -glucopyranosyl-(1!2)]-
copyranosyl. Thus, the structure of boromoenoside A (1),
was assigned as 3-O- -[
-arabinopyranosyl-(1!6)-[
-glucopyranosyl]-28-O-[ -glucopyr-
-glucopyranosyl-(1!2)]- -glucopyranosyl]-
b-D-
b-D-glu-
3
their pyranose form. The relative large
JH-1,H-2 values of these
b
-
D
sugar moieties (6.1–8.2 Hz) in their pyranose form indicated a
anomeric configuration for all Glc and Xyl moieties, and an
b
a
b
D
a
-
L
b-D-
anomeric configuration for Ara (Mimaki et al., 2004; Ten e´ et al.,
xylopyranosyl-(1!2)]-
b
-
b-D
13
2
011). The observation of the C NMR signals at
d
89.5 (C-3 of
anosyl-(1!6)-[
b
-
D
b-D
aglycone) and 176.9 (C-28 of the aglycone) demonstrated that 1 is
a bidesmosidic glycoside of oleanolic acid (Sahu and Achari, 2001;
Ten e´ et al., 2011) with sugar chains linked to C-3 and C-28 of the
aglycone through an ether and ester bond, respectively. The
linkage points of the sugar units to each other and to C-3 of the
aglycone were established from the HMBC correlations of signals at
oleanolic acid.
1
3
The analysis of C NMR spectra showed that the trisaccharide
chain was common to all four saponins and placed at C-3 of the
aglycone. It will not be further discussed in the structural
elucidation of compounds 2–4.
+
The HRESIMS spectrum ([M+NH
4
]
at m/z 1386.6575) of
d
4.84 (H-1 of Glc1) with
Ara) with 70.1 (C-6 of Glc) and
of Glc). This was supported by the NOESY correlations of signals at
d
89.5 (C-3 of the aglycone),
d
4.93 (H-1 of
boromoenoside B (2), obtained as a white amorphous powder,
was identical to that obtained for compound 1, suggesting that
these compounds were isomers with C64H O31 formula. Com-
104
d
d
5.38 (H-1 of Xyl) with
d
83.3 (C-2
d
4.84 (H-1 of Glc) with
d
3.16 and
d
1.18 (H-3 and H
3
-23 of the
parison of their NMR spectra showed that the sugar directly
Table 2
1
13
a
NMR spectroscopic data (500 MHz for H and 125 MHz for C) for the sugar moieties of compounds 1–5 (
d
in ppm and J in Hz).
Position
1
2
3
4
d
C
d
H
d
C
d
H
d
C
d
H
d
C
d
H
3
-O-sugars
Glc-1
104.9
83.3
77.7
72.2
76.7
70.1
4.84 (d, 7.6)
4.19
105.0
83.2
77.7
72.2
76.7
69.9
4.84 (d, 7.4)
4.19
105.0
83.3
77.7
72.2
76.7
70.0
4.84 (d, 7.4)
4.19
105.0
83.3
77.7
72.2
76.7
70.0
4.84 (d, 7.3)
4.19
2
3
4
5
6
4.14
4.11
4.14
4.14
4.08
4.09
4.08
4.08
4.06
4.07
4.06
4.06
4.24; 4.85
4.25; 4.84
(brd, 10.4)
4.93 (d, 6.5)
4.48
4.25; 4.86
4.26; 4.84
(
t, 10.9; 7.6)
Ara-1
105.6
72.5
71.9
74.6
64.2
106.3
73.1
78.7
71.8
66.7
4.93 (d, 6.5)
4.49
105.6
72.4
71.6
74.6
64.2
106.2
73.1
78.4
71.8
66.6
105.6
72.5
71.9
74.7
64.1
106.3
73.2
78.2
71.8
66.6
4.94 (d, 6.5)
4.49
105.6
72.5
71.9
74.6
64.2
106.3
73.1
78.7
71.8
66.6
4.94 (d, 6.4)
4.49
2
3
4.08
4.09
4.08
4.08
4
4.20
4.20
4.20
4.20
5
4.43; 4.74 (d, 9.8)
5.38 (d, 7.4)
4.15
4.43; 4.72 (d, 10.3)
5.37 (d, 7.7)
4.11
4.44; 4.71
5.37 (d, 7.5)
4.15
4.43; 4.74
5.39 (d, 7.5)
4.15
Xyl-1
2
3
4
5
4.04
4.04
4.03
4.04
4.08
4.09
4.08
4.08
4.32; 3.78
4.33; 3.77
(brd, 10.3)
4.34; 3.78
(d, 10.3)
4.34; 3.78
(
d, 11.0)
2
8-O-Sugars
0
Glc -1
93.8
78.5
77.6
72.0
76.0
69.3
104.9
75.5
78.4
72.1
78.7
62.9
6.16 (d, 8.2)
93.7
78.5
77.6
70.7
77.8
62.9
104.9
75.7
78.2
71.4
76.2
69.3
6.14 (d, 8.2)
4.39
93.9
79.2
77.8
70.9
78.7
62.2
104.9
73.4
78.4
69.3
79.4
63.0
6.24 (d, 8.2)
4.52
93.7
75.4
78.2
71.6
76.6
69.7
104.9
75.7
78.2
69.3
78.7
62.9
6.22 (d, 8.0)
4.04
2
3
4
5
6
4.42
4.21
4.30
4.14
4.10
4.24
4.26
4.30
4.24
4.06
3.95
3.95
4.06
4.35; 4.65 (brd, 11.0)
4.37; 4.49
5.69 (d, 7.7)
4.08
4.38; 4.45
5.77 (d, 7.8)
4.09
4.37; 4.73
5.77 (d, 7.4)
4.08
00
Glc -1
5.71 (d, 7.7)
4.09
2
3
4
5
6
4.27
4.27
4.27
4.30
4.09
4.15
4.35
4.15
3.91
4.04
3.94
3.91
4.31; 4.43
4.35; 4.65
4.37; 4.51
4.38; 4.50
(
brd, 10.7)
000
Glc -1
105.6
75.2
78.7
69.3
78.3
63.0
5.02 (d, 7.7)
4.04
105.5
75.2
78.4
69.3
78.1
62.9
5.02 (d, 7.7)
4.02
2
3
4
5
6
4.20
4.18
4.34
4.35
4.00
3.98
4.37; 4.53
4.39; 4.52
Assignments were based on the HMBC, HSQC, COSY and DEPT experiments.
a
1
Overlapped H NMR signals are reported without designated multiplicity.

4
0
O.P. Not e´ et al. / Phytochemistry Letters 11 (2015) 37–42
2
9
30
2
0
1
9
21
22
1
8
1
26
2
1
1
13
17
16
2
5
1
4
O
1
4
9
15
2
3
10
8
2
8
2
7
5
7
6
O
H O
6
O
2
4
O
23
O
H O
6
O
O
O
H O
H O
H O
H O
2
O
H O
H O
H O
OH
1
2
OH
1
Ara
1
Glc
Glc''
1
Glc'
O
O
H O
H O
H O
O
O
OH
1
H O
H O
Xyl
OH
1
Glc'''
NOESYcorrelations
HMBC correlations
Fig. 2. Key HMBC and NOESY correlations for compound 1.
0
0
attached at C-28 (Glc ) in 2 was monosubstituted at its C-2
Glc ) as assigned in the case of 3. Therefore, the sugar sequence at
position, instead of been disubstituted at its C-2 and C-6 positions
as described in the case of 1 (Table 1). The presence of only one
C-28 of the aglycone was determined as
(1!6)- -glucopyranosyl. Consequently, the structure of boro-
moenoside D was elucidated as 3-O- -[ -arabinopyranosyl-
(1!6)-[ -xylopyranosyl-(1!2)]- -glucopyranosyl]-28-O-[
-glucopyranosyl-(1!6)- -glucopyranosyl]-oleanolic acid.
Compounds 1–4 were evaluated for their inhibitory effect on
the survival of high grade human brain tumor cells, including the
U-87 MG human glioblastoma cell line and the TG1 glioblastoma
cancer stem-like cells isolated from a patient. None of the isolated
compounds showed positive activity in our assay. In addition, to
the best of our knowledge, our study is the first phytochemical
study of A. boromoensis and represents a valuable contribution to
the chemotaxomy of Albizia genus, which is known to be a rich
source of triterpenoid saponins.
b-D-glucopyranosyl-
b-D
0
glycosylation shift at
glycosylation shift at
one glycosylation shift at
d
78.5 (C-2 of Glc ), the absence of
b a-L
0
d
62.9 (C-6 of Glc ) and the presence of
69.3 (C-6 of Glc ) and the absence of any
glycosylation shift for Glc were evidences for the linear sequence
established for the sugar moiety attached at C-28 of the aglycone
for compound 2. Therefore, the sugar sequence at C-28 of the
b
-D
b-
D
b-
0
0
d
0
D
b-D
00
aglycone was determined as
copyranosyl-(1!2)- -glucopyranosyl. Consequently, the struc-
ture of compound was elucidated as 3-O- -[
arabinopyranosyl-(1!6)-[ -xylopyranosyl-(1!2)]- -gluco-
pyranosyl]-28-O-[ -glucopyranosyl-
-glucopyranosyl-(1!6)-
1!2)- -glucopyranosyl]-oleanolic acid.
HRESIMS of boromoenoside C (3), obtained as a white,
amorphous powder, indicated a C58 26 molecular formula, as
] ion at m/z 1224.6789 in the positive
b-
D-glucopyranosyl-(1!6)-
b-D-glu-
b-D
2
b a-L-
b
-
D
b-D
b-D
b-D
(
b-D
94
H O
+
deduced from the [M+NH
4
3. Experimental
ion mode. The difference between compounds 2 and 3 (162 amu
lower) was assigned to one glucose moiety to account for the
3.1. General experimental procedures
1
13
similarities between their H and C NMR spectra (Table 2).
Instead of a trisaccharide moiety at C-28 of oleanolic acid,
compound 3 displayed signals for a disaccharide unit attached
at C-28 of the aglycone according to the analysis of 2D NMR
experiments. Accordingly, boromoenoside C (3) was elucidated
Optical rotations were measured on a Jasco P-2000 polarimeter.
1
13
H NMR (500 MHz) and C NMR (125 MHz) spectra were recorded
at room temperature in pyridine-d5 using a Bruker AVANCE III
500 spectrometer. Chemical shifts aregiven in (ppm) value relative
d
to be 3-O-
1!2)]-
-glucopyranosyl]-oleanolic acid.
The HRESIMS spectrum ([M+NH
boromoenoside D (4), obtained as a white amorphous powder,
was identical to that obtained for compound 3, suggesting that the
b
-[
a
-
L
-arabinopyranosyl-(1!6)-[
b
-
D
-xylopyranosyl-
to TMS as internal standard. HRESIMS spectra were recorded on a
microTOF ESI-TOF mass spectrometer (Agilent) operating in positive
mode. Analytical HPLC was performed on Varian 920-LC apparatus
equipped with an autosampler, a pump, a diode array detector
(DAD), and Galaxie software. Semipreparative HPLC was performed
on a Gilson apparatus equipped with Trilution LC software using a
(
b
b
-
D
-glucopyranosyl]-28-O-[
b
-
D
-glucopyranosyl-(1!2)-
-D
+
4
]
at m/z 1386.6575) of
compounds were isomers with C58
their NMR spectra showed that compound
compound 3 only by the substitution pattern of the sugar chain
H
94
O
26 formula. Comparison of
differs from
Nucleodur 100-5 C18ec (21 mm ꢀ 250 mm, 5
mm) column pur-
chased from Machery-Nagel (Germany). Thin layer chromatography
(TLC) was performed on precoated silica gel plates (60 F254, Merck)
4
at C-28 of the aglycone. This difference was evidenced by the
using the system solvent n-BuOH–AcOH–H O, 65:15:25 as eluent.
The spots were observed after spray with Komarowsky reagent, a
2
0
carbon signal at
d
69.7 (C-6 of Glc ) in 4, which was downfield
shifted of+7.2 ppm compared to the carbon signal at
d
62.2 (C-6 of
(5:1) mixture of p-hydroxybenzaldehyde (2% in MeOH) and (50%

O.P. Not e´ et al. / Phytochemistry Letters 11 (2015) 37–42
41
+
H
2
O in EtOH). Vacuum–liquid chromatography (VLC) was carried
out using RP-18 silica gel 60 (25–40 m) and silica gel 60 (15–
m and 40–63 m).
Tables 1 and 2; HRESIMS m/z: 1224.6787 [M+NH
58 94
C H O26, 1224.6869).
4
]
(calcd. for
m
4
0
m
m
3.8. Acid hydrolysis of compounds and determination of the absolute
3.2. Plant material
configuration of the sugar residues
The roots of A. boromoensis were harvested at Nkolbisson,
Each saponin (2 mg) was hydrolyzed with 2 mL of 2 M HCl at
85 8C during 2 h. After cooling, the solvent was removed under
reduced pressure. The sugar mixture was extracted from the
Yaound e´ peripheral quarter, in Cameroon in October 2011 under
the guidance of Dr. Paul Nana, botanist of the National Herbarium
of Cameroon (NHC), where one specimen (47196/HNC) was
deposited.
aqueous phase (10 mL) and washed with CH
combined CH Cl extracts were washed with water to give after
evaporation the aglycone moiety. The sugars were first analyzed
by TLC over silica gel (CHCl –MeOH–H O, 8:5:1) by comparison
2
Cl
2
(3 ꢀ 5 mL). The
2
2
3
.3. Extraction and isolation
3
2
with standard samples. The absolute configuration of each
monosaccharide was determined from GC–MS analysis of their
trimethylsilylated derivatives by comparison with authentic
samples using the method previously described (Chaabi et al.,
The air-dried, powdered roots of A. boromoensis (200 g) were
extracted with 70% EtOH in soxhlet apparatus. The resulting
hydroalcoholic solution was then evaporated to dryness under
reduce pressure to yield brown residue (4.83 g). This residue was
2010). The following sugars were detected:
and -arabinose.
D-glucose, D-xylose,
suspended in 200 mL of H
2
O and partitioned with n-BuOH sat. H
2
O
L
(
3 ꢀ 300 mL). The n-BuOH soluble phase was evaporated to
dryness affording 6.14 g of brown gum residue which was taken
in a minimum of water (10 mL) and then submitted to vacuum–
3.9. Evaluation of inhibitory effect of saponins
liquid chromatography (VLC) using RP-18 silica gel 60 (25–40
eluted with a gradient of MeOH–H O (100% H
O ! 100%MeOH).
The 70–85% MeOH fraction (600.5 mg) was subjected to VLC using
silica gel 60 (15–40 m) eluted with CHCl –MeOH–H O (80:20:2,
0:30:5, 60:33:7, and 60:40:10) to give four main subfractions
Bfr1–Bfr4). Subfraction Bfr4 (126.3 mg) was purified by semi-
preparative HPLC using gradient system of CH CN–H O (20 mL/
min) to yield compounds 1 (t , 13.20 min, 7.9 mg), 2 (t , 13.68 min,
.5 mg), 3 (t , 16.16 min, 7.2 mg), and 4 (t , 16.63 min, 5.3 mg).
m
m)
Effect of chemical compounds on glioblastoma cells’ ATP level
was assessed using the CellTiter Glo reagent (Promega). Cells were
2
2
seeded in 50
50 000 cells/well) into 96-well opaque bottom plates (Greiner,
Courtaboeuf, France). 50 L of pure compounds 1–4 (or 50 L of
mL of their respective media (between 30 000 and
m
3
2
7
(
m
m
Tamoxifen) dissolved at a given concentration in the respective cell
culture medium, were added to the cell suspension. Cell were
3
2
R
R
2
incubated at 37 8C, 5% CO and ATP levels were measured 24 h later
8
R
R
according to the manufacturer’s instructions. Luminescence was
TM
measured using a Victor 3 plate reader (PerkinElmer). Relative
3
.4. 3-O-
b
-[
-glucopyranosyl]-28-O-[
-glucopyranosyl-(1!2)]- -glucopyranosyl]-oleanolic acid (1)
a
-
L
-arabinopyranosyl-(1!6)-[
b
-
D
-xylopyranosyl-
ATP levels in each well were determined by calculating the
percentage of luminescent signal in the well with respect to the
average signal measured in negative control wells (without
compounds). Tamoxifen was used as positive control.
(
1!2)]-
b
-D
b
-
D
-glucopyranosyl-(1!6)-[
b-
D
b-D
2
D
5
1
White amorphous powder; ½
aꢁ
ꢂ14.4 (c 0.01, MeOH); H NMR
13
(
C
5
D
5
N, 500 MHz) and C NMR (C
Tables 1 and 2; HRESIMS m/z: 1386.6789 [M+NH
31, 1386.6905).
5
D
5
N, 125 MHz) data, see
Acknowledgement
+
4
] (calcd. for
C
64
H
104
O
The authors are grateful to Dr. Victor Nana of the National
Herbarium of Cameroon (NHC) for the identification and collection
of plant.
3
.5. 3-O-
1!2)]-
-glucopyranosyl-(1!2)-
b
b
-[
-glucopyranosyl]-28-O-[
-glucopyranosyl]-oleanolic acid (2)
a
-L
-arabinopyranosyl-(1!6)-[
b-D-xylopyranosyl-
-glucopyranosyl-(1!6)-
(
-D
b
-
D
b-
D
b-D
References
2
D
5
1
White amorphous powder; ½
aꢁ
ꢂ17.5 (c 0.01, MeOH); H NMR
13
Abdel-Kader, M., Hoch, J., Berger, J.M., Evans, R., Miller, J.S., Wisse, J.H., Mamber,
S.W., Dalton, J.M., Kingston, D.G., 2001. Two bioactive saponins from Albizia
subdimidiata from the Suriname rainforest. J. Nat. Prod. 64, 536–539.
Arbonnier, M., 2009. Arbres arbustes et lianes des zones d’Afrique de l’Ouest.
MNHN, Quæ, Paris, pp. 383.
(
C
5
D
5
N, 500 MHz) and C NMR (C
Tables 1 and 2; HRESIMS m/z: 1386.6787 [M+NH
31, 1386.6905).
5 5
D N, 125 MHz) data, see
+
4
] (calcd. for
64 104
C H O
Cao, S., Norris, A., Miller, J.S., Ratovoson, F., Razfitsalama, J., Andriantsiferana, R.,
Rasamison, V.E., Tendyke, K., Suh, T., Kingston, D.G.I., 2007. Cytotoxic triterpe-
noid saponins of Albizia gummifera from Madagascar rain forest. J. Nat. Prod. 70,
361–366.
Chaabi, M., Chabert, P., Vonthron-S e´ n e´ cheau, C., Weniger, B., Ouattara, M., Corst-
jens, H., Sente, I., Declercq, L., Lobstein, A., 2010. Acylated flavonol pentaglyco-
sides from Baphia nitida leaves. Phytochem. Lett. 3, 70–74.
Haddad, M., Miyamoto, T., Laurens, V., Lacaille-Dubois, M.A., 2003. Two new
biologically active triterpenoidal saponins acylated with salicylic acid from
Albizia adianthifolia. J. Nat. Prod. 66, 372–377.
Krief, S., Thoison, O., Sevenet, T., Wrangham, R.W., Lavaud, C., 2005. Triterpenoid
saponins anthranilates from Albizia grandibracteata leaves ingested by primates
in Uganda. J. Nat. Prod. 68, 897–903.
Liu, R., Ma, S., Yu, S., Pei, Y., Zhang, S., Chen, X., Zhang, J., 2009. Cytotoxic oleanane
triterpene saponins from Albizia chinensis. J. Nat. Prod. 72, 632–639.
Melek, F.R., Miyase, T., Ghaly, N.S., Nabil, M., 2007. Triterpenoid saponins with N-
acetyl sugar from the bark of Albizia procera. Phytochemistry 68, 1261–1266.
Mimaki, Y., Yokosuka, A., Hamanaka, M., Sakuma, C., Yamori, T., Shibata, Y., 2004.
Triterpene saponins from the roots of Clematis chinensis. J. Nat. Prod. 67,
1511–1516.
3
.6. 3-O-
b
-[
a
-
L
-arabinopyranosyl-(1!6)-[
b-D-xylopyranosyl-
-glucopyranosyl-(1!2)-
(
1!2)]-
b
-D
-glucopyranosyl]-28-O-[
b
-
D
b-
D
-glucopyranosyl]-oleanolic acid (3)
2
D
5
1
White amorphous powder; ½
a
ꢁ
ꢂ2.2 (c 0.01, MeOH); H NMR
13
(C
5
D
5
N, 500 MHz) and C NMR (C
5
D
5
N, 125 MHz) data, see
+
Tables 1 and 2; HRESIMS m/z: 1224.6789 [M+NH
4
] (calcd. for
58 94
C H O
26, 1224.6869).
3
.7. 3-O-
b
-[
a
-
L
-arabinopyranosyl-(1!6)-[
b-D-xylopyranosyl-
(
1!2)]-
b
-D
-glucopyranosyl]-28-O-[
b
-
D-glucopyranosyl-(1!6)-
b-
D
-glucopyranosyl]-oleanolic acid (4)
2
D
5
1
White amorphous powder; ½
a
ꢁ
ꢂ5.3 (c 0.01, MeOH); H NMR
13
5 5 5 5
(C D N, 500 MHz) and C NMR (C D N, 125 MHz) data, see

4
2
O.P. Not e´ et al. / Phytochemistry Letters 11 (2015) 37–42
Miyase, T., Melek, F.R., Ghaly, N.S., Warashina, T., El-Kady, M., Nabil, M., 2010.
Not e´ , O.P., Tapondjou, A., Mitaine-offer, A., Miyamoto, T., Pegnyemb, D.E., Lacaille-
Dubois, M.A., 2013. Triterpenoid saponins from Piptadeniastrum africanum
(Hook F.) Brenan. Phytochem. Lett. 6, 505–510.
Echinocystic acid 3,16-O-bisglycosides from Albizia procera. Phytochemistry 71,
1
375–1380.
Nigam, S.K., Gopal, M., Uddin, R., Yoshikawa, K., Kawamoto, M., Arihara, S., 1997.
Pithedulosides A-G, oleanane glycosides from Pithecellobium dulce. Phytochem-
istry 44, 1329–1334.
Not e´ , O.P., Mitaine-offer, A.C., Miyamoto, T., Paululat, T., Mirjolet, J.F., Duchamp, O.,
Pegnyemb, D.E., Lacaille-Dubois, M.A., 2009. Cytotoxic acacic acid glycosides
from the roots of Albizia coriaria. J. Nat. Prod. 72, 1725–1730.
Sahu, N.P., Achari, B., 2001. Advances in structural determination of saponins and
terpenoid glycosides. Curr. Org. Chem. 5, 315–334.
Ten e´ , M., Chabert, P., Not e´ , O., Kenla, N.J.T., Tane, P., Lobstein, A., 2011. Triterpenoid
saponins from Cylicodiscus gabunensis. Phytochem. Lett. 4, 89–92.
Zheng, L., Zheng, J., Zhao, Y., Wang, B., Wu, L., Liang, H., 2006. Three anti-tumor
saponins from Albizia julibrissin. Bioorg. Med. Chem. Lett. 16, 2765–2768.