Triterpenoid saponins and C-glycosyl flavones from stem bark of Erythrina abyssinica Lam and their cytotoxic effects

Article abstract of DOI:10.1016/j.phytol.2015.05.013

Abstract Six new compounds including two oleanane-type triterpenoid saponins (1, 2) and four C-glycosyl flavones (3-6), along with a known saponin (7), three di-C-glycosyl flavones (8-10) and a glycosyl auronol (11), were isolated from the stem bark of Erythrina abyssinica Lam. The structures of the new compounds, identified as 3-O-[α-l-rhamnopyranosyl-(1 → 2)-β-d-galactopyranosyl-(1 → 2)-β-d-glucuronopyranosyl]-22-O-β-d-glucopyranosyl sophoradiol (1), 3-O-[α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl]-22-O-β-D-glucopyranosyl sophoradiol (2), 6-C-β-glucopyranosyl-8-C-β-quinovopyranosyl apigenin (3), 6-C-β-quinovopyranosyl-8-C-β-glucopyranosyl apigenin (4), 8-C-[6″-O-α-l-rhamnopyranosyl-(1? → 6″]-β-glucopyranosyl 7,4′-dihydroxyflavone (5) and 8-C-[6″-O-β-d-xylopyranosyl-(1?; → 6″)]-β-glucopyranosyl 7,4′-dihydroxyflavone (6), were determined by comprehensive spectroscopic analysis, including 1D and 2D NMR techniques, mass spectrometry and acid hydrolysis. These new compounds together with the known saponins 7 were evaluated for their cytotoxic activity against MCF-7 (estrogen dependent) and MDA-MB-231 (estrogen independent) cell lines. The new saponin 2 exhibited the highest cytotoxic activity among tested compounds, exerting a selective inhibitory effect against the proliferation of MCF-7 cells, with lower IC₅₀ value (12.90 μM) than that of the positive control, resveratrol (13.91μM). Structure-activity relationship of these compounds is also discussed.

Full text of DOI:10.1016/j.phytol.2015.05.013

Phytochemistry Letters 13 (2015) 59–67
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Triterpenoid saponins and C-glycosyl flavones from stem bark of
Erythrina abyssinica Lam and their cytotoxic effects.
b
a
b
Andy J. Pérez a, , Emad M. Hassan , Łukasz Pecio , Elsayed A. Omer ,
*
Malgorzata Kucinska c, Marek Murias c, Anna Stochmal a
Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100, Puławy,
a
Poland
b
Medicinal and Aromatic Plants Research Department, National Research Centre, 12311, Dokki, Cairo, Egypt
Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, 60-631 Poznan, Poland
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Six new compounds including two oleanane-type triterpenoid saponins (1, 2) and four C-glycosyl
flavones (3–6), along with a known saponin (7), three di-C-glycosyl flavones (8–10) and a glycosyl
auronol (11), were isolated from the stem bark of Erythrina abyssinica Lam. The structures of the new
Received 2 March 2015
Received in revised form 5 May 2015
Accepted 13 May 2015
compounds, identified as 3-O-[
onopyranosyl]-22-O- -d-glucopyranosyl sophoradiol (1), 3-O-[
copyranosyl-(1 !2)- -d-glucuronopyranosyl]-22-O- -glucopyranosyl
-glucopyranosyl-8-C- -quinovopyranosyl-8-C-
syl apigenin (4), 8-C-[600-O- -glucopyranosyl 7,40-dihydroxyflavone (5)
a
-l-rhamnopyranosyl-(1 !2)-
b
-d-galactopyranosyl-(1 !2)-
-l-rhamnopyranosyl-(1 !2)-
sophoradiol (2),
b-d-glucur-
Available online 27 May 2015
b
b
a
b
-d-glu-
b
-D
6-C-
Keywords:
b
b
-quinovopyranosyl apigenin (3), 6-C-
b
b-glucopyrano-
Erythrina abyssinica
Triterpenoid saponins
C-glycosyl flavones
Structural elucidation
Cytotoxic activity
a
-l-rhamnopyranosyl-(1000 !600)]-
b
and 8-C-[600-O-
b
-d-xylopyranosyl-(1000 !600)]-
b
-glucopyranosyl 7,40-dihydroxyflavone (6), were deter-
mined by comprehensive spectroscopic analysis, including 1D and 2D NMR techniques, mass
spectrometry and acid hydrolysis. These new compounds together with the known saponins 7 were
evaluated for their cytotoxic activity against MCF-7 (estrogen dependent) and MDA-MB-231 (estrogen
independent) cell lines. The new saponin 2 exhibited the highest cytotoxic activity among tested
compounds, exerting a selective inhibitory effect against the proliferation of MCF-7 cells, with lower IC50
Structure-activity relationship
value (12.90
of these compounds is also discussed.
ã2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
mM) than that of the positive control, resveratrol (13.91 mM). Structure–activity relationship
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.1.
2.2.
Characterization of compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Cytotoxicity results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.
Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.1.
3.2.
3.3.
3.4.
3.5.
3.6.
General experimental procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Plant material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Extraction and isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Acid hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Determination of the absolute configuration of sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Cell culture and cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
* Corresponding author. Tel.: +48 8188 63421x206; fax: +48 8188 63421x295.
E-mail address: aperez@iung.pulawy.pl (A.J. Pérez).
http://dx.doi.org/10.1016/j.phytol.2015.05.013
1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

60
A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
1. Introduction
Erythrina abyssinica Lam. is a medium-sized tree native to
Eastern and Southern Africa, where is considered as a medicinal
plant. It is member of the genus Erythrina (Fabaceae), which
comprises over 100 species widely distributed in tropical and
subtropical regions worldwide (Bisby et al., 1994). The bark of this
plant is extensively used in traditional medicine of Eastern Africa
for the treatment of various diseases such as malaria, microbial
infections, intestinal worms, cough and inflammations (Owuor
et al., 2006; Orwa et al., 2009).
Based on the common use in folk medicine several phytochem-
ical studies on the stem bark of E. abyssinica have been carried out
in the last two decades, giving rise to the isolation of different
secondary metabolites such as prenylated flavanones, chalcones
and pterocarpans, which have been found to possess a wide range
of biological activities including anti-plasmodial, inhibition of the
protein tyrosine phosphatase 1B (PTP1B) and cytotoxicity (Ichi-
maru et al., 1996; Moriyasu et al., 1998; Yenesew et al., 2004; Cui
et al., 2007, 2008a,b; Nguyen et al., 2009). For various reasons,
these preceding researches have been focused toward the medium
polar constituents of the E. abyssinica stem bark, while the
knowledge concerning to the most polar secondary metabolites
remains limited up to now. In contrast, the stem bark from other
Erythrina species have been previously studied for their polar
constituents, resulting in the isolation of other classes of secondary
metabolites such as triterpenoid saponins (Kouam et al., 1991,
2007, 2008; Mbafor et al., 1997), also found to exert a wide range of
pharmacological properties including cytotoxicity (Podolak et al.,
2010), and most recently C-glycosyl flavones (De Oliveira et al.,
2014). Though flavonoids most often are distinguished by their
antioxidant, antiallergic and anti-inflammatory activities, some
representative cases such as quercetin, catechin, gallocatechin and
apigenin have been investigated as potential chemopreventive or
chemotherapeutic agents against cancer (Di Carlo et al., 1999;
Marchand, 2002).
In the present work, we report the isolation and characteriza-
tion of six new compounds, including two oleanane-type
triterpenoid saponins (1 and 2), two 6,8-di-C-glycosyl flavones
(3 and 4) and two 8-C-glycosyl 7,40-dihydroxyflavones (5 and 6),
together with five further known compounds (7–11) from the polar
extracts of E. abyssinica stem bark (Fig. 1). In addition, the cytotoxic
activities against MCF-7 (estrogen dependent) and MDA-MB-231
(estrogen independent) breast adenocarcinoma cell lines were
evaluated for seven of the isolated compounds.
Fig. 1. Chemical structures of the isolated compounds (1–11) from E. abyssinica
stem bark.
(9), vicenin-2 (10) (Xie et al., 2003) and hovetrichoside C (11)
(Yoshikawa et al., 1998).
2. Results and discussion
Compound 1 was obtained as a white amorphous solid and its
molecular formula was assigned as C54H88O22 by HRESI-TOFMS
analysis (m/z 1087.5677 [M – H]–, calcd for C54H87O22, 1087.5689).
In the negative ESI-MS/MS spectrum, the deprotonated molecule
[M – H]– at m/z 1087 suffered the successive neutral losses of H2O
(18 Da) and CO2 (44 Da) to produce the m/z 1069 and 1025 ions,
respectively, which could indicate the presence of a carboxylic
group. Further fragment ions at m/z 879 and 717 indicated the
neutral losses of a deoxyhexose unit (146 Da) and a hexose unit
(162 Da), respectively. The successive neutral loss of 114 Da to
generate the ion at m/z 603, suggested the presence of a hexuronic
acid unit. It could be originated from the ion at m/z 717,
2.1. Characterization of compounds
The dried stem bark of E. abyssinica was extracted with 70%
EtOH and the concentrated extract was suspended in water and
successively partitioned with CHCl3 and n-BuOH. The n-BuOH
extract was subjected to multiple fractionation procedures to yield
a total of 11 compounds (Fig. 1), comprising two new triterpenoid
saponins (1 and 2, abyssaponins A and B), four new C-glycosyl
flavones (3–6, abyssinosides A–D) and five known compounds
(7–11). Their structures were elucidated on the basis of spectro-
scopic data obtained from 1H, 13C, 2D (HSQC, HMBC, DQF-COSY,
TOCSY, ROESY, HSQC-TOCSY), 1D-ROESY (250 ms) and 1D-TOCSY
(30, 60, 120 ms) NMR experiments, UV, HRESI-TOFMS, ESI-MS/MS
and acid hydrolysis. The known compounds were identified as 3-O-
corresponding to
a molecular rearrangement derived from
hexuronic acid after losses of water and carbon dioxide (Gülcemal
et al., 2013). Finally, the fragment ion at m/z 441 suggested the loss
of an additional hexose unit and it may be identified as being due to
the aglycone ion. 1H and 13C NMR data for the aglycone moiety of 1
[
a
-l-rhamnopyranosyl-(1 !2)-
-d-glucuronopyranosyl]-22-O-
nol B (7) (Simonet et al., 1999), schaftoside (8), isoschaftoside
b
-d-galactopyranosyl-(1 !2)-
(Table 1) suggested a
secondary alcoholic groups and consequently, a bidesmosidic
D
12-oleanene skeleton with two glycosylated
b
b-d-glucopyranosyl soyasapoge-

A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
61
Table 1
NMR spectroscopic data (500 MHz, C5D5N/D2O, 9:1) for the aglycone moiety and sugar units of compounds 1 and 2a.
Abyssaponin A (1)
dC, type
Abyssaponin B (2)
position
dH (J in Hz)
dC, type
dH (J in Hz)
1ax
1eq
2ax
2eq
3
38.7, CH2
0.78b
38.6, CH2
0.78b
1.34b
1.34b
26.3, CH2
1.83b
2.07 m
3.22 dd (11.8, 4.5)
–
26.2, CH2
1.86 m
2.07 m
3.23 dd (11.9, 4.3)
90.1, CH
39.5, C
90.2, CH
39.5, C
4
–
5ax
6ax
6eq
7ax
7eq
8
9
10
11
12
55.6, CH
18.2, CH2
0.68 brd (11.6)
55.5, CH
18.2, CH2
0.70 brd (11.5)
1.22b
1.25b
1.42b
1.45b
33.0, CH2
1.41b
32.9, CH2
1.43b
1.22b
1.26b
39.6, C
47.7, CH
36.6, C
23.5, CH2
122.4, CH
144.2, C
42.0, C
25.9, CH2
28.3, CH2
37.2, C
–
1.50b
39.6, C
47.6, CH
36.5, C
23.5, CH2
122.3, CH
144.2, C
42.0, C
25.8, CH2
28.3, CH2
37.2, C
–
1.51b
–
–
1.74b (2H)
1.74b (2H)
5.17 t (3.4)
–
5.17 t (3.8)
–
13
14
15
16
–
–
0.89b, 1.65b
1.46b, 1.77b
–
0.91b, 1.66b
1.47b, 1.77b
–
17
18
45.6, CH
46.2, CH2
2.17 brd (13.6)
1.75b
0.96 dd (13.3, 2.4)
–
45.6, CH
46.2, CH2
2.17 brd (13.6)
1.75b
19ax
19eq
20
21ax
21eq
22eq
23
24
25
26
27
28
29
30
0.97 brd (12.6)
–
30.3, C
37.2, CH2
30.3, C
37.2, CH2
1.70 dd (13.2, 7.6)
1.50b
3.76 dd (7.6, 3.2)
1.31 s
1.12 s
0.77 s
0.84 s
1.14 s
1.09 s
0.89 s
1.70b
1.50b
82.4, CH
28.1, CH3
16.5, CH3
15.4, CH3
16.9, CH3
25.2, CH3
20.9, CH3
32.3, CH3
28.4, CH3
82.4, CH
28.0, CH3
16.4, CH3
15.4, CH3
16.9, CH3
25.2, CH3
20.9, CH3
32.2, CH3
28.4, CH3
3.77 dd (7.3, 2.9)
1.26 s
1.09 s
0.77 s
0.86 s
1.16 s
1.06 s
0.90 s
1.13 s
1.14 s
Abyssaponin A (1)
Abyssaponin B (2)
position
dC, type
dH (J in Hz)
dC, type
dH (J in Hz)
3-O-b-d-GlcA
3-O-b-d-GlcA
1
2
3
4
5
6
105.2, CH
78.4, CH
78.1, CH
73.1, CH
77.0, CH
172.5, C
4.97 d (7.5)
4.46 dd (7.5, 9.1)
4.57 dd (9.1, 9.6)
4.40 dd (9.6, 9.7)
4.61 d (9.7)
-
105.3, CH
77.7, CH
78.1, CH
73.1, CH
77.0, CH
172.5, C
4.96 d (7.2)
4.52 dd (7.2, 9.4)
4.55 dd (9.4, 9.0)
4.37 dd (9.0, 9.6)
4.61 d (9.6)
-
Abyssaponin A (1)
Abyssaponin B (2)
position
dC, type
dH(Jin Hz)
dC, type
dH (Jin Hz)
b-d-Gal
b-d-Glc
1
2
3
4
5
6
102.3, CH
76.4, CH
75.6, CH
70.1, CH
76.2, CH
61.9, CH2
5.55 d (7.6)
101.6, CH
78.6, CH
78.8, CH
72.2, CH
77.9, CH
63.0, CH2
5.67 d (7.5)
4.18 dd (7.5, 8.7)
4.12 dd (8.7, 8.7)
3.87b
4.51 dd (7.6, 9.5)
4.03 dd (9.5, 3.5)
4.31 d (3.5)
3.83 dd (6.1, 5.2)
4.27 dd (11.2, 5.2)
4.37 dd (11.2, 6.1)
b
3.84
4.16b
4.51 d (12.8)
Abyssaponin A (1)
Abyssaponin B (2)
position
dC, type
dH(Jin Hz)
dC, type
dH (Jin Hz)
a
-l-Rha
a-l-Rha
1
2
3
4
5
6
101.8, CH
71.9, CH
72.1, CH
73.9, CH
69.1, CH
18.6, CH3
6.14 d (1.6)
101.7, CH
71.9, CH
72.0, CH
73.8, CH
69.0, CH
18.6, CH3
6.24 d (1.5)
4.72 dd (1.6, 3.4)
4.68 dd (3.4, 9.3)
4.29 dd (9.3, 9.3)
4.98 dq (9.3, 6.2)
1.70 d (6.2)
4.70 dd (1.5, 3.4)
4.65 dd (3.4, 9.3)
4.31 dd (9.3, 9.3)
4.98 dq (9.3, 6.2)
1.74 d (6.2)
Abyssaponin A (1)
Abyssaponin B (2)
position
dC, type
dH(Jin Hz)
22-O- -d-Glc
dC, type
dH (Jin Hz)
22-O- -d-Glcꢀ
b
b

62
A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
Table 1 (Continued)
Abyssaponin A (1)
Abyssaponin B (2)
position
dC, type
dH(Jin Hz)
22-O- -d-Glc
dC, type
dH (Jin Hz)
22-O- -d-Glcꢀ
b
b
1
2
3
4
5
6
102.1, CH
74.8, CH
78.2, CH
71.5, CH
77.9, CH
62.5, CH2
4.79 d (7.8)
102.1, CH
74.8, CH
78.2, CH
71.5, CH
77.8, CH
62.5, CH2
4.79 d (7.7)
3.96 dd (7.8, 9.0)
4.24 dd (9.0, 9.1)
4.13 dd (9.1, 9.3)
3.90 ddd (9.3, 5.5, 2.5)
4.29 dd (12.2, 5.5)
4.48 dd (12.2, 2.5)
3.97 dd (7.7, 9.0)
4.24 dd (9.0, 9.0)
4.13 dd (9.0, 9.3)
3.90 ddd (9.3, 5.2, 2.6)
4.29 dd (12.0, 5.2)
4.48 dd (12.0, 2.6)
aAssignments were confirmed by DQF-COSY, 2D-TOCSY, 2D-ROESY, HSQC, HSQC-TOCSY, HMBC, 1D-TOCSY and 1D-ROESY experiments
boverlapped with other signals.
saponin. A careful analysis of the correlations in 2D HMBC and 2D
ROESY spectra (Fig. 2), allowed to determine the position and
orientation of substituents, as well as the relative configurations of
chiral centers, leading therefore to the sapogenin structure of 1 as
correlations in the HSQC spectrum with carbons at d 102.1, 105.2,
102.3 and 101.8, respectively (Table 1). Individual sugar units were
identified by a combination of DQF-COSY,1D-TOCSYand 1D-ROESY
experiments. The latter two spectra were acquired from the
selective excitation of anomeric protons or others better coupled
with the spin system and less overlapped. In this way, spin systems
olean-12-ene-3
et al., 1996).
b,22b-diol, which is known as sophoradiol (Miyao
Additionally, the 1H NMR spectrum of
1
exhibited four
for a -galactopyranosyl unit were
b-glucopyranosyl unit and for a
b
anomeric signals at
d
4.79, 4.97, 5.55 and 6.14, and these showed
detected for the anomeric signals at d 4.79 and 5.55, respectively.
Fig. 2. Key HMBC and ROESY correlations for compounds 1,3 and 5.

A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
63
Consistent with the above mentioned ESI-MS/MS analysis, the
signal at 4.97 revealed a typical spin system of a -glucopyr-
anosiduronic acid unit, which was confirmed by the presence of a
carboxylic carbon signal in 13C NMR spectrum at
172.5 (C-6GlcA
and by its long-range HMBC correlations with H-4GlcA 4.40, dd,
J = 9.6, 9.7 Hz) and H-5GlcA 4.61, d, J = 9.7 Hz). Inefficient
magnetization transfer beyond H-2Rha 4.72, dd, J = 1.6, 3.4 Hz)
in 1D-TOCSY (120 ms) for the anomeric signal at 6.14 was
observed. This finding, together with the presence of a methyl
doublet at 1.70 (J = 6.2 Hz), is characteristic of a rhamnopyranosyl
unit. A further selective TOCSY experiment on this methyl (H-6Rha
allowed a sequential assignment of the signals from H-6Rha to
H-2Rha. The anomeric configuration of the rhamnose unit was
deduced from the H-1Rha/C-1Rha direct coupling value
(J = 173.7 Hz), measured from F1-coupled HSQC spectrum
(Agrawal, 1992). Based on the correlations observed in HSQC
and HSQC-TOCSY spectra of 1, the 13C signals for each sugar unit
were assigned. The sequence and linkage sites into the sugar chain
and with the aglycone moiety were established by inter-glycosidic
HMBC/ROESY correlations (Fig. 2), which were observed between
577 [M – H]–) showed the most abundant fragments at m/z 487 and
457, deriving from the loss of 90 and 120 Da, which is typical for the
fragmentation of a hexose unit attached to the aglycone through a
CꢀꢀC bond (Vukics and Guttman, 2010). Minor fragments at m/z
503 and 473 were also observed, corresponding to the loss of
74 and 104 Da due to the cleavage of a deoxyhexose unit CꢀꢀC
bound to the aglycone (Vukics and Guttman, 2010). On the other
hand, the ESI-MS/MS spectrum of 4 (m/z 577 [M – H]–) showed
almost the opposite behavior. The most abundant fragments were
observed at m/z 503, 473 and 457. The latter ([M – H – 120]–), is also
typical in the fragmentation of a deoxyhexose unit (Vukics and
Guttman, 2010), and was the base ion peak due to the contribution
of both sugar units. Moreover, other important ions that could be
observed in both 3 and 4, were those due to the aglycone (A = 270,
apigenin) plus the residues of the sugars that remain linked to it,
i.e. A + 113 (m/z 383) and A + 83 (m/z 353) from the loss of 194 and
224 Da, respectively (Ferreres et al., 2003; Vukics and Guttman,
2010). Similarly, the ion [M – H – 18]–was detected in both
compounds, but it was more abundant in 4. Therefore, it can be
hypothesized that the structure of 3 corresponds to 6-C-hexosyl-8-
C-deoxyhexosyl apigenin, while 4 could be the positional isomer of
3, namely 6-C-deoxyhexosyl-8-C-hexosyl apigenin.
d
b
d
)
(
d
(d
(d
d
d
)
a
H-1Rha
and C-2GlcA
90.1)/H-3 (
ROESY correlations between H-1Glc
3.76) of the aglycone moiety, confirmed that 1 is a bidesmosidic
(d
6.14) and C-2Gal
78.4)/H-2GlcA
3.22) of the aglycone moiety. In addition, the HMBC/
4.79) and C-22 ( 82.4)/H-22
(
d
76.4)/H-2Gal
(
d
4.51), H-1Gal
(d 5.55)
(d
(d
4.46), H-1GlcA
(d
4.97) and C-3 (d
d
The 1H NMR spectrum of 3 and 4, in C2D6SO (Table 2), showed
(d
d
signals at
6.68 (s, H-3), which are characteristics of the apigenin aglycone.
This, together with their carbon signals at 181.9/181.8 (C-4),
d
7.96/7.95 (d, H-20,60), 6.92/6.95 (d, H-30,50), and 6.75/
(d
saponin. A d configuration for glucuronic acid, galactose and
glucose units and l for rhamnose unit were established after the
acid hydrolysis of 1, followed by derivatization of sugars and UPLC-
UV-SRM/MS analysis (Tanaka et al., 2007; Pérez et al., 2014). Based
on the evidence outlined above, the structure of 1 was finally
d
158.9/159.2 (C-5), 161.1/160.7 (C-7), and 161.1/160.7 (C-40)
confirmed the apigenin skeleton bearing a carbonyl and three
hydroxyl groups. Substitution at C-6 and C-8 was inferred, as no
corresponding aromatic proton signals were observed at these
positions. In addition, 1H NMR spectrum of 3 displayed two
determined as 3-O-[
a
-l-rhamnopyranosyl-(1 !2)-
b-d-galacto-
-d-glucopyr-
pyranosyl-(1 !2)-
b
-d-glucuronopyranosyl]-22-O-
b
anomeric signals at
HSQC spectrum with carbons at
1D TOCSY, DQF COSYand 1D ROESY spectra, they were identified as
being due to a -glucopyranosyl unit and a 6-deoxy- -glucopyr-
anosyl unit ( -quinovose). Long-range correlations in the HMBC
spectrum from H-1Glc 4.76) to C-6 ( 107.9) and C-5 ( 158.9) and
from H-1Quin 4.82) to C-8 ( 103.1) and C-9 ( 154.8) indicated
d
4.76 and 4.82, which were correlated in the
anosyl sophoradiol and it was named abyssaponin A.
d
74.0 and 74.0, respectively. Using
The molecular formula of compound 2 was determined to be
the same as that of 1, by HRESI-TOFMS analysis (m/z 1087.5664
[M ꢀ H]–, calcd for C54H87O22, 1087.5689). It also contains the same
aglycone moiety as 1 (sophoradiol), according to their NMR
spectroscopic data (Table 1), which were almost superimposable.
Although the ESI-MS/MS spectrum of 2 showed fragmentation
pattern identical to 1, their NMR data for the sugar portion
(Table 1) showed differences. The 1H NMR spectrum of 2 exhibited
b
b
b
(
d
d
d
(d
d
d
that the C-6 and C-8 positions of the apigenin were bonded to a
glucose and a quinovose, respectively (Fig. 2). Thus, the structure of
3 was identified as 6-C-
syl apigenin and was named abyssinoside A.
Likewise, 1H NMR spectrum of 4 showed two anomeric signals
4.78 and 4.94, which were identified as being due to a
-quinovopyranosyl unit and a -glucopyranosyl unit, respective-
ly. Consistent with the aforementioned MS/MS analysis, the long-
range HMBC correlations from H-1Quin 4.78) to C-6 ( 107.9) and
C-5 ( 159.2) and from H-1Glc 4.94) to C-8 ( 103.4) and C-9 (
154.1) suggested a connectivity opposed to that of 3. Therefore, the
structure of 4 was elucidated as 6-C- -quinovopyranosyl-8-C-
-glucopyranosyl apigenin and was named abyssinoside B.
The molecular formula of compound 5 was determined to be
b-glucopyranosyl-8-C-b-quinovopyrano-
four anomeric signals at
d 4.79, 4.96, 5.67 and 6.24. However, the
latter two appear approximately at 0.1 ppm downfield shifted
compared with 1. The selective TOCSY and ROESY experiments on
at
d
these two signals, revealed the presence of a
5.67) instead of -galactopyranosyl and a
unit ( 5.67). A rigorous study of the HMBC/ROESY correlations led
to the sequence of the sugar chain as being the same as in 1. Thus,
the structure of 2 was identified as 3-O-[ -l-rhamnopyranosyl-
-d-glucuronopyranosyl]-
-d-glucopyranosyl sophoradiol and was named abyssapo-
b
-glucopyranosyl unit
b
b
(d
b
a
-rhamnopyranosyl
d
(d
d
d
(d
d
d
a
(1 !2)-
b
-d-glucopyranosyl-(1 !2)-
b
b
22-O-
b
b
nin B.
Compounds 3 and 4 were found to be structural isomers, giving
identical cationized molecules [M + Na]+ in their HRESI-TOFMS
corresponding to the molecular formula C27H30O14Na. Their UV
spectra showed absorption maxima at 225, 270 and 335 nm,
characteristic for flavones and similar to those of apigenin (Mabry
et al., 1970). Their ESI-MS/MS spectra showed typical fragmenta-
tion patterns of di-C-glycosyl flavones (Ferreres et al., 2003; Vukics
and Guttman, 2010), however certain differences were observed.
Taking into account that the fragmentation of 6,8-di-C-glycosyl
flavones occurs preferentially at position 6 in relation to the sugar
unit at C-8 (Ferreres et al., 2003), a comparative study of the
relative abundance of the fragment ions in both isomers allowed us
to draw important conclusions regarding the type of sugar and
position of the C-glycosylation. The ESI-MS/MS spectrum of 3 (m/z
C27H30O13 according to the HRESI-TOFMS spectrum (m/z 585.1573
[M + Na]+, calcd for C27H30O13Na, 585.1584). Its UV spectrum
showed absorption maxima at 220 and 335 nm, characteristic of
7,40-dihydroxyflavone (Mabry et al., 1970). The ESI-MS/MS
spectrum of 5 (m/z 561 [M – H]–) showed a fragmentation pattern
typical for
a mono-C-glycosylated flavone. The presence of
fragment ions at m/z 325 [(M – H – 146) – 90] and 295 [(M – H –
146) – 120]– indicated that 5 is a glycoside with hexose attached
through a CꢀꢀC bond to aglycone core and a deoxyhexose unit
connected through O-glycosidic bond to hexose. The fact that ion
[(M – H – 146) – 120]– was more abundant (base peak) than [(M –
H – 146) – 90] –implied that C-8 is the glycosylation position
(Ferreres et al., 2003). An additional fragment ion at m/z 267
[(M – H – 146) – 148]– was observed, which belong to the aglycone

64
A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
Table 2
NMR spectroscopic data (500 MHz, C2D6SO) for compounds 3–6a.
Abyssinoside A (3)c
Abyssinoside B (4)d
Abyssinoside C (5)e
Abyssinoside D (6)e
position
dC, type
dH (J in Hz)
dC, type
dH (J in Hz)
dC, type
dH (J in Hz)
dC, type
dH (J in Hz)
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
163.3, C
102.4, CH
181.9, C
158.9, C
107.9, C
161.1, C
103.1, C
154.8, C
103.1, C
121.5, C
128.5, CH
115.7, CH
161.1, C
–
163.6, C
102.3, CH
181.8, C
159.2, C
107.9, C
160.7, C
103.4, C
154.1, C
103.4, C
121.3, C
128.1, CH
115.5, CH
160.7, C
115.5, CH
128.1, CH
–
162.0, C
103.8, CH
175.8, C
124.7, CH
114.5, CH
161.2, C
112.3, C
155.6, C
115.7, C
–
162.1, C
–
6.75 s
–
6.68
–
s
6.59 s
–
103.8, CH
175.8, C
124.8, CH
114.2, CH
160.7, C
112.4, C
155.6, C
116.0, C
6.59 s
–
–
–
7.82 d (8.7)
6.92 d (8.7)
7.82 d (8.7)
6.92 d (8.7)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
122.1, C
–
122.1, C
–
7.96 d (8.7)
6.92 d (8.7)
–
7.95 d (8.6)
6.95 d (8.6)
–
127.6, CH
115.4, CH
160.1, C
115.4, CH
127.6, CH
7.89 d (8.4)
6.93 d (8.4)
–
127.7, CH
115.4, CH
160.1, C
115.4, CH
127.7, CH
7.90 d (8.7)
6.94 d (8.7)
–
115.7, CH
128.5, CH
6.92 d (8.7)
7.96 d (8.7)
6.95 d (8.6)
7.95 d (8.6)
6.93 d (8.4)
7.89 d (8.4)
6.94 d (8.7)
7.90 d (8.7)
6-C-
b
-Glc
6-C-
4.78 d (9.8)
3.93 m
3.30 dd (8.7, 8.7)
3.08 dd (8.7, 8.9)
3.37 m
b
-Quin
8-C-
b
-Glc
8-C-b-Glc
4.96 d (9.8)
100
200
300
400
500
600
74.0, CH
71.5, CH
78.2, CH
69.5, CH
81.0, CH
60.2, CH2
4.76 d (9.7)
73.5, CH
70.9, CH
78.2, CH
75.1, CH
76.1, CH
17.4, CH3
73.9, CH
71.1, CH
78.3, CH
70.4, CH
79.9, CH
67.1, CH2
4.97 d (9.8)
3.94 dd (9.8, 8.7)
3.41 dd (8.7, 8.7)
3.35 dd (8.7, 8.5)
3.55b
73.8, CH
71.0, CH
78.3, CH
70.7, CH
79.7, CH
68.4, CH2
3.66b
3.96 dd (9.8, 8.9)
3.28b
3.40b
3.32b
3.41b
3.29b
3.57 ddd (9.1, 6.3, 2.2)
3.65 dd (11.2, 6.3)
4.08 dd (11.2, 2.2)
3.58 dd (12.0, 4.0)
3.66 dd (12.0, 2.1)
1.25 d (6.1)
3.55b
3.98 d (10.8)
8-C-
4.82 d (9.5)
3.85 dd (9.5, 8.7)
3.28 dd (8.7, 8.7)
3.14 dd (8.7, 9.0)
3.36 m
b
-Quin
8-C-
b
-Glc
a
-l-Rha
b-d-Xyl
1000
2000
3000
4000
74.0, CH
71.5, CH
78.4, CH
75.8, CH
76.4, CH
18.3, CH3
74.1, CH
71.3, CH
78.2, CH
69.9, CH
81.1, CH
60.6, CH2
4.94 d (9.0)
100.4, CH
69.9, CH
70.4, CH
71.8, CH
67.9, CH
17.2, CH3
4.59 brs
102.8, CH
70.0, CH
72.1, CH
66.6, CH
64.0, CH2
4.19 d (5.9)
3.42 m
3.79b
3.62 dd (1.6, 3.5)
3.41 dd (8.1, 8.1)
3.48 dd (8.1, 9.2)
3.41b
3.65 dd (11.5, 4.2)
3.78 d (11.5)
3.42b
3.36b
3.17 dd (9.3, 9.3)
3.45 dq (9.3, 6.2)
1.13 d (6.2)
3.64 m
5
ax/5000
3.35b
5eq/6000
1.23 d (5.6)
3.69 dd (11.8, 4.2)
a
Assignments were confirmed by 1H-1H COSY, 1D-TOCSY, 1D-ROESY, HSQC, HSQC-TOCSY and HMBC experiments;
b
c
Overlapped with other signals;
Acquired at 37 ꢁC;
d
e
Acquired at 100 ꢁC;
Acquired at 90 ꢁC.
(A = 254; 7,40-dihydroxyflavone) plus the residue of sugar that
(1000 !600)]-
b
-glucopyranosyl 7,40-dihydroxyflavone and was
named abyssinoside C.
remains linked to it (A + 13) (Waridel et al., 2001). Thus, it can be
hypothesized that the structure of 5 corresponds to 8-C-(O-
deoxyhexosyl)-hexosyl 7,40-dihydroxyflavone.
Compound 6 has molecular formula C26H28O13, that suggests
lack of a methyl group (14 Da) in relation to compound 5. Its UV
spectrum showed absorption maxima identical to 5, which
suggested that 7,40-dihydroxyflavone is also the aglycone moiety
of 6. The ESI-MS/MS spectrum showed fragmentation pattern
similar to that of 5, indicative of a hexose unit as the C-glycosyl
sugar, but bearing a pentose unit O-glycosylated instead of a
deoxyhexose unit. The 1H and 13C NMR assignments for the
aglycone moiety of 6 were almost superimposable with those of 5
(Table 2), which confirmed the 7,40-dihydroxyflavone aglycone.
The anomeric region of the 1H NMR spectrum of 6 displayed two
1
The H NMR spectrum of 5 (Table 2) confirmed the 7,40-
dihydroxyflavone moiety as aglycone, as is evident from the
presence of H-20/60 and H-30/50 aromatic protons of the B ring at
d
7.89 and 6.93, respectively, as well as a singlet at
doublets at 7.82 (H-5, J = 8.7 Hz) and 6.92 (H-6, J = 8.7 Hz).
Furthermore, 13C NMR spectrum (Table 2) showed signals at
d 6.59 (H-3) and
d
d
175.8 (C-4), 161.2 (C-7) and 160.1 (C-40), which also confirm the
7,40-dihydroxyflavone skeleton bearing
a
carbonyl and two
hydroxyl groups. Two anomeric signals at
d 4.59 and 4.97 were
also observed in the 1H NMR spectrum of 5. The first one was
signals at
spectrum with carbons at
experiment for the anomeric signal at
characteristic of a -xylopyranose unit, later confirmed by the
d
4.19 and 4.96, which showed correlation in the HSQC
102.8 and 73.8, respectively. 1D-TOCSY
4.19 showed a spin system
identified as being due to an O-linked
a
-l-rhamnopyranose, later
d
confirmed by UPLC-UV-SRM/MS analysis of the arylthiocarbamate
derivative obtained after the acid hydrolysis of 5. The second
d
b
anomeric proton displayed a HSQC correlation with carbon at
73.9 (C-1Glc) and its spin system was identified as being due to a C-
linked -glucopyranose. Finally, cross peaks in the HMBC
spectrum confirmed the hypothesized structure (Fig. 2). The
correlations between the anomeric proton at 4.97 and carbons at
161.2 (C-7), 112.3 (C-8) and 155.6 (C-9) proved a C-8 glycosidic
d
UPLC-UV-SRM/MS analysis of the derivative obtained after the acid
hydrolysis of 6. The other anomeric signal was identified as being
b
due to
a b-glucopyranose. Finally, the connectivities were
established by the long-range correlations observed in the HMBC
d
spectrum and the structure of 6 was elucidated as 8-C-[600-O-
b-d-
d
xylopyranosyl-(1000!600)]-
b
-glucopyranosyl 7,40-dihydroxyflavone
linkage. The anomeric proton of rhamnose (
d
4.59) correlated with
and was named abyssinoside D.
C-6 of glucose (d 67.1), suggesting the involvement of C-1Rha and C-
6Glc in an O-glycosidic linkage between the two sugar moieties,
which was confirmed by the observed ROESY correlation between
2.2. Cytotoxicity results
H-1Rha and 2H-6 of glucose (
d
3.55, 3.98) (Fig. 2). Therefore, the
-l-rhamnopyranosyl-
Although the triterpenoid saponins are known to possess a
wide range of biological activities, they have been more extensively
structure of 5 was elucidated as 8-C-[600-O-
a

A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
65
Table 3
mode with the following parameters: spray voltage 3.9 kV,
capillary voltage ꢀ47 V, tube lens offset ꢀ60 V and capillary
temperature 240 ꢁC. UV spectra were simultaneously obtained by a
PDA detector in-line. Semi-preparative HPLC in isocratic mode was
performed on a chromatographic system equipped with Gilson
321 pump, a Gilson GX-271 liquid handler with a 2 mL sample loop,
a Gilson Prep ELSTM II detector and a semi-preparative reversed
Effect of compounds 1–7 on the growth inhibition of MCF-7 and MDA-MB-231 cell
lines.
MCF-7
IC50
MDA-MB-231
IC50 M)
compound
(
mM)
95% C.I.
(
m
95% C.I.
1
2
3
4
5
6
7
24.52
12.90
41.93
36.01
70.98
41.95
15.50
13.91
17.37–34.37
9.65–16.16
61.41
74.54
20.51
33.24
54.79
45.67
31.02
19.3
46.67–71.85
56.65–87.21
16.35–26.38
29.57–37.89
43.82–72.42
36.64–58.98
25.8–37.63
17.66–21.18
32.49–56.53
30.94–42.68
53.95–54.30
31.97–58.07
13.15–18.17
11.22–16.89
phase Atlantis Prep T3, column (250 mm ꢂ 10 mm i.d., 5
mm,
Waters, Milford, MA).
3.2. Plant material
Resveratrol
The stem bark of E. abyssinica Lam. was collected in May 2012 in
Kadogli, Nuba Mountain, Southern Kordofan State, Sudan (about
900 km West to Khartoum Town). The plant material was
authenticated by Prof. Dr. S. A. Kawashty at the Herbarium of
National Research Centre (NRC), Cairo, Egypt. A voucher specimen
(No. 0001) was deposited at the Herbarium of the NRC, Cairo,
Egypt. The stem bark (500 g) was dried at room temperature and
then finely powdered for the further processing.
IC50 = the concentration of compound that cause a 50% reduction in cell growth after
72 h of incubation; C.I. = confidence interval.
reported for their cytotoxicity (Sparg et al., 2004; Podolak et al.,
2010). Thus, the isolated saponins (1, 2 and 7) together with the
new C-glycosyl flavones (3–6) were evaluated for their cytotoxic
activity against the proliferation of MCF-7 (estrogen dependent)
and MDA-MB-231 (estrogen independent) breast adenocarcinoma
cell lines. The viability of MCF-7 cells was in general more affected
by saponins, especially 2 and 7, while C-glycosyl flavones were
inactive (Table 3). The new compound 2 exhibited the most
3.3. Extraction and isolation
The dried and powdered stem bark (500 g) was extracted with
70% EtOH at room temperature (1 L ꢂ 48 h ꢂ 5). The combined
extracts were concentrated under reduced pressure. Crude extract
(21 g) was suspended in distilled water and successively extracted
with CHCl3 and n-BuOH. After removing the solvent, 2.1 g of
significant cytotoxic activity with an IC50 value (12.9
than that of resveratrol (13.91 M). It is interesting to note that
saponin 1 resulted moderately active (IC50 = 24.52 M), suggesting
that the exchange of a glucose unit by a galactose in the sugar chain
has detrimental effect on cytotoxicity. Nevertheless, the
hydroxylation at C-24 seems to slightly enhance the cytotoxic
activity, as compound 7 showed lower IC50 value (15.5 M) than
that of 1. Contrary, both saponins and C-glycosyl flavones were
inactive against the proliferation of MDA-MB-231 cells, with the
exception of 3, one of the major constituents of E. abyssinica stem
bark, which exerted a moderate cytotoxic activity (IC50 = 20.51
mM) lower
m
m
a
n-BuOH extract were fractionated on a 10 cm ꢂ 6 cm, 40–63
mm
LiChroprep RP-18 glass column (Merck, Darmstadt, Germany),
previously conditioned with water and eluted with MeOHꢀꢀH2O
(10 to 100%, v/v) to give nine fractions Fr1–Fr9. Fraction Fr2
(51.5 mg) was applied onto a 40 cm ꢂ 3.2 cm, i.d. glass column
(Millipore Corp., Bedford, MA) packed with Sephadex LH-20
(Sigma–Aldrich, Steinheim, Germany) and eluted with isocratic
MeOHꢀꢀH2O (80:20, v/v) at a flow rate of 1 mL/min. Six milliliter
fractions were collected and checked by TLC on Si 60 F254 silicagel
plates (Merck, Darmstadt, Germany), developed with ethyl
acetate–formic acid–acetic acid–water (100:11:11:26, v/v/v/v).
Plates were observed under 254 nm and 366 nm UV light and then
sprayed with Liebermann–Burchard reagent and heated at 130 ꢁC.
Fractions with similar profiles were combined to obtain Fr2-1
(4 mg) and Fr2-2 (41.8 mg). Compound 11 (21.0 mg, tR = 33.5 min)
m
m
M). A notable selectivity of the cytotoxic compound 2, which
despite of its significant activity against the proliferation of
MCF-7 cells, was the least active compound against MDA-MB-
231 cells (IC50 = 74.54 mM), was observed. These findings could
suggest that indeed compound 2 may act as an antiestrogen
substance. However, this cannot be solely confirmed with the
present investigation and thus further studies to this regard are
needed.
was obtained from Fr2-2 by semi-preparative HPLC on
a
C18 column (flow rate, 3 mL/min; 30 ꢁC; CH3CNꢀꢀH2OꢀꢀFA,
12:88:0.2, v/v/v). Fraction Fr3 (135.7 mg) was separated on a
Sephadex LH-20 column under the same conditions as described
above, to give fractions Fr3-1 (28.0 mg), Fr3-2 (57.8 mg) and Fr3-3
(27.7 mg). Compounds 6 (2.9 mg, tR = 14.4 min) and 10 (13.4 mg,
tR = 18.5 min) were obtained from Fr3-1 and Fr3-2 respectively, by
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a JASCO P-2000 digital
polarimeter. 1D and 2D NMR spectra were recorded on a Bruker
Avance III HD AscendTM-500 spectrometer equipped with 5 mm 1H
semi-preparative
HPLC
(flow
rate,
3 mL/min;
30 ꢁC;
{
109Ag-31P} broadband inverse (BBI) z-gradient probe. 1H
CH3CNꢀꢀH2OꢀꢀFA, 14:86:0.2, v/v/v). Fractionation of Fr4
(83.4 mg) on Sephadex LH-20 gave fractions Fr4-1 (34.2 mg) and
Fr4-2 (43.8 mg). Compound 5 (5.6 mg, tR = 20.3 min) was obtained
from Fr4-1 by semi-preparative HPLC (flow rate, 3 mL/min; 30 ꢁC;
CH3CNꢀꢀH2OꢀꢀFA, 14:86:0.2, v/v/v). Fraction Fr5 (315.2 mg) was
subjected to Sephadex LH-20 column chromatography to yield
fractions Fr5-1 (26.3 mg), Fr5-2 (12.8 mg), Fr5-3 (198.0 mg), Fr5-4
(13.1 mg) and Fr5-5 (13.8 mg). Fr5-3 was then purified by column
chromatography using a 30 cm ꢂ 1.6 cm, i.d. glass column (Milli-
(500.18 MHz) and 13C (125.77 MHz) NMR spectra for saponins
were recorded in C5D5N at 25 ꢁC and in C2D6SO at 37, 90 or 100 ꢁC
for the C-glycosyl flavones. Adiabatic pulse sequences using
gradients were applied. Chemical shifts are given on the
d scale
referenced to residual pyridine, dH 8.74, 7.58, 7.22 and dC 150.35,
135.91, 123.87 or to residual DMSO, dH 2.50 and dC 39.51. Exact
masses were determined on a Q-TOF ESI (Waters SYNAPT G2-S
HDMS, Manchester, UK) high resolution mass spectrometer
(HRESI-TOFMS). Mass spectra for saponins were recorded in
negative ion mode and in positive ion mode for the flavonoids. The
fragmentation patterns of saponins and flavonoids were obtained
using a Thermo LCQ Advantage Max (Thermo, Waltham, MA) ion-
trap mass spectrometer coupled with a Surveyor HPLC system. The
mass spectrometer was operated in the negative electrospray
pore Corp., Bedford, MA) packed with 25–40 mm LiChroprep RP-18
(Merck, Darmstadt, Germany) and eluted with isocratic
MeOHꢀꢀH2OꢀꢀFA (40:60:0.5, v/v/v) at a flow rate of 1 mL/min,
to obtain fractions Fr5-3-1 (95.8 mg) and Fr5-3-2 (66.3 mg).
Compounds 8 (7.0 mg, tR = 26.7 min) and 9 (6.3 mg, tR = 22.7 min)
were obtained from Fr5-3-1 by semi-preparative HPLC (flow rate,

66
A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
5 mL/min; 30 ꢁC; MeOHꢀꢀH2OꢀꢀFA, 29.5:70.5:0.2, v/v/v). Com-
pound 4 (9 mg, tR = 31.9 min) was yielded from Fr5-3-2 by semi-
preparative HPLC (flow rate, 3 mL/min; 35 ꢁC; CH3CNꢀꢀH2OꢀꢀFA,
15.5:84.5:0.2, v/v/v). The fractionation of Fr6 (227.0 mg) on
Sephadex LH-20 column yielded fractions Fr6-1 (16.7 mg), Fr6-2
(100.8 mg) and Fr6-3 (18.9 mg). Fr6-2 was subjected to column
120]–, 267 (19) [(M – H – 132) – 148]– (A + 13). For 1H and 13C NMR
data see Table 2.
3.4. Acid hydrolysis
It was carried out to obtain the hydrolysable sugar residues of
compounds 1, 2, 5 and 6. These compounds (1 mg each) were
treated with 2 M HCl in 1,4-dioxane/H2O (1:1, v/v, 2 mL) at 80 ꢁC for
3 h. After cooling, the solvent was removed with a stream of N2. The
dry residues were suspended in water and aglycones were
extracted with ethyl acetate (3 ꢂ 2 mL). The aqueous layers
containing sugars were neutralized with Amberlite IRA-400
(OH– form), dried under N2 and stored prior to analysis.
chromatography on 25-40 mm LiChroprep RP-18, under the same
conditions as described above, to afford two further fractions,
Fr6-2-1 (17.4 mg) and Fr6-2-2 (61.7 mg). Compound 3 (19.9 mg,
tR = 37.7 min) was obtained from Fr6-2-2 by semi-preparative HPLC
(flow rate, 3 mL/min; 35 ꢁC; CH3CNꢀꢀH2OꢀꢀFA, 16.5:83.5:0.2, v/v/
v). Separation of Fr9 (53.1 mg) on LiChroprep RP-18 (25-40 mm)
column and then semi-preparative HPLC (flow rate, 3 mL/min;
30 ꢁC; CH3CN–H2O–FA, 29.5:70.5:0.2, v/v/v) gave compounds 1
(6.2 mg, tR = 20.6 min), 2 (2.6 mg, tR = 21.6 min) and 7 (3.8 mg,
3.5. Determination of the absolute configuration of sugars
tR = 17.1 min).
25
3.4. Abyssaponin A (1): white amorphous solid; [
a
]
D –132.0
The absolute configurations of monosaccharide constituents of
compounds 1, 2, 5 and 6 were determined according to the method
reported by Tanaka et al. (2007) with the modifications described
(MeOH, c 0.1). HRESI-TOFMS, m/z 1087.5677 [M – H]– (calcd for
C54H87O22, 1087.5689). ESI-MS in negative ion mode (% base peak),
m/z 1087 (100) [M – H]–, which was fragmented in the MS/MS to
give m/z 1069 (100) [(M – H) – 18]–, 1025 (31) [(M – H) – 18 – 44]–,
879 (31) [(M – H) – 18 – 44 – 146]–, 717 (3) [(M – H) – 18 – 44 – 146 –
162]–, 603 (4) [(M – H) – 18 – 44 – 146 – 162 – 114]–, 441 (2) [(M –
H) – 18 – 44 – 146 – 162 – 114 – 162]–. 1H and 13C NMR data see
previously by us (Pérez et al., 2014). The derivatives of
acid, -galactose, -glucose and -rhamnose in compound 1,
-glucuronic acid, -glucose and -rhamnose in compound 2,
-rhamnose in compound 5 and -xylose in compound 6 were
identified by comparison of their retention times (Rt) with those of
authentic sugars (Sigma–Aldrich, Steinheim, Germany) treated in
D-glucuronic
D
D
L
D
D
L
L
D
Table 1.
25
3.5. Abyssaponin B (2): white amorphous solid; [
a
]
D – 127.0
the same way as samples (Rt:
-glucuronic acid 10.14 min, -galactose 9.93 min,
-glucose 10.03 min,
-rhamnose 9.80 min, -xylose 10.70 min, L
D
-glucuronic acid 10.26 min,
-galactose
-rhamnose
-xylose
(MeOH, c 0.1). HRESI-TOFMS, m/z 1087.5664 [M – H]– (calcd for
L
D
L
C
54H87O22, 1087.5689). ESI-MS in negative ion mode (% base peak),
10.03 min,
11.39 min,
10.56 min).
D
-glucose 10.24 min,
L
L
m/z 1087 (100) [M – H]–, which was fragmented in the MS/MS to
give m/z 1069 (100) [(M – H) – 18]–, 1025 (36) [(M – H) – 18 – 44]–,
879 (32) [(M – H) – 18 – 44 – 146]–, 717 (5) [(M – H) – 18 – 44 – 146 –
162]–, 603 (3) [(M – H) – 18 – 44 – 146– 162 – 114]–, 441 (1) [(M – H)
D
D
3.6. Cell culture and cytotoxicity
– 18–44–146–162–114–162]–. 1H and 13C NMR data see Table 1.
25
3.6. Abyssinoside A (3): orange amorphous solid; [
a
]
D +59.7
The MCF-7 (estrogen dependent) and MDA-MB-231 (estrogen
independent) breast adenocarcinoma cell lines were obtained
from European Collection of Cell Cultures (ECACC). The cells were
cultured in DMEM without phenol red, supplemented with 10%
(MeOH, c 0.1). For UV spectrum see Fig S47 (Supplementary Data).
HRESI-TOFMS, m/z 601.1523 [M + Na]+ (calcd for C27H30O14Na,
601.1533). ESI-MS in negative ion mode (% base peak), m/z 577
(100) [M – H]–, which was fragmented in the MS/MS to give m/z
559 (11) [(M – H) – 18]–, 503 (1) [(M – H) – 74]–, 487 (40) [(M – H) –
90]–, 473 (8) [(M – H) – 104]–, 457 (100) [(M – H) – 120]–, 383 (6)
[(M – H) – 194]– (A [270,aglycone apigenin] + 113), 353 (16) [(M –
fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine
at 37 ꢁC, in a humidified atmosphere containing 5% CO2. All
reagents, cell culture media and supplements used in cytotoxicity
study were obtained from Sigma–Aldrich.
H) – 224]–(A + 83). For 1H and 13C NMR data see Table 2.
The effect of tested compounds (1–7) on the growth and
proliferation of breast cancer cells was determined by MTT assay
(Berridge et al., 2005). Briefly, 2 ꢂ104 cells per well were plated in
96-well microtiter plate and treated with tested compounds for
25
3.7. Abyssinoside B (4): yellow amorphous solid; [a] D + 44.2
(MeOH, c 0.1). For UV spectrum see Fig S59 (Supplementary Data).
HRESI-TOFMS, m/z 601.1524 [M + Na]+ (calcd for C27H30O14Na,
601.1533). ESI-MS in negative ion mode (% base peak), m/z 577
(100) [M – H]–, which was fragmented in the MS/MS to give m/z
559 (30) [(M – H) – 18]–, 503 (67) [(M – H) – 74]–, 487 (7) [(M – H) –
90]–, 473 (78) [(M – H) – 104]–, 457 (100) [(M – H) – 120]–, 383 (44)
[(M – H) – 194]–(A + 113), 353 (44) [(M – H) – 224]– (A + 83). For 1H
72 h in concentration range 1–100
mM. At the end of treatment, the
medium was aspirated and 170 L of reaction solution containing
m
methylthiazolydiphenyl-tetrazolium bromide (MTT) solution
(0.5 mg/mL) in culture medium was added to each well. The cells
were incubated 2 h, and centrifuged at 1300 rpm for 3 min. After
careful removal of the MTT solution, 0.1 mL of dimethyl sulfoxide
was added to each well, and plates were shaken for 15 min. The
absorbance was measured at 570 nm with plate reader (Elx-800,
Biotek Instruments, USA). Cell viability was calculated as a
percentage of the control. The dose response curves were fitted
to a Hill equation using a non-linear least squares method using
GraphPad Prism version 6.00 for Windows (GraphPad Software, La
Jolla California USA). Resveratrol, a cell cycle arrest-inducing
compound, was included in the assays as a positive control (Joe
et al., 2002; Perrone et al., 2005).
and 13C NMR data see Table 2.
25
3.8. Abyssinoside C (5): pale yellow amorphous solid; [
a
]
–
D
31.9 (MeOH, c 0.1). For UV spectrum see Fig S73 (Supplementary
Data). HRESI-TOFMS, m/z 585.1573 [M + Na]+ (calcd for
C
27H30O13Na, 585.1584). ESI-MS in negative ion mode (% base
peak), m/z 561 (100) [M – H]–, which was fragmented in the MS/MS
to give m/z 325 (4) [(M – H – 146) – 90]–, 295 (100) [(M – H – 146) –
120]–, 267 (14) [(M – H – 146) – 148]– (A [254,aglycone 7,40-
dihydroxyflavone] + 13). For 1H and 13C NMR data see Table 2.
25
3.9. Abyssinoside D (6): dark yellow amorphous solid; [
a
]
–
D
27.6 (MeOH, c 0.1). For UV spectrum see Fig S86 (Supplementary
Data). HRESI-TOFMS, m/z 571.1415 [M + Na]+ (calcd for
Appendix A. Supplementary data
C
26H28O13Na, 571.1428). ESI-MS in negative ion mode (% base
peak), m/z 547 (100) [M – H]–, which was fragmented in the MS/MS
to give m/z 325 (13) [(M – H – 132) – 90]–, 295 (100) [(M – H – 132) –
Supplementary data associated with this article include copies
of NMR (1H, 13C, HSQC, HMBC, ROESY, DQF-COSY, HSQC-TOCSY,
selective TOCSY and ROESY), HRESI-TOFMS, ESI-MS/MS spectra for

A.J. Pérez et al. / Phytochemistry Letters 13 (2015) 59–67
67
the new compounds (1–6) and UV spectra for compounds 3–6.
Graphics showing the effect of compounds 1–7 over the viability of
MCF-7 and MDA-MB-231 cell lines for 24 and 72 hours of
incubation.
Kouam, J., Noundou, X.S., Mabeku, L.B.K., Lannang, A.M., Choudhary, M.I., Fomum, Z.
T., 2007. Sigmoiside E: A new antibacterial triterpenoid saponin from Erythrina
sigmoidea (Hua). Bull. Chem. Soc. Ethiop. 21, 373–378.
Kouam, J., Meli, A.L., Choudhary, M.I., Fomum, Z.T.Z., 2008. Sigmoiside F and
propyloxyamyrin, two new triterpenoid derivatives from Erythrina sigmoidea.
Naturforsch 63b, 101–104.
Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The systematic identification of
flavonoids. Springer-Verlag, New York.
Acknowledgments
Marchand, L.L., 2002. Cancer preventive effects of flavonoids - a review. Biomed.
Pharmacother 56, 296–301.
Mbafor, J.T., Ndom, J.C., Fomum, Z.T., 1997. Triterpenoid saponins from Erythrina
sigmoidea. Phytochemistry 44, 1151–1155.
This research was supported by the European Union’s Seventh
Framework Programme project EMAP (Contract 247548).
Miyao, H., Sakai, Y., Takeshita, T., Kinjo, J., Nohara, T., 1996. Triterpene saponins from
Arbus cantoniensis (Leguminosae). I. 1) Isolation and characterization of four
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Nguyen, P.H., Le, T.V.T., Thuong, P.T., Dao, T.T., Ndinteh, D.T., Mbafor, J.T., Kang, K.W.,
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.
phytol.2015.05.013.
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