New Cytotoxic Triterpenoid Saponins from the Roots of Albizia gummifera (J.F.Gmel.) C.A.Sm.

Article abstract of DOI:10.1002/cbdv.201700260

As part of our search for new bioactive saponins from Cameroonian medicinal plants, two new oleanane-type saponins, named gummiferaosides D and E (1 and 2), along with one known saponin, julibroside J₈ (3), were isolated from the roots of Albizia gummifera. Their structures were established on the basis of extensive 1D- and 2D-NMR (¹H- and ¹³C-NMR, DEPT, COSY, TOCSY, NOESY, HSQC, HSQC-TOCSY, and HMBC) and HR-ESI-MS studies, and by chemical evidence. The apoptotic effect of saponins 1?–?3 was evaluated on the A431 human epidermoid cancer cell. Flow cytometric analyses showed that saponins 1?–?3 induced apoptosis of human epidermoid cancer cell (A431) in a dose-dependent manner.

Full text of DOI:10.1002/cbdv.201700260

Article Type: Full Paper
New Cytotoxic Triterpenoid Saponins from the
Roots of Albizia gummifera C.A. Smith
Line Made Simo a,b, Olivier Placide Noté* a,b, Joséphine Ngo Mbingb, Sarah Ali Aouazoua,
Dominique Guillaumec, Christian Dominique Mullera, Dieudonné Emmanuel Pegnyembb, and
Annelise Lobsteina
a Laboratoire d’Innovation Thérapeutique, UMR 7200, Faculté de Pharmacie, CNRS-
Université de Strasbourg, 74 route du Rhin, F-67401 Illkirch Cedex, France
b Laboratoire de Pharmacochimie des Substances Naturelles, Département de Chimie
Organique, Faculté de Sciences, Université de Yaoundé I, BP 812, Yaoundé,
Cameroun(oliviernote1@yahoo.fr (Olivier Noté))
cLaboratoire de Chimie Thérapeutique, UMR7312, Université de Reims, 51 rue
Cognacq-Jay, 51100 Reims, France
Abstract
As part of our search of new bioactive saponins from Cameroonian medicinal
plants, two new oleanane-type saponins, named gummiferaosides D–E (1–2), along
with one known saponin, julibroside J8 (3), were isolated from the roots of Albizia
gummifera. Their structures were established on the basis of extensive 1D and 2D
NMR (1H-, 13C-NMR, DEPT, COSY, TOCSY, NOESY, HSQC, HSQC-TOCSY and
HMBC) and HRESIMS studies, and by chemical evidence. The apoptotic effect of
saponins 1–3 was evaluated on the A431 human epidermoid cancer cell. Flow
cytometric analyses showed that saponins 1–3 induced apoptosis of human
epidermoid cancer cell (A431) in a dose-dependent manner.
Keywords:Albizia gummifera; Mimosaceae ; Triterpenoid saponins; apoptosis of human epidermoid cancer cell A431.
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Introduction
The genus Albizia comprises about 150 species widely distributed in the tropics,
with the greatest diversity in Africa and South America [1]. Triterpenoid glycosides
commonly described in Albizia genus are complex acacic acid-type saponins, some
of which have been reported to inhibit the growth of tumor cells, and thus appear as
a new potential class of anticancer natural triterpenoid saponins [2]. However, the
proapoptotic function of acacic acid-type saponins isolated from Albizia genus has
only been reported for adianthifoliosides A and D, isolated from Albizia adianthifolia
[3], and very recently for zygiaosides A-B isolated from Albizia zygia [4]. These
saponins share some structural features: they all have acacic acid asaglycone unit,
oligosaccharide moieties at C-3 and C-28, and an acyl group at C-21.
As part of our continuing search of new apoptosis-inducing triterpenoid
saponins from Cameroonian Albizia genus [4, 5], we investigated the saponins
content of the roots of Albizia gummifera. It is an umbrella-like crown tree about 50 m
high of the legume family growing naturally in Nigeria, Cameroon, Democratic
Republic of Congo, Angola, Ethiopia, Kenya, and Madagascar where plant parts are
used in traditional medicine [6]. In Kenya a bark infusion is taken to treat malaria and
the pounded bark is used as a snuff to treat headache, while the pounded roots are
added to a bath to treat skin diseases, and an extract of crushed pods is drunk to
treat stomach-ache. In eastern DR Congo a bark maceration is used as a body wash
and drink to treat psoriasis. The roots and leaves are purgative and are used in
Madagascar to treat diarrhoea and eye troubles. Its leaf decoction is reputed to have
antitussive activity and is administered to treat asthma [6]. The In vitro antimalarial
activity of the extracts of Albizia gummifera has also been reported [7]. Previous
phytochemical studies reported the presence of triterpenoid saponins from stem
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bark [8] and roots [9] of A. gummiferagrowing in Ethiopia and Madagascar,
respectively.
In the present investigation of the roots of A. gummifera growing in Cameroon,
we report the isolation and structural characterization of two new oleanane-type
saponins, named gummiferaosides D–E (1–2), along with one known saponin,
julibroside J8 (3). The isolated saponins (1‒3) were evaluated for their pro-apoptotic
effect on the A431 human epidermoid cancer cell, results are reported herein.
Results and Discussion
The air-dried powdered roots of A. gummifera (300 g) were extracted with aq-
EtOH 70% using a soxhlet apparatus. After evaporation of the solvent, the resulting
brown residue was partitioned between water and water-saturated n-BuOH. The n-
BuOH fraction was then submitted to vacuum-liquid chromatography (VLC) on
reversed-phase silica gel yielding three main fractions which were subjected to VLC
on silica gel. Purification of the eluated subfractions by Semprep-HPLC afforded two
new triterpenoid saponins, named gummiferaosides D‒E (1‒2), together with known
julibroside J8 (3) (Scheme 1).
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30
29
OH
O
20
17
19
21
O
12
26
22
18
15
11
25
13
14
16
O
1
4
O
9
6
OH
8
7
2
10
28
27
5
3
OH
O
OH
O
O
OH
23
24
O
O
HO
HO
O
O
HO
O
HO
HO
HO
O
O
O
O
O
OR1
HO
HO
O
O
O
OH
OH
HO
HO
O
HO
O
O
HO
O
OH
O
HO
HO
OH
O
OH
HO
OH
HO
OR2
MT-Qui
R1
(1) Glc
R2
MT-Qui
MT-Qui
H
(2)
(3)
H
H
Scheme 1. Structures of the isolated saponins (1–3)
Gummiferaoside D (1) was obtained as a white, amorphous powder. Its positive
HR-ESI-MS gave a pseudo-molecular ion peak at m/z 2663.2630 [M + NH4]+(calcd
2663.2552), consistent with a molecular formula of C124H196O60. Upon acid hydrolysis
with 2.0 M HCl, 1 gave an acacic acid lactone unit, identified with an authentic
sample, together with glucose (Glc), xylose (Xyl), arabinofuranose (Araf), fucose
(Fuc), rhamnose (Rha), and quinovose (Qui), which were identified by co-TLC with
authentic samples. The absolute configuration of these sugar residues was
determined to be D for Glc, Xyl, Qui, and Fuc, and L for Araf and Rha based on GC
1
analysis of their trimethylsilyl thiazolidine derivatives [10]. H-NMR spectrum of 1
showed seven angular methyl groups as singlets at δ (H) 0.99, 1.06, 1.07, 1.11,
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1.18, 1.30, and 1.90 (each 3H, s), one olefinic proton at δ (H) 5.65 (1H, brs), and
sugar proton signals at δ (H) 4.87–6.30. Its 13C-NMR spectrum showed two olefinic
carbon signals at δ (C) 122.8 and 143.1, suggesting that 1 was an oleanane type
triterpenoid saponin. 1D (1H-, 13C-NMR, DEPT) and 2D (COSY, HSQC, NOESY,
HSQC-TOCSY, and HMBC) NMR techniques permitted the unambiguous
1
assignment of all H- and 13C-NMR signals of the aglycone of 1 (Table 1). This
aglycon was thus recognized to be acacic acid (3β, 16α, 21β-trihydroxyolean-12-
ene-28-oic acid) by comparison of its 1H- and 13C-NMR signals with those reported in
the literature [11–20].
13
Table 1. C-NMR (125 MHz) and 1H-NMR (500 MHz) data of the Aglycone Part of
Compounds 1–2 (in pyridine-d5; δ in ppm)a.
1
2
DEPT
δ (C)
38.6
26.6
88.3
39.4
55.8
18.4
33.4
39.9
46.9
36.8
23.2
122.8
143.1
41.8
35.7
73.6
51.4
40.5
47.6
35.2
76.9
36.2
27.9
16.7
15.6
17.1
27.0
174.2
28.9
18.9
δ (H) (multiplicity)
δ (C)
38.6
26.6
88.1
39.4
55.7
18.4
33.3
39.9
46.9
36.8
23.3
122.8
144.9
41.8
35.7
73.6
51.4
40.7
47.6
35.2
76.7
36.2
28.0
17.1
15.7
16.9
27.0
174.2
28.9
18.9
δ (H) (multiplicity)
CH2 (1)
CH2 (2)
CH(3)
C (4)
CH(5)
CH2 (6)
CH2 (7)
C (8)
CH(9)
C (10)
1.61; 1.19
2.29; 1.96
3.54
1.58; 1.19
1.93; 2.30
3.62
-
-
0.99
0.99
1.70; 1.48
1.70; nd
-
1.71; 1.53
1.74; nd
-
1.93
-
2.08; nd
5.64 (brs)
-
1.93
-
2.08; nd
5.65 (brs)
-
CH2 (11)
CH(12)
C (13)
C (14)
-
-
CH2 (15)
CH(16)
C (17)
CH(18)
CH2 (19)
C (20)
CH(21)
CH2 (22)
CH3 (23)
CH3 (24)
CH3 (25)
CH3 (26)
CH3 (27)
C (28)
2.27; 2.05
5.25
2.26; 2.05
5.24
-
-
3.45
3.45
2.96; 1.42
2.98; 1.45
-
-
5.35
5.35
2.75; 2.20
1.30 (s)
1.18 (s)
0.99 (s)
1.07 (s)
1.90s
-
2.72; 2.19
1.33 (s)
1.18 (s)
0.99 (s)
1.05 (s)
1.89 (s)
-
CH3 (29)
CH3 (30)
1.06 (s)
1.11 (s)
1.05 (s)
1.10 (s)
Assignments based on the HMBC, HSQC, COSY, HSQC-TOCSY, NOESY, and DEPT
experiments. nd, not determined
a) Overlapped proton NMR signals are reported without designated multiplicity.
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Substitutions at C-3 and C-28 of acacic acid were evidenced by the observed
glycosylation-induced shifts of C-3 at δ 88.3 and C-28 at δ 174.2. Substitution at C-
21 was ascertained from the acylation-induced shift observed for C-21 at δ 76.9. All
of these data established that 1 was a 21-acyl 3, 28-bidesmosidic acacic acid
derivative; sugar chains being linked to C-3 and C-28 through an ether and ester
bond, respectively.
1
The H-NMR spectrum of 1 showed eleven anomeric protons at δ (H) 4.91 [d, J
= 7.7 Hz, glucose (GlcI)], 4.99 [d, J = 8.1 Hz, fucose (Fuc)], 5.45 [d, J = 8.0 Hz,
glucose (GlcII)], 5.08 [d, J = 7.3 Hz, xylose (Xyl)], 6.09 [d, J = 8.1 Hz, glucose
(GlcIII)], 5.89 [brs, rhamnose (Rha)], 5.36 [d, J = 7.7 Hz, glucose (GlcIV)], 6.30 [d, J
= 6.8 Hz, arabinofuranose (Araf)], 4.90 [d, J = 7.7 Hz, quinovose (QuiI)], 4.87 [d, J =
7.7 Hz, quinovose (QuiII)], and 4.96 [d, J = 8.1 Hz, quinovose (QuiIII)], which
correlated with eleven anomeric carbon atom signalsat δ (C) 104.7, 103.2, 105.5,
106.6, 95.5, 101.8, 105.6, 110.8, 99.1, 99.1, and 96.7, respectively, in the HSQC
1
13
spectrum. The H- and C-NMR data (Tables 2-3) of the monosaccharide residues
were assigned starting, either from the readily identifiable anomeric proton of each
hexosyl or pentosyl unit, or from the CH3-proton doublet of each 6-deoxyhexosyl
unit, by means of COSY, HSQC-TOCSY, HSQC, NOESY and HMBC spectra. Data
indicated that these eleven sugar residues were in their pyranose form. Based on the
coupling constants of the anomeric protons and the chemical shifts of the anomeric
carbons, the anomeric configuration of the sugar moieties were determined as β for
the glucose, xylose, fucose, and quinovose moieties and α for the rhamnose and
arabinofuranose moieties.
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Table 2.13C-NMR (125 MHz) and 1H-NMR (500 MHz) Data of the Sugar Moieties Attached at C-3 and
C-28 of Compounds 1–2(in pyridine-d5; δ in ppm)a.
1
2
Position
DEPT
δ (C)
δ (H) (J in Hz)
δ (C)
δ (H) (J in Hz)
3-O-sugars
GlcI
````
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH2 (6)
104.7
4.91 (d, J= 7.7)
106.7
4.93 (d, J= 7.7)
82.5
78.3
71.6
76.3
69.9
4.27
4.15
4.05
75.8
78.3
71.6
77.1
69.8
4.06
4.19
4.18
4.08
4.10
4.74; 4.36
4.77; 4.39
Fuc
Glc II
Xyl
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH3 (6)
103.2
81.8
75.0
71.9
70.6
18.0
4.99 (d, J= 8.1)
4.46
103.3
82.0
75.0
71.3
71.4
17.2
5.03 (d, J= 7.5)
4.47
4.10
3.97
3.69
4.04
4.16
3.77
1.37 (d, J= 6.6)
1.51 (d, J= 6.6)
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH2 (6)
105.5
75.4
77.3
71.1
77.0
62.5
5.45 (d, J= 8.0)
4.02
4.15
3.79
3.75
4.44; 4.30
CH (1)
CH (2)
CH (3)
CH (4)
CH2 (5)
106.6
75.4
78.1
70.7
67.0
5.08 (d, J= 7.3)
4.05
4.00
4.10
4.48; 3.60
106.9
75.8
78.2
70.6
67.0
5.10 (d, J= 7.0)
4.04
4.08
4.18
4.49; 3.61
28-O-sugars
Glc III
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH2 (6)
95.5
76.0
78.1
75.1
79.2
62.5
6.09 (d, J= 8.1)
4.05
95.6
76.8
77.2
71.0
78.5
61.6
6.08 (d, J= 7.9)
4.09
4.16
4.10
3.96
4.16
4.18
3.98
4.50; 4.29
4.35; 4.18
Rha
Glc VI
Araf
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH3 (6)
101.8
70.5
81.8
79.2
69.1
18.4
5.89 (brs)
5.21
4.96
4.51
4.58
101.7
70.4
82.0
79.0
69.0
18.9
5.91 (brs)
5.19
4.92
4.50
4.57
1.63 (d, J= 6.5)
1.78 (d, J= 5.6)
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH2 (6)
105.6
75.2
77.4
71.0
78.2
62.3
5.36 (d, J= 7.7)
4.00
105.7
75.3
78.1
71.6
78.4
62.5
5.36 (d, J= 7.0)
3.99
4.15
4.12
3.92
4.23
4.09
3.99
4.50; 4.28
4.51; 4.19
CH (1)
CH (2)
CH (3)
CH (4)
CH2 (5)
110.8
84.3
78.2
85.2
61.7
6.30 (d, J= 6.8)
5.01
4.83
4.77
4.27; 4.18
111.0
84.3
78.3
85.2
62.3
6.29 (d, J= 7.7)
5.00
4.82
4.76
4.27; 4.17
Assignments were based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not
determined. a)Overlapped proton NMR signals are reported without designated multiplicity.
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Table 3.13C-NMR (125 MHz) and 1H-NMR (500 MHz) Data of the Quinovosyl Moieties attached at C-
21 of compounds 1–2 (in pyridine-d5; δ in ppm)a.
Quinovose
(Qui)
1
2
DEPT
δ (C)
δ (H) (J in Hz)
δ (C)
δ (H) (J in Hz)
QuiI
CH (1)
99.1
75.4
76.2
77.3
70.2
18.7
4.90 (d, J = 7.7)
99.1
75.4
76.2
77.1
70.2
18.7
4.90 (d, J = 7.7)
CH (2)
CH (3)
CH (4)
CH (5)
CH3 (6)
4.02
4.21
5.36
4.03
4.21
5.35
3.70
3.70
1.37 (d, J = 6.5)
1.37 (d, J = 6.6)
QuiII
QuiIII
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH3 (6)
99.1
75.5
76.2
77.4
72.6
18.9
4.87 (d, J = 7.7)
99.1
75.5
76.2
77.4
72.6
18.9
4.86 (d, J = 7.7)
4.02
4.23
5.37
3.75
4.03
4.21
5.37
3.75
1.63 (d, J = 6.5)
1.62 (d, J = 6.2)
CH (1)
CH (2)
CH (3)
CH (4)
CH (5)
CH3 (6)
96.7
75.6
75.7
76.9
72.4
18.7
4.96 (d, J = 8.1)
96.7
75.6
75.8
76.9
72.4
18.7
4.97 (d, J = 7.9)
4.08
4.23
3.75
4.02
4.24
3.75
3.73
3.73
1.60 (d, J = 6.5)
1.60 (d, J = 6.2)
Assignments based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not
determined. a)Overlapped proton NMR signals are reported without designated multiplicity
In addition, the 1H-NMR spectrum of compound 1 exhibited three olefinic proton
signals at δ (H) 7.06 (d, J = 7.3 Hz), 7.00 (d, J = 7.7 Hz), and 7.06 (d, J = 7.3 Hz),
and three groups of one-substituted olefinic proton signals, one group at δ (H) 6.24
(dd, J = 11.1; 17.6Hz), 5.24 (d, J = 11.1 Hz) and 5.44 (d, J = 17.6 Hz), another group
at δ (H) 6.27 (dd, J = 11.1; 17.6 Hz), 5.25 (d, J = 11.1 Hz) and 5.48 (d, J = 17.6 Hz),
and the last group at δ (H) 6.21 (dd, J = 11.1; 17.9 Hz), 5.30 (d, J = 11.1 Hz) and
5.38 (d, J = 17.9 Hz), indicating that compound 1 had three monoterpenoid units [16]
(Table 4).
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Table 4.13C-NMR (125 MHz) and 1H-NMR (500 MHz) Data of the Monoterpene Moieties Attached at
C-21 of Compounds 1–2 (in pyridine-d5; δ in ppm)a.
Monoterpene
(MT)
1
2
DEPT
δ (C)
δ (H) (J in Hz)
δ (C)
δ (H) (J in Hz)
MT1
MT2
MT3
C (1)
C (2)
167.5
133.6
142.6
23.5
40.8
79.2
-
-
167.4
128.6
142.6
23.5
40.3
79.2
-
-
CH (3)
CH2 (4)
CH2 (5)
C (6)
CH(7)
CH2 (8)
7.06 (t, J= 7.3)
2.46; nd
7.05 (t, J= 7.7)
2.68; 2.45
1.82; 1.73
1.78; nd
-
-
145.0
114.7
6.24 (dd, J= 11.1; 17.6)
5.44 (d, J= 17.6);
5.24 (d, J= 11.1)
4.74 (brs)
144.9
114.7
6.24 (dd, J= 11.2; 17.8)
5.44 (d, J= 17.8);
5.25 (d, J= 11.2)
4.72 (brs)
CH2 (9)
CH3 (10)
56.0
23.6
56.0
23.6
1.51(s)
1.51 (s)
C (1)
C (2)
167.3
127.9
142.8
23.6
40.6
79.3
-
-
167.3
127.9
142.7
23.7
40.6
79.3
-
-
CH (3)
CH2 (4)
CH2 (5)
C (6)
CH(7)
CH2 (8)
7.00 (t, J= 7.7)
7.00 (t, J= 7.7)
2.68; 2.45
1.82; 1.73
-
2.45; nd
1.76; nd
-
143.8
115.0
6.27 (dd, J= 11.1; 17.6)
5.43 (d, J= 17.6);
5.25 (d, J= 11.1)
1.90 (s)
143.8
114.9
6.22 (dd, J= 11.1; 17.8)
5.42 (d, J= 17.8);
5.29 (d, J= 11.1)
1.92 (s)
CH3 (9)
CH3 (10)
12.5
23.6
12.5
23.7
1.54 (s)
1.54 (s)
C (1)
C (2)
CH (3)
CH2 (4)
CH2 (5)
C (6)
167.0
127.7
143.0
23.5
40.3
79.5
-
-
167.0
127.9
143.0
23.6
40.8
79.5
-
-
7.06 (t, J= 7.3)
7.07 (t, J= 7.3)
2.66; 2.44
1.83; 1.73
-
2.45; nd
1.80; nd
-
CH(7)
CH2 (8)
143.6
115.3
6.21(dd, J= 11.1; 17.9)
5.38 (d, J= 17.9);
5.30 (d, J= 11.1)
1.96 (s)
143.7
115.3
6.00 (dd, J= 6.9; 17.4)
5.42 (d, J= 17.4);
5.29 (d, J= 6.9)
1.95 (s)
CH3 (9)
CH3 (10)
12.5
23.6
12.5
23.4
1.58 (s)
1.57 (s)
Assignments based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not determined.
a)Overlapped proton NMR signals are reported without designated multiplicity
Extensive analysis of 1D and 2D NMR spectra of 1, revealed that the sugar moiety at
C-3 was identical to that of albizoside B [12], julibroside J31 [15], albizoside D [16],
and julibroside J32 [17], and that the sugar moiety at C-28 was identical to that of
julibrosides J16 and J17 [13], J14 and J28 [14], J32, J35, and J36 [15], adianthifoliosides
A–F [18,19], and coriarioside A [20]. Hence, the units attached at C-3 and C-28 of
the aglycon of 1 were establishedas β-D-xylopyranosyl-(1→2)-β-D-fucopyranosyl-
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(1→6)-[β-D-glucopyranosyl-(1→2)]-β-D-glucopyranoside, and α-L-arabinofuranosyl-
(1→4)-[β-D-glucopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→2)-β-D-
glucopyranoside, respectively. By comparing the 13C NMR data of 1 with those
published in the literature for related compounds, it was found that the 13C NMR data
of 1 were almost superimposable with those of albizoside D [16] and julibroside J36
[17], except for the signals of the monoterpene-quinovosyl moiety. Indeed, the
difference was the observation of the signals of one additional monoterpene-
quinovosyl moiety in compound 1 in comparison to albizoside D [16] and julibroside
J36 [17]. This was supported by the mass spectrum of 1, which displayed 312 mass
units more than that of albizoside D [16] and julibroside J36 [17], accounting for the
mass of one quinovose and one monoterpene unit. In addition, the monoterpene-
quinovosyl moiety of 1, differs from that of zygiaoside A [5], gummiferaoside C [9],
lebbeckoside A [11], coriariosides A [20], C and D [21], only by the additional
presence of one hydroxyl group attached to C-9 of the monoterpenoid unit directly
attached to C-21 of the aglycon as evidenced by the signals of C-2 (δ 133.6) and of
C-9 (δ 56.0) of a 9-hydroxy-monoterpenoid unit. Furthermore, the 13C NMR data of
compound 1 showed that the signals of C-5 and C-10 of the monoterpene moieties
in 1 were quite similar to those of the outer monoterpene moiety of albizoside C [12]
and of Julibroside III [14] indicating the (S) absolute configuration at C-6 of the three
monoterpene moieties. Extensive analysis of 1D and 2D NMR spectra of 1 allowed
to identify the monoterpene-quinovosyl moiety attached at C-21 of 1 as {(2E,6S)-2-
hydroxymethyl-6-methyl-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(β-D-
quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(β-D-
quinovopyranosyl)octa-2,7-dienoyl]-β-D-quinovopyranosyl}-2,6-dimethylocta-2,7-
dienoyl}. Accordingly, the structure of gummiferaoside D (1) was elucidated as 3-O-[
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β-D-xylopyranosyl-(1→2)-β-D-fucopyranosyl-(1→6)-[β-D-glucopyranosyl-(1→2)]-β-D-
glucopyranosyl]-21-O-{(2E,6S)-2-hydroxymethyl-6-methyl-6-O-{4-O-[(2E,6S)-2,6-
dimethyl-6-O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-
O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-β-D-quinovopyranosyl}-2,6-dimethylocta-
2,7-dienoyl}acacic acid 28-O- α-L-arabinofuranosyl-(1→4)-[β-D-glucopyranosyl-
(1→3)]-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester.
Gummiferaoside E (2), obtained as a white amorphous powder, exhibited in its
HRESIMS spectrum (positive-ion mode) a pseudo-molecular ion peak at m/z
2502.1972 [M +NH4]+ (calcd 2502.2135), consistent with a molecular formula of
C118H186O55. As already observed for 1, acid hydrolysis of 2 also afforded an acacic
acid lactone unit, identified by co-TLC with authentic sample, together with D-glucose,
D-xylose, D-fucose, D-quinovose, L-arabinofuranose and L-rhamnose units which
were identified by GC analysis of their trimethylsilyl thiazolidine derivatives
(Experimental Section). Comparison of 1D and 2D NMR data of 2 with those of 1
indicated that 2 had also acacic acid as aglycon (Table 1).The observation of
glycosylation- and acylation-induced shifts at δ (C) 88.1 (C-3 of aglycon), 76.7 (C-21
of aglycon), and 174.2 (C-28 of aglycon) in the 13C-NMR spectrum of 2 suggested
that it should be also a 21-acyl 3,28-bidesmosidic acacic acid derivative with sugar
chains linked to C-3 and C-28 through an ether and ester bond, respectively, and
1
with an acyl group attached at C-21. The H-NMR spectrum of 2 showed ten
anomeric protons signals at δ (H) 4.93 [d, J = 7.7 Hz, glucose (GlcI)], 5.03 [d, J = 7.5
Hz, fucose (Fuc)], 5.10 [d, J = 7.0 Hz, xylose (Xyl)], 6.08 [d, J = 7.9 Hz, glucose
(GlcII)], 5.91 [brs, rhamnose (Rha)], 5.36 [d, J = 7.0 Hz, glucose (GlcIII)], 6.29 [d, J =
7.7 Hz, arabinofuranose (Araf)], 4.90 [d, J = 7.7 Hz, quinovose (QuiI)], 4.86 [d, J =
7.7 Hz, quinovose (QuiII)], and 4.97 [d, J = 7.9 Hz, quinovose (QuiIII)], which
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correlated with ten anomeric carbon atom resonances at δ (C) 106.5, 103.1, 106.9,
95.6, 101.7, 105.7, 111.0, 99.1, 99.1, and 96.7, respectively in the HSQC spectrum
(Tables 2-3). Comparison of their NMR data revealed that the units attached at C-21
and C-28 of the aglycon of 2 were identical to those of 1, and the two saponins differ
only by the sugar chain attached at C-3 of the aglycon, as suggested by the mass
spectrum of 2, which displayed 162 mass units less than that of 1, accounting for the
mass of one hexose unit, assigned to one glucopyranosyl moiety to account for the
1
similarities between their H and 13C NMR spectra.Therefore, the moieties at C-21
and C-28 of 2 were established as {(2E,6S)-2-hydroxymethyl-6-methyl-6-O-{4-O-
[(2E,6S)-2,6-dimethyl-6-O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-
dimethyl-6-O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-β-D-quinovopyranosyl}-2,6-
dimethylocta-2,7-dienoyl}andα-L-arabinofuranosyl-(1→4)-[β-D-glucopyranosyl-
(1→3)]-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside
moiety,
respectively.
Extensive analysis of 1D and 2D NMR spectra of 2 allowed us to identify the unit
attached at C-3 as β-D-xylopyranosyl-(1→2)-β-D-fucopyranosyl-(1→6)-β-D-
glucopyranoside, a unit identical to that of lebbeckosides A and B [11].
Consequently, the structure of gummiferaoside E (2) was established as 3-O-[β-D-
xylopyranosyl-(1→2)-β-D-fucopyranosyl-(1→6)-β-D-glucopyranosyl]-21-O-{(2E,6S)-2-
hydroxymethyl-6-methyl-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(β-D-
quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(β-D-
quinovopyranosyl)octa-2,7-dienoyl]-β-D-quinovopyranosyl}-2,6-dimethylocta-2,7-
dienoyl}acacic acid 28-O-α-L-arabinofuranosyl-(1→4)-[β-D-glucopyranosyl-(1→3)]-α-
L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester.
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Compound 3, isolated as an amorphous powder, was identified as 3-O-[β-D-
xylopyranosyl-(1→2)-β-D-fucopyranosyl-(1→6)-β-D-glucopyranosyl]-21-O-{(2E,6S)-2-
hydroxymethyl-6-methyl-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(β-D-
quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(β-D-
quinovopyranosyl)octa-2,7-dienoyl} acacic acid 28-O-α-L-arabinofuranosyl-(1→4)-[β-
D-glucopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl
ester,
named Julibroside J8 by comparison of its spectra data with those reported in the
literature [22].
The pro-apoptotic activity of compounds 1-3 was evaluated using Annexin V-
FITC binding assay on the A431 human epidermoid cancer cell. In result, percentage
of apoptotic cells following 24 h of treatment was increased in a concentration-
dependent manner for A431 cell (Figure1). Compounds 2 and 3 displayed the most
promising proapoptotic activity since computed concentration of each compound to
induced half-maximal effects (EC50) on cell apoptosis were determined as being 158
µM, 13 µM, and 23 µM for compounds 1-3, respectively.
Figure 1. Effects of compounds 1- 3 on the apoptosis rate.
Cells were exposed to compounds at the indicated concentrations and incubated for 24h. Cell
apoptosis rate was assessed by flow cytometry using the Annexin V-FITC/PI staining assay (n=3).
*P<0.05 and ***P<0.001 vs. vehicle-treated cells.
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Conclusions
This study unveiled the presence, in the roots of Albizia gummifera growing in
Cameroon, of two new acacic acid-type saponins, among which one molecule,
gummiferaoside E (2), having a promising proapoptotic function towards the A431
human epidermoid cancer cell. It is worthy to note that the proapoptotic function of
acacic acid-type saponins from Albizia genus had been, so far, only reported for
adianthifoliosides A and D [3] and zygiaosides A and B [4]. Our study confirms that
acacic acid, oligosaccharide moieties at C-3 and C-28 and an acyl group at C-21
constitute a pharmacophore compatible with proapoptotic function. Interestingly,
substitution of C-2 of GlcI seems to inhibit proapoptotic function since compound 1
(EC50 158 µM), having a tetrasaccharide moiety at C-3 of aglycone, with a
substituted GlcI at its C-2 position, is less active than compound 2 (EC5013 µM),
which has a linear trisaccharide moiety at the same position of the aglycone.
Nevertheless, further studies are necessary to support this conclusion.
Gummiferaoside E (2), being the most active compound, might therefore be highly
useful to further decipher its mechanism of action and to obtain new structure-activity
relationship data of the related triterpenoid saponins towards the A431 human
epidermoid cancer cell. It will be also of high interest to evaluate the effect of
Gummiferaoside E (2) on other types of cancer cells.
Experimental Section
General
1
Optical rotations were measured on a Jasco P-2000 polarimeter. H NMR (500
MHz) and 13C NMR (125 MHz) spectra were recorded at room temperature in
pyridine-d5 using a Bruker 500 MHz spectrometer. Chemical shifts are given in δ
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(ppm) value relative 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 Nucleodur 100-5 C18ec (21 x 250 mm, 5 µm) column
purchased from Machery-Nagel (Germany). Thin layer chromatography (TLC) was
performed on precoated silica gel plates (60 F254, Merck) using the system solvent n-
BuOH-AcOH-H2O, 65:15:25 as eluent. The spots were observed after spray with
Komarowsky reagent, a (5:1) mixture of p-hydroxybenzaldehyde (2% in MeOH) and
(Ethanolic H2SO4 50%). Vacuum-liquid chromatography (VLC) was carried out using
RP-18 silica gel 60 (25-40 µm) and silica gel 60 (15-40 µm and 40-63 µm).
Plant material.
The roots of Albizia gummifera were harvested at Nkolbisson, Yaoundé
peripheral quarter, in Cameroon in November 2013 under the guidance of Mr. Victor
Nana, botanist of the National Herbarium of Cameroon (NHC), where a voucher
specimen (31315/HNC) was deposited.
Extraction and isolation.
Air-dried and finely powdered roots of A. gummifera (300 g) were extracted with
70% EtOH in soxhlet apparatus. The hydroalcoholic solution was then evaporated to
dryness under reduced pressure to yield a brown residue (6.80 g). The residue was
suspended in 200 mL of water and partitioned between H2O and satured n-BuOH
(3x 300 mL). The n-BuOH phase was evaporated to dryness affording 4.90 g of a
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brown gum which was taken in a minimum amount of water (10 mL) and then
submitted to vacuum-liquid chromatography (VLC) using RP-18 (25-40 µm) eluting
with H2O, 50% MeOH, and 100% MeOH. The 100% MeOH extract was evaporated
to dryness affording 3.90 g crude saponin mixture that was then submitted to VLC
using silica gel 60 (15-40 µm), eluted with CHCl3-MeOH-H2O (80:20:2, 70:30:5,
60:33:7, and 60:40:10) to give three main fractions (G1- G3). Fraction G2 (700.1 mg)
was subjected to VLC on silica gel 60 (15-40 µm), eluted with CHCl3-MeOH-H2O
(80:20:2, 70:30:5, 60:33:7, and 60:40:10) affording three main subfractions (G21-
G23). Subfraction G23 (175.4 mg) was purified by semipreparative HPLC using
gradient system of CH3CN-H2O (20 ml/min) to yield compounds 1 (tR, 9.30 min, 5.8
mg) and 2 (tR, 17.10 min, 6.1 mg), while subfraction G21 (130.5 mg), purified in the
same conditions, yielded compound3 (tR, 14.97 min, 6.7 mg).
Gummiferaoside D (= 3-O-[ β-D-xylopyranosyl-(1→2)-β-D-fucopyranosyl-(1→6)-
[β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl]-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-
2,6-dimethyl-6-O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-
6-O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-β-D-quinovopyranosyl}-2,6-
dimethylocta-2,7-dienoyl}acacic
acid
28-O-
α-L-arabinofuranosyl-(1→4)-[β-D-
glucopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester (1).
White amorphous powder.1H- and 13C-NMR (pyridine-d5): for aglycon, sugars and
monoterpenoid moieties, see Tables 1-4. Positive HR-ESI-MS: m/z 2663.2630 [M +
NH4]+ (calcd 2663.2552)
Gummiferaoside E (=3-O-[β-D-xylopyranosyl-(1→2)-β-D-
fucopyranosyl-(1→6)-β-D-glucopyranosyl]-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-
dimethyl-6-O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-
O-(β-D-quinovopyranosyl)octa-2,7-dienoyl]-β-D-quinovopyranosyl}-2,6-dimethylocta-
2,7-dienoyl} acacic acid 28-O-α-L-arabinofuranosyl-(1→4)-[β-D-glucopyranosyl-
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(1→3)]-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester (2).White amorphous
powder.1H- and13C-NMR (pyridine-d5): for aglycon, sugars and monoterpenoid
moieties, see Tables 1-4. Positive HR-ESI-MS: m/z 2502.1972 [M +NH4]+ (calcd
2502.2135)
Acidic hydrolysis of isolated saponins.
Each saponin (2 mg) was hydrolyzed with 2 ml of 2 M HCl at 85 °C during 2 h.
After cooling, the solvent was removed under reduced pressure. The sugar mixture
was extracted from the aqueous phase (10 ml) and washed with CH2Cl2 (3x 5 ml).
Combined CH2Cl2 extracts were washed with water to give after evaporation the
aglycon moiety, which was compared with authentic standard sample by Co-TLC.
The sugars were first analyzed by TLC over silica gel (CHCl3–MeOH–H2O, 8:5:1) by
comparison 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 [10]. The following sugars were detected: D-glucose, D-xylose, D-fucose,
D-quinovose, L-arabinofuranose and L-rhamnose.
Cell lines and culture conditions.
The A431 human epidermoid cancer cell, was purchased from ATCC (LGC
Standards, Molsheim, France), and cultivated in DMEM-based media (Sigma-Aldrich,
Saint-Quentin-Fallavier, France), supplemented with 10% (v/v) fetal bovine serum
(BioWhittaker, Verviers, Belgium), 2 mMultraglutamine, 50 μM non-essential amino
acids, 50 U/ml penicillin and 50 µg/ml streptomycin (Sigma-Aldrich). The culture was
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kept at 37°C in a humidified incubator equilibrated with 5% CO2. Before confluency
adherent cells were trypsinized and subcultured twice a week.
Pro-apoptotic evaluation.
Cells were treated with the three different compounds (1–3) in ranging
concentrations (1 to 15µM) then collected for apoptosis induction estimation. A
minimum of 5,000 cells was acquired per sample and analyzed on the InCyte
software (Guava/Millipore/Merck, CA, USA). Apoptosis rates were assessed by
capillary cytometry (Guava EasyCyte Plus, Millipore Merck) using Annexin V-FITC
(ImmunoTools, Germany) and PI (MiltenyiBiotec Inc., USA) according to the
manufacturer’s recommendations. Gates were drawn around the appropriate cell
population using a forward scatter (FSC) versus side scatter (SSC) acquisition dot
plot to exclude debris. Final concentration of DMSO applied to cells during
incubation with tested drugs was always 0.5%. In the tested setup that concentration
had no adverse effects on cell viability, nor cell morphology. To discriminate between
negative and positive events in the analysis, a non-stained control sample from each
culture condition always accompanied acquisition of the stained cells to define their
cut off. Negative control, i.e. sample with cells without compounds but with the same
amount of DMSO as for diluted compounds, as well as positive control with 50μM
Celastrol, a natural pentacyclic triterpenoid (Enzo Life Sciences, Farmingdale, US),
were included in each experiment. Cytometers performances are checked weekly
using the Guava easy Check Kit 4500-0025 (Merck/Millipore/Guava Hayward, CA,
USA).
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Statistical analysis
Data, presented as bar graphs, were expressed as means S.E.M. of at least
three independent experiments. Statistical evaluation was performed with the one-
way ANOVA test followed by the post-hoc Bonferroni test using GraphPad Prism
software (Prism version 5.04 for Windows, GraphPad Software, CA, USA); a p-value
less than 0.05 was considered as significant (*), less than 0.01 very significant (**)
and less than 0.001 highly significant (***).
Acknowledgements
We thank the World Academy of sciences (TWAS) (grant no: 3240277733) for
financial support of this work. The authors are also grateful to Mr. Victor Nana of the
National Herbarium of Cameroon (NHC) for the identification and collection of plant.
Author Contribution Statement
Conceived and designed the experiments: Line Made Simo, Olivier Placide Noté,
Joséphine Ngo Mbing, Sarah Ali Aouazou
Performed the experiments: Line Made Simo, Olivier Placide Noté, Sarah Ali Aouazou
Analyzed the data: Line Made Simo, Olivier Placide Noté, Joséphine Ngo Mbing, Sarah Ali
Aouazou
Wrote the paper: Line Made Simo, Olivier Placide Noté, Joséphine Ngo Mbing, Sarah Ali
Aouazou
Revised the article for intellectual content: Dominique Guillaume, Christian Dominique
Muller, Dieudonné Emmanuel Pegnyemb, and Annelise Lobstein
Final approval of the version to be published: Dominique Guillaume, Christian Dominique
Muller, Dieudonné Emmanuel Pegnyemb, and Annelise Lobstein
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