Glaucasides A-C, three saikosaponins from Atriplex glauca L. var. ifiniensis (Caball) Maire

Article abstract of DOI:10.1002/mrc.2696

From the roots of Atriplex glauca L. var. ifiniensis (Caball) Maire (syn. of Atriplex parvifolia Lowe var. genuina Maire), three new saikosaponins designated as glaucasides A-C (1-3) were isolated together with the known 3-O-β-D-glucopyranosyl-(1 → 2)-β-D-galactopyranosyl-saikogenin F (4). The structures of the new compounds were elucidated by extensive analysis of one-dimensional and two-dimensional NMR spectroscopy, FABMS, HR-ESIMS and chemical evidence as 13β,28-epoxy-16β,21β-dihydroxyolean-11-en- 3β-yl O-β-D-[2-O-sulfate]-glucopyranosyl-(1 → 2)-α-L-arabinopyranoside (1), 13β,28-epoxy-16β,21β- dihydroxyolean-11-en-3β-yl O-β-D-[2-O-sulfate]-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl 21-O-{4-(secbutylamido)-butanoyl ester} (2) and 3-O-β-D-glucopyranosyl-(1 → 2)-β-D-galactopyranosyl saikogenin G (3). The cytotoxic activities of these compounds were evaluated against the HT-29 and HCT 116 human colon cancer cell lines. Copyright

Full text of DOI:10.1002/mrc.2696

MRC Letters
Received: 27 July 2010
Revised: 3 October 2010
Accepted: 7 October 2010
Published online in Wiley Online Library: 3 January 2011
(wileyonlinelibrary.com) DOI 10.1002/mrc.2696
Glaucasides A–C, three saikosaponins
from Atriplex glauca L. var. ifiniensis (Caball)
Maire
Aymen Jabrane,a,b Hichem Ben Jannet,c Tomofumi Miyamoto,d
Chiaki Tanaka,d Jean-Franc¸ois Mirjolet,e Olivier Duchamp,e
b
a∗
´
Fethia Harzallah-Skhiri and Marie-Aleth Lacaille-Dubois
From the roots of Atriplex glauca L. var. ifiniensis (Caball) Maire (syn. of Atriplex parvifolia Lowe var. genuina Maire), three
new saikosaponins designated as glaucasides A–C (1–3) were isolated together with the known 3-O-β-D-glucopyranosyl-
(1 → 2)-β-D-galactopyranosyl-saikogenin F (4). The structures of the new compounds were elucidated by extensive
analysis of one-dimensional and two-dimensional NMR spectroscopy, FABMS, HR-ESIMS and chemical evidence as
13β,28-epoxy-16β,21β-dihydroxyolean-11-en-3β-yl O-β-D-[2-O-sulfate]-glucopyranosyl-(1 → 2)-α-L-arabinopyranoside (1),
13β,28-epoxy-16β,21β-dihydroxyolean-11-en-3β-yl O-β-D-[2-O-sulfate]-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl 21-O-
{4-(secbutylamido)-butanoyl ester} (2) and 3-O-β-D-glucopyranosyl-(1 → 2)-β-D-galactopyranosyl saikogenin G (3). The
cytotoxic activities of these compounds were evaluated against the HT-29 and HCT 116 human colon cancer cell lines.
c
Copyright ꢀ 2011 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: NMR; 1H; 13C; 2D NMR; triterpene saponins; glaucasides; saikosaponins; Amaranthaceae; Atriplex
Introduction
and HMBC) spectroscopy and mass spectrometry. The cytotoxicity
of these compounds was evaluated against two human colon
cancer cell lines (HT-29 and HCT 116).
Saikosaponins is a group of oleanane derivatives which are found
as natural constituents in a large number of plant families. Var-
ious pharmacological properties have been observed in some
saikosaponins from medicinal plants including cytotoxic,[1,2] anti-
inflammatory,[3] estrogen-like,[4] immunosuppressive activities,[5]
as well as anti-viral, liver protection effects and apoptosis in-
duction in human hepatoma cell lines.[6–8] The genus Atriplex
L. (Amaranthaceae) is a medium-size cosmopolitan genus com-
prising about 270 species.[9] The genus is widely distributed in
the deserts and semideserts of North and South America, South
Australia and South Central Asia and several temperate zones
of the Northern hemisphere.[10] Previous chemical investigations
of Atriplex species revealed the presence of tannins, flavonoids,
alkaloids, proteins and amino acids as well as saponins.[11] Sev-
eral species are used to treat fungal infections, bronchitis and
diabetes in some countries.[12] In continuation of our investiga-
tions on the secondary metabolites of Tunisian plants,[13–16] we
investigated Atriplex glauca L. var. ifiniensis (Caball) Maire (syn.
of Atriplex parvifolia Lowe var. genuina Maire), a shrub up to
50 cm which is widely distributed in Tunisia.[17] A literature sur-
vey showed that no significant chemical and biological work has
been done on A. glauca. In this article, we report the isolation and
structure determination of three new saikosaponins (1–3), along
with one known compound 3-O-β-D-glucopyranosyl-(1 → 2)-β-
D-galactopyranosyl saikogenin F (4),[18] (Fig. 1) isolated for the first
time in this plant. The structures of the compounds were eluci-
datedmainlyby600 MHzNMRanalysisincludingone-dimensional
(1D) and two-dimensional (2D) NMR (COSY, TOCSY, NOESY, HSQC
Results and Discussion
Glaucaside A (1), a yellow amorphous powder, had the molecular
formula C41H66O16S as determined by HR-ESIMS (positive-ion
mode), showing apseudomolecularionpeakat m/z 869.3973[M+
Na]+ (calcd for 869.3969). Its FABMS (negative-ion mode) showed
a quasi-molecular ion peak at m/z 845 [M − H]−, indicating a
molecularweightof846. Anotherfragmentionpeakwasobserved
∗
Correspondence to: Marie-Aleth Lacaille-Dubois, Laboratoire de Pharmacog-
´
´
´ ˆ
´
nosie, UnitedeMolecules d’Interet Biologique, UMIB UPRES-EA 3660, Facultede
´
Pharmacie, Universite de Bourgogne, F-21079 Dijon Cedex, France.
E-mail: m-a.lacaille-dubois@u-bourgogne.fr
´
´
´ ˆ
a
LaboratoiredePharmacognosie, UnitedeMoleculesd’InteretBiologique, UMIB
´
´
UPRES-EA 3660, FacultedePharmacie, UniversitedeBourgogne, F-21079 Dijon
Cedex, France
´
´
b
c
Institut Superieur de Biotechnologie de Monastir, Universite de Monastir, 5000
Monastir, Tunisie
`
Laboratoire de Chimie des Substances Naturelles et de Synthese Organique,
´
´
FacultedesSciencesdeMonastir,UniversitedeMonastir,5000Monastir,Tunisie
d
e
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka
812-8582, Japan
Oncodesign, 20 rue Jean Mazen, BP 27627, 21076 Dijon Cedex, France
c
Magn. Reson. Chem. 2011, 49, 83–89
Copyright ꢀ 2011 John Wiley & Sons, Ltd.

A. Jabrane et al.
29
30
OR
20
28
19
18
21
22
O
12
26
17
13
11
9
25
H
14
16
1
4
15
OH
10
8
2
3
H
27
OH
5
7
Ara
6
O
O
2
H
24
23
HO
1
O
OH
O
HO
HO
2
1
Glc
OSO3H
R
O
1 H
2
S
C
2'
4'
8'
1'
3'
6'
7'
N
H
9'
O
10'
S=
29
30
20
28
19
18
21
22
O
12
26
17
13
11
25
H
14
9
16
1
4
15
Gal
OH
10
8
2
H
27
OH
OH
3
5
7
6
O
O
H
24
HO
23
OH
3
O
OH
O
HO
HO
OH
Glc
Figure 1. Structure of compounds 1–3.
atm/z765[(M−H)−80]− correspondingtothelossofonesulfated
group.
carbons. The presence of the two hydroxyl groups attached
to C-16 and C-21 was evidenced by the signals observed in
the HSQC spectrum at δH/δC 4.48/65.3 and δH/δC 4.06/72.3,
respectively (Table 1). The existence of the 13,28-epoxy group
was demonstrated by the HMBC cross-peaks H-28α/C-16 and H-
28β/C-16, H-28β/C-17, H-28β/C-18. The deshielded signal at δC
88.6 (C-3) in comparison with the known triterpenes suggested
a glycosidic linkage at this position. After extensive 2D NMR
analysis, the structure of the aglycon of 1 was thus recognized
to be closely related to aglycon of rotundioside Q characterized
as(3β,16α,21α)-13,28-epoxy-16,21-dihydroxyolean-11-en-3-yl.[19]
To establish the relative configuration of all chiral centers, a NOESY
analysis was done. The cross-peaks δH 3.16/δH 0.62 (H-3/H-5)
and δH 3.16/δH 0.78 (H-3/H-1α) confirmed the β configuration
of the C-3 OH. The NOe cross-peak δH 1.78/δH 0.62 (H-9/H-5),
δH 1.78/δH 1.02 (H-9/Me-27) and δH 4.48/δH 1.02 (H-16/Me-27)
evidenced the configuration of H-5, H-9, Me-27 and H-16 to be
α and consequently the 16 β-OH configuration. Furthermore, the
NOe cross-peaks at δH 4.06/δH4.48 (H-21/H-16) and δH 4.48/δH
The 1H NMR spectra of compound 1 showed characteristic
signals of a triterpenoid aglycon linked to a disaccharide chain
(Tables 1 and 2). The structure of the aglycon moiety was
determined on the basis of spectroscopic data and by comparison
with those of known triterpenes.[19–21] The 1H NMR spectrum of
1 showed signals for seven tertiary methyl groups as singlets
at δH 0.77, 0.92, 1.02, 1.05, 1.15, 1.19 and 1.21, two olefinic
protons at δH 5.85 (1H, d, J = 10.5 Hz) and 5.58 (1H, dd, J = 2.5,
10.5 Hz) and two oxygen-bearing methylenic protons at δH 3.32
(1H, d, J = 7 Hz) and δH 4.30 (1H, d, J = 7 Hz), suggesting
a saikogenin-type aglycon. The 13C NMR spectrum contained
signals for 41 carbons, 30 of them for the aglycon part. The DEPT
and HMQC experiments allowed the assignment of six quaternary
carbons methylenic protons δC 39.2, 36.2, 36.6, 41.7, 45.2, 48.8),
two ethylenic carbons (δC 130.5 and 132.1), one oxygen-bearing
quaternary carbon, one oxygen-bearing methylenic carbon (δC
71.8) as well as seven methylene, seven methyl and three methine
c
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Copyright ꢀ 2011 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2011, 49, 83–89

Glaucasides A–C, three saikosaponins from Atriplex glauca
Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) data of the aglycon part of 1–3 in pyridine-d5 (δ in ppm, J in Hz)a
Aglycon
1
2
3
Positions
δC
δH
δC
δH
δC
δH
1
38.4
25.8
88.6
39.2
54.8
17.0
31.3
41.7
52.4
36.2
132.1
130.5
83.6
45.2
35.9
65.3
48.8
51.5
34.1
36.6
72.3
37.0
27.6
15.8
17.9
19.5
20.4
71.8
17.7
30.1
0.78, 1.66 br d (12.8)
38.4
25.7
88.6
39.2
54.8
17.0
31.3
41.7
52.4
35.9
132.4
130.1
83.5
45.7
35.6
64.7
48.5
50.7
36.7
35.9
76.0
37.0
27.6
15.8
17.6
19.5
20.2
71.8
18.3
29.2
0.78, 1.64
38.1
25.6
82.5
43.7
48.0
17.2
31.1
42.2
53.0
35.7
132.2
130.4
83.9
45.6
35.2
64.0
46.9
52.2
37.3
31.1
34.2
25.3
63.7
12.3
18.2
19.5
20.4
73.0
33.2
28.4
0.97, 1.75 br d (12.6)
2
1.80, 1.98
1.78, 2.00
2.00, 2.30 br dd (3.3, 13.1)
3
3.16 dd (4.0, 10.0)
3.16 dd (4.0, 10.0)
4.14 t
–
4
–
–
5
0.62 br d (11.0)
0.62 br d (11.0)
1.51 br s
b
6
1.30, 1.38
1.30, 1.38
1.52, –
7
1.28, 1.14
1.26, 1.13
1.21, 1.49
8
–
–
–
9
1.78
1.76
1.98
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
–
–
–
5.85 d (10.5)
5.87 d (10.5)
5.95 d (10.5)
5.58 dd (2.5, 10.5)
5.58 dd (2.5, 10.5)
5.65 dd (2.6, 10,5)
–
–
–
–
–
–
1.70 dd (5.2, 12.0), 1.96
1.67 dd (5.2, 12.0), 1.94
1.68 dd (5.2, 5.7), 2.02
4.48 br d (8.5)
4.44
4.50
–
–
1.98
–
1.99
1.96
1.90 t (13.6), 1.39
1.35, 1.90 t (14.7)
–
1.31 t (11.4), 1.82 t (13.3)
–
–
4.06 t (9.7)
5.26 dd (4.3, 12.6)
2.67 dd (4.5, 12.6), 1.36
1.13 s
1.17 br dd (12.8), 1.58 dd (3.1, 3.3)
2.90 dd (4.0, 12.6), 1.57 t (12.0)
2.27, 2.48 br d (13.3)
3.78, 4.39
1.07 s
1.15 s
0.92 s
0.90 s
0.77 s
0.76 s
0.95 s
1.21 s
1.20 s
1.38 s
1.02 s
0.96 s
1.08 s
3.32 d (7.0), 4.30 d (7.0)
1.05 s
3.33, 4.31
0.91 s
3.33 d (6.6), 4.37
0.91 s
1.19 s
0.92 s
0.88 s
Acyl
1ꢁ
173.2
31.9
25.4
38.4
–
b
2ꢁ
2.48 qr (7.6),
2.00, 1.98
–
3ꢁ
4ꢁ
3.52 qn (6.0), 3.45 qn (6.0)
8.63 br s
5ꢁ
6ꢁ
177.3
42.5
27.3
11.9
17.7
7ꢁ
2.41 m
1.75 m, 1.38 m
0.84 t (7.5)
8ꢁ
9ꢁ
10ꢁ
1.18 d (7.0)
a Assignments were based on HMBC, HSQC, COSY, TOCSY, NOESY and DEPT experiments. Overlapped proton NMR signals are reported without
designated multiplicity.
b Not determined.
1.90 (H-21/H-19α) indicated the configuration of the OH group
at C-21 to be β (Fig. 2). Other cross-peaks H-1β/H-11, H-7/H-
15α, H-12/H-18, H-18/H-30, H-24/H-6β, H-26/H-15β were also
observed, confirmingthestereochemistryoftheaglyconparttobe
(3β,16β,21β)-13,28-epoxy-16,21-dihydroxyolean-11-en-3-yl). This
aglycon differed from that of rotundioside Q by the configuration
at C-16 and C-21.
The 1H NMR spectrum showed two anomeric proton signals at
δH 4.90 (1H, d, J = 5.2 Hz) and 5.28 (1H, d, J = 7.6 Hz), giving
correlations in the HSQC spectrum, with two anomeric carbon
signals at δC 104.0 and 102.0, respectively, indicating the presence
of two sugars. The ring protons of the monosaccharide residues
were assigned starting from the readily identifiable anomeric pro-
tons by means of COSY, TOCSY, HSQC and HMBC experiments
(Table 2). Evaluation of spin–spin couplings and chemical shifts
allowedtheidentificationofoneα-arabinopyranosyl(Ara)andone
β-glucopyranosyl(Glc)moiety.Therelativelylarge3JH−1,H−2 values
of the anomeric protons of Glc (J = 7.6 Hz) and Ara (J = 5.2 Hz)
indicated a β-anomeric orientation of Glc and α-orientation of
Ara.[22,23] The D and L configuration of Glc and Ara, respectively
c
Magn. Reson. Chem. 2011, 49, 83–89
Copyright ꢀ 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

A. Jabrane et al.
Table 2. 1H NMR (600 MHz) and 13C NMR (150 MHz) data of the sugar moieties of compounds 1–3 in pyridine-d5 (δ in ppm, J in Hz)a
1
2
3
Positions
δC
δH
δC
δH
δC
δH
3-O
1
Ara
104.0
79.5
72.2
67.7
64.6
Ara
104.0
79.0
72.3
67.6
64.2
Gal
104.2
82.7
75.6
69.8
76.6
61.4
4.90 d (5.2)
4.52 t (7.1)
4.90 d (5.2)
4.52 t (7.1)
4.45
5.02 d (7.6)
4.60 t (7.8)
4.12 t (7.1)
4.51
2
3
4.45 dd (3.0, 7.1)
4.42 br s
4
4.42 br s
5
3.68 d (9.7), 4.18
3.69 d (9.7), 4.18
3.92 t (5.9)
4.42, 4.40
6
Glc
Glc
Glc
1
2
3
4
5
6
102.0
81.3
77.1
71.0
77.2
62.1
5.28 d (7.6)
4.95 t (8.2)
102.0
81.3
77.0
71.0
77.2
62.1
5.28 d (7.6)
4.94 t (8.2)
4.31 t (8.0)
106.1
76.7
78.1
71.3
78.2
62.0
5.27 d (7.6)
4.10 t (8.5)
4.13
4.32 t (8.0)
4.08 br d (8.8)
3.75 m
4.07 dd (3.3, 9.6)
3.75 m
4.28
3.76 m
4.36, 4.39
4.36 br d (10.0), 4.16
4.36 br d (10.0), 4.16
a Assignments were based on HMBC, HSQC, COSY, TOCSY, NOESY and DEPT experiments. Overlapped proton NMR signals are reported without
designated multiplicity.
at m/z 1038.5068 [M + Na]+ (calcd for 1038.5072), consistent
with the molecular formula C50H81NO18S. Its FABMS (negative-ion
mode) showed a quasi-molecular ion peak at m/z 1014 [M − H]−,
indicating a molecular weight of 1015 followed by a fragment ion
peak at m/z 827 [(M − H) − 185]− corresponding to the loss of a
C9H16NO3 moiety. The presence of a sulfate group was ascertained
by the presence of peaks at m/z 97 [OSO3H]− and m/z 80 [SO3]−.
A detailed comparison of the 1H and 13C NMR chemical shifts
of 1 and 2 obtained by extensive 2D NMR analysis revealed that
all signals corresponding to the aglycon and sugar moieties in
2 are superimposable with those of 1 except those at C-21. The
deshielding observed in the HSQC spectrum of 2 at δH 5.26 (dd,
J = 4.3, 12.6)/δC 76.0 Agly H/C-21 suggested an acylation at this
position. The 13C NMR and DEPT spectra of 2 revealed that the
presence of 41 carbons corresponding to glaucaside A and 9
additional carbon signals, including two methyl groups (δC 11.9,
17.8), four methylenes (δC 25.4, 27.3, 31.9, 38.4), one methine
(δC 42.5), one ester carbonyl (δC 173.2) and an amide carbonyl
(δC 177.3), corresponding to the partial sequence a (C-1ꢁ –C-4ꢁ),
12
30
20
H
11
H
18
1
H
29
19
O
13
2
3
22
9
OH
21
17
10
26
25
H
28
H
25
CH3
8
H
14
O
5
15
4
16
H
24
HO
7
6
23
Figure 2. Some key NOESY correlations for 1.
was determined by gas chromatography (GC) analysis.[24,25] The
sequence of the disaccharide chain of 1 was determined by HMBC
and NOESY experiments. The correlations observed in the NOESY
spectrum between δH 4.90 (1H, d, J = 5.0 Hz, Ara H-1) and δH
3.16 (1H, dd, J = 4.0, 10.0 Hz, Agly H-3) suggested Ara to be
linked at C-3 of the aglycon. The correlation in the HMBC spec-
trum, between the 1H NMR signal at δH 4.52 (Ara H-2) and the
13C NMR signal at δC 102.0 (Glc C-1), revealed a (1 → 2) linkage
between these two sugars, which was confirmed by the cross-
peak in the NOESY spectrum between δH 4.52 (Ara H-2) and δH
5.28 (Glc H-1). The deshielding observed in the HSQC spectrum
for Glc H-2/C-2 at δH 4.95 (1H, t, J = 8.5 Hz)/δC 81.3 showed that
this position was substituted, but the substituent does not con-
tain carbon atoms. It was suggested that the substituent was a
SO3H group. This was confirmed by FABMS, which presented a
fragment ion at m/z 765 [(M − H) − 80]−. On the basis of the
above results, the structure of compound 1 was elucidated as
13β,28-epoxy-16β,21β-dihydroxyolean-11-en-3β-yl O-β-D-[2-O-
sulfate]-glucopyranosyl-(1 → 2)-α-L-arabinopyranoside, named
glaucaside A.
1
b (NH-C-6ꢁ) and c (C-10ꢁ –C-7ꢁ –C-8ꢁ –C-9ꢁ) (Fig. 1). In the H NMR
spectrum, the signal at δH 8.63 (br s) was attributed to the proton
of the amide function (NH–C O). The connection among partial
sequence a, b and c was deduced from the 2D NMR spectra (COSY,
TOCSY and HMBC) (Fig. 3). The COSY and TOCSY spectra revealed
the presence of two spin systems, namely H-10ꢁ –H-7ꢁ –H-8ꢁ –H-9ꢁ
characteristic of a terminal sec-butyl moiety (c) and H-2ꢁ –H-4ꢁ
characteristic of a butanoyl moiety (a). The HMBC correlations
between δH 3.52 (H-4ꢁ), 2.41 (H-7ꢁ), 1.18 (H-10ꢁ) and δC 177.3 (C-6ꢁ)
establishedthecompletestructureoftheacylgrouptobe21-O-{4-
(secbutylamido)-butanoyl}ester(Fig. 3).Thiswassupportedbythe
observation of a fragment ion at m/z 827 [(M − H) − 185]− in the
FAB mass spectrum. Therefore, the structure of 2 was elucidated
as 13β,28-epoxy-16β,21β-dihydroxyolean-11-en-3β-yl O-β-D-[2-
O-sulfate]-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl 21-O-{4-
(secbutylamido)-butanoyl ester}.
Glaucaside C (3), a white amorphous powder, exhibited in the
HR-ESIMS (positive-ion mode) a pseudomolecular ion peak at
m/z 819.4503 [M + Na]+ (calcd for 819.4507), consistent with
Glaucaside B (2), a yellow amorphous powder, exhibited in
the HR-ESIMS (positive-ion mode) a pseudomolecular ion peak
c
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Copyright ꢀ 2011 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2011, 49, 83–89

Glaucasides A–C, three saikosaponins from Atriplex glauca
O
C
29
30
6'
4'
O
3'
20
28
8'
2'
N
H
19
1'
7'
9'
21
22
H
O
O
18
10'
17
16
13
26
25
H
14
15
1
4
9
OH
8
7
10
2
3
H
27
OH
5
Ara
6
O
O
H
24
23
HO
2
1
O
OH
O
HO
HO
2
OSO3H1
: 1H-1H COSY and TOCSY
: HMBC
Glc
Figure 3. Representative 1H–1H COSY, TOCSY and HMBC correlations for 2.
the molecular formula C42H68O14. Its FABMS (negative-ion mode)
showedaquasi-molecularionpeakatm/z 795[M− H]−, indicating
a molecular weight of 796. Another fragment ion peak was
observed at m/z 634 [(M − H) − 162]− corresponding to the
loss of a one hexosyl moiety. A comparison of the 13C NMR spectra
of the aglycon part of 3 and1 showed the absence of signals due to
the tertiary methyl group at δC 27.6 and to the hydroxyl group at δC
72.3assignabletoC-23andC-21andtheappearanceofsignalsdue
toahydroxymethylgroupat δC 63.7andmethylenicsignalsatC-21
(Table 1). Extensive 2D NMR analysis allowed the identification of
the aglycon part of 3 as 13β,28-epoxy-16α,23-dihydroxyolean-11-
en-3β-yl, named saikogenin G.[20]
The presence of two sugar residues was confirmed from the
observation of two anomeric 1H NMR signals at δH 5.02 (d,
J = 7.6 Hz) and 5.27 (d, J = 7.6 Hz), giving HSQC correlations with
two anomeric 13C NMR signals at δC 104.2 and 106.1, respectively.
Complete assignments of each sugar moiety were achieved by
extensive 1D and 2D NMR analysis, allowing the identification
of one β-glucopyranosyl (Glc) and one β-galactopyranosyl (Gal)
units in 3 (Table 2). The D configuration of Glc and Gal was
determined by GC.[24,25] The sequence of the oligosaccharide
chain of 3 was achieved by HMBC and NOESY experiments. The
HMBC correlations between the anomeric 1H NMR signal at δH 5.27
(Glc H-1) and δC 82.8 (Gal C-2) and between δH 5.02 (d, J = 7.6 Hz,
Gal H-1) and δC 82.5 (Agly C-3) proved the sequence of sugar chain
at C-3 to be β-D-glucopyranosyl-(1 → 2)-β-galactopyranosyl. This
sequence was confirmed by the observation of NOESY cross-peaks
betweenδH 5.27(GlcH-1)andδH 4.60(GalH-2)andbetweenδH 5.02
(Gal H-1) and δH 4.14 (Agly H-3). Thus, the structure of glaucaside
C (3) was concluded to be 3-O-β-D-glucopyranosyl-(1 → 2)-β-D-
galactopyranosyl saikogenin G, a 16-epimer of 4.[18]
with an IC50 of 24.34 and 27.23 µM on HT-29 and HCT 116 cells,
respectively, while compounds 1 and 2 were considered inactive
with IC50 > 100 µ M.
Experimental
General
Optical rotations were recorded on an AA-OR automatic po-
larimeter. HR-ESIMS (positive-ion mode) was obtained on a
Q-TOF1-micromassspectrometerandFABMS(negative-ionmode)
was performed on a JEOL-SX-102 instrument. GC analysis was car-
ried out on a Termoquest gas chromatograph using a DB-1701
capillarycolumn(30 m×0.25 mm, i.d.)(J&WScientific);detection,
FID; detector temperature, 250 ◦C; injection temperature, 230 ◦C;
initial temperature was maintained at 80 ◦C for 5 min and then
raised to 270 ◦C at the rate of 15 ◦C/min; carrier gas, He. Isolations
were carried out using a medium-pressure liquid chromatog-
raphy (MPLC) system [Gilson M 305 pump, 25 SC head pump,
M 805 manometric module, Bu¨chi glass column (460 × 25 mm
and 460 × 15 mm), Bu¨chi precolumn (110 × 15 mm)] on silica
gel 60 (Merck, 15–40 µm) and reversed-phase RP-18 silica gel
(Silicycle 75–200 µm), and with a Gilson Pump Manager C-605
having two pumps (2× Bu¨chi pump modul C-601) and one frac-
tion collector (Bu¨chi C-660). Vacuum-liquid chromatography (VLC)
was performed on a reversed-phase RP-18 silica gel (Silicycle,
75–200 µm) (12 × 6 cm). Thin-layer chromatography (TLC, Sili-
cycle) and high-performance thin-layer chromatography (HPTLC,
Merck) were performed on precoated silica gel plates 60 F254. The
spray reagent for saponins was the Komarowsky reagent, which
is a mixture (5 : 1) of p-hydroxybenzaldehyde (2% in MeOH) and
ethanolic H2SO4 (50%).
The cytotoxicity of 1–4, was evaluated against the HT-29 and
HCT 116 human colon cancer cell lines by the MTT assay,[26] using
paclitaxel as the positive control. Among them, the most active
compound on both cell lines was the known saikosaponin 3-
O-β-D-glucopyranosyl-(1 → 2)-β-D-galactopyranosyl saikogenin
F (4) with an IC50 of 7.08 and 9.47 µM on HT-29 and HCT 116
cells, respectively. Compound 3 exhibited a moderate cytotoxicity
NMR spectroscopy
The NMR spectra were recorded on a Varian UNITY Inova 600
spectrometer equipped with 5-mm probes. Samples were dis-
solved in C5D5N and transferred into 5-mm NMR tubes (Shigemi).
c
Magn. Reson. Chem. 2011, 49, 83–89
Copyright ꢀ 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

A. Jabrane et al.
1
The H and 13C NMR spectra (at 600 and 150 MHz, respectively)
(positive-ion mode) m/z 1038.5068 [M + Na]+ (calcd for
1038.5072); FABMS (negative mode) m/z 1014 [M − H]−, 827 [(M
− H) − 185]−, 97 [OSO3H]−, 80 [SO3]−.
were measured at 303 K. Chemical shifts are given on the δ scale
and referenced to the residual solvent signals (δH = 7.19, 7.59,
8.50; δC = 123.5, 135.9, 150.3). Coupling constants (J) are in Hz.
For 2D experiments, Varian software using pulse field gradient
was applied. The pulse conditions in C5D5N were as follows: for
the 1H NMR spectrum, observation frequency (OF) = 599.88 MHz,
acquisition time (AQ) = 4.199 s, relaxation delay (RD) = 5.0 s, 90
pulse width = 10.0 µs, spectral width (SW) = 7804.1 Hz, Fourier
transform (FT) size = 65 536; for the 13C NMR spectrum, OF =
150.854 MHz, AQ = 0.453 s, RD = 1.547 s, 90 pulse = 15.8 µs, SW
= 36 182.7 Hz, line broadening (LB) = 1.0 Hz, FT size = 65 536; for
the COSY spectrum, AQ = 0.131, F2 = 2048, F1 = 256, RD = 0.369,
SW = 7798.8 Hz; for the NOESY spectrum, AQ = 0.131, F2 = 2048,
F1 = 256, RD = 0.369, SW = 7804.1 Hz, mixing time = 500 ms;
for the TOCSY spectrum, AQ = 0.131, F2 = 2048, F1 = 256, RD =
0.369, SW = 7804.1 Hz, mixing time = 60 ms; for the HSQC spec-
trum, AQ = 0.131, RD = 0.369, F1 = 36 182.7 Hz, F1 = 7804.1 Hz
and for the HMBC spectrum, AQ = 0.131, SF = 599.880 MHz, RD
= 0.369, DE = 50 ms, F1 = 36 182.7 Hz, F1 = 7804.1 Hz.
Glaucaside C (3): white amorphous powder; [α]21 +31.5
D
(c 0.20, MeOH); 1H NMR (pyridine-d5, 600 MHz) and 13C NMR
(pyridine-d5, 150 MHz) see Tables 1 and 2; HR-ESIMS (positive-ion
mode) m/z 819.4503 [M + Na]+ (calcd for 819.4507); FABMS
(negative mode) m/z 795 [M − H]−, 634 [(M − H) − 162]−.
Acid hydrolysis and GC analysis
Each compound (2 mg) was hydrolyzed with 2 N aq. CF3COOH
(5 ml) for 3 h at 95 ◦C. After extraction with CH2Cl2 (3 × 5 ml),
the aqueous layer was repeatedly evaporated to dryness with
MeOH until neutral and then analyzed by TLC over silica
gel (CHCl3 –MeOH–H2O, 8 : 5 : 1) by comparing with authen-
tic samples. Furthermore, the residue of sugars was dissolved
in anhydrous pyridine (100 µl) and L-cysteine methyl ester hy-
drochloride (0.06 mol/l) was added. The mixture was stirred
at 60 ◦C for 1 h, then 150 µl of HMDS–TMCS (hexmethyldisi-
lazane–timethylchlorosilane 3 : 1) was added and the mixture was
stirred at 60 ◦C for another 30 min. The precipitate was centrifuged
off and the supernatant was concentrated under an N2 stream. The
residue was partitioned between n-hexane and H2O (0.1 ml each),
and the hexane layer (1 µl) was analyzed by GC.[24,25] D-glucose,
D-galactose and L-arabinose were detected by co-injection of
the hydrolyzate with standard silylated samples. Identification
of D-glucose and L-arabinose was carried out for 1 and 2 giv-
ing peaks at 18.33 and 12.00 min, respectively. Identification of
D-glucose and D-galactose was carried out giving peaks at 18.31
and 15.80 min, respectively for 3 and 4.
Plant material
The roots of A. glauca was collected around salt marshes near
Monastir, located in the east Mediterranean coast of Tunisia in
June 2008 and identified by Professor Fe´thia Harzallah-Skhiri,
botanist of the Higher Institute of Biotechnology of Monastir
(ISBM), where a voucher specimen (AP 18-6-08) was deposited.
Extraction and isolation
Fresh roots (1.2 kg) were refluxed two times with MeOH–H2O
(5 : 5, 3 l × 2) for 3 h and evaporated in vacuo yielding an aqueous
extract that was partitioned successively with CHCl3 (3 l) and
H2O-satd n-BuOH (2 l), yielding after evaporation of the solvents
the corresponding green CHCl3 extract (15.4 g) and a red n-BuOH
extract (12.6 g). A 9-g aliquot of the n-BuOH residue was submitted
to VLC on RP-18 silica gel using H2O (1000 ml), MeOH–H2O
mixtures(5 : 5,1250 ml)andfinallyMeOH(1000 ml)aseluents.After
evaporation of the solvents, three fractions were obtained: API
(2.35 g), APII (2.18 g) and APIII (1.80 g). APIII (1.30 g) was submitted
to VLC on normal silica gel using a mixture CHCl3 –MeOH (9 : 1 to
0 : 10) affording 11 fractions. F-1 (13 mg), F-2 (62 mg), F-3 (240 mg),
F-4 (110 mg), F-5 (43 mg), F-6 (67 mg), F-7 (67 mg), F-8 (33 mg), F-9
(69 mg), F-10 (50 mg) and F-11 (9 mg). F-3 (240 mg) was purified by
CC on Sephadex LH-20 eluted with MeOH and then by successive
MPLC on RP-18 silica gel eluted with MeOH–H2O (5 : 5 to 3.5 : 7.5),
yielding 3 (24 mg) and 4 (15 mg). APII (800 mg) was submitted
to VLC on normal silica gel using a mixture CHCl3 –MeOH–H2O
(6 : 3 : 1) to give eight fractions: Fꢁ-1 (17 mg), Fꢁ-2 (165 mg), Fꢁ-3
(169 mg), Fꢁ-4 (35 mg), Fꢁ-5 (30 mg), Fꢁ-6 (120 mg), Fꢁ-7 (48 mg) and
Fꢁ-8 (31 mg). Fꢁ-4 (35 mg) was purified by MPLC on RP-18 silica gel
eluted with MeOH–H2O (6 : 4 to 3 : 7) to give 1 (5 mg) and 2 (5 mg).
MTT cytotoxicity assay
The bioassay was carried out according to the method described
by Carmichael et al.[26] with two human colon cancer cell lines
(HCT 116 and HT-29). Paclitaxel was used as a positive control and
exhibited IC50 values of 1.60 and 2.05 nM against HT-29 and HCT
116, respectively.
Acknowledgement
The authors are thankful to IFC-Tunisie (Institut Franc¸ais de
Coope´ration en Tunisie) for a scholarship to A. J.
Supporting information
Supporting information may be found in the online version of this
article.
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