Unusual fernane and gammacerane glycosides from the aerial parts of Spergula fallax

Article abstract of DOI:10.1021/np4008415

The aerial parts of Spergula fallax afforded four glycosides (1-4) based on two new triterpene aglycones (1a and 2a), along with the known hopane glycoside succulentoside A. Compound 1 was identified as belonging to the fernane class, unusual migrated hopane triterpenoids, mainly isolated from ferns and only rarely from higher plants. Compounds 2-4 were assigned as gammacerane glycosides, having as aglycone a hydroxylated derivative of tetrahymanol. The structures of the isolated compounds 1-4 and their aglycones 1a and 2a obtained by acid hydrolysis were elucidated by spectroscopic data interpretation. The growth inhibitory activity of the isolated compounds and their aglycones was evaluated against the HeLa and DLD-1 cancer cell lines.

Full text of DOI:10.1021/np4008415

Article
pubs.acs.org/jnp
Unusual Fernane and Gammacerane Glycosides from the Aerial Parts
of Spergula fallax
Arafa I. Hamed,†,⊥ Milena Masullo,‡,⊥ Lukasz Pecio,§ Dario Gallotta,‡ Usama A. Mahalel,†
Sylwia Pawelec,§ Anna Stochmal,§ and Sonia Piacente*,‡
†Department of Botany, Faculty of Science, Aswan University, Aswan 81528, Egypt
‡Dipartimento di Farmacia, Universita
̀
degli Studi di Salerno, Via Giovanni Paolo II n. 132, 84084 Fisciano, Salerno, Italy
§Department of Biochemistry and Crop Quality, Institute of Plant Cultivation and Environmental Science, ul. Czartoryskich 8, 24-100
Pulawy, Poland
S
* Supporting Information
ABSTRACT: The aerial parts of Spergula fallax afforded four glycosides (1−4) based
on two new triterpene aglycones (1a and 2a), along with the known hopane glycoside
succulentoside A. Compound 1 was identified as belonging to the fernane class,
unusual migrated hopane triterpenoids, mainly isolated from ferns and only rarely from
higher plants. Compounds 2−4 were assigned as gammacerane glycosides, having as
aglycone a hydroxylated derivative of tetrahymanol. The structures of the isolated
compounds 1−4 and their aglycones 1a and 2a obtained by acid hydrolysis were
elucidated by spectroscopic data interpretation. The growth inhibitory activity of the isolated compounds and their aglycones was
evaluated against the HeLa and DLD-1 cancer cell lines.
s part of an ongoing investigations of wild medicinal plants
growing in the Egyptian desert, a phytochemical study of
peaks at m/z 835 [M + Na − 42]+, ascertaining the presence of
an acetyl moiety, at m/z 673 [M + Na − 42 − 162]+, and at m/
z 541 [M + Na − 42 − 162 − 132]+, ascribable to the loss of
both a hexose and a pentose moiety.
A
Spergula fallax L. was carried out. The genus Spergula
(Caryophyllaceae) comprises 14 species distributed in temper-
ate regions (Central India, North Africa, Canary Islands, and
South America).1−3 A previous phytochemical investigation on
the Peruvian Spergularia (syn. Spergula) ramosa led to the
isolation of oleanane glycosides.1
The 13C NMR spectrum of 1 showed 43 carbon signals, of
which 30 were due to the aglycone moiety and 11 to a
saccharide portion made up of two sugar units (Tables 1 and
1
2). The H NMR spectrum displayed signals for six tertiary
Spergula fallax (syn. Spergularia flaccida and Spergularia
fallax), is an annual herb distributed in the Egyptian desert,
known by the Arabic name “Gileglaag”.4,5 The aerial parts of
the plant are used as a diuretic, while the seeds are usually dried
and ground into a meal and then used with flour for making
bread.6
From the aerial parts of S. fallax four glycosides with new
aglycones (1−4), along with the known hopane glycoside
succulentoside A,7 were isolated. Compound 1 was identified as
a fernane derivative. Fernanes are unusual triterpenoids
belonging to the migrated hopane class and mainly isolated
previously from ferns.8 Compounds 2−4 were assigned as
gammacerane glycosides, possessing the same aglycone, a new
hydroxylated derivative of tetrahymanol.9
methyl groups at δ 0.87, 0.95, 1.07, 1.11, 1.17, and 1.34, a
secondary methyl at δ 1.30 (d, J = 8.0 Hz), an olefinic proton at
δ 5.83 (s), four oxygen-bearing methine protons at δ 3.09 (d, J
= 10.8 Hz), 3.68 (m), 4.31 (dd, J = 11.0, 2.0 Hz), and 4.80 (dd,
J = 7.0, 2.0 Hz), and one methyl signal at δ 2.16 (s), ascribable
to an acetyl group (Tables 1 and 2). In the 13C NMR spectrum,
signals for methyl groups at δ 15.2, 16.9, 17.4, 17.6, 22.0, 22.4,
and 27.9, two olefinic carbons at δ 124.0 and 171.7, two
carboxyl carbons at δ 171.5 and 182.6, and a carbonyl group at
δ 201.3 suggested the occurrence of a triterpene derivative. The
structural units of rings A and B were established by HMBC
correlations between the methyl signal at δ 1.07 (Me-24) and
1.34 (Me-23) with the carbon resonances at δ 94.0 (C-3) and
65.4 (C-5). A COSY correlation between the proton
resonances at δ 3.09 (H-3) and 3.68 (H-2) revealed the
presence of two hydroxy groups at C-2 and C-3, oriented as α
and β, respectively, as deduced by the coupling constant of H-3
(d, J = 10.8 Hz) and by the ROESY correlations between H-2
and Me-25 and from H-3 to H-5 and Me-23. The HMBC
RESULTS AND DISCUSSION
■
The structures of 1−4 were elucidated by spectroscopic
methods including 1D- (1H, 13C, and TOCSY) and 2D-NMR
(DQF-COSY, HSQC, HMBC, and ROESY) experiments as
well as ESIMS analysis.
The HRMALDITOFMS of 1 (m/z 877.4192 [M + Na]+,
calcd for C43H66O17Na, 877.4198) supported a molecular
formula of C43H66O17. The ESIMS showed a major ion peak at
m/z 877 [M + Na]+. Analysis of the ESIMS2 data of 1 showed
Special Issue: Special Issue in Honor of Otto Sticher
Received: October 8, 2013
Published: February 14, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
657
dx.doi.org/10.1021/np4008415 | J. Nat. Prod. 2014, 77, 657−662

Journal of Natural Products
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experiment, which showed a correlation between H-19 (δ 4.31)
and Me-27 (δ 1.11). The ROESY correlation between H-20α
(δ 4.80) and the methyl doublet at δ 1.30, along with the
absence of ROESY correlations between this methyl doublet
and H2-16 (δ 1.61), supported the presence of this methyl
group at Me-30.8 On the basis of this evidence, the aglycone of
1 was established as 2α,3β,19β,20β-tetrahydroxyfern-7-en-6-
oxo-29-oic acid.
1
The H NMR spectrum in the saccharide portion of 1
exhibited signals ascribable to two anomeric protons at δ 4.54
(d, J = 5.2 Hz) and 4.83 (d, J = 8.0 Hz), which correlated in the
HSQC spectrum to the corresponding carbon resonances at δ
103.4 and 103.0. The NMR data (HSQC, HMBC, COSY, 1D-
TOCSY) indicated the presence of α-arabinopyranosyl (δ 4.54)
and β-glucopyranosyl (δ 4.83) units. The configurations of the
arabinose and glucose units were established as L and D,
respectively, after hydrolysis of 1 with 1 N HCl, trimethylsi-
lation, and GC analysis.11 An unambiguous determination of
the linkage site was obtained from the HMBC spectrum, which
showed key correlation peaks between the proton signal of H-
1′ (δ 4.54) and the 13C NMR resonance of C-3 (δ 94.0) and
between the proton signal of H-1″ (δ 4.83) and the 13C NMR
resonance of C-4′ (δ 77.2). Moreover, the downfield shift
observed for H-2′ (δ 4.91), indicative of an esterification of the
hydroxy group (Table 2), was observed. In the HMBC
spectrum, correlations between the proton signal at δ 4.91
(H-2′) and the carbon resonance at δ 171.5 and between the
proton signal at δ 2.16 and the same carbon revealed the
presence of the acetyl group at C-2′. Therefore, compound 1
was determined as 3-O-[β-D-glucopyranoside-(1→4)-O-α-L-(2-
O-acetyl)-arabinopyranosyl]-2α,3β,19β,20β-tetrahydroxyfern-7-
en-6-oxo-29-oic acid.
correlations between the olefinic proton at δ 5.83 (s) (H-7) and
the carbon resonances at δ 65.4 (C-5) and 201.3 (C-6) allowed
the double bond to be located at C-7 and the carbonyl group at
C-6. Analysis of NMR data (HSQC, HMBC, COSY, and 1D-
TOCSY) suggested that compound 1 is a triterpene derivative,
belonging to either the arborane or fernane class.9 The
differences between arborane and fernane triterpenoids are
the opposite configurations at C-8, C-13, C-14, C-17, C-18, and
C-21. The relationship between Me-25 and H-8 is syn-1,3-
diaxial in arboranes and anti-1,3-diaxial in fernanes.10 The two
skeletons can be distinguished based on a ROESY correlation
between H-8 and Me-25, which is diagnostic for arborane
derivatives, while not expected for fernane derivatives.
In the absence of a H-8 resonance, to establish the relative
configuration of the triterpene skeleton of 1, the ROESY
correlations starting from H-9 were investigated. The
correlations observed between H-9 (δ 3.09) and Me-27 (δ
1.11), as well as from H-18 (δ 1.80) to Me-26 (δ 1.17) and H-
21 (δ 2.26), together with the absence of ROESY correlations
between H-9 and Me-25 (δ 0.95) and from Me-28 (δ 0.87) to
H-18 and H-21, indicated that 1 is a fernane-type triterpenoid.
The long-range correlation between the secondary methyl
proton at δ 1.30 (Me-30) and the carboxylic group at δ 182.6
revealed that one of the methyl groups of the isopropyl
function connected to C-21 in a fernane-type skeleton is
substituted by a carboxylic group. Moreover, the further
secondary hydroxy group functions at δ 4.31 and 4.80 could
be located at C-19 and C-20, respectively, on the basis of COSY
correlations between the proton signal at δ 4.31 (H-19, dd, J =
11.0, 2.0 Hz) and the proton resonance at δ 4.80 (H-20, dd, J =
7.0, 2.0 Hz), which, in turn, correlated with a methine signal at
δ 2.26 (H-21, d, J = 7.0 Hz). The β-orientation of the OH
groups at C-19 and C-20 was deduced from the coupling
constants of the protons of ring E and confirmed by a ROESY
The HRMALDITOFMS of 2 (m/z 647.4142 [M + Na]+,
calcd for C35H60O9Na, 647.4135) supported a molecular
formula of C35H60O9. The ESIMS showed a major ion peak
at m/z 647, which was assigned to [M + Na]+. The MS/MS
spectrum of this ion showed a peak at m/z 515 [M + Na −
132]+, corresponding to the loss of a pentose unit.
The 13C NMR spectrum of 2 showed 35 carbon signals, of
which 30 were assigned to the aglycone moiety and five to a
sugar unit (Tables 1 and 2). The 1H NMR spectrum displayed
signals for eight tertiary methyl groups at δ 0.93, 0.98, 1.02,
1.05, 1.12, 1.13, 1.31, and 1.40 and five oxygen-bearing methine
protons at δ 2.88 (d, J = 9.7 Hz), 3.12 (dd, J = 11.6, 4.4 Hz),
3.62 (m), 4.00 (m), and 4.02 (ddd, J = 10.8, 9.5, 4.5 Hz) (Table
1). These signals, along with the carbon resonances in the 13C
NMR spectrum for the methyl groups at δ 15.6, 17.0, 17.2, 18.2
(3C), 31.2, and 31.7, suggested the presence of a triterpene
derivative. In the HSQC spectrum, four methine correlations at
δH 0.89/δC 61.3 (H-5), δH 1.19/δC 60.1 (H-17), δH 1.30/δC
50.3 (H-9), and δH 1.35/δC 50.4 (H-13) were observed. The
chemical shifts of the A, B, and C rings were similar to those
reported for an oleanane derivative,12 but differences for the
13C NMR resonances of the D and E rings were observed. In
particular, in the HMBC spectrum, correlations between the
methyl signal at δ 1.13 (Me-27) and the carbon resonance at δ
50.4 allowed a methine function to be assigned to C-13. A
further correlation between the methyl signal at δ 1.02 and the
latter 13C NMR resonance allowed the tertiary methyl group to
be located at C-18 (40.9). A long-range correlation between the
methyl signal at δ 1.02 and the methine carbon at δ 60.1
permitted the location of a methine function at C-17. The
remaining methyl groups at δ 1.05 (Me-29) and 1.40 (Me-30)
658
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1
Table 1. 13C and H NMR Data (J in Hz) of the Aglycone Moieties of Compounds 1, 1a, 2, 2a, and 3 (600 Mz, δ ppm, in
a
CD3OD)
1
1a
2
2a
3
4
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
44.4
2.00 dd (12.5,
4.2), 1.39, t
(12.5)
45.5
1.95 dd (12.5,
4.2), 1.41, t
(12.5)
39.9 1.72, 1.00, m
39.7 1.71, 1.00, m
39.9 1.72, 1.00, m
39.8 1.72, 1.00, m
2
3
66.3
94.0
3.68, m
68.5
83.8
3.65, m
27.5 1.69, 1.61, m
27.4 1.64, 1.60, m
27.5 1.69, 1.61, m
27.5 1.70, 1.61, m
3.09, d (10.8)
2.97, d (10.8)
79.4 3.12, dd
(11.6, 4.4)
79.5 3.12, dd
(11.6, 4.4)
79.7 3.13, dd (11.6,
4.4)
79.8 3.12, dd (11.6,
4.4)
4
5
6
40.1
65.4
201.3
39.9
66.3
202.5
40.0
40.2
40.5
40.5
2.45, s
2.40, s
61.3 0.89, d (10.8)
61.5 0.88, d (10.8)
61.6 0.89, d (10.8)
61.4 0.89, d (10.8)
68.9 4.02, ddd
(10.8, 9.5,
4.5)
68.9 4.00, ddd
(10.8, 9.5,
4.5)
68.9 4.01, ddd (10.8, 68.8 4.00, ddd (10.8,
9.5, 4.5)
9.5, 4.5)
7
124.0 5.83, s
171.7
124.3 5.83, s
172.0
45.8 1.58 (2H), m
43.5
45.5 1.59 (2H), m
43.5
45.8 1.58 (2H), m
43.1
45.7 1.58 (2H), m
43.1
8
9
50.0
45.2
16.6
32.2
36.5
44.2
29.6
3.09, m
50.3
45.7
16.4
32.5
37.2
44.6
30.0
3.10, m
50.3 1.30, m
50.0
50.4 1.32, m
50.2
50.7 1.31, m
50.4
50.7 1.31, m
50.1
10
11
12
13
14
15
1.85, 1.61, m
1.89, 1.79, m
1.87, 1.63, m
1.90, 1.80, m
22.2 1.65, 1.35, m
22.3 1.66, 1.34, m
50.4 1.35, m
43.1
22.0 1.65, 1.35, m
23.4 1.66, 1.34, m
50.4 1.35, m
43.1
22.2 1.70, 1.36, m
22.1 1.64, 1.34, m
50.1 1.37, m
43.6
22.4 1.70, 1.37, m
22.1 1.65, 1.34, m
50.1 1.37, m
43.6
1.72, 1.64, m
1.61 (2H), m
1.73, 1.65, m
1.62 (2H), m
44.1 2.00, 1.64, m
37.4 1.67, 1.32, m
44.1 2.01, dd (12.7,
3.5), 1.64, m
43.9 2.04, dd (12.7,
3.5), 1.65, m
16
17
18
19
34.9
42.1
57.0
77.2
35.2
42.4
57.1
77.5
80.8 4.00, m
60.1 1.19, d (10.8)
40.9
69.3 4.00, m
61.3 0.98, d (10.8)
40.2
80.5 4.03, m
60.5 1.21, d (10.8)
41.2
80.5 4.02, m
60.0 1.21, d (10.8)
41.2
1.80, d (11.0)
1.79, d (11.0)
4.31, dd (11.0,
2.0)
4.31, dd (11.0,
2.0)
47.9 2.00, 0.90, m
47.7 1.98, 0.90, m
45.6 2.17, dd (4.4,
13.0), 1.00, m
45.6 2.17, dd (4.4,
13.0) 1.00, m
20
92.8
4.80, dd (7.0, 2.0)
93.3
4.78, dd (7.0, 2.0)
68.7 3.62, m
69.2 3.63, m
78.6 3.76, td (4.4,
9.7, 11.5)
78.6 3.76, td (4.4,
9.7, 11.5)
21
22
23
24
25
26
27
28
29
30
57.6
36.9
27.9
16.9
15.2
22.0
22.4
17.6
182.6
17.4
2.26, d (7.0)
2.63, d (8.0)
1.34, s
58.5
37.4
29.1
16.5
15.4
22.5
22.6
17.3
183.3
17.4
2.25, d (7.0)
2.63, d (8.0)
1.35, s
84.6 2.88, d (9.7)
40.8
84.5 2.88, d (9.7)
41.0
82.2 3.05, d (9.7)
41.1
82.2 3.05, d (9.7)
41.0
31.2 1.31, s
15.6 0.98, s
17.2 0.93, s
18.2 1.12, s
18.2 1.13, s
18.2 1.02, s
17.0 1.05, s
31.7 1.40, s
31.8 1.31, s
16.1 0.98, s
17.0 0.93, s
18.2 1.13, s
18.2 1.13, s
18.3 0.99, s
17.0 1.02, s
31.8 1.35, s
31.8 1.31, s
15.5 0.98, s
17.4 0.93, s
18.3 1.12, s
18.5 1.14, s
18.2 1.01, s
17.5 1.08, s
32.1 1.43, s
31.6 1.31, s
15.4 0.98, s
17.3 0.93, s
18.3 1.12, s
18.4 1.13, s
18.2 1.01, s
17.6 1.08, s
32.3 1.44, s
1.07, s
1.12, s
0.95, s
0.95, s
1.17, s
1.19, s
1.11, s
1.12, s
0.87, s
0.88, s
1.30, d (8.0)
1.31, d (8.0)
a
The chemical shift values of the aglycone moiety of 4 were superimposable on those of 3.
1
Table 2. 13C and H NMR Data (J in Hz) of the Sugar Portions of Compounds 1−4 (600 Mz, δ ppm, in CD3OD)
1
2
3
4
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
α-Ara (at C-3)
103.4 4.54, d (5.2)
α-Ara (at C-16)
105.6 4.38, d (5.2)
α-Ara (at C-16)
105.8 4.38, d (5.2)
α-Ara (at C-16)
105.8 4.38, d (5.2)
1′
2′
3′
4′
5′
74.5
73.7
77.2
64.5
4.91, dd (8.0, 5.2)
72.2
74.3
68.7
3.64, dd (8.0, 5.2)
72.2
74.4
68.8
3.64, dd (8.0, 5.2)
72.8
72.9
72.5
3.58, dd (8.0, 5.2)
3.70, dd (8.0, 3.0)
3.88, m
3.56, dd (8.0, 3.0)
3.84, m
3.56, dd (8.0, 3.0)
3.84, m
3.69, dd (8.0, 3.0)
5.02, m
4.18, dd (12.5, 3.0), 3.43, 65.6
dd (12.5, 2.6)
3.83, dd (12.5, 3.0), 3.52, 65.7
dd (12.5, 2.6)
3.83, dd (12.5, 3.0), 3.52, 64.0
dd (12.5, 2.6)
3.88, dd (12.5, 3.0), 3.61,
dd (12.5, 2.6)
COCH3
COCH3
171.5
21.0
172.2
2.16, s
20.7
2.12, s
β-Glc (at C-4Ara
103.0 4.83, d (8.0)
)
β-Glc (at C-20)
β-Glc (at C-20)
1″
2″
3″
4″
5″
6″
102.4 4.35, d (7.9)
102.4 4.35, d (7.9)
74.1
77.5
71.1
77.5
64.5
3.24, dd (9.0, 8.0)
3.38, dd (9.0, 9.0)
3.29, dd (9.0, 9.0)
3.38, m
74.6
77.5
71.4
77.5
62.5
3.21, dd (9.0, 7.9)
3.38, dd (9.0, 9.0)
3.31, dd (9.0, 9.0)
3.32, m
74.7
77.5
71.3
77.7
3.21, dd (9.0, 7.9)
3.38, dd (9.0, 9.0)
3.31, dd (9.0, 9.0)
3.31, m
3.91, dd (12.0, 2.5), 3.66,
dd (12.0, 4.5)
3.89, dd (11.0, 2.2), 3.68, 62.5
dd (11.0, 5.0)
3.88, dd (11.0, 2.2), 3.67,
dd (11.0, 5.0)
659
dx.doi.org/10.1021/np4008415 | J. Nat. Prod. 2014, 77, 657−662

Journal of Natural Products
Article
were located at C-22 on the basis of the HMBC correlations
between their proton resonances (δ 1.05 and 1.40) and the
carbon resonance at δ 60.1 (C-17). Thus, compound 2 was
identified as a gammacerane derivative.9 The relative
configuration was determined from the ROESY spectrum.
The usual trans junction of rings A and B was assumed with H-
5α and Me-25β. A ROESY correlation between Me-25 and Me-
26 showed that these two methyl groups are on the same side
(β) of the main plane of the molecule, whereas correlations of
the methine protons H-5 and H-9 with Me-27 showed that
these are all on the opposite side (α) of the molecule. The D
and E ring junctions were established as trans, as determined by
the presence of ROESY correlations between Me-28 (δ 1.02)
and Me-27 (δ 1.13), Hβ-17 (δ 1.19), and Hβ-13 (δ 1.35). This
relative configuration reported for compound 2 was in
agreement with that reported for other gammacerane
derivatives.9 HMBC experiments allowed a secondary hydroxy
group function to be located at C-3 on the basis of the
correlations between the methyl groups at δ 0.98 (Me-24) and
1.31 (Me-23) and the 13C NMR resonance at δ 79.4. Thus, the
aglycone of 2 was identified as a tetrahymanol derivative.9,13
The COSY correlation between the proton resonances at δ 0.89
(H-5) and 4.02 permitted a hydroxy group to be assigned at C-
6. The coupling constant of H-6 (ddd, J = 10.8, 9.5, 4.5 Hz)
suggested an α-orientation of this hydroxy group,14 as
confirmed by ROESY correlations between the proton
resonances at δ 4.02 (H-6) and the methyl signals at δ 0.93
(Me-25) and 0.98 (Me-24). The COSY correlation between
the oxygen-bearing methine proton at δ 4.00 (m) and the
methine proton at δ 1.19 (H-17) allowed a hydroxy group to be
proposed at C-16, with an α-orientation, as deduced by the
coupling constant of H-17 (d, J = 10.8 Hz) and by the ROESY
correlations between the proton resonances at δ 4.00 (H-16)
and 1.40 (Me-30). A further secondary hydroxy group was
assigned to C-21 on the basis of the HMBC correlations of the
methyl protons at δ 1.05 (Me-29) and 1.40 (Me-30) with the
carbon resonance at δ 84.6. In a COSY experiment the
corresponding proton at δ 2.88 (H-21) correlated with the
proton at δ 3.62 (H-20), allowing a final aglycone secondary
hydroxy group to be located at C-20. The coupling constant of
H-21 (d, J = 9.7 Hz) revealed a trans-diaxial orientation with
the proton at δ 3.62 (H-20), and ROESY correlations between
the proton at δ 2.88 (H-21) and the methyl signal at δ 1.40
(Me-30) and the protons at δ 3.62 (H-20) and the methyl
signal at δ 1.05 (Me-29) allowed the proposal of a β- and an α-
orientation for the hydroxy groups at C-20 and C-21,
respectively. On the basis of the above information, the
aglycone of 2 was determined as 6α,16α,20β,21α-tetrahydrox-
ytetrahymanol.
C41H70O14Na, 809.4663). A [M + Na]+ ion peak at m/z 809
could be observed in the ESIMS of 3, and the MS/MS of this
ion showed peaks at m/z 647 [M + Na − 162]+ and at m/z 515
[M + Na − 162 − 132]+, corresponding to the loss of a hexose
1
and a pentose unit, respectively. The H and 13C NMR signals
of the aglycone of 3 assigned from the 2D-NMR spectra were
almost superimposable on those of 2, except for the downfield
1
shift of H-20 (δH 3.76/δC 78.6) (Table 1). The H NMR
spectrum of the sugar region, in comparison with that of 2,
displayed an additional anomeric signal at δ 4.35 (d, J = 7.9
Hz), which was assigned unambiguously from HSQC, COSY,
HMBC, and 1D-TOCSY experiments to a β-glucopyranosyl
unit (Table 2). The D configuration of the glucose unit and the
L configuration of the arabinose unit were established after
hydrolysis of 3 with 1 N HCl, trimethylsilation, and GC
analysis.11 The linkage site of the β-glucopyranosyl unit was
obtained from the HMBC spectrum, which showed a key
correlation peak between the proton signal of H-1″ (δ 4.35)
and the carbon resonance of C-20 (δ 78.6). Therefore, the
structure of compound 3 was determined as 16-O-α-L-
arabinopyranosyl-20-O-β-D-glucopyranosyl-6α,16α,20β,21α-tet-
rahydroxytetrahymanol.
The HRMALDITOFMS of 4 (m/z 851.4774 [M + Na]+,
calcd for C43H72O15Na, 851.4769) supported a molecular
formula of C43H72O15. The ESIMS showed a major ion peak at
m/z 851 [M + Na]+. The MS/MS of this ion showed peaks at
m/z 809 [M + Na − 42]+, corresponding to the loss of an
acetyl unit, at m/z 647 [M + Na − 42 − 162]+, corresponding
to the loss of a hexose unit, and at m/z 515 [M + Na − 42 −
1
162 − 132]+, due to further loss of a pentose unit. The H
NMR spectrum of 4 was almost superimposable on that of 3,
except for the presence of a signal at δ 2.12, corresponding to
an acetyl function. The NMR data of the sugar moieties of 4
were superimposable on those of 3 except for the downfield
shift observed for H-4′ (δ 5.02) indicative of the esterification
of the hydroxy group (Table 2). In the HMBC spectrum,
correlations between the proton signal at δ 5.02 (H-4′) and the
carbon resonance at δ 172.2 and between the proton signal at δ
2.12 and the same carbon revealed the presence of an acetyl
group at C-4′. Therefore, compound 4 was determined as 16-
O-(4-O-acetyl)-α-L-arabinopyranosyl-20-O-β-D-glucopyranosyl-
6α,16α,20β,21α-tetrahydroxytetrahymanol.
Additionally, succulentoside A was isolated and was
1
identified by comparison of its H and 13C NMR data with
values reported in the literature.7 Since the aglycones of 1 and
2−4 have not been reported before, compounds 1 and 2 were
submitted to acid hydrolysis to afford the corresponding
aglycones, 1a and 2a, which were characterized by NMR
spectroscopy (Table 1) and ESIMS analysis.
1
The H NMR spectrum of 2 displayed in the saccharide
region an anomeric proton at δ 4.38 (d, J = 5.2 Hz), which was
correlated by a HSQC experiment to the corresponding carbon
resonance at δ 105.6. The 2D-NMR data showed the presence
of an α-arabinopyranosyl unit (Table 2) that could be located
at C-16 on the basis of the HMBC correlation between the
proton signal of H-1′ (δ 4.38) and the carbon resonance of C-
16 (δ 80.8). The L configuration of the arabinose unit was
established after hydrolysis of 2 with 1 N HCl, trimethylsilation,
and analysis by GC.11 Hence, the structure of compound 2 was
proposed as 16-O-α-L-arabinopyranosyl-6α,16α,20β,21α-tetra-
hydroxytetrahymanol.
Due to their scarce occurrence in Nature, very little is known
on the biological activity of fernane and gammacerane
derivatives. Antitumor-promoting activity has been reported
for fernanes and gammaceranes isolated from ferns,15 and
inhibitory effects on DNA topoisomerases in vitro have been
reported for fernane-type triterpenoids isolated from the genus
Euphorbia.16 Therefore, the growth inhibitory effects of
compounds 1−4, succulentoside A, and the aglycones 1a and
2a were tested against the HeLa (human epitheloid cervix
carcinoma) and DLD-1 (colorectal adenocarcinoma) cancer
cell lines. Only compound 4 showed weak activity, with IC50
value of 52 μM in both HeLa cells and DLD-1 cells.
The molecular formula of 3 was established as C41H70O14 by
HRMALDITOFMS (m/z 809.4669 [M + Na]+, calcd for
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Journal of Natural Products
Article
i.d. × 5 cm), eluted with CHCl3−MeOH (15:2), to obtain compounds
1a and 2a, respectively.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO DIP 1000 polarimeter. IR measurements
were obtained on a Bruker IFS-48 spectrometer. NMR experiments
were performed on a Bruker DRX-600 spectrometer (Bruker BioSpin
GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI
CryoProbeat 300 K. All 2D-NMR spectra were acquired in CD3OD
(99.95%, Sigma-Aldrich), and standard pulse sequences and phase
cycling were used for DQF-COSY, HSQC, and HMBC spectra. The
NMR data were processed using UXNMR software. Exact masses were
measured using a Voyager DE mass spectrometer. Samples were
analyzed by matrix-assisted laser desorption ionization time-of-flight
(MALDITOF) mass spectrometry. A mixture of analyte solution and
α-cyano-4-hydroxycinnamic acid (Sigma) was applied to the metallic
sample plate and dried. Mass calibration was performed with the ions
from the ACTH (fragment 18−39) at 2465.1989 Da, with α-cyano-4-
hydroxycinnamic acid at 190.0504 Da as an internal standard.
Plant Material. The aerial parts of Spergula fallax were collected at
Elba-Mountain, South-Eastern Desert, Aswan, Egypt, in April 2011,
and identified by Prof. N. Kadry (Sohag University, Egypt) according
to Boulos.2 A representative voucher specimen (no. 10415) was
deposited at the Botany Department Herbarium, Faculty of Science,
Aswan University, Aswan, Egypt.
Compound 1a: amorphous, yellow solid; [α]25 −8.5 (c 0.10
D
1
MeOH); IR (KBr) νmax 3445, 2935, 1660, 1650 cm−1; H and 13C
NMR (CD3OD, 600 MHz), see Table 1; HRMALDITOFMS [M +
Na]+ m/z 541.3138 (calcd for C30H46O7Na, 541.3131).
Compound 2a: amorphous, white solid; [α]25 +10.2 (c 0.10
D
MeOH); IR (KBr) νmax 3440, 2940 cm−1; 1H and 13C NMR (CD3OD,
600 MHz) data, see Table 1; HRMALDITOFMS [M + Na]+ m/z
515.3716 (calcd for C30H52O5Na, 515.3712).
Determination of Sugar Configuration. The configurations of
sugar units of compounds 1 and 2 were established after hydrolysis of
1−4 with 1 N HCl, trimethylsilation, and determination of the
retention times by GC operating in the experimental conditions
previously reported by De Marino et al.10 The peaks of the hydrolysate
of 1 were detected at 14.72 min (D-glucose) and at 8.94 and 9.81 min
(L-arabinose). Retention times for authentic samples after being
treated in the same manner with 1-(trimethylsilyl)imidazole in
pyridine were detected at 14.71 min (D-glucose) and 8.92 and 9.80
(L-arabinose).
Cancer Cell Lines and Treatment. HeLa and DLD-1 cells, from
American Type Culture Collection, were grown in RPMI-1640
medium and DMEM, respectively, as previously reported.17
HeLa and DLD-1 cells, growing as monolayers, were plated one day
before the beginning of treatment at a density of 1.3 × 105/mL. After
72 h of incubation with different concentrations of each test
compound, cell viability was determined by an MTT ([3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]) assay.18
Mitochondria in living cells transform MTT to formazan salts,
which, upon solubilization with DMSO, can be read at 550 nm using a
microplate reader (LabSystems, Vienna, VA, USA). Data were
subtracted of the corresponding appropriate blank. The number of
viable cells in treated samples was calculated as a percentage of control
samples containing equal amounts of vehicle (DMSO). Etoposide was
used as a positive control (IC50 values of 16 and 20 μM against HeLa
and DLD-1 cells, respectively). To exclude any interference of test
compounds with the tetrazolium salt-based assay, cell growth
inhibition was randomly verified also by cytometric count (trypan
blue exclusion test).
Extraction and Isolation. The aerial parts of S. fallax (190 g)
were powdered and extracted exhaustively with 80% MeOH (3 × 1.5
L) for 20 days by maceration at room temperature. The crude extract
was concentrated under reduced pressure to a syrupy consistency (23
g). A 14 g aliquot from the crude extract was dissolved in a small
quantity of H2O and loaded on a water-preconditioned short flash C18
column (6 × 10 cm, LiChroprep, RP-18, 40−60 μm, Merck). Six
fractions (1000 mL) were collected: 100% H2O (S-1, 550 mg), 20%
MeOH (S-2, 624 mg), 40% MeOH (S-3, 226 mg), 60% MeOH (S-4,
349 mg), 80% MeOH (S-5, 500 mg), and 100% MeOH (1.411 g),
respectively. Fraction S-3 was loaded on a small C18 column (3 × 30
cm, LiChroprep, RP-18, 25−40 μm, Merck) and eluted with a gradient
of 20−25% MeOH to give compounds 2 (10 mg) and 3 (20 mg),
respectively. Fraction S-4 was loaded on a small C18 column (3 × 30
cm, LiChroprep, RP-18, 25−40 μm, Merck) and eluted with a gradient
of 30−35% MeOH to give compounds 4 (12 mg) and 1 (10 mg),
respectively. Fraction S-5 was fractioned on an RP-18 column (50 × 2)
eluted with 60−70% MeOH as mobile phase to yield succulentoside A
(40 mg).
Statistical Analysis. IC50 data reported are the mean values SD
of at least two experiments performed in duplicate.
Compound 1: amorphous, yellow solid; [α]25 −36.0 (c 0.13
D
MeOH); IR (KBr) νmax 3430, 2930, 1665, 1650 cm−1; H and 13C
1
ASSOCIATED CONTENT
■
NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar
portion, see Tables 1 and 2, respectively; HRMALDITOFMS [M +
Na]+ m/z 877.4192 (calcd for C43H66O17Na, 877.4198).
S
* Supporting Information
1H and 13C NMR, HSQC, HMBC, COSY, and ROESY spectra
of compounds 1−4 and their aglycones 1a and 2a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Compound 2: amorphous, white solid; [α]25 +20.7 (c 0.20
D
MeOH); IR (KBr) νmax 3450, 2945 cm−1; 1H and 13C NMR (CD3OD,
600 MHz) data of the aglycone moiety and the sugar portion, see
Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z
647.4142 (calcd for C35H60O9Na, 647.4135).
AUTHOR INFORMATION
Compound 3: amorphous, white solid; [α]25D +32.0 (c 0.04
MeOH); IR (KBr) νmax 3450, 2945 cm−1; 1H and 13C NMR
(CD3OD, 600 MHz) data of the aglycone moiety and the sugar
portion, see Tables 1 and 2, respectively; HRMALDITOFMS [M +
Na]+ m/z 809.4669 (calcd for C41H70O14Na, 809.4663).
■
Corresponding Author
*Tel: +39 089969763. Fax: +39 089969602. E-mail: piacente@
unisa.it.
Compound 4: amorphous, white solid; [α]25 +5.23 (c 0.19
Author Contributions
D
⊥Arafa I. Hamed and Milena Masullo contributed equally to
this work.
MeOH); IR (KBr) νmax 3450, 2945 cm−1; 1H and 13C NMR (CD3OD,
600 MHz) data of the aglycone moiety and the sugar portion, see
Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z
851.4774 (calcd for C43H72O15Na, 851.4769).
Notes
The authors declare no competing financial interest.
Acid Hydrolysis. Compounds 1 (5.3 mg) and 2 (5.0 mg) were
treated with 2 M HCl in 1,4-dioxane−H2O (1:1, v/v, 1.5 mL) at 80 °C
for 3 h. On cooling, solvent was eliminated with a stream of N2, each
dry residue was suspended in water, and the aglycones were extracted
with ethyl acetate (3 × 1.5 mL). After evaporating the solvent,
aglycones were purified by silica gel column chromatography (1.5 cm
ACKNOWLEDGMENTS
■
Part of the work has been supported by FP7 EC project
“PROFICIENCY” (contract no. 245751).
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Journal of Natural Products
Article
DEDICATION
■
Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich,
Switzerland, for his pioneering work in pharmacognosy and
phytochemistry.
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