New compounds from the Red Sea marine sponge Echinoclathria gibbosa

Article abstract of DOI:10.1016/j.phytol.2014.04.008

Three new compounds; β-sitosterol-3-O-(3Z)-pentacosenoate (1), 5α-pregna-3β-acetoxy-12β,16β-diol-20-one (2), and echinoclathriamide ((R)-2′-hydroxy-N-((2S,3S,4R)-1,3,4-trihydroxy-19- methylicosan-2-yl)heptadecanamide) (3), together with two known compound

Full text of DOI:10.1016/j.phytol.2014.04.008

Phytochemistry Letters 9 (2014) 51–58
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
New compounds from the Red Sea marine sponge Echinoclathria
gibbosa
a
a
b
b,c,
Gamal A. Mohamed , Ali E.E. Abd-Elrazek , Hashim A. Hassanean , Diaa T.A. Youssef *,
Rob van Soest
d
a
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
c
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
d
Naturalis Biodiversity Center, Department of Marine Zoology, P.O.Box 9517, 2300 RA Leiden, The Netherlands
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new compounds;
b
-sitosterol-3-O-(3Z)-pentacosenoate (1), 5
a
-pregna-3
b
-acetoxy-12
b
,16
b
-
Received 23 December 2013
Received in revised form 13 March 2014
Accepted 2 April 2014
0
diol-20-one (2), and echinoclathriamide ((R)-2 -hydroxy-N-((2S,3S,4R)-1,3,4-trihydroxy-19-methylico-
san-2-yl)heptadecanamide) (3), together with two known compounds; thymine (4) and uracil (5) were
isolated from the EtOAc fraction of the Red Sea sponge Echinoclathria gibbosa. Their structures were
unambiguously established on the basis of 1D and 2D NMR spectroscopy, in addition to mass
spectrometry. The total MeOH extract (TME) and its fractions were evaluated for their antimicrobial,
anti-inflammatory, antipyretic, and hepato-protective activities. The in vitro growth inhibitory activity of
the isolated compounds was evaluated against three human cancer cell lines including the A549 non-
small cell lung cancer (NSCLC), U373 glioblastoma (GBM), and PC-3 prostate cancer cell lines. Compound
Available online 19 April 2014
Keywords:
Echinoclathria gibbosa
Pregnane
Ceramide
1
showed weak activity against the three cancer cell lines.
Anti-inflammatory
Hepato-protective
Cancer growth inhibitory activity
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1
. Introduction
Marine sponges are a rich source of interesting class of
its fractions were evaluated. The in vitro growth inhibitory activity
of the isolated compounds was evaluated against three human
cancer cell lines; NSCLC, GBM, and PC-3. Compound 1 showed
weak activity against the tested cancer cell lines, while 2–5 were
inactive.
compound metabolites with unique chemical features and
pronounced biological activities (Blunt et al., 2013). The genus
Echinoclathria has been subjected to extensive research leading to
the isolation and identification of a wide variety of novel structures
2. Results and discussion
(
Sarma et al., 2005). Previous phytochemical studies of genus
Echinoclathria resulted in the isolation of steroids (Li et al., 1993)
and pyridine alkaloids (Kitamura et al., 1999; Dembitsky et al.,
Compound 1 was isolated as a white greasy powder. It gave
positive Liebermann–Burchard’s test (Schmidt, 1964), suggesting
its steroidal or triterpenoidal nature. The HRFABMS spectrum of 1
2
005; Ueoka et al., 2009). In this paper, we describe the isolation
and structural elucidation of three new compounds from the EtOAc
fraction of the Red Sea sponge E. gibbosa; -sitosterol-3-O-(3Z)-
+
showed pseudo-molecular ion peak at m/z 777.7415 [M+H] , that
b
is consistent with a formula of C54
96 2
H O . The IR spectrum of 1
pentacosenoate (1), 5
a
-pregna-3
b
-acetoxy-12
b
,16
b
-diol-20-one
showed characteristic absorption bands indicating the presence of
0
ꢀ1 ꢀ1
an ester group (1720 cm ), unsaturation (1632 cm ), and long
(
2), and echinoclathriamide ((R)-2 -hydroxy-N-((2S,3S,4R)-1,3,4-
ꢀ1
1
trihydroxy-19-methylicosan-2-yl)heptadecanamide) (3), together
with two known compounds; thymine (4) and uracil (5) (Fig. 1). In
addition, the antimicrobial, anti-inflammatory, antipyretic, and
hepato-protective activities of the total MeOH extract (TME) and
aliphatic chain (724 cm ). The H NMR spectrum (Table 1)
displayed signals for sterol and fatty acid moieties (Qi et al., 2003;
H
Sayed et al., 2007). The appearance of six methyl groups at d 0.68–
1.02 including two tertiary methyl groups at
and 1.02 (3H, s, H-19), a methine proton at
H
and an olefinic proton at d 5.34 (1H, m, H-6) suggesting the
d
H
0.68 (3H, s, H-18)
d
H
H
d 4.59 (1H, m, H-3),
*
Corresponding author. Tel.: +966 548535344; fax: +966 26951696.
presence of sitosteryl moiety. Furthermore, 1 displayed signals for
0
E-mail addresses: dyoussef@kau.edu.sa, diaa22@yahoo.com (Diaa T.A. Youssef).
a primary methyl group at
d
H
0.89 (3H, t, J = 6.1 Hz, H-25 ), two
http://dx.doi.org/10.1016/j.phytol.2014.04.008
874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1

5
2
G.A. Mohamed et al. / Phytochemistry Letters 9 (2014) 51–58
Fig. 1. Structures of the isolated compounds.
0
0
13
fatty acid. The C NMR and HSQC spectra displayed 54 signals, 29
olefinic protons at
a large number of methylene protons at
the presence of an aliphatic fatty acid moiety. The H– H COSY
Table 1) revealed different spin-coupling systems for sterol and
d
H
5.38 (2H, each dt, J = 7.5, 6.0 Hz, H-3 , 4 ), and
d
H
1.22–1.38 suggesting
of them were attributed to sitosteryl moiety with olefinic carbons
1
1
at
in addition to other carbon signals were in full agreement with
those previously reported for -sitosterol (Sayed et al., 2007; Bagri
et al., 2009). They also showed an ester carbonyl group at 173.2,
a terminal methyl group at 14.1, olefinic carbons at
29.9, and large number of methylene groups at
C C
d 139.7 (C-5) and 122.5 (C-6). A carbinol carbon at d 73.6 (C-3),
(
b
d
C
Table 1
d
d
122.5 and
C
C
NMR data of compound 1 (CDCl
3
, 400 and 100 MHz).
1
C
d 29.1–29.7
a
characteristic for the unsaturated fatty acid moiety. The geometry
of the double bond was deduced to be Z from the coupling constant
value (J = 6.0 Hz) (Vlahov, 2009). The attachment of the fatty acid
to sterol moiety at C-3 was confirmed from HMBC cross-peak
No.
d
H
[mult., J (Hz)]
d
C
(mult.)
HMBC
1
2
3
4
5
6
7
8
9
2.10 m, 1.21 m
1.90 m, 1.79 m
4.59 m
39.3 (CH
31.7 (CH
2
)
)
–
–
2
0
73.6 (CH)
38.1 (CH
139.7 (C)
122.5 (CH)
31.9 (CH
1
0
2.30 dd (14.5, 7.2)
–
2
)
3, 5, 6
between H-3 and C-1 , as well as the downfield shift of H-3 (
d
H
–
4
.59) (Fig. 2). Furthermore, the HMBC correlation correlations from
5.34 m
–
0
0
0
0
H-2 to C-4 and H-5 to C-3 confirmed the position of the double
2.12 m, 2.16 m
1.21 m
2
)
–
0
0
bond between C-3 and C-4 . This was further secured by the
fragement ion peaks at m/z 99 (4%) and 86 (13%) in the GCMS of
fatty acid methyl ester (FAME). Alkaline hydrolysis (Sayed et al.,
31.8 (CH)
50.0 (CH)
36.3 (C)
–
1.11 m
–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
1
2
3
4
5
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
–
–
1.51 m
21.0 (CH
39.7 (CH
42.3 (C)
2
)
)
–
2007) of 1 afforded b-sitosterol and FAME. b-Sitosterol was
2.00 m, 1.15 m
–
2
–
confirmed by co-TLC with authentic sample and the GCMS
–
+
1.20 m
56.7 (CH)
–
molecuar ion peak at m/z 414 (100%) [M] . The FAME was
1.61 m
24.3 (CH
28.2 (CH
2
)
)
–
identified to be (3Z)-pentacosenoic acid methyl ester by the GCMS
1.62 m, 1.41 m
1.09 m
2
–
+
molecular ion peak at m/z 394 [M] (100%). The fragment ion peaks
56.0 (CH)
–
at m/z 295 and 99 in the GCMS spectrum confirmed the position of
0.68 s
11.8 (CH
19.3 (CH
3
)
)
13, 14
5, 9, 10
0
0
double bond between C-3 and C-4 (Fig. 3). From the above
mentioned data and by comparison with literature (Sayed et al.,
1.02 s
3
1.42 m
37.0 (CH)
0.95 d (6.2)
1.11 m
18.9 (CH
36.6 (CH
26.3 (CH
3
2
2
)
)
)
–
–
–
–
–
–
–
–
–
–
2007; Bagri et al., 2009), 1 was deduced as b-sitosterol-3-O-(3Z)-
pentacosenoate and considered a new natural product.
1.20 brs
Compound 2 was isolated as white needles. It gave positive
Salkowski’s and Liebermann–Burchard’s tests (Schmidt, 1964),
suggesting its steroidal or triterpenoidal nature. Compound 2
1.0 m
46.3 (CH)
29.5 (CH)
1.02 m
0.86 d (7.2)
0.84 d (7.2)
1.54 m, 1.68 m
0.82 t (6.8)
–
19.3 (CH
19.6 (CH
25.0 (CH
12.3 (CH
173.2 (C)
34.7 (CH
3
3
2
3
)
)
)
)
36 5
possesses a molecular formula C23H O as confirmed from the
1
HRESIMS molecular ion peak at m/z 392.2571. The H NMR
9
0
0
0
0
0
spectrum (Table 2) revealed characterisitic signals for two acetyls
0
0
0
0
0
2.26 d (7.6)
5.38 dt (7.6, 6.0)
5.38 dt (7.5, 6.0)
2.01 m
2
)
1 , 4
at
d
H
2.30 (3H, s, H-21) and 1.91 (3H, s, 3-OAc), two methyls at
d
d
H
H
129.9 (CH)
122.5 (CH)
–
5
0
4
.99 (3H, s, H-19) and 0.84 (3H, s, H-18), downfield methines at
.86 (1H, m, H-16), 4.31 (1H, m, H-17), 4.26 (1H, m, H-12), and 3.91
2
3
1
3
0
0
0
0
(1H, m, H-3). The C NMR and HSQC spectra (Table 2) showed the
presence of 23 carbon resonances including four methyls, three
6 –23
1.22–1.38 m
1.49 brs
29.1–29.7 (CH
2
)
–
–
–
4
5
22.7 (CH
14.1 (CH
2
)
)
0.89 t (6.1)
3
oxygenated methines at
C
d 73.1 (C-12), 72.8 (C-16), and 71.9 (C-3),
a
five methines, seven methylenes, and four quaternary carbons two
Signals were assigned from multiplicity-edited HSQC.

G.A. Mohamed et al. / Phytochemistry Letters 9 (2014) 51–58
53
Fig. 2. Some important COSY and HMBC correlations of 1–3.
1
13
of them were attributed to ketone (
d
C
213.8) and acetoxy carbonyls
169.8). In the HMBC experiment (Fig. 2), the correlations of H-
6to C-17, C-14, and C-13 and H-12 to C-14, C-13, and C-11
The structural elucidation and complete H and C assignments
1
1
(
d
C
were achieved by 2D NMR techniques ( H– H COSY, HSQC, and
1
1
HMBC) and chemical methods. The H NMR spectrum (Table 3)
confirmed the position of OH groups at C-16 and C-12, respectively.
In addition, the cross peaks from H-3 to carbonyl of acetoxy and H-
revealed the presence of an amide moiety, as reflected by a proton
signal at
d
H
13
7.31 (1H, d, J = 8.4 Hz, 2-NH) and a signal at
d
C
174.6 (C-
0
2
1 to C-17 confirmed the attachment of acetate and acetyl groups
1 ) in the C NMR spectrum (Table 3). Also, it showed signals for an
at C-3 and C-17, respectively. The relative stereochemistry at C-3,
C-12, C-16 and C-17 was determined based on comparison of the
H and C NMR chemical shifts with related compounds in
literature isolated from the same Echinoclathria (Li et al., 1993;
Nohara et al., 2008). Based on the analysis of the previously
mentioned data and by comparison with literature (Li et al., 1993;
isopropyl group, as shown by the signals at
22.3 and 0.74, 0.78 (2d, 3H each, J = 6.7 Hz, H-20, 21)/
presence of carbons resonating at 60.9 (C-1), 75.5 (C-3), 71.6 (C-
4), and 71.5 (C-2 ) in the C NMR spectrum indicated the presence
of hydroxymethyl (CH O) and three oxymethine groups (CHO),
respectively, which resonate in the H NMR spectrum at
(1H, dd, J = 6.2, 10.8 Hz, H-1A) and 3.64 (1H, dd, J = 6.2, 10.8 Hz, H-
1B) for the hydroxymethyl group, and at 3.96, 3.49, and 3.48
(each 1H, m) for the oxymethines. In addition, the H NMR
d
H
1.42 (1H, m, H-19)/
d
C
d
C
22.0. The
1
13
d
C
0
13
2
1
H
d 3.77
Nohara et al., 2008), 2 was determined to be 5
acetoxy-12 ,16 -diol-20-one.
a-pregna-3b-
b
b
d
H
1
Compound 3 was isolated as white amorphous powder. The
HRESIMS spectrum showed the characteristic molecular ion peak
at m/z 628.5811 [M+H] which in conjunction with 1D and 2D NMR
spectrum showed three hydroxy groups at
J = 6.4 Hz, 2 -OH), 4.38 (1H, d, J = 4.4 Hz, 1-OH), and 4.16 (1H, t,
J = 6.4 Hz, 3-OH). All the previous signals, in addition to the cluster
d
H
4.48 (1H, d,
+
0
was compatible with the molecular formula C38
H
77NO
5
(Fig. 3).
m/ z 416
HO
m/z 141
m/ z 269
m/ z 85
m/z 57
O
m/z 269
NH OH
mlz 343
m/z 43
m/ z 358
HO
m/ z 141
OH
m/ z 358
A
m/z 285
m/z 252
O
O
m/z 57
OH
B
m/z 99
m/z 295
m/z 253
m/z 211
m/z 183
O
m/z 57
O
m/z 86
C
Fig. 3. Possible fragmentation patterns of 3 (A) and FAMEs of 3 (B) and 1 (C).

5
4
G.A. Mohamed et al. / Phytochemistry Letters 9 (2014) 51–58
Table 2
n-hexane layer afforded FAME, which was subjected to GC-MS and
NMR data of compound 2 (DMSO-d
6
, 400 and 100 MHz).
1
+
H NMR analysis. It afforded a molecular ion peak at m/z 300 [M]
a
1
No.
d
H
[mult., J (Hz)]
d
C
(mult.)
HMBC
in GCMS spectrum. Its H NMR spectrum showed a methoxy group
at 3.79 (OCH , s), a methine proton 4.19 (1H, m) and a cluster
of methylene groups at 1.25 (m), and a terminal methyl at
0.88 (3H, t, J = 6.4 Hz) suggesting the methyl ester of 2-hydroxy
heptadecanoic acid. The presence of methyl group at 0.88 (3H, t,
d
H
3
d
H
1
2
3
4
5
6
7
8
9
1.94 m, 1.08 m
2.01 m, 1.49 m
3.91 m
37.2 (CH
32.3 (CH
2
)
)
–
2
–
d
H
d
H
71.9 (CH)
38.3 (CH
46.7 (CH)
3-OAc, 5
2.18 m
2
)
–
d
H
1.47 m
3, 7
J = 6.4 Hz) in FAME supported that isopropyl group present in
sphingosine base. The MeOH layer provided a C21 sphingosine with
an isopropyl terminus identified by the EIMS ion peaks at m/z 359
2.19 m, 1.51 m
1.39 m, 0.92 m
1.59 m
38.7 (CH
32.3 (CH
2
)
)
–
2
–
35.0 (CH)
55.1 (CH)
35.6 (C)
–
+
+
1.13 m
–
[
M] and 43 [MꢀC18
H38NO
3
] due to loss of the terminal isopropyl
(Fig. 3). This
1
1
1
1
1
1
1
1
1
1
2
2
3
0
–
–
group and its molecular formula had to be C21H45NO
3
1
1.88 m, 1.63 m
4.26 m
28.9 (CH
2
)
–
was further confirmed by the presence of fragment ion peak at m/z
2
73.1 (CH)
50.4 (C)
11, 13, 14
+
3
–
–
358 (100%) [MꢀC17
H
33
O
2
]
in the ESIMS of 3. The relative
4
0.78 m
50.7 (CH)
–
stereochemistry of 3 was determined to be D-erythro, as the
5
2.27 m, 1.21 m
4.86 m
38.5 (CH
2
)
13, 17
naturally occurring phytosphingosine base, while the absolute
6
72.8 (CH)
67.5 (CH)
13, 14, 17, 20
0
stereochemistry at C-2, C-3, C-4 and C-2 were determined as
7
4.31 m
13, 14, 16
0
0
2
S,3S,4R,2 R from the chemical shifts of H-2, H-3, H-4 and H-2 ,
which were in good agreement with those reported in the
literature, in addition to the [ value (Ibrahim et al., 2008a,b,
009; Elkhayat et al., 2013). Accordingly, the structure of
8
0.84 s
13.5 (CH
14.2 (CH
3
)
)
13
9
0.99 s
3
5, 9
–
0
–
213.8 (C)
30.6 (CH
22.3 (CH
a]
D
1
2.30 s
3
)
)
17, 20
20
2
-OAc
1.91 s
3
0
compound 3 was assigned as (R)-2 -hydroxy-N-((2S,3S,4R)-1,3,4-
trihydroxy-19-methylicosan-2-yl)heptadecanamide, and named
echinoclathriamide. This is the first isolation of ceramide from the
sponge Echinoclathria gibbosa.
169.8 (C)
a
Signals were assigned from multiplicity-edited HSQC.
of methylene groups at
methyl at
methine group at
presence of a ceramide moiety (Ibrahim et al., 2008a,b, 2009;
d
H
1.00–1.30 (m)/
0.81 (3H, t, J = 6.4 Hz), and a nitrogen bearing
51.4 (C-2)/ 4.04 (1H, m, H-2) suggested the
C
d 28.9–29.3, a terminal
The known compounds were identified through the analysis of
the spectroscopic data and comparison of their data with those in
the literature as thymine (4) (Tilvi et al., 2010) and uracil (5)
d
C
13.5/
d
H
d
C
d
H
(
Hisamoto et al., 2003).
1
1
Elkhayat et al., 2013). In H– H COSY, two spin systems were
observed (Fig. 2), the first spin system assigned to the sphingosine
base and the second to the saturated fatty acyl moiety. Direct
correlations of protons to their respective carbons were revealed in
the multiplicity-edited HSQC spectrum. The structure was
confirmed by HMBC experiment (Table 3). The attachment of
sphingosine base to the fatty acyl moiety was confirmed by the
To evaluate the biological activities of the Red Sea sponge E.
gibbosa, different extracts of the sponge were evaluated in a panel
of different biological screen including antimicrobial, anti-inflam-
matory, antipyretic, hepato-protective. In addition, the pure
compounds were evaluated for their cancer growth inhibitory
activity against three cell lines. In all screens both positive and
negative controls were used. TME and EtOAc fraction exhibited
potent antimicrobial activity against the tested strains (Table 4).
They showed high activity against P. aeruginosa at all concentra-
tions and against S. marcescens at concentrations of 25 and 50 mg/
0
HMBC correlation of H-2 to C-1 . H-1 showed cross peaks to C-2
0
0
0
and C-3, and H-3 to C-1 (
1
d
C
174.6). Also, 2 -OH correlated with C-
0
0
0
, C-2 , and C-3 confirming the 2-OH fatty acyl moiety. The length
and branching pattern of the long chain base (LCB) and fatty acid
were determined on the basis of the results of its methanolysis
3
mL. The CHCl fraction showed moderate activity against S. auerus
and B. cereus at all concentrations and no activity against P.
aeruginosa and S. marcescens. Aqueous fraction showed moderate
1
followed by EI, GCMS, and H NMR analyses of the methanolysis
products (Ibrahim et al., 2008a,b, 2009; Elkhayat et al., 2013). The
3
activity against E. coli and P. aeruginosa. CHCl and EtOAc fractions
were the most active fractions against the tested fungal strains.
They showed high activity against F. oxysporum (conc. 100 mg/mL).
Aqueous fraction showed no antifungal activity. Anti-inflammato-
ry activity of the different extracts was measured against
carrageenan-induced edema (Table 5). All the tested fractions
gave anti-inflammatory activity at both concentrations. TME and
Table 3
NMR data of compound 3 (DMSO-d
6
, 400 and 100 MHz).
a
No.
1
d
H
[mult., J (Hz)]
d
C
(mult.)
HMBC
2, 3
3.77 dd (6.2, 10.8)
60.9 (CH
2
)
3
.64 dd (6.2, 10.8)
3
CHCl fraction at doses of 100 and 200 mg/kg gave potent activity.
0
2
3
4
5
4.04 m
51.4 (CH)
75.5 (CH)
71.6 (CH)
1, 4, 1
These results may be attributed to the presence of sterols and
saponins in the extracts. All tested fractions showed a well-marked
antipyretic activity (Table 6) at doses of 100 and 200 mg/kg. They
reduce yeast-induced fever compared with indomethacin (8 mg/
kg) with maximum activity after 3 h. In the present study,
3.49 m
1, 4, 5
3.48 m
–
1.68 m, 1.34 m
1.00–1.30 m
1.42 m
32.3 (CH
2
)
–
6
1
2
2
1
2
3
4
1
1
2
1
3
2
–18
28.9–29.3 (CH
22.3 (CH)
2
)
)
–
9
0
20, 21
–
0.78 d (6.7)
0.74 d (6.7)
–
22.0 (CH
22.0 (CH
3
)
)
4
administration of the TME decreased the CCl induced elevated
1
0
3
enzyme levels in animals (Table 7). This suggests the maintenance
of structural integrity of hepatocytic cell membrane or regenera-
tion of damaged liver cells by the extract. Decrease in serum
bilirubin after treatment with the extract in liver damage indicates
the effectiveness of the extract in normal functional status of the
liver. Thus, it is evident that TME had remarkable hepato-
174.6 (C)
71.5 (CH)
34.1 (CH
–
–
0
0
0
0
3.96 dd (4.8, 8.4)
1.75 m, 1.52 m
1.00–1.30 m
1.34 m
2
)
1 , (CH
2
)
n
0
–15
28.9–29.3 (CH
2
–
–
–
–
1
3
0
6
7
24.6 (CH
2
)
)
0
0.81 t (6.4)
7.31 d (8.4)
4.38 t (4.4)
4.16 d (6.4)
4.48 d (6.4)
13.5 (CH
3
-NH
-OH
–
–
–
–
4
protective effect in CCl -induced liver damage. The determination
-OH
of the in vitro growth inhibitory activity using MTT colorimetric
assay revealed that compound 1 displayed weak in vitro growth
inhibitory activity against the tested cell lines, with magnitude of
0
0 0
0
-OH
1 , 2 , 3
a
Signals were assigned from multiplicity-edited HSQC.

G.A. Mohamed et al. / Phytochemistry Letters 9 (2014) 51–58
55
Table 4
Antimicrobial activity results.
c
Tested strains
Inhibition zone diameter (mm/sample)
CHCl EtOAc
a
b
3
Aqueous
25
TME
25
Chol.
Clot.
25
50
100
25
50
100
50
100
50
100
25
25
S. aureus
10
16
11
0
9
16
10
0
10
16
13
0
11
15
10
11
0
12
16
12
11
10
0
12
12
11
12
0
9
0
0
0
9
8
0
0
0
0
0
0
0
0
0
9
0
0
0
0
0
0
0
0
12
10
14
10
11
0
10
16
11
9
22
27
25
15
30
–
–
–
B. cereus
14
E. coli
10
8
12
–
P. aeurginosa
S. marscescens
C. albicans
G. candidum
F. oxysporum
A. flavus
11
–
0
0
0
0
11
0
–
10
10
15
16
13
17
12
12
16
20
16
17
14
14
19
24
26
22
0
13
14
20
23
22
22
0
0
0
0
22
23
18
32
30
30
13
15
15
14 p.i.
17
14
17
19
18
18
0
12
13
16
18
16
15
13
14
18
20
17
–
0
11
–
0
12
–
S. brevicaulis
T. rubrum
0
12 p.i.
15 p.i.
–
0
–
P.i. = Partial inhibition.
a
Cholroamphenicol as antibacterial standard.
Clotrimazole as antifungal standard.
Conc (mg/mL).
b
c
Table 5
Results of the anti-inflammatory activity.
Group
Dose (mg/kg)
Thickness of the right paw (mm) after injection
1
h
2 h
3 h
4 h
Control
–
8
8.2 ꢁ 0.04
7.9 ꢁ 0.01
5.7 ꢁ 0.08 (27.9)
7.8 ꢁ 0.03
8.2 ꢁ 0.04
Indomethacin
5.9 ꢁ 0.09 (28.1)
5.3 ꢁ 0.08 (32.1)
4.4 ꢁ 0.14 (45.1)
CHCl
3
200
6.7 ꢁ 0.09 (18.3)
6.8 ꢁ 0.10 (17.1)
5.9 ꢁ 0.15 (26.6)
6.0 ꢁ 0.15 (24.1)
5.8 ꢁ 0.15 (25.6)
6.0 ꢁ 0.18 (23.1)
5.8 ꢁ 0.15 (29.3)
6.0 ꢁ 0.16 (26.8)
100
EtOAc
TME
200
00
5.9 ꢁ 0.08 (28.1)
6.1 ꢁ 0.08 (25.1)
5.8 ꢁ 0.08 (26.6)
6.0 ꢁ 0.11 (24.1)
5.3 ꢁ 0.08 (32.1)
5.5 ꢁ 0.09 (29.4)
5.2 ꢁ 0.07 (36.6)
5.5 ꢁ 0.10 (32.9)
1
200
00
6.8 ꢁ 0.15 (17.1)
7.1 ꢁ 0.15 (14.4)
6.7 ꢁ 0.08 (15.2)
6.9 ꢁ 0.10 (12.7)
5.8 ꢁ 0.08 (25.6)
6.0 ꢁ 0.09 (21.8)
5.0 ꢁ 0.13 (39.1)
5.0 ꢁ 0.07 (29.0)
1
S.E., standard error; n, number of animals; Data are expressed as mean ꢁ S.E., n = 6.
Differences with respect to the control group were evaluated using the Student’s t-test (P < 0.001).
effects that ranged between those of cisplatin and carboplatin
3. Experimental
3.1. General experimental procedures
(
Table 8), while compounds 2–5 were inactive.
In conclusion, this study resulted in the identification of five
compounds, including three new ones, from the EtOAc fraction of
the Red Sea sponge E. gibbosa. Their structures were unambigu-
ously established on the basis of 1D and 2D NMR spectroscopy, in
addition to mass spectrometry. The TME and its fractions were
evaluated for their antimicrobial, anti-inflammatory, antipyretic,
and hepato-protective activities. Compound 1 showed weak in
vitro growth inhibitory activity against three human cancer cell
lines.
Melting points were obtained in an Electrothermal 9100 Digital
Melting Point (England). Optical rotations were measured with a
Perkin-Elmer 241 automatic polarimeter (Perkin-Elmer Inc, MA,
USA). IR was measured with a Shimadzu Infrared-400 spectropho-
tometer (Japan). Optical rotation was recorded on a Perkin-Elmer
Model 341 LC Polarimeter. ESIMS spectra were obtained with a
Thermofinnigan LCQ DECA mass spectrometer coupled to an
Table 6
Results of antipyretic activity.
Group
Dose (mg/kg)
Average rectal temperature (8C)
1
h
2 h
3 h
4 h
Control
–
8
38.7 ꢁ 0.05
38.2 ꢁ 0.04
38.2ꢁ 0.02
37.8ꢁ 0.03
38.2 ꢁ 0.06
37.6 ꢁ 0.04
39.4 ꢁ 0.03
38.4 ꢁ 0.03
a
Indomethacin
a
CHCl
3
200
38.4 ꢁ 0.04
38.5 ꢁ 0.03
37.7ꢁ 0.02
37.8ꢁ 0.03
37.7 ꢁ 0.03
37.7 ꢁ 0.04
38.8 ꢁ 0.02
38.8 ꢁ 0.04
a
100
a
EtOAc
TME
200
00
38.0 ꢁ 0.03
38.1 ꢁ 0.04
37.8ꢁ 0.02
37.8ꢁ 0.03
37.7 ꢁ 0.03
37.7 ꢁ 0.03
38.8 ꢁ 0.02
38.8 ꢁ 0.03
a
1
200
00
38.2 ꢁ 0.07
38.3 ꢁ 0.05
38.2ꢁ 0.05
38.2ꢁ 0.06
38.1 ꢁ 0.03
38.2 ꢁ 0.05
38.8 ꢁ 0.04
38.9 ꢁ 0.02
1
S.E., standard error; n, number of animals; Data are expressed as mean ꢁ S.E., n = 6.
Differences with respect to the control group were evaluated using the Student’s t-test (P < 0.001).

5
6
G.A. Mohamed et al. / Phytochemistry Letters 9 (2014) 51–58
Table 7
ectosomal subtylostyles, in two or three size categories, but these
are not clearly differentiated measuring about 125–360 ꢂ 1–4 m.
Microseleres are very thin, shallow-curved toxas, up to 20 m in
Results of Hepato-protective activity.
m
Group
SGOT
SGPT
Total bilirubin
m
length. The specimen was compared with a slide of the Berlin
Museum type and found to conform closely to it. The voucher is
registered in the collections of the Netherlands Centre of
Biodiversity Naturalis under number ZMA Por. 19793. Another
voucher was kept at the Red Sea Invertebrates Collection at the
Department of Pharmacognosy of Suez Canal University under the
code no. RS-46.
Control (normal)
21.16 ꢁ 0.95
68.80 ꢁ 2.52
27.40 ꢁ 1.36
44.60 ꢁ 4.08
35.86 ꢁ 0.50
53.34 ꢁ 3.02
44.06 ꢁ 1.21
45.70 ꢁ 0.94
0.41 ꢁ 0.03
a
a
a
CCl
4
treated
1.98ꢁ 0.12
a,b
a,b,c
a,b
a,b
a,b
Silymarin treated
TME
0.81 ꢁ 0.06
b
0.65 ꢁ 0.06
S.E., standard error; n = 6.
a
Significant from control at P < 0.05.
b
c
Significant from CCl
4
at P < 0.05.
Significant from silymarin at P < 0.05.
3.3. Extraction and isolation
Agilent 1100 HPLC system equipped with a photodiode array
detector. EIMS was recorded on a Jeol the mass route JMS.600 H
mass spectrometer. HRESI and FABMS were recorded on a LTQ
Orbitrap and an API 2000 (ThermoFinnigan, Bremen, Germany)
mass spectrometers, respectively. GCMS was recorded on gas
chromatograph (GC)–mass spectrometer (MS), Clarus 500 GC/MS
The freeze-dried sponge of E. gibbosa (300 g) was extracted with
MeOH (4ꢂ 2 L). The combined MeOH extracts were concentrated
under reduced pressure to give a reddish brown residue (8.2 g).
This residue was suspended in 500 mL distilled H
2
O and subjected
to solvent fractionation using n-hexane, CHCl , and EtOAc, which
3
were separately concentrated to give 1.0, 1.4, 1.9, and 2.9 g,
(
PerkinElmer, Shelton, CT). The software controller/integrator was
Turbo Mass, version 4.5.0.007 (PerkinElmer). An Elite 5 GC MS
m, Perkin Elmer)
respectively. The n-hexane soluble fraction (1.0 g) was chromato-
graphed on SiO
n-hexane:EtOAc gradient. The fractions eluted with n-hexane:E-
tOAc (95:5) was subjected to SiO column using n-hexane:EtOAc
gradient elution to afford compound 1 (14 mg, white greasy
powder). The CHCl fraction (1.4 g) was chromatographed on SiO
2
column (100 g ꢂ 50 cm ꢂ 3 cm) and eluted with
capillary column (30-mm ꢂ 0.25-mm ꢂ 0.5
m
was used. The carrier gas was helium (purity 99.9999%) at a flow
rate of 2 mL/min (32 p.s.i., flow initial 55.8 cm/s, split; 1:40).
Temperature conditions were: inlet line temperature, 200 8C;
source temperature, 150 8C; trap emission, 100 8C; and electron
energy, 70 eV. The column temperature program was: 50 8C for
2
3
2
column (120 g ꢂ 50 cm ꢂ 3 cm) and eluted with n-hexane:EtOAc
gradient. The fractions eluted with n-hexane:EtOAc (90:10) was
5
min, increased to 220 8C (rate, 20 8C/min), and held for 5 min. The
injector temperature was 220 8C. MS scan was from 50 to 650 m/z.
D and 2D NMR spectra (chemical shifts in ppm, coupling
constants in Hz) were recorded on Bruker BioSpin GmbH
00 MHz Ultrashield spectrometer using standard Bruker software
and CDCl and DMSO-d as solvents, with TMS as the internal
further chromatographed over SiO
as an eluent to give 3 (11 mg, white amorphous powder). The
EtOAc fraction (1.9 g) was subjected to VLC using CHCl :MeOH
gradient elution to give 3 sub-fractions (I–III). Sub-fraction I
(340 mg) was chromatographed on SiO column
followed by
:MeOH gradient. The fractions eluted with CHCl :MeOH
2
column using n-hexane:EtOAc
1
3
4
2
(
80 g ꢂ 50 cm ꢂ 2 cm) and eluted with CHCl
3
,
3
6
reference. Column chromatographic separation was performed on
silica gel 60 (0.04–0.063 mm). TLC was performed on pre-coated
CHCl
3
3
(95:5) subjected to repeated purification steps through small
TLC plates with silica gel 60
chromatograms were developed using the following solvent
systems: n-hexane: EtOAc (95:5, S ), n-hexane:EtOAc (90:10,
), and CHCl :MeOH (93:7, S ).
F
254 (0.2 mm, Merck). The
columns afforded compound 2 (4 mg, white needles). Sub-fraction
III (700 mg) was chromatographed on SiO
2
column
(100 g ꢂ 50 cm ꢂ 3 cm) and eluted with CHCl
3
:MeOH gradient
yielded 4 (7 mg, white needles) and 5 (8 mg, white powder).
1
S
2
3
3
3.2. Sponge material
3.4. Alkaline hydrolysis of compound 1
The sponge E. gibbosa (Keller, 1889) was collected from
A solution of 1 (5 mg) in 3% KOH/MeOH (4 mL) was left to stand
for 15 min at room temperature then neutralized with 1 N HCl/
Hurghada at the Egyptian Red Sea coast at depth of 30 m. It forms
a mass of long branches, which anastomose infrequently. The
length of the branches in the voucher sample is up to 20 cm,
thickness varies due to the irregular outline of the branches from 1
to 2 cm. The surface is pitted and clathrate. The live color is blood-
red. The skeleton is a square- to round-meshed reticulation of
spongin fibers, cored by 1–5 spicules in cross section. Meshes
MeOH. The solution was extracted with CHCl
evaporated and the residue obtained was chromatographed on a
SiO column using n-hexane:EtOAc gradient to furnish -sitosterol
and (3Z)-pentacosenoate methyl ester. -Sitosterol was identified
3
. The solvent was
2
b
b
by GCMS analysis and co-TLC with authentic sample. The (3Z)-
pentacosenoate methyl ester was identified by GCMS (Sayed et al.,
2007).
measure 150–300
mm in diameter, while connecting fibers
measure 10–25 m in diameter. Surface skeleton is a tangential
m
arrangement of loose styles. Spicules are choanosomal styles and
3.5. Methanolysis
Table 8
Compound 3 (5 mg) was subjected to methanolysis by boiling
In vitro cancer growth inhibitory activity of compounds 1–5.
2
with 5 mL of 1 N HCl in MeOH at 80 8C for 18 h. 10 mL cold H O was
added to the reaction mixture, followed by extraction with n-
hexane (3ꢂ 20 mL) to yield FAME. After removal of n-hexane under
Sample
IC50
(
m
M)
A549 NSCLC U373 GBM PC-3 Prostate cancer Mean ꢁ SEM
2
reduced pressure, the residue was suspended in H O, and extracted
1
64
>100
>100
>100
>100
4
64
>100
>100
>100
>100
64
28 ꢁ 6
>100
>100
>100
>100
–
several times with EtOAc to yield sphingosine base (Ibrahim et al.,
008a,b; Ibrahim et al., 2009; Elkhayat et al., 2013).
2
>100
>100
>100
>100
NT
2
3
4
5
3.6. Spectral data
a
Cisplatin
0.4
38
a
Carboplatin
90
>100
>64
b
-Sitosterol-3-O-(3Z)-pentacosenoate (1). White greasy pow-
a
Positive anticancer controls.
der; R
f
= 0.62 (S
1
); [a
]
D
3
ꢀ31.6 (c 0.1, CHCl ); IR gmax; 2920, 1720,

G.A. Mohamed et al. / Phytochemistry Letters 9 (2014) 51–58
57
ꢀ
1
1
7
2
632, 724 cm ; HRFABMS: m/z 777.7415 (calcd for C54
77.7410); GCMS of -sitosterol: m/z 414 (100%) 396, 381, 329,
73, 255, 231; GCMS of FAME: m/z (rel. int.) 394 [M] (100) 295
H
97
O
2
,
Group I: Normal control received distilled water (1 mL/kg) daily
for 5 days and olive oil (1 mL/kg, intraperitoneal) on days 2 and 3.
b
+
4
Group II: CCl control received distilled water (1 mL/kg) daily
(
31), 265 (37), 211 (43), 183 (7), 163 (17), 99 (13), 57 (61); NMR
data; see Table 1.
-Pregna-3 -acetoxy-12
dles; R = 0.25 (S ); [ +71.4 (c 0.1, MeOH); m.p. 201–202 8C; IR
max; 3385, 2932, 1742, 1468, 1075 cm ; HRESIMS: m/z 393.2571
calcd for C23 , 393.2563); NMR data, see Table 2.
Echinoclathriamide (3). White amorphous powder; R
); [ +19.4 (c 0.1, CHCl ); IR max; 3435, 2921, 2849, 1651,
540, 1468, 1261, 1077 cm ; HRESIMS: m/z 628.5811 (calcd for
for 5 days and CCl
days 2 and 3.
Group III: Treated with silymarin orally through intragastric
feeding tube at dose of 50 mg/kg.
Groups IV: Treated with TME orally through intra-gastric
feeding tube at dose of 100 mg/kg.
4
:olive oil (1:1, 1 mL/kg, intraperitoneal) on
5a
b
b
,16
b
-diol-20-one (2). White nee-
f
3
a]
D
ꢀ
1
g
(
37 5
H O
f
= 0.41
Biochemical estimations: The rats were sacrificed on day 6 and
blood was collected from orbital sinus in plain tubes. The serum
was obtained by centrifugation and serum samples were taken for
biochemical assays; namely glutamate oxaloacetate transaminase
(GOT), and glutamate pyruvate transaminase (GPT). The data were
expressed as mean ꢁ SE (n = 6). Results were analyzed statistically
by one-way ANOVA followed by comparison using Prism software
(S
2
a
]
D
3
1
g
ꢀ
1
C
+
38 5
H78NO , 628.5802); GCMS of FAME: m/z 300 [M] ; EIMS of LCB:
m/z 359 (100%); NMR data, see Table 3.
3
.7. Antimicrobial assay
(
Graph Pab. Ver. 3.0).
The organisms used in this study were Staphylococcus aureus
(AUMC No. B-54), and Bacillus cereus (AUMC No. B-5) as Gram-
3.8.5. Cancer growth inhibitory
positive bacteria, Escherichia coli (AUMC No. B-53), Pseudomonas
aeurginosa (AUMC No. B-73), and Serratia marscescens (AUMC No.
B-55) as Gram-negative bacteria, Candida albicans (AUMC No. 418),
Geotrichium candidum (AUMC No. 226), Fusarium oxysporum
Cancer growth inhibitory activity was examined against the
A549 NSCLS (non-small-cell lung cancer), U373 GBM (glioblasto-
ma), and PC-3 (prostate cancer) cell lines as described earlier
(
Mijatovic et al., 2008; Ibrahim et al., 2012a,b).
(AUMC No. 5119), Aspergillus flavus (AUMC No. 1276), Scopular-
iopsis brevicaulis (AUMC No. 729), and Trichophyton rubrum (AUMC
No. 1804) as fungi. They were obtained from the Assiut University
Mycology Center, Assiut (http://www.aun.edu.eg/aumc/Cata-
log.htm). The procedure was carried out as previously described
Conflict of interest
There is no conflict of interest associated with the authors of
this paper.
(
Bonev et al., 2008; Ibrahim et al., 2012a,b).
Acknowledgments
3.8. Pharmacological studies
We would like to express our deep thanks to Prof. Robert Kiss
for conducting the cancer growth inhibitory assays and Mr. Volker
Brecht for measuring NMR and MS spectroscopic data.
3
.8.1. Animals
Adult male albino rats (120–150 g body weight) were used. All
animal procedures were conducted in accordance with the
internationally accepted principles for laboratory animals’ use
and care as found in the European Community Guidelines and
Institutional Ethical Committee Approval was obtained. The study
protocol was approved by the Animal Ethical Committee of Assiut
University. The animals were housed under standardized
environmental conditions in the pre-clinical Animal House,
Pharmacology Department, Faculty of Medicine, Assuit Universi-
ty. The animals were fed with standard diet and free access to
water. They were kept at 24–28 8C, 60–70% relative humidity, 12 h
day and night cycle for one week to acclimatize to the
environmental conditions.
References
Adams, S.S., Hebborn, P., Nicholson, J.S., 1968. Some aspects of the pharmacology of
ibufenac, a non-steroidal anti-inflammatory agent. J. Pharm. Pharmacol. 20,
305–312.
Ali, A.A., Sayed, H.M., Ibrahim, S.R.M., Zaher, A.M., 2013. Chemical constituents,
antimicrobial, analgesic, antipyretic, and anti-inflammatory activities of Eu-
phorbia peplus L. Phytopharmacology 4, 69–80.
Bagri, P., Ali, M., Sultana, S., Aeri, V., 2009. New sterol esters from the flowers of
Punica granatum Linn. J. Asian Nat. Prod. Res. 11, 710–715.
Bergmeyer, H.U., Bernt, E., 1974. 2nd ed. Methods of Enzymatic Analysis, 2nd ed.,
Academic Press, New York, , pp. 735–739.
Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G., Prinsep, M.R., 2013. Marine
natural products and the previous report in this series. Nat. Prod. Rep. 30, 237–
323.
Bonev, B., Hooper, J., Parisot, J., 2008. Principles of assessing bacterial susceptibility
to antibiotics using the agar diffusion method. J. Antimicrob. Chemother. 61,
3
.8.2. Anti-inflammatory activity
The anti-inflammatory activity was evaluated in adult albino
1295–1301.
rats by carageenin-induced rat hind paw edema method according
to the published procedures (Winter et al., 1962; Ali et al., 2013).
The percentage of edema inhibition (% of change) was calculated
Bosma, A., Brouwer, A., Seifert, W.F., Knook, D.L., 1988a. Synergism between ethanol
and carbon tetrachloride in the generation of liver fibrosis. J. Pathol. 156, 15–21.
Bosma, A., 1988b. Synergism between ethanol and carbon tetrachloride in the
generation of liver fibrosis. J. Pathol. 156, 15–21.
(
Table 5).
Dembitsky, V.M., Gloriozova, T.A., Poroikov, V.V., 2005. Novel antitumor agents:
marine sponge alkaloids, their synthetic analogs and derivatives. Mini Rev. Med.
Chem. 5, 319–336.
Elkhayat, E.S., Mohamed, G.A., Ibrahim, S.R.M., 2013. Ceramides, activity and
structure elucidation. Curr. Bioactive Compds. 8, 370–409.
3
.8.3. Antipyretic activity
The antipyretic activity was screened in adult albino rats by
Hisamoto, M., Kikuzaki, H., Ohigashi, H., Nakatani, N., 2003. Antioxidant compounds from
the leaves of Peucedanum japonicum Thunb. J. Agric. Food Chem. 51, 5255–5261.
Ibrahim, S.R.M., Mohamed, G.A., Elkhayat, E.S., Gouda, Y.G., Proksch, P., 2008a.
Strepsiamide A–C, new ceramides from the marine sponge Strepsichordaia
lendenfeldi. Nat. Prod. Commun. 3, 205–209.
using yeast-induced hyperpyrexia as previously described (Adams
et al., 1968; Ibrahim et al., 2012a,b). Rectal temperature of each rat
was recorded after 1, 2, 3, and 4 h from administration of tested
fractions.
Ibrahim, S.R.M., Ebel, R.A., Ebel, R., Proksch, P., 2008b. Acanthomine A, a new
pyrimidine-b-carboline alkaloid from the sponge Acanthostrongylophora ingens.
Nat. Prod. Commun. 3, 175–178.
3.8.4. Hepato-protective activity
The hepato-protective activity was determined as previously
Ibrahim, S.R.M., Mohamed, G.A., Al-Musayeib, N.M., 2012a. New constituents from
the Egyptian Iris germanica L. rhizomes. Molecules 17, 2587–2598.
Ibrahim, S.R.M., Mohamed, G.A., Fouad, M.A., El-Khayat, E.S., Proksch, P., 2009.
Iotrochotamides I and II new ceramides from the Indonesian sponge Iotrochota
purpurea. Nat. Prod. Res. 23, 86–92.
outlined (Bosma et al., 1988a,b; Bergmeyer and Bernt, 1974;
Mohamed et al., 2009). The animals were divided into four groups
each of six animals.

5
8
G.A. Mohamed et al. / Phytochemistry Letters 9 (2014) 51–58
Ibrahim, S.R.M., Mohamed, G.A., Shaala, L.A., Banuls, L.M., Van Goietsenoven, G.,
Kiss, R., Youssef, D.T.A., 2012b. New ursane-type triterpenes from the root barks
of Calotropis procera. Phytochem. Lett. 5, 490–495.
Qi, S.H., Wu, D.G., Ma, Y.B., Luo, X.D., 2003. The chemical constituents of Munronia
Henryi. J. Asian Nat. Prod. Res. 5, 215–221.
Sarma, N.S., Krishna, M.S.R., Rao, S.R., 2005. Sterol ring system oxidation pattern in
marine sponges. Mar. Drugs 3, 84–111.
Keller, C., 1889. Die Spongienfauna des rothen Meeres (I. H a¨ lfte)., Z. Wiss. Zool. 48.
3
11-405, pls XX–XXV.
Sayed, H.M., Mohamed, M.H., Farag, S.F., Mohamed, G.A., Proksch, P., 2007. A new
steroid glycoside and furochromones from Cyperus rotundus L. Nat. Prod. Res.
21, 343–350.
Kitamura, A., Tanaka, J., Ohtani, I.I., Higa, T., 1999. Echinoclathrines A-C: a new class
of pyridine alkaloids from an Okinawan sponge Echinoclathria sp. Tetrahedron
5
5, 2487–2492.
Li, H.Y., Matsunaga, S., Fusetani, N., Fujiki, H., Murphy, P.T., Willis, R.H., Baker, J.T.,
993. Echinoclasterol sulfate phenethylammonium salt, a unique steroid sul-
fate from the marine sponge, Echinoclathria subhispida. Tetrahedron Lett. 34,
733–5736.
Schmidt, J., 1964. Organic Chemistry, 8th ed. Oliver and Boyed, Edinburgh and
London 318, 673.
Tilvi, S., Moriou, C., Martin, M.T., Gallard, J.F., Sorres, J., Patel, K., Petek, S., Debitus, C.,
Ermolenko, L., Al-Mourabit, A., 2010. Agelastatin E, Agelastatin F, and benzos-
ceptrin C from the marine sponge Agelas dendromorpha. J. Nat. Prod. 73, 720–
723.
1
5
Mijatovic, T., De Neve, N., Gailly, P., Mathieu, V., Haibe-Kains, B., Bontempi, G.,
Lapeira, J., Decaestecker, C., Facchini, V., Kiss, R., 2008. The nucleolus and cMyc:
potential targets of cardenolide-mediated anti-tumor activity. Mol. Cancer
Ther. 7, 1285–1296.
Mohamed, G.A., Abdel-Lateff, A., Fouad, M.A., Ibrahim, S.R.M., Elkhayat, E.S., Okino,
T., 2009. Chemical composition and hepato-protective activity of Imperata
cylindrica Beauv. Pharmacog. Mag. 4, 28–36.
Ueoka, R., Ito, A., Izumikawa, M., Maeda, S., Takagi, M., Shin-ya, K., Yoshida, M.,
VanSoest, R.W., Matsunaga, S., 2009. Isolation of azaspiracid-2 from a marine
sponge Echinoclathria sp. as a potent cytotoxin. Toxicon 53, 680–684.
1
3
Vlahov, G., 2009. C nuclear magnetic resonance spectroscopy to determine fatty
acid distribution in triacylglycerols of vegetable oils with ‘‘high–low oleic acid’’
and ‘‘high linolenic acid’’. Open Magn. Reson. J. 2, 8–19.
Nohara, T., Iwakawa, E., Matsushita, S., Fujiwara, Y., Ikeda, T., Miyashita, H., Ono, M.,
Yoshimitsu, H., 2008. A Pregnane glycoside from Overripe Tomato. Chem. Pharm.
Bull. 56, 1013–1014.
Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-induced edema in hind paw
of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 111,
544–547.