New ceramides and isoflavone from the Egyptian Iris germanica L. rhizomes

Article abstract of DOI:10.1016/j.phytol.2013.04.009

Two new ceramides, irisamides A (1) and B (2), together with a new isoflavone, iridin S (3) have been isolated from the MeOH extract of the rhizomes of Iris germanica L. (Iridaceae). Their structures were established by UV, IR, HRESIMS, 1D and 2D NMR experiments, in addition to comparison with literature data. The isolated compounds were tested for their cytotoxic activity against different cancer cell lines.

Full text of DOI:10.1016/j.phytol.2013.04.009

Phytochemistry Letters 6 (2013) 340–344
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
New ceramides and isoflavone from the Egyptian Iris germanica L. rhizomes
Gamal A. Mohamed a,b, Sabrin R.M. Ibrahim c, Samir A. Ross d,e,
*
a Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
b Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
c Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
d National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
e Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new ceramides, irisamides A (1) and B (2), together with a new isoflavone, iridin S (3) have been
isolated from the MeOH extract of the rhizomes of Iris germanica L. (Iridaceae). Their structures were
established by UV, IR, HRESIMS, 1D and 2D NMR experiments, in addition to comparison with literature
data. The isolated compounds were tested for their cytotoxic activity against different cancer cell lines.
ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Received 4 January 2013
Received in revised form 8 April 2013
Accepted 10 April 2013
Available online 1 May 2013
Keywords:
Iris germanica L.
Iridaceae
Irisamides
Iridin S
Cytotoxicity
1. Introduction
2. Results and discussion
Iris germanica L. (Iridaceae) is widely distributed in most
parts of the world, and also cultivated as an ornamental plant
(Rahman et al., 2003a; Choudhary et al., 2005). The plant has
been used as antispasmodic, anti-inflammatory, emmengogue,
stimulant, diuretic, and violently cathartic (Rahman et al.,
2003b; Asghar et al., 2009). Concerning the diversity of chemical
classes of I. germanica L., several previous reports were cited:
flavonoids (Rahman et al., 2003a; Choudhary et al., 2005; Akashi
et al., 2005; Asghar et al., 2009, 2010; Ibrahim et al., 2012;
Eckhard et al., 2003; Orhan et al., 2003), triterpenes (Ito et al.,
1995; Orhan et al., 2002), benzene and benzoquinones deriva-
tives (Asghar et al., 2009, 2010). Our previous phytochemical
study of the methanol extract of the rhizomes led to the isolation
of different sterols and phenolic compounds (Ibrahim et al.,
2012). In continuation of our investigation, we report herein the
isolation and structural elucidation of two new ceramides and a
new isoflavone (Fig. 1). Their structures were verified by various
spectroscopic methods. All the isolated compounds were
evaluated for their cytotoxic activity.
Compound 1 was isolated as a white amorphous powder. The
1H and 13C NMR spectra revealed the presence of two amide
moieties, as reflected by the exchangeable proton signals at
(1H, d, J = 8.8 Hz) and 8.45 (1H, d, J = 8.8 Hz), and the carbon signals
at C 175.3 and 173.4 and were confirmed by IR absorption bands at
3345, 1628, and 1542 cmꢀ1. The observed terminal methyl groups
at dH 0.84 (3H, t, J = 7.4 Hz, H-260)/ C14.3 (C-260), 0.86 (3H, t,
dH 8.65
d
d
J = 7.1 Hz, H-23)/dC 11.6 (C-23), and 0.88 (3H, d, J = 6.8 Hz, H-24)/
19.4 (C-24) were assigned to normal and ante-iso type side chains
(Table 1) (Inagaki et al., 2006; Tanaka et al., 1998). The presence of
carbons resonating at d
C 62.2/62.1 (C-1), 72.5 (C-20), 76.7/76.8 (C-
3), and 73.0/73.1 (C-4) in the DEPT and 13C NMR spectra indicated
the presence of hydroxymethyl and three oxygenated methine
groups, respectively, which appear in the 1H NMR spectrum at dH
4.52 (1H, dd, J = 10.7, 4.7 Hz, H-1A) and 4.42 (1H, dd, J = 10.7,
4.7 Hz, H-1B) for the hydroxymethyl group, and
4.28 (each 1H, m) for the oxygenated methines. In addition, the 1H
spectrum showed four OH moieties at H 7.65, 6.71, 6.50, and 6.21
dH 4.73, 4.62, and
d
(each 1H, brs), which were confirmed by the IR absorption band at
3415 cmꢀ1. All the previous signals, in addition to the cluster for
methylene groups at dC 29.6–30.4 and dH 1.20–1.33 and methine
groups at dH/dC 5.11/53.0 and 5.08/53.8 suggested the presence of a
* Corresponding author at: Department of Pharmacognosy, School of Pharmacy,
The University of Mississippi, University, MS 38677, USA. Tel.: +1 662 915 1031;
fax: +1 662 915 7989.
ceramide structure (Shin and Seo, 1995). All the NMR data
indicated the ceramide was present in mixture form. This was
confirmed from HRESIMS that showed pseudo-molecular ion
E-mail address: sross@olemiss.edu (S.A. Ross).
1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2013.04.009

G.A. Mohamed et al. / Phytochemistry Letters 6 (2013) 340–344
341
R
26`
11`
13`
14
15`
17`
19`
21`
21
23`
O
7`
9`
1`
5`
3`
14`
15
18`
20`
20
4`
5
6`
8`
10`
12`
16`
22`
24`
23
2`
25`
OH
NH
22
HO
11
19
7
9
13
17
3
1
16
8
18
6
2
4
10
12
OH
1
2
R= H
24
R= OH
OH
HO
O
HO
8
O
O
7
OH
2
2`
3
OCH3
OCH3
1`
3`
4`
6
H3CO
5
O4
OH
6`
5`
OCH3
3
Fig. 1. Structures of the isolated compounds.
peaks at m/z 780.7732 [M+H]+ and 796.7675, which in combina-
were identified as hexacosanoic acid methyl ester (C27H54O2) and
(R)-2-hydroxyhexacosanoic acid methyl ester (C27H54O3). Accord-
ingly, the molecular formula of the base had to be C24H51NO3,
which was confirmed by the fragment ion peaks at m/z 422.1032
[M-C22H48NO4]+ and 380.2456 [M-C24H51NO4]+ in the HRESIMS
spectrum (Fig. 3). From the above mentioned data, compound 1 is a
ceramide mixture has two fatty acids, one hydroxylated and the
other non-hydroxylated, as well as phytosphingosine with a
terminal ante-iso moiety. The relative stereochemistry of 1 was
tion with 13C and DEPT data indicated a molecular formula of
C50H101NO4 and C50H101NO5 with one degree of unsaturation due
to the carbonyl carbon of the amide group. The difference between
the two compounds is 16 mass unit this means that one hydroxyl
group. Although the number of carbons of the alkyl chains
remained unknown, a 2-amino-1,3,4-triol phytosphingosine de-
rivative was established in the structure on the basis of 2D NMR
spectroscopy, including 1H–1H COSY (Fig. 2), where the observed
cross peaks between the H-2 proton at
H 8.45/8.65, 4.52/4.42, and 4.73 for amide (NH), methylene (H-1),
and methine (H-3), respectively. Other COSY correlations include
the H-3 with H-4 proton at H 4.28, which further had cross peaks
with the H-5 protons at dH 2.27 and 1.95. The H-20 protons at dH
2.46/4.62 also showed a cross peak with H-30 protons at dH 1.82/
2.02, 2.22. The length of the base and the structure of the fatty acid
methyl esters (FAMs) were determined by methanolysis using HCl-
MeOH (Hattori et al., 1998; Tanaka et al., 1998). FAMs were
extracted with n-hexane and subjected to GC–MS analysis, which
afforded molecular ion peaks at m/z 410 [M]+ and 426 [M]+, which
d
H 5.08/5.11 with protons at
determined to be D-erythro, as the naturally occurring phyto-
d
sphingosine base (Inagaki et al., 2006; Tanaka et al., 1998; Kawano
et al., 1988; Sugiyama et al., 1988; Muralidhar et al., 2003), while
the absolute stereochemistry at C-2, C-3, and C-4 was determined
as 2S,3S,4R, since 1H and 13C NMR spectral data of 1 [
dH 4.52 (dd,
d
J = 4.7, 10.7 Hz, H-1A), 4.42 (dd, J = 4.7, 10.7 Hz, H-1B)/dC 62.2 (C-
1), dH 5.08 (m, H-2)/dC 53.8 (C-2), dH 4.73 (m, H-3)/dC 76.7 (C-3),
and H-4 at dH 4.28 (m, H-4)/dC 73.0 (C-4)] and were in a good
agreement with those of (2S,3S,4R)-
tricosanoyl)-2-amino-18-methyl-isocosan-1,3,4-triol
D
-erythro-N-(20R-hydroxy-
isolated
from marine sponge Strepsichordaia lendenfeldi [dH 4.52 (dd,
Table 1
NMR spectroscopic data of compounds 1 and 2 (C5D5N, 500 and 125 MHz).
No.
1
2
dH [mult., J (Hz)]
dC (mult.)
1H–1H COSY
dH [mult., J (Hz)]
dC (mult.)
1H–1H COSY
1
4.52 dd (4.7, 10.7) 4.42 dd (4.7, 10.7)
62.2 (CH2)
53.8 (CH)
76.7 (CH)
73.0 (CH)
33.9 (CH2)
26.6 (CH2)
29.6–30.4 (CH2)
25.8 (CH2)
37.3 (CH2)
36.8 (CH)
11.6 (CH3)
19.4 (CH3)
173.4 (C)
34.2 (CH2)
26.4 (CH2)
29.6–30.4 (CH2)
32.0 (CH2)
22.8 (CH2)
14.3 (CH3)
–
2
4.52 dd (4.7, 10.7) 4.42 dd (4.7, 10.7)
62.1 (CH2)
53.0 (CH)
76.8 (CH)
73.1 (CH)
35.7 (CH2)
26.7 (CH2)
29.6–30.4 (CH2)
27.4 (CH2)
37.3 (CH2)
34.6 (CH2)
11.6 (CH3)
19.4 (CH3)
175.3 (C)
72.5 (CH)
36.9 (CH2)
29.6–30.4 (CH2)
32.1 (CH2)
23.0 (CH2)
14.3 (CH3)
–
2
2
5.08 m
1, 3, NH
5.11 m
1, 3, NH
2, 4
3, 5
4, 6
5
3
4.73 m
2, 4
4.73 m
4
4.28 m
3, 5
4.28 m
5
1.95, 2.27 m
1.68 m
4, 6
1.95, 2.27 m
1.70 m
6
5
22, (CH2)12
1.28–1.33 m
1.25 m
–
1.28–1.33 m
1.25 m
–
19
20, 21
20, 21
21
20
1.25 m
21
20, 22
22
23
–
30
20
1.25 m
21
1.66 m
1.66 m
20, 22
22
23
0.86 t (7.1)
0.88 d (6.8)
–
0.86 t (7.1)
0.88 d (6.8)
–
24
23
10
20
2.46 t (7.6)
1.82 m
4.62 m
30
20
–
30
2.02, 2.22 m
1.24–1.27 m
1.44 m
(CH2)20
240
250
260
OH
20-OH
NH
1.20–1.24 m
1.44 m
–
–
–
1.12 m
–
1.12 m
–
0.84 t (7.4)
6.21, 6.50, 6.71 brs
–
–
0.84 t (7.4)
6.21, 6.50, 6.71 brs
7.65 brs
–
–
20
–
20
–
–
8.45 d (8.8)
–
2
8.65 d (8.8)
–
2

342
G.A. Mohamed et al. / Phytochemistry Letters 6 (2013) 340–344
Table 2
OH
NMR spectroscopic data of compound 3 (DMSO-d6, 500 and 125 MHz).
O
20
No.
dH [mult., J (Hz)]
dC (mult.)
HN
2
8.51 s
155.4 (CH)
125.9 (C)
180.5 (C)
152.9 (C)
132.5 (C)
156.6 (C)
94.0 (CH)
152.9 (C)
104.5 (C)
122.0 (C)
106.4 (CH)
152.4 (C)
141.4 (C)
152.4 (C)
106.4 (CH)
59.9 (CH3)
60.3 (CH3)
60.6 (CH3)
55.8 (CH3)
–
OH
3
–
HO
4
–
12
5
–
OH
6
–
7
–
Fig. 2. Important 1H–1H COSY correlations of 2.
8
6.70 s
9
–
10
–
10
–
J = 4.4, 10.7 Hz, H-1A), 4.43 (dd, J = 4.7, 10.7 Hz, H-1B)/dC 62.1 (C-
1), dH 5.10 (m, H-2)/dC 53.0 (C-2), dH 4.36 (m, H-3)/dC 76.7 (C-3),
and dH 4.28 (m, H-4)/dC 73.1 (C-4)], and further confirmed by
20
6.91 brs
–
30
40
–
50
–
comparing the optical rotation [a] value (+10.7) (Natori et al.,
D
60
6.91 brs
3.71 s
3.80 s
3.78 s
3.58 s
12.92 s
5.12 d (7.5)
5.35–4.50
1994; Zhang et al., 2005; Radhika et al., 2004; Elkhayat et al., 2012).
Accordingly, the structure of compound 1 was assigned as N-
((2S,3S,4R)-1,3,4-trihydroxy-21-methyltricosan-2-yl)hexacosana-
mide, and named irisamide A (1), where is the other was assigned
as (2R)-2-hydroxy-N-((2S,3S,4R)-1,3,4-trihydroxy-21-methyltri-
cosan-2-yl)hexacosanamide, and named irisamide B (2).
Iridin S (3) was isolated as a yellow amorphous powder
(MeOH). The UV spectrum of 3 showed absorption bands at 211,
261, and 329 nm, suggesting its isoflavone nature and further
40-OCH3
30-OCH3
50-OCH3
6-OCH3
5-OH
100
100.1 (CH)
73.1 (CH)
77.3 (CH)
69.6 (CH)
76.7 (CH)
60.6 (CH2)
200
300
400
500
600
established by the singlet signal at dH 8.51 for H-2 (Harborne et al.,
1975; Mabry et al., 1970). The IR spectrum showed absorption
bands at 3437, 2949, 1651, and 1059 cmꢀ1, indicating the presence
of OH, aromatic C–H,
in the molecule. Compound 3 possessed a molecular formula
25H28O13 by HRESIMS that showed a pseudomolecular ion peak at
m/z 537.1537 [M+H]+. The 1H NMR spectrum showed resonances
for 28 protons; four aromatic protons singlets at dH 8.51 (s, H-2),
6.91 (brs, H-20, 60), 6.70 (s, H-8), four methoxy group signals at dH
3.58 (6-OCH3), 3.71 (40-OCH3), 3.78 (50-OCH3), and 3.80 (30-OCH3),
and a hydrogen bonded phenolic hydroxyl group at dH 12.92 (5-
OH) (Table 2). Furthermore, the 1H NMR spectrum displayed
a
,
b
-unsaturated carbonyl, and C–O functions
The cytotoxic effect of the isolated compounds was tested
against L5178Y, PC12, and Hela cell lines. It was found that
irisamides A (1) and B (2) were active against L5178Y and Hela cell
lines, while they were found to be inactive against PC12. Iridin S (3)
was inactive against the three cell lines. Irisamides A and B gave
76% and 63% growth suppression against the L5178Y and HeLa cell
C
lines at a concentration of 12.67
m
M as well as 68% and 49% at a
concentration of 3.81
tively.
m
M; ED50(s) of 0.91 and 2.40 M, respec-
m
anomeric proton signal at dH 5.12 (d, J = 7.5 Hz) indicated
b-
configuration of the glucosidic linkage. The 13C NMR showed
twenty five carbon signals, their multiplicities were determined
through DEPT. Considering the UV, 1H NMR, and 13C NMR results,
the four methoxyl groups could be placed at C-6, C-30, C-40, and C-50
as well as the glucose moiety at C-7. Acid hydrolysis of 3 yielded
5,7-dihydroxy- 6,30,40,50-tetramethoxy isoflavone (aglycone) and
3. Experimental
3.1. General experimental procedures
Optical rotation was measured on a Perkin-Elmer Model 341 LC
polarimeter. HRESIMS was recorded on a LTQ Orbitrap (Thermo-
Finnigan, Bremen, Germany). Melting points was determined
using an Electrothermal 9100 Digital Melting Point apparatus
(Electrothermal Engineering Ltd., Essex, England). UV spectra
were recorded in absolute MeOH on a Shimadzu 1601 UV/VIS
spectrophotometer. The IR spectra were measured on a Shimadzu
Infrared-400 spectrophotometer (Kyoto, Japan). 1D and 2D NMR
spectra (chemical shifts in ppm, coupling constants in Hz) were
recorded on Bruker BioSpin GmbH 500 MHz Ultrashield spec-
trometer using standard Bruker software and DMSO-d6, and
C5D5N as solvents, with TMS as the internal reference. Solvents
were distilled prior spectroscopic measurements. A GCMS was
b-D-glucose. The sugar in the aqueous layer was identified by co-PC
with authentic sample. The glycosylation at position C-7 was
established by UV spectral data of aglycone with diagnostic shift
reagents. The bathochromic shift of the UV absorption band II; 39
and 22 nm upon addition of AlCl3 and NaOAc, respectively,
confirmed the presence of 5- and 7-OH groups, respectively. On
the basis of the above evidences, and by comparing the spectral
data with literature (Asghar et al., 2009), the structure of 3 was
elucidated as 5-hydroxy-6,30,40,50-tetramethoxy-7-O-
b-D-gluco-
pyranosyl isoflavone and considered as a new natural product,
for which we proposed the name iridin S.
m/z 380
m/z 422
OH
O
OH
HN
HO
m/z 779
m/z 240
m/z 126
OH
Fig. 3. Possible fragmentation pattern of 1 and 2.

G.A. Mohamed et al. / Phytochemistry Letters 6 (2013) 340–344
343
performed on Clarus 500 GCMS (Perkin Elmer, Shelton, CT). The
software controller/integrator was Turbo Mass, version 4.5.0.007
(Perkin Elmer). An Elite 5MS GC capillary column (30 ꢁ
residue was suspended in H2O and extracted with EtOAc to yield
the phytosphingosine moiety (Elkhayat et al., 2012).
0.25 mm ꢁ 0.5
m
m, Perkin Elmer) was used. The carrier gas was
3.5. Acid hydrolysis
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 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. Column chro-
matographic separations were performed on silica gel 60 (0.04–
0.063 mm) and Sephadex LH-20 (0.25–0.1 mm, Merck). TLC was
performed on precoated TLC plates with silica gel 60 F254 (layer
thickness 0.2 mm, Merck). The solvent systems used for TLC
analyses were CHCl3:MeOH (97:3, S1), CHCl3:MeOH (90:10, S2),
and n-BuOH:acetone:formic acid:H2O (60:17:8:15, S3). The
compounds were detected by UV absorption at lmax 255 and
366 nm followed by spraying with anisaldehyde/H2SO4 reagent
and heating at 110 8C for 1–2 min.
A solution of the isolated glycoside 3 (3 mg in 5 mL MeOH) was
treated with 3% H2SO4 (1.5 mL) and heated at 100 8C for 1 hr. The
aglycone was extracted with EtOAc, concentrated under reduced
pressure, purified on Sephadex LH-20 column using MeOH. The
sugar in the aqueous layer was identified by co-PC (paper
chromatography) with an authentic sample using solvent system
S3 (El-Shanawany et al., 2012).
3.6. Spectral data
Irisamides A (1) and B (2). White amorphous powder (MeOH);
Rf = 0.84 (S1); [a]
D + 10.7 (C 0.2, CHCl3); IR vmax (cmꢀ1): 3415,
3345, 2937, 1628, 1542, 1065; HRESIMS: m/z 780.7732 (calcd for
C50H101NO4, 780.7731) and 796.7675 (calcd for C50H101NO5,
796.7679); NMR spectral data, see Table 1.
Iridin S (3). Yellow amorphous powder (MeOH); Rf = 0.67 (S2);
3.2. Plant material
m.p. 203–205 8C (dec.); UV (MeOH)
(cmꢀ1): 3437, 2949, 1651, 1059; HRESIMS: m/z 537.1537 (calcd for
25H29O13 537.1529); NMR spectral data, see Table 2.
lmax nm: 211, 261, 329; IR vmax
The rhizomes of I. germanica L. were collected in April 2007
from the growing plants at the botanical garden of Faculty of
Agriculture, Assiut University. The plant material was kindly
identified by Prof. Dr. A. Fayed, Professor of Plant Taxonomy,
Faculty of Science, Assiut University. A voucher specimen was
deposited in herbarium of the Department of Pharmacognosy,
Faculty of Pharmacy, Al-Azhar University, Assiut under the
registration number IG-1-2007.
C
3.7. Cytotoxicity test
Mouse lymphoma (L5178Y), rat brain (PC12) and human cervix
(Hela) cancer cell lines were used. All cells were mycoplasma-free
and cultures were propagated under standardized conditions
(Kreuter et al., 1992). The cytotoxicity was evaluated by the
[3H] thymidine assay at concentrations of 3.81 and 12.67
(Carmichael et al., 1987).
mM
3.3. Extraction and Isolation
The air-dried powdered rhizomes of I. germanica L. (320 g)
were extracted with MeOH 70% (6 ꢁ 2 L) at room temperature to
afford a dark brown residue (18.1 g). The total MeOH extract was
subjected to normal phase vacuum liquid chromatography (VLC)
using n-hexane, CHCl3, EtOAc, and MeOH to afford 4 fractions; n-
hexane IG-1 (3.5 g), CHCl3 IG-2 (4.0 g), EtOAc IG-3 (3.2 g), and
MeOH IG-4 (5.3 g). Fraction IG-1 (3.5 g) was subjected to VLC by
gradient elution using n-hexane:EtOAc as solvents, and four
subfractions were obtained; IG-1-A (720 mg, n-hexane, 100%), IG-
1-B (650 mg, n-hexane:EtOAc, 75:25), IG-1-C (820 mg, n-hex-
ane:EtOAc, 50:50), and IG-1-D (960 mg, EtOAc, 100%). Subfraction
IG-1-D (960 mg) was chromatographed over silica gel column
several times (200 g ꢁ 50 ꢁ 3 cm) using n-hexane:EtOAc gradient
to afford compounds 1 and 2 (41 mg). EtOAc fraction IG-3 (3.2 g)
was subjected to Sephadex LH-20 column chromatography
(150 g ꢁ 100 ꢁ 5 cm) using MeOH as an eluent to afford four
subfractions IG-3-A (0.45 g), IG-3-B (0.51 g), IG-3-C (0.75 g), and
IG-3-D (0.68 g). Subfraction IG-3-A and IG-3-B were investigated
previously (Ibrahim et al., 2012). A sephadex LH-20 column
chromatorgraphy (100 g ꢁ 50 ꢁ 3 cm) of subfraction IG-3-C
(0.75 g) using MeOH gave impure compound 3 which was further
purified by repeated chromatography on silica gel column
(50 g ꢁ 50 ꢁ 2 cm) using CHCl3:MeOH gradient to afford com-
pound 3 (9 mg).
4. Conclusions
Three new compounds: irisamides A (1) and B (2) in addition to
iridin S (3) were isolated from the rhizomes of I. germanica L.
growing in Egypt and evaluated for their cytotoxic activity using
different cancer cell lines.
References
Akashi, T., Ishizaki, M., Aoki, T., Ayabe, S., 2005. Isoflavonoid production by
adventitious-root culture of Iris germanica (Iridaceace). Plant Biotechnol. 22,
207–215.
Asghar, S.F., Aziz, S., Rehman, H.U., Ahamed, I., Hussein, H., Rahman, A.U., Choudh-
ary, M.I., 2009. Secondary metabolites isolated from Iris germanica. Rec. Nat.
Prod. 3, 139–152.
Asghar, S.F., Rehman, H.U., Rahman, A.U., 2010. Phytochemical investigation of Iris
germanica. Nat. Prod. Res. 24, 131–139.
Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evaluation
of a tetrazolium-based semi-automated colorimetric assay: assessment of radio
sensitivity. Cancer Res. 47, 943–946.
Choudhary, M.I., Naheed, S., Jalil, S., Alam, J.M., Rahman, A.U., 2005. Effect of
ethanolic extract of Iris germanica on lipid profile of rats fed on a high-fat diet.
J. Ethnopharmacol. 98, 217–220.
Eckhard, W., Frederik, S.J., Karin, K., Jutta, K., Norbert, F., Clarissa, G., 2003. Cancer
chemoprotective in vitro activities of isoflavones isolated from Iris germanica.
Planta Med. 69, 15–20.
El-Shanawany, M.A., Sayed, H.M., Ibrahim, S.R.M., Fayed, M.A.A., Radwan, M.M.,
Ross, S.A., 2012. A new isoflavone from Blepharis ciliaris of an Egyptian origin.
Med. Chem. Res. 19, 2346–2350.
Elkhayat, E.S., Mohamed, G.A., Ibrahim, S.R.M., 2012. Activity and structure eluci-
dation of ceramides. Curr. Bioactive Compd. 8, 370–409.
3.4. Methanolysis
Harborne, B., Mabry, T.J., Mabry, H., 1975. The Flavonoids, vols. 1 and 2 Academic
Press, New York, NY, USA, , pp. 50–55, 743–761.
Hattori, T., Adachi, K., Shizuri, Y., 1998. New ceramide from marine sponge Haliclona
koremella and related compounds as antifouling substances against microalgae.
J. Nat. Prod. 61, 823–826.
Irisamides (8 mg) were subjected to methanolysis by boiling
with 5 mL of 1 N HCl in MeOH at 80 8C for 18 hr. Cold water
(10 mL) was added to the reaction mixture, which was extracted
with n-hexane to yield the corresponding fatty acid methyl
esters. After removal of MeOH under reduced pressure, the
Ibrahim, S.R.M., Mohamed, G.A., Al-Musayeib, N.M., 2012. New constituents from
the rhizomes of Egyptian Iris germanica L. Molecules 17, 2587–2598.

344
G.A. Mohamed et al. / Phytochemistry Letters 6 (2013) 340–344
Inagaki, M., Ikeda, Y., Kawatake, S., Nakamura, K., Tanaka, M., Misawa, E., Yamada,
M., Higuchi, R., 2006. Isolation and structure of four new ceramides from the
Starfish Luidia maculate. Chem. Pharm. Bull. 54, 1647–1649.
Ito, H., Miyake, Y., Yoshida, T., 1995. New piscicidal triterpenes from Iris germanica.
Chem. Pharm. Bull. 43, 1260–1262.
Kawano, Y., Higuchi, R., Isobe, R., Komori, T., 1988. Isolation and structure of six new
cerebrosides. Liebigs Ann. Chem. 19–24.
Kreuter, M.H., Robitzki, A., Chang, S., Steffen, R., Michaelis, M., Kljajic, Z., Bachmann,
M., Schroeder, H.C., Mueller, W.E.G., 1992. Production of cytotoxic agent aero-
plysinin by the sponge Verongia aerophoba in in vitro culture. Comp. Biochem.
Physiol. 101 C, 183–187.
Orhan, I., Sener, B., Hashimoto, T., Asakawa, Y., Ozgu¨ven, M., Ayanog˘lu, F., 2002.
Iristectorone K, a novel monocyclic triterpene ester from Iris germanica rhi-
zomes growing in Turkey. Fitoterapia 73, 316–319.
Radhika, P., Rao, V.L., Laatsch, H., 2004. Chemical constituents of a marine soft coral
of the genus Lobophytum. Chem. Pharm. Bull. 52, 1345–1348.
Rahman, A.U., Nasim, S., Baig, I., Orhan, I., Sener, B., Ayanoglu, F., Choudhary, I.M.,
2003a. Isoflavonoid glycosides from the rhizomes of Iris germanica. Helv. Chim.
Acta 86, 3354–3362.
Rahman, A.U., Nasim, S., Biag, I., Jalil, S., Orhan, I., Sener, B., Choudhary, M.I., 2003b.
Anti-inflammatory isoflavonoids from the rhizomes of Iris germanica. J. Ethno-
pharmacol. 86, 177–180.
Mabry, T.J., Markham, K.R., Thomas, T.B., 1970. The Systematic Identification of
Flavonoids. Springer-Verlag, Berlin, Germany, pp. 35–61.
Shin, J., Seo, Y., 1995. Isolation of new ceramides from the Gorgorian Acabaria
undulata. J. Nat. Prod. 58, 948–953.
Muralidhar, P., Krishna, N., Kumar, M.M., Rao, C.B., Rao, D.V., 2003. New sphingo-
lipids from marine sponge Iotrochota baculifera. Chem. Pharm. Bull. 51,
1193–1195.
Sugiyama, S., Honda, M., Komori, T., 1988. A symmetric synthesis of phytosphin-
gosine and phytosphingosine anhydro base: assignment of the absolute ste-
reochemistry. Liebigs Ann. Chem. 619–625.
Natori, T., Morita, M., Akimoto, K., Koezuka, Y., 1994. Agelasphins, novel antitumor
and immunostimulatory cerebrosides from the marine sponge Agelas maur-
itianus. Tetrahedron 50, 2771–2784.
Tanaka, I., Matsuoka, S., Murata, M., Tachibana, K., 1998. A new ceramide with a
novel branched-chain fatty acid isolated from the Epiphytic Dinoflagellate
Coolia monotis. J. Nat. Prod. 61, 685–688.
¨
Orhan, I., Nasim, S., Tener, B., Ayanoglu, F., Ozgu¨ven, M., Choudhary, I.M., Rahman,
Zhang, G., Ma, X., Zhang, C., Su, J., Ye, W., Zhang, X., Yao, X., Zeng, L., 2005. Two new
ceramides from the marine sponge Ircinia fasciculate. Helv. Chem. Acta 88,
885–890.
A.U., 2003. Two isoflavones and bioactivity spectrum of the crude extract if Iris
germanica rhizomes. Phytother. Res. 17, 575–577.