Urgineaglyceride A: A new monoacylglycerol from the Egyptian Drimia maritima bulbs

Article abstract of DOI:10.1080/14786419.2014.927468

One new compound, (2S)-1-O-(Z)-tetracos-6-enoate glycerol (1) named urgineaglyceride A, along with six known compounds, 3,5,7,3′,5′-pentahydroxydihydroflavonol (2), stigmasterol (3), (25S)-5β-furostane-3β-22α-26-triol (4), scillaridin A (5), (2S)-(+)-2-hydroxynaringenin-4′-O-β-d-glucopyranoside (6) and quercetin-3′-O-β-d-glucopyranoside (7), were isolated from the EtOAc fraction of Drimia maritima (L.) Stearn bulbs. Their structures were secured based on their IR, UV, 1D and 2D NMR data, in addition to HR-MS data and comparison with the literature data. The isolated compounds were evaluated for their in vitro growth inhibitory activity against A549 non-small cell lung cancer (NSCLC), U373 glioblastoma (GBM) and PC-3 prostate cancer cell lines. Compounds 2 and 3 displayed variable activities against the tested cancer cell lines. Compound 2 was a selective inhibitor of the NSCLC cell line with an IC₅₀ of 2.3 μM, whereas 3 was selective against GBM with IC₅₀ of 0.5 μM and against PC-3 with 2.0 M.

Full text of DOI:10.1080/14786419.2014.927468

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Urgineaglyceride A: a new
monoacylglycerol from the Egyptian
Drimia maritima bulbs
Gamal A. Mohameda, Sabrin R.M. Ibrahimb, Lamiaa A. Shaalacd,
Khalid Z. Alshalie & Diaa T.A. Yousseffg
a Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar
University, Assiut Branch, Assiut 71524, Egypt
b Department of Pharmacognosy, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt
c Suez Canal University Hospital, Suez Canal University, Ismailia
41522, Egypt
d Natural Products Unit, King Fahd Medical Research Center, King
Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
e Department of Medicine, Faculty of Medicine, King Abdulaziz
University, Jeddah 21589, Kingdom of Saudi Arabia
f Department of Natural Products, Faculty of Pharmacy, King
Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
g Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal
University, Ismailia 41522, Egypt
Published online: 18 Jun 2014.
To cite this article: Gamal A. Mohamed, Sabrin R.M. Ibrahim, Lamiaa A. Shaala, Khalid Z. Alshali
& Diaa T.A. Youssef (2014) Urgineaglyceride A: a new monoacylglycerol from the Egyptian Drimia
maritima bulbs, Natural Product Research: Formerly Natural Product Letters, 28:19, 1583-1590,
DOI: 10.1080/14786419.2014.927468
To link to this article: http://dx.doi.org/10.1080/14786419.2014.927468
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Natural Product Research, 2014
Vol. 28, No. 19, 1583–1590, http://dx.doi.org/10.1080/14786419.2014.927468
Urgineaglyceride A: a new monoacylglycerol from the Egyptian Drimia
maritima bulbs
Gamal A. Mohameda, Sabrin R.M. Ibrahimb, Lamiaa A. Shaalacd, Khalid Z. Alshalie and
Diaa T.A. Yousseffg*
aDepartment of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524,
Egypt; Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt;
b
d
cSuez Canal University Hospital, Suez Canal University, Ismailia 41522, Egypt; Natural Products Unit,
King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia;
eDepartment of Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Kingdom of
Saudi Arabia; fDepartment of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah
g
21589, Kingdom of Saudi Arabia; Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal
University, Ismailia 41522, Egypt
(Received 3 April 2014; final version received 20 May 2014)
One new compound, (2S)-1-O-(Z)-tetracos-6-enoate glycerol (1) named urgineagly-
ceride A, along with six known compounds, 3,5,7,30,50-pentahydroxydihydroflavonol
(2), stigmasterol (3), (25S)-5b-furostane-3b-22a-26-triol (4), scillaridin A (5), (2S)-
(þ)-2-hydroxynaringenin-40-O-b-D-glucopyranoside (6) and quercetin-30-O-b-D-
glucopyranoside (7), were isolated from the EtOAc fraction of Drimia maritima (L.)
Stearn bulbs. Their structures were secured based on their IR, UV, 1D and 2D NMR
data, in addition to HR-MS data and comparison with the literature data. The isolated
compounds were evaluated for their in vitro growth inhibitory activity against A549
non-small cell lung cancer (NSCLC), U373 glioblastoma (GBM) and PC-3 prostate
cancer cell lines. Compounds 2 and 3 displayed variable activities against the tested
cancer cell lines. Compound 2 was a selective inhibitor of the NSCLC cell line with an
IC50 of 2.3 mM, whereas 3 was selective against GBM with IC50 of 0.5 mM and against
PC-3 with 2.0 mM.
Keywords: Drimia maritima; monoacylglycerol; urgineaglyceride A; in vitro growth
inhibitory activity
1. Introduction
Drimia maritima (L.) Stearn (syn. Urginea maritima) (Asparagaceae) is a native plant found in
the Mediterranean area, North Africa and India. It is known in Arabic as Basal Farion, Onsul,
Basal el-far, Samm el-far and Ishkil (Boulos 2002). It is used as a cardiotonic diuretic for the
treatment of cardiac marasmus and oedema (Iizuka et al. 2001; Metin & Bu¨ru¨n 2010; Bozcuk
et al. 2011). It is also used in bronchitis, bronchial asthma, whooping cough and cancer (Duke &
Ayensu 1985; Bown 1995). Externally, the bulb is applied for skin problems such as injury,
haemorrhoids, warts, dandruff and seborrhoea (Metin & Bu¨ru¨n 2010). It exhibits insecticidal
and cytotoxic effects (Iizuka et al. 2001; Boulos 2002; Metin & Bu¨ru¨n 2010; Bozcuk et al. 2011;
El-Seedia et al. 2013). Previous phytochemical studies of D. maritima bulbs resulted in the
isolation of cardiac glycosides (Patel et al. 1986; Kopp et al. 1996; Krenn et al. 2000; Iizuka et al.
2001; Metin & Bu¨ru¨n 2010; El-Seedia et al. 2013), anthocyanins, lignans, flavonoids, fatty acids
*Corresponding author. Email: dyoussef@kau.edu.sa
q 2014 Taylor & Francis

1584
G.A. Mohamed et al.
and polysaccharides (Fernandez et al. 1972; Vega et al. 1972; Fernandez et al. 1975; Iizuka et al.
2001). Bioassay-directed fractionation of the EtOAc fraction of D. maritima bulbs using
different cancer cell lines (A549 non-small cell lung cancer (NSCLC), U373 glioblastoma
(GBM) and PC-3 prostate cancer) led to the isolation of one new compound, (2S)-1-O-(Z)-
tetracos-6-enoate glycerol (1) named urgineaglyceride A, together with six known compounds,
3,5,7,30,50-pentahydroxydihydroflavonol (2) (Wang et al. 2008), stigmasterol (3) (Mohamed &
Ibrahim 2007), (25S)-5b-furostane-3b-22a-26-triol (4) (Yang et al. 2002; Jin et al. 2004),
scillaridin A (5) (Koorbanally et al. 2004), (2S)-(þ)-2-hydroxynaringenin-40-O-b-D-
glucopyranoside (6) (Kim et al. 2000; Ti et al. 2011) and quercetin-30-O-b-D-glucopyranoside
(7) (Malikov & Yuldashev 2002) (Figure 1). Compounds 4 and 7 are reported here for the first
time from D. maritima and 2 and 6 for the first time from this family. This study reports the
isolation and structure characterisation of the isolated compounds using spectroscopic analyses,
especially 1D and 2D NMR studies and HRMS. In addition, the in vitro growth inhibitory
activity of the isolated compounds was also evaluated.
OH
O
1`
13
O
HO
O
O
OH
1
HO
2
3
OH
OH
HO
OH
OH
1
2
3
O
O
OH
O
H
H
5
H
OH
HO
4
OH
OH
HO
O
HO
OH
OH
OH
HO
O
O
O
OH
OH
HO
O
O
HO
O
OH
O
OH
OH
6
Figure 1. Structures of the isolated compounds 1–7.
7

Natural Product Research
1585
2. Results and discussion
Compound 1 was obtained as colourless oil. The HR-ESI-MS spectrum showed a molecular ion
peak at m/z 441.3852 [M þ H]þ, which is consistent with a formula of C27H52O4. This requires
two double bond equivalents. The IR spectrum revealed absorption bands at 3435 (OH) and 1742
(CvO) cm21. The 1H and 13C NMR spectra for 1 revealed the presence of a fatty acid moiety as
reflected by the signals at dC 174.3 (s, C10), dH 2.35 (2H, t, J ¼ 7.6 Hz, H-20)/dC 34.1 (t, C-20),
dH 1.30–1.25 (m, (CH2)n)/dC 29.7–29.1 (t, (CH2)n), dH 0.88 (3H, t, J ¼ 7.2 Hz, H-24)/dC 14.1
(q, C-240) and confirmed by the observed 1H–1H COSY and HMBC correlations
(Supplementary Figure S7). The proton signals at dH 5.34 (m, H-60) and 5.33 (m, H-70)
correlated with the carbon signals at dC 129.7 (C-60) and 130.0 (C-70) in the HSQC spectrum,
respectively, indicating the presence of an olefinic double bond in 1. The position of the double
bond was established by the HMBC correlations from H-50 to C-60 and C-70 andþfrom H-60 and
H-70 to C-50 and confirmed by the fragment ion peaks at m/z 350 [M-C3H7O3] and 265 [M-
C8H15O4]þ in the ESI-MS spectrum (Supplementary Figure S8). The geometry of the double
bond between C-60 and C-70 was deduced to be Z from the 1H–1H coupling constant of 7.6 Hz
between H-60 and H-70 (Bankeu et al. 2010). The signals at dC 65.2 (C-1)/dH 4.19 (1H, dd,
J ¼ 11.6 and 4.8 Hz, H-1a) and 4.15 (1H, dd, J ¼ 11.6 and 4.8 Hz, H-1b), dC 63.3/dH 3.69 (1H,
dd, J ¼ 12.0 and 6.4 Hz, H-3a) and 3.60 (1H, dd, J ¼ 12.0 and 6.4 Hz, H-3b) and dC 70.3/dH 3.93
(1H, m, H-2) indicated the presence of an asymmetrical substituted glycerol moiety (Vlahov
1999). This was supported by the fragment ion peaks at m/z 350 [M-glycerol]þ, 423 [M-H2O]þ
and 406 [M-2H2O] in the ESI-MS spectrum, also was confirmed by the HMBC correlations
of H-1 and H-3 with C-2 and H-2 with C-1 and C-3. The attachment of the fatty acid moiety
at C-1 of glycerol was confirmed by the HMBC correlation of H-1 with C-10. According to
the ESI-MS spectrum, the fatty acid moiety should be (Z)-tetracos-6-enoic acid. This was
established by alkaline hydrolysis of 1 using NaOMe–MeOH (Kim et al. 2005; Al-Musayeib
et al. 2013). The resulting fatty acid methyl ester (FAME) was extracted with n-hexane and
subjected to GC–MS analysis, which afforded molecular ion peak at m/z 380 [M]þ and was
identified as (Z)-tetracos-6-enoic acid methyl ester (C25H48O2). The stereochemistry at C-2
1
was assigned as 2S based on REOESY experiment as well as comparison of H and 13C
NMR chemical shifts and optical rotation with the reported data for glyceride analogue
(Dharma et al. 1985; Ibrahim et al. 2010; Ibrahim 2014) (Supplementary Figures S1–S6).
Thus, the structure of 1 was elucidated as (2S)-1-O-(Z)-tetracos-6-enoate glycerol and a
trivial name urgineaglyceride A was given.
The known compounds were identified by analysing the spectroscopic data (1D, 2D NMR
and MS) and comparing their data with those in the literature as 3,5,7,30,50-pentahydroxydihy-
droflavonol (2) (Wang et al. 2008), stigmasterol (3) (Mohamed & Ibrahim 2007), (25S)- 5b-
furostane-3b-22a-26-triol (4) (Yang et al. 2002; Jin et al. 2004), scillaridin A (5) (Koorbanally
et al. 2004), (2S)-(þ)-2-hydroxynaringenin-40-O-b-D-glucopyranoside (6) (Kim et al. 2000; Ti
et al. 2011) and quercetin-30-O-b-D-glucopyranoside (7) (Malikov & Yuldashev 2002).
Compounds 4 and 7 are reported here for the first time from the plant, and 2 and 6 for the first
time from the family.
The determination of the in vitro growth inhibitory activity using the 3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay for n-
BuOH, EtOAc and EtOAc subfractions, as well as the isolated pure compounds, revealed
that the subfractions UM-2 to UM-7 showed significant activity against A549 NSCLC, PC-3
prostate cancer and U373 GBM cell lines, with IC50 values from 0.02 to 3.7 mg/mL (Table 1).
Concerning the pure compounds, 2 and 3 displayed in vitro growth inhibitory activity in the
three cancer cell lines under study, with magnitude of effects that ranged between those of
cisplatin (IC50 from 0.4 to 4.0 mM, Table 1). Compound 2 has selectivity against A549 NSCLC

1586
G.A. Mohamed et al.
Table 1. In vitro growth inhibitory activity of the tested fractions and compounds 1–7.
IC50 (mM)
Sample
A549 NSCLC
U373 GBM
PC-3 prostate cancer
Mean ^ SEM
EtOAca
n-BuOHa
UM-1a
UM-2a
UM-3a
UM-4a
UM-5a
UM-6a
UM-7a
UM-8a
UM-9a
1
0.009
0.03
.100
0.006
0.005
0.006
0.01
0.2
1
3
2
41
2.3
10
0.04
0.1
0.06
0.2
.100
0.2
0.01
0.08
0.2
1
4
11
0.04 ^ 0.01
0.11 ^ 0.05
.100
0.08 ^ 0.06
0.02 ^ 0.01
0.04 ^ 0.02
0.17 ^ 0.08
1.7 ^ 1.2
3.7 ^ 1.4
15 ^ 8
12 ^ 6
.100
.100
4.4 ^ 0.6
88.6
.100
.100
0.03
0.03
0.04
0.3
4
6
30
22
11
.100
.100
0.5
.100
.100
NT
.100
.100
2
.100
.100
NT
2
3
4
5
6
7
38.6
.100
NT
.100
4
–
.100
–
53.8
0.4
.100
NT
Cisplatinb
Note: NT, not tested.
a The growth inhibitory effects of the tested fractions are expressed as mg/mL.
b The cisplatin-related data have already been published (Ibrahim et al. 2012; Ibrahim et al. 2014).
at a concentration 2.3 mM. However, 1 and 4 displayed moderate activity against A549 NSCLC
model (Table 1), while 7 displayed moderate in vitro growth inhibitory activity against U373
GBM cell line. In addition, 5 had no activity towards the different cell lines.
3. Experimental
3.1. General experimental procedures
Melting points were obtained in an Electrothermal 9100 Digital Melting Point (Electrothermal
Engineering, Southend-on-Sea, Essex, UK). The UV spectra were carried out in MeOH (Merck,
Darmstadt, Germany) using a Perkin-Elmer Lambda 25 UV/VIS spectrophotometer (Perkin-
Elmer, Waltham, MA, USA). IR was measured with a Shimadzu Infrared-400 spectropho-
tometer (Shimadzu, Kyoto, Japan). Optical rotation was recorded on a Perkin-Elmer Model 341
LC Polarimeter (Perkin-Elmer). HR-ESI-MS was determined with a Micromass Qtof 2 mass
spectrometer (Micromass, Manchester, UK). EI-MS spectra were recorded on JEOL JMS-SX/
1
SX 102A mass spectrometer (ThermoFinnigan, Bremen, Germany). H and 13C NMR spectra
were measured on a Bruker DRX 400 spectrometer (Bruker, Rheinstetten, Germany). 1D NMR
spectroscopic data were measured at a temperature of 24.28C and 2D NMR (1H–1H COSY,
ROESY, HSQC and HMBC) spectroscopic data were measured at a temperature of 278C. A
GC–MS was performed on Clarus 500 GC–MS (Perkin-Elmer, Shelton, CT, USA). The
software controller/integrator was Turbo Mass, version 4.5.0.007 (Perkin-Elmer). An Elite 5MS
GC capillary column (30 mm £
0.25 mm £ 0.5 mM, Perkin-Elmer) was used. The carrier gas was helium (purity 99.9999%) at a
flow rate of 2 mL/min (32 psi, flow initial 55.8 cm/s, split; 1:40). Temperature conditions were as
follows: inlet line temperature, 2008C; source temperature, 1508C; trap emission, 1008C and

Natural Product Research
1587
electron energy, 70 eV. The column temperature program was as follows: 50 8C for 5 min,
increased to 2208C (rate, 208C/min) and held for 5 min. The injector temperature was 2208C. MS
scan was from 50 to 650 m/z. Vacuum liquid chromatography (VLC) was performed using silica
gel 60 (0.04–0.063 mm; 500 g; Merck). Column chromatographic separations were performed
on silica gel 60 (0.04–0.063 mm; Merck) and Sephadex LH-20 (0.25–0.1 mm, Merck). TLC
analyses were conducted on pre-coated silica gel F254 aluminium sheets (Merck). Compounds
were detected by spraying the sheets with an anisaldehyde/H2SO4 reagent followed by heating at
1108C for 1–2 min. The solvent systems used for TLC analyses were CHCl3–MeOH (95:5,
Figure S1), CHCl3–MeOH (90:10; Figure S2) and n-BuOH–acetone–formic acid–H2O
(60:17:8:15, Figure S3).
3.2. Plant materials
The fresh bulbs of D. maritima (red squill) were collected in July 2009 from the desert of
Matrouh, Egypt. The plant material was kindly identified by Prof. Dr A. Fayed (Professor of
Plant Taxonomy, Faculty of Science, Assiut University, Egypt). A voucher specimen has been
deposited at the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Suez Canal
University, Ismailia, Egypt (Registration code DY-UM-2009).
3.3. Extraction and isolation
The fresh bulbs were cleaned from sandy soil, cut into small slices (3 kg) and extracted with
MeOH (5 L £ 4) at r.t. The combined extracts were concentrated under reduced pressure to
afford a dark reddish residue (140.0 g). Eighty grams of the latter was suspended in distilled
water (400 mL) and then successively partitioned between EtOAc (800 mL £ 4) and n-
BuOH (800 mL £ 3). Each fraction was concentrated under reduced pressure to give EtOAc
(16.5 g) and n-BuOH (48.0 g). The EtOAc fraction was subjected to silica gel VLC using
n-hexane–EtOAc gradients, nine subfractions were collected as follows: UM-1 (0.9 g,
n-hexane–EtOAc; 90:10), UM-2 (1.3 g, n-hexane–EtOAc; 80:20), UM-3 (1.1 g, n-hexane–
EtOAc; 70:30), UM-4 (1.9 g, n-hexane–EtOAc; 60:40), UM-5 (0.95 g, n-hexane–EtOAc;
50:50), UM-6 (1.8 g, n-hexane–EtOAc; 40:60), UM-7 (2.1 g, n-hexane–EtOAc; 30:70), UM-
8 (1.9 g, n-hexane–EtOAc; 10:90) and UM-9 (1.9 g, EtOAc; 100%) (1 L for each fraction
was collected and evaporated to afford corresponding weight). The in vitro growth inhibitory
activity of EtOAc subfractions (Table 1) were subjected to different chromatographic
techniques to isolate compounds 1–7. Fraction UM-2 (1.3 g) was subjected to silica gel
column chromatography (80 g £ 50 cm £ 2 cm) using n-hexane–EtOAc (98:2 to 90:10) to
afford compounds 3 (70 mg, colourless needles) and 4 (20 mg, colourless needles). Fraction
UM-3 (1.1 g) was chromatographed over silica gel column (70 g £ 50 cm £ 2 cm) using n-
hexane–EtOAc in order of increasing polarity to afford compound 5 (18 mg, colourless
needles). Compounds 3–5 were re-crystallised from MeOH–Me2CO (1:1) to furnish
colourless needles. Similarly, fraction UM-4 (1.9 g) was subjected to silica gel column
chromatography (100 g £ 50 cm £ 2 cm) using n-hexane–EtOAc gradients to afford
compound 1 (28 mg, colourless oil). Repeated silica gel column chromatography of fraction
UM-5 (0.95 g) using CHCl3–MeOH (96:4 to 90:10) gave compound 2 (15 mg, yellow
crystals). Fraction UM-6 (1.8 g) was subjected to silica gel column chromatography (90 g £
50 cm £ 2 cm) using CHCl3–MeOH (95:5 to 85:15) to afford compound 6 (2.9 mg, yellow
crystals). Fraction UM-7 (2.1 g) was chromatographed over Sephadex LH-20 column (0.25–
0.1 mm; 100 g £ 50 cm £ 3 cm) using MeOH as an eluent to give impure 7, which was further
purified on a silica gel column (30 g £ 50 cm £ 2 cm) using CHCl3–MeOH (90:10 to 80:20)
to give 7 (28 mg, yellow amorphous powder).

1588
G.A. Mohamed et al.
3.4. Alkaline hydrolysis of compound 1
A solution of 1 (10 mg) in 3% NaOMe–MeOH (2 mL) was stirred at 408C for 2 h. The reaction
mixture was neutralised with 2 N HCl in MeOH and partitioned between MeOH and n-hexane.
The n-hexane layer was concentrated under reduced pressure to yield FAME, which was
analysed as (Z)-tetracos-6-enoic acid methyl ester by GC–MS from the molecular ion peaks at
m/z 380 [M]þ (Kim et al. 2005; Al-Musayeib et al. 2013).
3.5. Acid hydrolysis
Solutions of the isolated glycosides (6 and 7) (5 mg in 10 mL MeOH) were treated with 3%
H2SO4 (1.5 mL) and heated at 1008C for 1 h. The aglycones were extracted with EtOAc,
concentrated under reduced pressure, purified on Sephadex LH-20 column using MeOH. The
sugars in the aqueous layer were identified by co-PC (paper chromatography) with authentic
materials using solvent system S3 (El-Shanawany et al. 2012).
3.6. Spectral data
(2S)-1-O-(Z)-Tetracos-6-enoate glycerol (1): Colourless oil; Rf 0.57, silica gel 60 F254 (Figure
S1); [a]D þ 14.88 (c ¼ 0.5, CHCl3); IR (KBr) gmax: 3435, 2966, 1742, 1472, 1072, 952 cm21
;
1H NMR (400 MHz, CDCl3): dH 4.19 (1H, dd, J ¼ 11.6 and 4.8 Hz, H-1A), 4.15 (1H, dd,
J ¼ 11.6 and 4.8 Hz, H-1B), 3.93 (1H, m, H-2), 3.69 (1H, dd, J ¼ 12.0 and 6.4 Hz, H-3A), 3.60
(1H, dd, J ¼ 12.0 and 6.4 Hz, H-3B), 2.35 (2H, t, J ¼ 7.6 Hz, H-20), 1.64 (2H, m, H-30), 2.01
(2H, m, H-50), 5.34 (1H, dt, J ¼ 7.6 and 6.8 Hz, H-60), 5.33 (1H, dt, J ¼ 7.6 and 6.8 Hz, H-70),
1.30–1.25 (2H £ 14, m, H-40, H-80-H-210), 0.88 (3H, t, J ¼ 7.2 Hz, H-240); 13C NMR
(100 MHz, CDCl3): dC 65.2 (C-1), 70.3 (C-2), 63.3 (C-3), 174.3 (C-10), 34.1 (C-20), 24.9 (C-30),
27.2 (C-50), 129.7 (C-60), 130.0 (C-70), 29.7–29.1 (CH2 £ 15, C-40, C-80-C-210), 31.9 (C-220),
22.7 (C-230), 14.1 (C-240); HR-ESI-MS m/z 441.3852 (calcd for C27H53O4 [M þ H]þ,
441.3866).
3.7. Determination of in vitro growth inhibitory activity (cytotoxicity)
The in vitro growth inhibitory activity of the n-BuOH and EtOAc fractions, as well as EtOAC
subfractions and isolated compounds was determined using the A549 NSCLC (Deutsche
Sammlung von Mikroorganismen and Zellkulturen (DSMZ); code ACC107), PC-3 prostate
cancer (DSMZ; code ACC465) and U373 GBM (European Collection of Cell Culture; code
89081403) cell lines. The cells were cultured in RPMI (Lonza, Braine-l’Alleud, Belgium)
media supplemented with 10% heat-inactivated foetal bovine serum (Lonza), 4 mM glutamine,
100 mg/mL gentamicin, 200 U/mL penicillin and 200 mg/mL streptomycin (Lonza). The overall
growth of the human cancer cell lines was determined using a colorimetric MTT assay (Sigma,
Diegem, Belgium) as previously detailed (Mijatovic et al. 2006; Ibrahim et al. 2012; Ibrahim
et al. 2014). Each experimental condition was performed in six replicates. Cisplatin (Sigma-
Aldrich, Bornem, Belgium) was used as a positive control. The results are presented in Table 1.
4. Conclusion
Investigation of the in vitro growth inhibitory activity of the EtOAc subfractions of D. maritima
bulbs afforded the isolation of one new and six known compounds. Their structures were
established by different spectroscopic analyses. The in vitro growth inhibitory activity of the n-
BuOH, EtOAc and EtOAc subfractions, as well as the isolated compounds was evaluated against

Natural Product Research
1589
three different human cancer cell lines. Compounds 4 and 7 are reported here for the first time
from the plant and 2 and 6 for the first time from this family.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S8.
Acknowledgements
We would like to express our deep thanks to Prof. Robert Kiss for conducting the in vitro growth inhibitory
assays and Mr Volker Brecht for acquiring NMR and MS spectroscopic data.
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