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Proceraside A, a new cardiac glycoside

from the root barks of Calotropis
procera with in vitro anticancer effects
a bc de a
S.R.M. Ibrahim , G.A. Mohamed , L.A. Shaala , L. Moreno , Y.
f f b
Banuls , R. Kiss & D.T.A. Youssef
a
Department of Pharmacognosy, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt
b
Department of Natural Products, Faculty of Pharmacy, King
Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
c
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar
University, Assiut Branch, Assiut 71524, Egypt
d
Natural Products Unit, King Fahd Medical Research Center, King
Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia
e
Suez Canal Hospital, Suez Canal University, Ismailia 41522, Egypt
f
Laboratoire de Cancérologie et de Toxicologie Expérimentale,
Faculté de Pharmacie, Université Libre de Bruxelles, Bruxelles,
Belgique
Published online: 31 Mar 2014.

To cite this article: S.R.M. Ibrahim, G.A. Mohamed, L.A. Shaala, L. Moreno, Y. Banuls, R. Kiss &
D.T.A. Youssef (2014) Proceraside A, a new cardiac glycoside from the root barks of Calotropis
procera with in vitro anticancer effects, Natural Product Research: Formerly Natural Product
Letters, 28:17, 1322-1327, DOI: 10.1080/14786419.2014.901323

To link to this article: http://dx.doi.org/10.1080/14786419.2014.901323

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 Natural Product Research, 2014
Vol. 28, No. 17, 1322–1327, http://dx.doi.org/10.1080/14786419.2014.901323

Proceraside A, a new cardiac glycoside from the root barks of Calotropis

procera with in vitro anticancer effects
S.R.M. Ibrahima, G.A. Mohamedb,c, L.A. Shaalad,e, L. Moreno Y. Banulsf, R. Kissf and
D.T.A. Youssefb*
a
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt;
b
Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589,
Kingdom of Saudi Arabia; cDepartment of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University,
Assiut Branch, Assiut 71524, Egypt; dNatural Products Unit, King Fahd Medical Research Center, King
Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia; eSuez Canal Hospital, Suez Canal
Downloaded by [Gamal Mohamed] at 11:14 28 July 2014

University, Ismailia 41522, Egypt; fLaboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté

de Pharmacie, Université Libre de Bruxelles, Bruxelles, Belgique
(Received 10 January 2014; final version received 3 March 2014)

We have studied the ethyl acetate fraction of the methanolic extract of the root barks of
Calotropis procera (Asclepiadaceae) from Egypt. Bioassay-directed fractionation and
final purification of the extract resulted in the identification of a new cardenolide
glycoside named proceraside A (1) together with two known compounds, frugoside (2)
and calotropin (3). Their structures were elucidated by extensive NMR studies and
mass spectrometric data. The in vitro cytotoxicity of the isolated compounds was
evaluated against A549 non-small cell lung cancer, U373 glioblastoma and PC-3
prostate cancer cell lines. They showed potent activity against the tested cancer cell
lines with IC50 ranging from 0.005 to 0.3 mg/mL. Cisplatin was used as positive
control.
Keywords: Calotropis procera; proceraside A; cytotoxicity

1. Introduction
Calotropis procera is a member of the plant family Asclepiadaceae, a shrub widely distributed in
West Africa, Asia and other parts of the tropics (Begum et al. 2011). The various parts of the
plant, i.e. flowers, stem, leaves, roots and milky latex, are used in the traditional systems of
medicine for the treatment of various ailments around the world. Calotropis seed extract
exhibited insecticidal activity (Begum et al. 2011). Its flower extract showed anthelmintic
activity (Iqbal et al. 2005). The latex of this plant has been shown to possess anti-inflammatory,
analgesic, antipyretic, anti-ulcers, anti-convulsivant, anti-diarrhoeal, hepatoprotective, cytotoxic
and cardio-protective properties (Iwalewa et al. 2005; Van Quaquebeke et al. 2005; Bharti et al.
2010; Moustafa et al. 2010). The root bark and leaves extracts showed antioxidant, anti-bacterial
and cytotoxic activities (Yesmin et al. 2008). Previous chemical investigations of C. procera
worldwide revealed the presence of cardenolides (Hanna et al. 1999; Van Quaquebeke et al.
2005; Moustafa et al. 2010), triterpenes (Ibrahim et al. 2012), flavonoids (Moustafa et al. 2010;
Shaker et al. 2010), sterols (Alam and Ali 2009) and fatty acids (Khanzada et al. 2008; Ibrahim
et al. 2012). Also, we have identified a novel cardenolide, 200 -oxovoruscharin from C. procera
plants growing in Burkina Faso, i.e. West Africa (Van Quaquebeke et al. 2005). Our previous

*Corresponding author. Email: dyoussef@kau.edu.sa

q 2014 Taylor & Francis

 Natural Product Research 1323

phytochemical study of the Egyptian C. procera root barks led to the isolation of different
ursane-type triterpenes (Ibrahim et al. 2012). Bioassay-directed fractionation of the EtOAc
fraction of C. procera root barks using A549 non-small-cell lung cancer (NSCLC), U373
glioblastoma (GBM) and PC-3 prostate cancer cell lines led to the isolation of one new
cardenolide glycoside named proceraside A (1) together with two known compounds, frugoside
(2) (Kiuchi et al. 1998; Maoyuan et al. 2008) and calotropin (3) (Abe et al. 1991) (Figure 1). This
study reports the full account of the structure characterisation of the isolated compounds. The
in vitro cytotoxicity of the isolated compounds was also evaluated.

2. Results and discussion

Compound 1 was obtained as colourless amorphous solid. The molecular formula was
established as C31H44O10 by HR-ESI-MS molecular ion peaks at m/z 599.2836 [M þ Na]þ and
577.2931[M þ H]þ. The IR spectrum showed the presence of hydroxyl (3450 cm21), carbonyl
(1745 and 1724 cm21) and C –O –C (1052 cm21) groups. The presence of a,b-unsaturated
Downloaded by [Gamal Mohamed] at 11:14 28 July 2014

g-lactone moiety was suggested from the UV absorption band at 217 nm (Siddiqui et al. 1997).
In the 1H and 13C NMR spectra, the characteristic signals of a lactone (dC 174.5), an olefinic
double bond (dH 5.90 (s, H-22)/dC 116.2 (C-22), 176.1 (C-20), an oxygenated methylene (dH
4.95, 4.86 (2H, each br d, J ¼ 18.0 Hz (H-21)/dC 73.0 (C-21)) and anomeric sugar signals (dH
4.50 (1H, d, J ¼ 8.0 Hz (H-10 )/dC 98.1 (C-10 )) indicated that 1 possesses an a,b-unsaturated g-
lactone cardenolide moiety with a sugar residue, which was consistent with the observed UV and
IR absorption bands (Cheenpracha et al. 2004). The carbons multiplicity was assigned from the
multiplicity-edited HSQC spectrum. The signals at dH 9.83 (1H, s, H-19) and 0.69 (3H, s, H-18)
in the 1H NMR spectrum correlated with carbon signals at dC 209.4 and 15.4 in the HSQC
spectrum, suggesting a cardenolide skeleton with an aldehyde and methyl at C-10 and C-13,
respectively. The position of these moieties was supported by the HMBC cross peaks of H-1,

O
23
O
22 O
20 21 O
18
12
17
19 11
OHC 14 HO
1 9 15
6` 10
4` 3
OH
5` O 6 OH
HO 5
1`O O
HO O
O
OH OH
O OH
1 2

O
O

OH
OH OHC
O
OH
H3C O O
H
3

Figure 1. Structures of the isolated compounds.

 1324 S.R.M. Ibrahim et al.

H-5 and H-9 to C-19 and H-18 to C-12, C-13, C-14 and C-17 (Supplementary Figure S6). The
13
C spectrum of 1 displayed five methine carbons of the aglycone moiety at dC 75.7 (C-3), 42.5
(C-5), 41.8 (C-8), 47.4 (C-9) and 49.9 (C-17), which correlated with the signals at dH 3.53
(H-3), 1.45 (H-5), 1.42 (H-8), 1.22 (H-9) and 2.71 (H-17) in HSQC spectrum, respectively. The
oxygenated quaternary carbon signal at dC 83.2 (C-14) was in accordance with that reported for
corotoxigenin (Abe et al. 1992; Karkare et al. 2007). In addition, the HMBC correlation of H-18
to C-14 suggested the assignment. The aglycone of 1 was established as 3-O-substituted
corotoxigenin on the basis of its 1D and 2D NMR data and by comparison with literature reports
(Abe et al. 1992; Karkare et al. 2007). Signals for acetyl group at dH 1.98/dC 21.0 (CH3CO) and
173.7 (CH3CO) were observed. In addition, a doublet signal at dH 1.19 (3H, d, J ¼ 6.3 Hz, H-60 )
indicated the presence of a 60 -deoxy hexose residue and further confirmed by the observed
fragment ion peak at 387.9 [M þ H-(deoxy-20 -O-acetyl-allopyranose)]þ in the ESI-MS
spectrum. The large coupling constant of anomeric proton (J ¼ 8.0 Hz) indicated a
b-configuration of glycosidic bond (Agrawal 1992). The HMBC correlations of H-10 /C-3 and
H-3/C-10 indicated the attachment of the sugar moiety at C-3. The placement of acetyl group at
Downloaded by [Gamal Mohamed] at 11:14 28 July 2014

C-20 of sugar was established by the HMBC correlation of H-20 to carbonyl carbon of the acetyl
group (dC 173.7) and further confirmed by the downfield shift of C-20 (dC 75.0) compared with
this in regular sugar (dC 72.5) (Kiuchi et al. 1998; Maoyuan et al. 2008). The full assignment of
all protonated carbons was accomplished by interpretation of the COSY, HSQC and HMBC
experiments (Supplementary Figure S6) which allowed the sequential identification of H-10 to
H-60 within the sugar unit that was determined as 60 -deoxy-20 -O-acetyl-allopyranose (Carter
et al. 1997; Maoyuan et al. 2008). On the basis of these findings, 1 was assigned as
corotoxigenin-3-O-b-(60 -deoxy-20 -O-acetyl)-allopyranoside and named proceraside A.
The known compounds were identified by analysis of the spectroscopic data (1D, 2D NMR
and MS) and comparison of their data with those in the literature to be frugoside (2) (Kiuchi et al.
1998; Maoyuan et al. 2008) and calotropin (3) (Abe et al. 1991).
Proceraside A (1) and frugoside (2) displayed similar in vitro cytotoxicity in the three cancer
cell lines analysed, while they appeared about 10 times less active than calotropin (3); these three
compounds displayed more important in vitro growth inhibitory effects than cisplatin used as a
reference compound (Table 1). The EtOAc extract displayed in vitro growth inhibitory effects
similar to those displayed by proceraside A (1) and frugoside (2) (Table 1).

3. Experimental
3.1. General experimental procedures
Optical rotation was measured on a Perkin-Elmer Model 341 LC polarimeter (Perkin-Elmer,
Waltham, MA, USA). Normal and HR-ESI-MS spectra were recorded on an LTQ Orbitrap and
an API 2000 (ThermoFinnigan, Bremen, Germany) mass spectrometers. UV spectra were
Table 1. Cytotoxicity results.
IC50 (mg/mL)
A549 U373 PC-3
Sample NSCLC GBM Prostate cancer Mean ^ SEM
EtOAc 0.1 0.2 0.4 0.23 ^ 0.09
1 0.3 0.05 0.06 0.14 ^ 0.08
2 0.2 0.03 0.05 0.09 ^ 0.05
3 0.005 0.005 0.005 0.005
Cisplatina 1.2 0.12 NT –
Notes: NT, not tested; –, not calculated.
a
The cisplatin-related data have already been published (Ibrahim et al. 2012).
 Natural Product Research 1325

recorded in absolute MeOH on a Shimadzu 1601 UV/VIS spectrophotometer. The IR spectra

were measured on a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). 1D
and 2D spectra were measured on a Bruker DRX 400 spectrometer (Bruker, Rheinstetten,
Germany). Chromatographic separations were performed using a silica gel 60 (0.04 – 0.063 mm;
Merck, Darmstadt, Germany), Sephadex LH-20 (0.25 –0.1 mm, Merck, Darmstadt, Germany)
and RP-18 (0.04 –0.063 mm; Merck, Darmstadt, Germany). TLC analyses were carried out on
pre-coated silica gel F254 aluminium sheets and RP-18 F254s glass plates (Merck, Darmstadt,
Germany). The 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; S1) and CHCl3 – MeOH (90:10; S2).

3.2. Plant material

The root barks of C. procera were collected in April 2009 from trees growing in Ismailia, Egypt.
The plant material was identified by Prof. Dr A. Fayed (Professor of Plant Taxonomy, Faculty of
Downloaded by [Gamal Mohamed] at 11:14 28 July 2014

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-CP-2009).

3.3. Extraction and isolation

The air-dried powdered root barks (1.2 kg) were extracted with MeOH (3.5 L £ 4). The
combined extracts were concentrated under reduced pressure to afford a dark brown residue
(55.0 g). The latter was suspended in distilled H2O (400 mL), then successively partitioned
between n-hexane, CHCl3, EtOAc and n-BuOH to give n-hexane (4.5 g), CHCl3 (3.2 g), EtOAc
(5.6 g) and n-BuOH (9.0 g) fractions, respectively. The active EtOAc fraction was subjected to
VLC using CHCl3 –MeOH gradients to afford four subfractions: A (1.1 g, CHCl3, 100%), B
(1.4 g, CHCl3 – MeOH, 75:25%), C (1.0 g, CHCl3 – MeOH, 50:50%) and D (1.3 g, MeOH,
100%). Subfraction A (1.1 g) was subjected to silica gel column chromatography (150 g £ 50
£ 3 cm) using CHCl3 – MeOH (98:2 to 92:8) to afford 3 (colourless needles, 23 mg). Subfraction
B (1.4 g) was chromatographed over silica gel column (200 g £ 50 £ 3 cm) using CHCl3 –
MeOH in the order of increasing polarity to give 1 (14 mg, colourless amorphous solid).
Subfraction D (1.3 g) was subjected to Sephadex LH-20 chromatography using MeOH as an
eluent to afford three major subfractions D-1 (0.37 g), D-2 (0.48 g) and D-3 (0.29 g). Subfraction
D-2 (0.48 g) was chromatographed on silica gel column (100 g £ 50 £ 3 cm) using CHCl3 –
MeOH gradients to give impure 2 which was further purified by repeated chromatography on
RP-18 column chromatography using MeOH – H2O gradients to afford 2 (27 mg, colourless
amorphous solid).

3.4. Determination of in vitro growth inhibitory activity (cytotoxicity) of the compounds in

cancer cell lines
The in vitro growth inhibitory activity of the ethyl acetate fraction and isolated compounds was
determined by means of the MTT colorimetric assay 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, ECACC;
code 89081403) cell lines. Cisplatin was used as a reference compound (Ibrahim et al. 2012). For
each extract and compound analysed, the concentration that reduced by 50% the growth of each
cancer cell line (IC50 concentration) was determined after having cultured the cancer cells for
72 h with the extract or the compound of interest. Each experimental condition was performed in
six replicates. The results are presented in Table 1.
 1326 S.R.M. Ibrahim et al.

3.5. Spectral data

Proceraside A: colourless amorphous solid (14 mg); Rf 0.79, silica gel 60 F254 (S1); [a ]D 28.6
(c ¼ 1.1, MeOH); UV (MeOH) lmax 217 (log 14.21) nm; IR (KBr) nmax 3450, 2925, 1745, 1724,
1265, 1052 cm21; 1H NMR (DMSO-d6, 400 MHz): dH 2.01 (1H, m, H-1A), 1.78 (1H, m, H-1B),
2.15 (1H, m, H-2A), 1.84 (1H, m, H-2B), 3.53 (1H, m, H-3), 1.81 (1H, m, H-4A), 1.09 (1H, m,
H-4B), 1.45 (1H, m, H-5), 2.26 (1H, m, H-6A), 1.53 (1H, m, H-6B), 2.13 (1H, m, H-7A), 1.84
(1H, m, H-7B), 1.42 (1H, m, H-8), 1.22 (1H, m, H-9), 1.62 (2H, m, H-11), 1.60 (1H, m, H-12A),
1.39 (1H, m, H-12B), 1.91 (1H, m, H-15A), 1.53 (1H, m, H-15B), 2.02 (1H, m, H-16A), 1.81
(1H, m, H-16B), 2.71 (1H, dd, J ¼ 9.8, 6.0 Hz, H-17), 0.69 (3H, s, H-18), 9.83 (1H, s, H-19),
4.95 (1H, br d, J ¼ 18.0 Hz, H-21A), 4.86 (1H, br d, J ¼ 18.0 Hz, H-21B), 5.90 (1H, s, H-22),
4.50 (1H, d, J ¼ 8.0 Hz, H-10 ), 4.04 (1H, dd, J ¼ 8.0, 2.8 Hz, H-20 ), 3.77 (1H, m, H-30 ), 3.14 (1H,
dd, J ¼ 8.8, 2.8 Hz, H-40 ), 3.51 (1H, dq, J ¼ 8.5, 6.3 Hz, H-50 ), 1.19 (3H, d, J ¼ 6.3 Hz, H-60 ),
1.98 (3H, s, 20 -CH3CO); 13C NMR (DMSO-d6, 100 MHz): dC 30.2 (C-1), 27.9 (C-2), 75.7 (C-3),
35.5 (C-4), 42.5 (C-5), 27.8 (C-6), 27.3 (C-7), 41.8 (C-8), 47.4 (C-9), 55.9 (C-10), 21.2 (C-11),
38.8 (C-12), 51.1 (C-13), 83.2 (C-14), 31.4 (C-15), 26.0 (C-16), 49.9 (C-17), 15.4 (C-18), 209.4
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(C-19), 176.1 (C-20), 73.0 (C-21), 116.2 (C-22), 174.5 (C-23), 98.1 (C-10 ), 75.0 (C-20 ), 72.6 (C-
30 ), 73.6 (C-40 ), 70.5 (C-50 ), 18.5 (C-60 ), 173.7 (20 -CH3CO), 21.0 (20 -CH3CO); HR-ESI-MS: m/z
599.2836 (calcd for C31H44O10Na [M þ Na]þ, 599.2832), 577.2931 [M þ H]þ (calcd for
C31H45O10 [M þ H]þ, 576.2935).

4. Conclusion
In conclusion, in this study one new cardiac glycoside, proceraside A (1), together with two
known compounds, frugoside (2) and calotropin (3), was isolated from the ethyl acetate fraction
of the methanolic extract of the root barks of C. procera. The isolated compounds showed potent
in vitro growth inhibitory activity against human A549 NSCLC, U373 GBM and PC-3 prostate
cancer cells.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1 – S6.

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