Bioorganic & Medicinal Chemistry Letters 24 (2014) 37773781

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Two new unusual monoterpene acid glycosides from Acacia cyclops

with potential cytotoxic activity
Amira Jelassi a, Afa Zardi-Bergaoui a, Aymen Ben Nejma a, Meriam Belaiba b, Jalloul Bouajila b,,
Hichem Ben Jannet a,
a
Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity (LR11SE39), Team: Medicinal Chemistry and Natural Products, Faculty of Science of Monastir,
University of Monastir, Avenue of Environment, 5019 Monastir, Tunisia
b
University of Toulouse, Faculty of Pharmacy of Toulouse, Laboratory of Molecular Interactions and Chemical and Photochemical Reactivities, UMR CNRS 5623,
University Paul-Sabatier, 118 Narbonne Road, F-31062 Toulouse, France

a r t i c l e

i n f o

Article history:
Received 21 April 2014
Revised 21 June 2014
Accepted 25 June 2014
Available online 1 July 2014
Keywords:
Acacia cyclops
Fabaceae
Pods
O-Heterosides
2D-NMR
Cytotoxic activity

a b s t r a c t
A phytochemical investigation of the Tunisian plant Acacia cyclops pods led to the isolation of two new
monoterpenoid glycosides, which have been designated Cyclopside 1 and Cyclopside 2. Their structures
were elucidated on the basis of extensive spectroscopic procedures including IR, MS and 2D-NMR. The
cytotoxic effect of the isolates was also evaluated in vitro against the human breast cancer (MCF-7)
and ovarian cancer (OVAR) cell lines. Results showed that the highest cytotoxic activity (90.88%) was
against MCF-7 cell line and was exhibited by the Cyclopside 1 at the concentration of 50 lg/mL.
2014 Elsevier Ltd. All rights reserved.

The genus Acacia is quite large and widespread in the warm arid
and sub-arid regions in the word. Little is known about the chemistry of most species. Acacia Mill. is the second largest genus belonging to the subfamily Mimosoideae of the family Fabaceae with
about 1350 species.1 The main use of Acacia species is as a fodder
source but they have a long history of traditional medicinal use
for the treatment of diarrhea, urinary tract infections, headaches,
sore throat, gastritis problems, tuberculosis and bronchial asthma.
Acacia has been also proven useful in high blood pressure, hypoglycemia, inammation, dysentery, colitis, leucorrhoea and leprosy.25
It was the case in Tunisia, whereby some Acacia species have been
used in folk medicine. According to information gathered from
herbalists, inhabitants of rural regions and traditional healers, in
south Tunisia, Acacia salicina, for example, is frequently used in
diverse applications for the treatment of inammatory diseases,
as a febrifuge, to treat cancer and to promote human fertility,
whereas in north Tunisia, the same species serves as a treatment
of diarrhea and rheumatism.6 Other authors have reported their
spasmogenic and vasoconstrictor actions7 and also their cytotoxic8
and antioxidant9 activities.
Corresponding authors. Tel.: +33 562256825; fax: +33 562256826 (J. Bouajila),
tel.: +216 73500279; fax: +216 73500278 (H. Ben Jannet).
E-mail addresses: jalloul.bouajila@univ-tlse3.fr (J. Bouajila), hich.benjannet@
yahoo.fr (H. Ben Jannet).
http://dx.doi.org/10.1016/j.bmcl.2014.06.075
0960-894X/ 2014 Elsevier Ltd. All rights reserved.

The Acacia genus is well documented as a good source of biologically active compounds such as alkaloids, cyanogenic glycosides,
cyclitols, fatty acids and seed oils, uoroacetate, terpenes (including essential oils, diterpenes, phytosterols), saponins, hydrolysable
tannins, avonoids and condensed tannins.10
Acacia cyclops A. Gunn. ex G. Don is a shrubby evergreen tree
(1.56 m high), originating in south-western Australian and it is
introduced in Tunisia with other Acacia species in the 18th century.
These plantations were created for several purposes such as their
use as fodder reserves particularly during periods of drought and
for dune stabilization.11
Study of this plant pods has been limited to certain botanical
and agronomical research. To the best of our knowledge, so far it
has not been the subject of any phytochemical investigation.
The research for anticancer agents from natural sources has
been successful worldwide, active constituents have been isolated
and are nowadays used to treat human cancer. The ethnopharmacological knowledge is helpful to lead the research for plants with
potential cytotoxic activity.12
The current study describes the isolation and the structure
elucidation of two new monoterpene acid glycosides named
Cyclopside 1 and Cyclopside 2 from the ethyl acetate extract of A.
cyclops pods.13 Their structures were established principally by
two-dimensional NMR spectroscopy. Furthermore, the cytotoxic

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A. Jelassi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 37773781

activity of the isolates against the human breast cancer (MCF-7)

and ovarian cancer (OVAR) cell lines was evaluated.
Cyclopside 1 (Fig. 1) was obtained as a yellow oily substance
from the ethyl acetate extract of Acacia cyclops pods, 69 mg;
Rf = 0.6 (H2O/MeOH 4:6). Its ES-HRMS gave [M+Na]+ and [M+H]+
molecular ion peaks at m/z 535.2710 and m/z 513.2708, respectively, suggesting a molecular formula of C26H40O10. The IR spectrum of 1 displayed absorption bands attributable to olenic
groups (mC@C 1645 cm1) and to carboxylic acid function (mOH
3418 cm1 and mCO 1694 cm1), conrmed by the 13C NMR spectrum showing the presence of a signal at dc 171.6 corresponding
to a carbonyl function (Table 1). Further analysis of the 1H NMR
spectrum of 1 (Table 1) pointed to the presence of four singlets
of methyl groups attributed to H9 (dH 1.78, s), H10 (dH 1.32, s),
H900 (dH 1.75, s) and H1000 (dH 1.22, s), eight olenic proton signals
between 5.01 and 6.75 ppm and four methylene groups H400 (dH
1.56, t, J = 7.56 Hz), H500 (dH 2.19, m,), H4 (dH 1.70, t, J = 7.28 Hz)
and H5 (dH 2.25, m). Furthermore, the anomeric signals indicative
of a sugar unit were observed at dH 4.36 (H10 , d, J = 7.76 Hz) and
dC 99.1 ppm in 1H and 13C spectra, respectively.
Full assignments of the 1H and 13C NMR spectra were consolidated through the use of 1H1H COSY, CHcorr, HMBC and NOESY
experiments. The identication of a glycopyranoside moiety was
reinforced by the presence of long-range correlation H20 C10 ,
H30 C20 , H40 C20 , H50 C60 , H60 C30 . The signicant HMBC correlation
(Fig. 2) between H10 (dH 4.36, d, J = 7.76 Hz) and C3 (dC 81) indicated
that the glycopyranose must be linked to the C3 of the linear chain.

In the NOE correlation, proton coupling constants and comparison

with reported literature data for glucoside analogues,1417 suggested that the sugar in 1 was a b-D-glucopyranose.
In fact, the structure of the C-1 linear chain was identied by
the correlation of H1a,1bH2, H4H5 and H5H6 in 1H1H COSY.
HMBC data (Fig. 2) conrmed the structure of the C-1 linear chain
due to the signicant long range couplings H1C3, H2C3, H4C3,
H5C4, H6C4 and H6C8. The detection of a duplication of the
signals in the 1H NMR spectrum suggested the presence of a second
C-100 linear chain fragment in the molecule which is in accordance
with the molecular formula deduced from the ES mass spectrum.
These results were evidenced by the HMBC correlation (Fig. 2)
between the proton H40 of the glucopyranosyl portion and the carbon C300 of the second monoterpene acid skeleton (C100 -linear chain).
The stereochemistry of the double bonds of Cyclopside 1 was
assigned as (6E, 600 Z). This was conrmed by the appearance of
the NOE cross peaks (Fig. 3) between H5H9 and H4H6 as well
as between H600 and both H400 and H900 .
Cyclopside 2 (Fig. 1) was obtained as a yellow oily substance
from the same ethyl acetate extract of A. cyclops pods, (60 mg);
Rf = 0.5 (H2O/MeOH 3:7). Its molecular formula was established
as C26H40O9, on the basis of the molecular ion at m/z 519.2758
and m/z 497.2760 displayed by ES-HRMS [M+Na]+ and [M+H]+,
respectively, and its NMR spectral data (Table 1). The comparison
of the spectral data of Cyclopsides 1 and 2 shows a close similarity
and the only difference noticed between the two chemical structures occurs in the nature of the sugar and the stereochemistry

1''
10''

HO

8''

2''

(Z)
3''

OH O
O

O
6

HO

(E)

1'

6'

9''

H
H

3'

10

7''
6''

4''

4'

5'

2'

HO

5''

OH

Compound 1
1
9

HO

(E)
8

10

1'

5'

H
H

6'
3'

2'

O
4'

OH

HO

H
10''

4''

6''
5''

3''
2''
1''

Compound 2
Figure 1. The structures of compounds 1 and 2 isolated from Acacia cyclops.

(E)

7''

9''

8''

OH

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A. Jelassi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 37773781

Table 1
H (400 MHz) and

13

C (100 MHz) NMR spectral data for compounds 1 and 2 in CD3OD (d in ppm)

Position

1
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
100
200
300
400
500
600
700
800
900
1000

dH (J en Hz)

dC

dH (J en Hz)

dC

5.13
5.19
6.05

1.70
2.25
6.75

1.78
1.32
4.36
3.23
3.54
4.76
3.35
3.48
5.01
5.17
5.85

1.56
2.19
6.75

1.75
1.22

115.3

5.08
5.19
5.97

1.69
2.27
6.74

1.78
1.30
4.33
3.22
3.48
4.56
3.39
1.04
5.01
5.17
5.85

1.56
2.19
6.74

1.76
1.22

115.2

(d, 11.08)
(d, 17.62)
(dd, 17.60; 11.00)

144.0
81.0
39.8
24.5
144.7
128.7
171.6
12.4
23.8
99.1
75.1
75.8
72.6
75.6
62.3
112.4

(t, 7.28)
(m)
(m)

(s)
(s)
(d, 7.76)
(t, 8.50)
(t, 9.32)
(t, 9.54)
(m)
(m)
(d, 10.80)
(d, 17.40)
(dd, 17.34; 10.83)

145.8
73.5
41.5
24.3
144.1
128.2
169.0
12.4
27.8

(t, 7.56)
(m)
(m)

(s)
(s)

(d, 11.68)
(d, 17.72)
(dd, 17.68; 11.04)
(t, 8.04)
(m)
(m)

(s)
(s)
(d, 7.76)
(t, 8.40)
(t, 9.24)
(t, 9.44)
(m)
(d, 6.00)
(d, 10.76)
(d, 17.36)
(dd, 17.34; 10.82)

145.8
73.5
41.6
24.1
144.1
128.3
168.9
12.5
27.8

(t, 7.50)
(m)
(m)

(s)
(s)

1''
10''

144.1
80.8
39.4
24.5
144.4
128.6
171.6
12.5
24.1
99.0
75.4
75.6
77.2
70.8
18.1
112.4

HO

8''

2''

(Z)
OH
O

O
6

HO

1'

(E)
2

7''
6''

4''

9''

H
H

3'

10

5''

4'

5'

2'

HO

6'

3''

OH

Compound 1
1
9

10

HO

(E)
8

1'

H
H

5'

O
2'

HO

6'
3'

O
4'

OH

4''

6''
5''

3''

7''

(E)

H
10''

2''
1''

Compound 2
Figure 2. Key HMBC (H ? C) correlations of compounds 1 and 2.

9''

8''

OH

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A. Jelassi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 37773781

1''
10''

HO

8''

2''

(Z)
3''

OH O
O

O
6

HO

1'

(E)
2

10

HO

6'

7''
6''

4''

9''

4'

5'

2'

5''

H
H

3'

OH

Compound 1
1
9

10

HO

(E)
8

7
6

1'

5'

H
H

6'
3'

2'

O
4'

OH

4''

7''

(E)

HO

6''
5''

3''

10''

2''

8''

OH

9''

1''

Compound 2
Figure 3. NOESY correlations of compounds 1 and 2.

Table 2
Cytotoxicity of compounds 1 and 2, expressed as growth inhibition ratio of tumor
cells in an effective concentration of 50 lg mL1
Compound

Concentration (lM)

Cyclopside 1
Cyclopside 2

98
100

Growth inhibition (%) in cell lines

MCF-7

OVAR

98.88
68.21

77.68
66.54

Cancer cell lines: MCF-7 a breast cancer cell line, OVAR an ovarian cancer cell line.

of the double bonds. The presence of the a-L-rhamnosyl unit was

evidenced by the detection of a doublet (3H, dH 1.04, J = 6 Hz) in
the 1H NMR spectrum and a signal at dc 18.1 ppm in the 13C
NMR spectrum arising from the methyl group (C60 ). These results
were reinforced by the HMBC correlations observed between
H60 C40 and H60 C50 . The stereochemistry of the double bonds in
Cyclopside 2 was determined as (6E, 600 E). This was conrmed by
the NOE cross peaks between H4H6, H5H9, H400 H600 and H500 H900
observed in the NOESY spectrum (Fig. 3).
Several biological activities have been reported from various
species of Acacia1822 but little is known about the anti-cancer
one and there is no information about the cytotoxicity of the
studied species. Compounds 1 and 2 were tested for their
cytotoxic activities in vitro towards the human breast cancer
(MCF-7) and ovarian cancer (OVAR) cell lines, according to the

method of Bendaoud et al.23 The results are shown in Table 2

and expressed in growth inhibition ratio of tumour cells. As
determined by the MTT essay, the most active compound was
Cyclopside 1 with inhibition percentage values of 98.88% and
77.68% against MCF-7 and OVAR, respectively, at the used concentration of 50 lg/mL. The toxic effect of Cyclopside 2 against
the same cell lines is 68.21% and 66.54%, respectively. The relative high activity of compound 1 compared to that of compound
2 towards the two used cell lines showed the clear contribution
of the nature of the glycoside moiety and the double bond
stereochemistry in this activity.
In summary, in continuation of our pharmacological
phytochemical integrated studies on the plants of the genus Acacia,
we have investigated Acacia cyclops which resulted in the isolation
of two new monoterpene acid glycosides (12) with selective cytotoxic activity against the human breast cancer (MCF-7) and ovarian
cancer (OVAR) cell lines. On the basis of these promising results,
Cyclopside 1 and Cyclopside 2 might possess benecial therapeutic
potential against various tumors.
Acknowledgment
The authors are grateful to Prof. Fethia Harzallah-Skhiri, High
Institute of Biotechnology of Monastir, Tunisia, for botanical identication and to Nabeul Forest Department (Ministry of Agriculture, Tunisia) for the supply of plant material.

A. Jelassi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 37773781

References and notes

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14.
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16.
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18.
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23.

3781

vacuum, was successively extracted with ethyl acetate and n-butanol to yield
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(MCF-7) and a human ovarian cancer cell (OVAR). Cells were distributed in 96well plates at 3  104 cells/well in 100 lL and then 100 lL of culture medium
containing sample at the concentration of 50 lg/mL were added. Cell growth
was estimated by the MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay. MTT is a yellow water-soluble tetrazolium salt.
Metabolically active cells are able to convert the dye to water-insoluble dark
blue formazan by reductive cleavage of the tetrazolium ring. The tested
compounds were re-suspended in the DMSO followed by dilution in the buffer
so that the DMSO does not exceed 1%. Tamoxifen was used as positive control.
The test was performed in triplicate.