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SHORT REPORT
The article was published by Academy of Chemistry of Globe Publications
www.acgpubs.org/RNP © Published 5/28/2013 EISSN:
1307-6167
Rec. Nat. Prod. 7:3 (2013) 245-249
Cytotoxic Activity of Laserpitium latifolium L. Extract and Its
Daucane and Phenylpropanoid Constituents
Višnja Popović
1*
, Arne Heyerick
2
, Silvana Petrović
1
, Serge Van
Calenbergh
3
, Izet Karalić
3
, Marjan Niketić
4
and Dieter Deforce
2
1
University of Belgrade - Faculty of Pharmacy, Department of Pharmacognosy, Vojvode Stepe 450,
11221 Belgrade, Serbia
2
Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences, Ghent
University, Harelbekestraat 72, 9000 Ghent, Belgium
3
Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University,
Harelbekestraat 72, 9000 Ghent, Belgium
4
Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia
(Received October 17, 2012; Revised March 11, 2013; Accepted April 9 , 2013)
Abstract: In the search for novel cytotoxic agents, sesquiterpenoids and phenylpropanoids have provided
interesting lead compounds. From the chloroform extract of the underground parts of Laserpitium latifolium, the
daucane sesquiterpenoids laserpitin and acetyldesoxodehydrolaserpitin, and the phenylpropanoids laserin and
latifolon were isolated as the major compounds. Acetyldesoxodehydrolaserpitin is identified for the first time in
the genus Laserpitium. Using a MTT and a sulforhodamine B (SRB) assay, the cytotoxic and antiproliferative
activity of the extract and compounds laserpitin, acetyldesoxodehydrolaserpitin and laserin was tested in two
closely related human breast adenocarcinoma cell lines, ie. the invasive MCF 7/6 and the non-invasive MCF
7/AZ. The IC
50
values of the extract were in the range of 184.72 – 397.16 µg/mL, with the most potent effect
observed in the MTT test on the MCF 7/6 line. Among the tested compounds, acetyldesoxodehydrolaserpitin
exerted a most potent, concentration-dependent effect (IC
50
values of 0.60 and 0.51 µM in the MCF 7/6 cell line,
and 2.29 µM and 31.87 µM in the MCF 7/AZ cell line in the MTT and SRB test, respectively). The effect of
laserin was more pronounced in the MTT test (IC
50
of 4.57 and 2.46 µM in the MCF 7/6 and MCF 7/AZ,
respectively).
Keywords: Daucane esters; phenylpropanoids; Laserpitium latifolium; cytotoxic activity.
1. Plant Source
Laserpitium latifolium L. (Apiaceae) is widely distributed European species, whose
underground parts
(roots and rhizomes)
, known as Radix Gentianae albae, were used in
traditional medicine for their sharp and bitter taste for preparing tonics for refreshing and
strengthening, as well as for their diuretic and carminative properties [1,2].
The underground
parts of L. latifolium, used in this investigation were collected at Mt. Gučevo (West-Serbia) in October
2008. The plant material was collected and identified by Dr. Marjan Niketić, and a voucher specimen
was deposited at the Natural History Museum in Belgrade under the accession number ko03102008.
*
Corresponding author: E- Mail: visnjap@pharmacy.bg.ac.rs; Phone +381 11 3951 319.
Cytotoxic activity of Laserpitium latifolium
246
2. Previous Studies
Previous chemical investigations of the extracts of underground parts of L. latifolium revealed
the presence of daucane derivatives such as laserpitin [3,4], phenylpropanoid derivatives such as
latifolon [4], while from extracts of herb were isolated sesquiterpene lactones of the guaianolide type
[5]. Daucane derivatives are a relatively small group of sesquiterpenoids commonly isolated from
Apiaceae species [6]. Some daucane esters exert cytotoxic activity on several human cancer cell lines
[7]. The phenylpropanoid laserin was isolated and identified as the bitter compound present in carrot,
Daucus carrota L., and has been shown to exhibit cytotoxic activity against HL-60 cells [8].
3. Present Study
The underground parts of L. latifolium (631.96 g) were extracted twice with CHCl
3
for 48 h at
room temperature, at a ratio of 100 g of powdered material in 1 L of CHCl
3
. The collected filtrates
were concentrated under vacuum to obtain a dark brown, gummy extract (31.78 g). Part of the crude
CHCl
3
extract (4.51 g) was solubilized in MeOH and filtrated in order to remove waxes and lipophilic
compounds. The obtained MeOH soluble fraction was dried under reduced pressure (4.46 g) and
further eluted with a stepwise gradient system with increasing polarity of a solvent mixture composed
of hexane-EtOAc (from 5:1 to pure EtOAc), followed by EtOAc-MeOH (from 1:1 to pure MeOH). In
total 120 fractions were collected, in a volume of 20 mL each. Separation was followed by TLC
analyses using vanillin-sulphuric acid for derivatization. The total extract and selected fractions were
further analyzed by HPLC using a gradient system optimized for terpenoids. The mobile phases
consisted of 0.025% HCOOH in H
2
O (solvent A) and 0.025% HCOOH in 50 : 50 v/v of H
3
CN and
MeOH (solvent B). The elution program was 0-3 min isocratic 35.0% A (65% of B), 3-25 min linear
gradient from 35 to 15% of A (85% of B), 25–28 min linear gradient from 15 to 0% of A (100% of B),
28–30 min isocratic 0% A (100% of B), 30-32 min linear gradient 0 to 35% A (65% of B) and 32-35
min isocratic 35% A (65 of B). The flow rate was 0.7 mL/min; the temperature was set at 35 °C; an
injection volume of 20 µL was applied and a detection wavelength of 225 nm was used.
From the CHCl
3
extract of L. latifolium underground parts, four compounds were isolated as
the main constituents: daucane esters laserpitin and acetyldesoxodehydrolaserpitin and
phenylpropanoids laserine and latifolone. The sturucture of all isolated compounds was elucidated by
1D (
1
H NMR,
13
C NMR) and 2D (gHSQC, gHMBC and COSY) NMR analyses, and further
confirmed by high resolution MS.
Latifolon (λ
max
=219; 298 nm; R
t
=6.47 min) was isolated as an amorphous solid, with the
molecular formula C
11
H
12
O
4
as assigned by HR-MS (m/z molecular ion peak at 209.0826 (calcd for
[M + H]
+
, 209.0736). Also its
1
H and
13
C NMR spectra were corresponding to the structure of
latifolon, previously isolated from L. latifolium root
[4].
Laserpitin (λ
max
=223 nm; R
t
=18.22 min) was isolated as a white crystalline powder and with
molecular formula C
25
H
38
O
7
, assigned on the basis of the m/z molecular ion peak at 451.2759 (calcd
for [M + H]
+
, 451.2690) in HR-MS. 1D and 2D NMR experiments allowed to identify characteristic
patterns of two angelic acid esters bounded to alcohol laserol (Figure 1). Interestingly, laserpitin was
the first isolated daucane derivative [6] whose structure, due to the complexity, was finally established
after several revisions [3].
Acetyldesoxodehydrolaserpitin (λ
max
=223 nm; R
t
=25.84 min) was isolated as a sticky,
colourless liquid. The molecular formula C
27
H
40
O
7
was assigned by HR-MS with m/z molecular ion
peak at 477.2843 (calcd for [M + H]
+
, 477.2847) and m/z for the pseudomolecular ion peak at
499.2658 (calcd for [M + Na]
+
, 499.2666). The NMR spectra allowed characteristic patterns for two
angeloyl and one acetyl ester moieties, bound to the daucane skeleton. The
1
H NMR spectrum matches
with that of the daucane ester acetyldesoxodehydrolaserpitin that was previously isolated only from
the roots of Ferula tingitana L. [9].
Laserin (λ
max
=223 nm; R
t
=20.86 min) was retrieved as a sticky, colourless liquid, with the
molecular formula C
21
H
26
O
7
derived from the pseudomolecular ion peaks at m/z 413.1584 and
Popović et al., Rec. Nat. Prod. (2013) 7:3 245-249
247
429.1335 (calcd for [M + Na]
+
, 413.1600 and for [M + K]
+
, 429.1300).
1
H and
13
C NMR data were
matching the previously reported spectra for laserin [8], isolated from L. archangelica Wulf. [10] and
L. garganicum (Ten.) Bertol. [11].
In the system for terpenoids, using isolated compounds as external standards, contents of
compounds were quantified, and it was determined that concentrations of laserpitin,
acetyldesoxodehydrolaserpitin, laserin and latifolon were respectively 17.69, 1.02, 0.72 and 0.64 mg
per g of the dry weight of L. latifolium underground parts.
The cytotoxic and antiproliferative activities of CHCl
3
L. latifolium extract and isolated
daucanes laserpitin and acetyldesoxodehydrolaserpitin and the phenylpropanoid laserin were
investigated using two in vitro colorimetric assays namely: MTT [12] (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide), for detecting the number of living, metabolically active cells, and
SRB [13] (sulforhodamine B) for measuring the total cellular protein content. Activity was tested on
the two human breast adenocarcinoma cell lines: MCF 7/6 and MCF 7/AZ, invasive and non-invasive
type, respectively. The L. latifolium CHCl
3
extract and the pure compounds were tested at five
concentrations between the following ranges: 7.5-750 µg/mL for the extract and 0.5-100 µM for
compounds. Vinblastine sulphate was used as a reference compound at five concentrations ranging
from 0.1 to 40 nM. The results expressed as IC
50
values (extract concentration required to inhibit 50%
of the cell growth) are given in Table 1. The negative controls were carried out by replacing the test
compounds by corresponding volumes of medium and these results were considered to represent
100% of cell growth.
The extract showed a concentration dependent cytotoxic and/or antiproliferative activity with
IC
50
values ranging from 184.72 to 397.16 µg/mL, and the highest effect was achieved in the MTT test
on the invasive MCF 7/6 cell line (Table 1).
Among the compounds tested, acetyldesoxodehydrolaserpitin (Figure 1) was the most active
compound, especially on highly invasive MCF 7/6 cell line in both assays tested. Determined IC
50
values were in the low micromolar range (0.51 and 0.60 µM at MCF 7/6 line in the SRB and the MTT
test, respectively and 2.29 µM on MCF 7/AZ in the MTT test), except IC
50
value of 31.87 µM in SRB
test at MCF 7/AZ cell line, where exerted cytotoxic and/or antiproliferative activity was weaker. The
cell growth inhibition caused by acetyldesoxodehydrolaserpitin (5 µM) and the reference compound
vinblastine sulphate (10 nM) are given in the Figure 2. The results are expressed as the mean values ±
SD of four independent replicates. Some compounds of the class of daucanes were previously shown
to, at least in part, contribute to the cytotoxic activity of several Ferula species, such as
elaeochytrin A, 6-antraniloyl daucane ester isolated from F. elaeochytris Korovin that was the most
active compound in a MTT assay against two resistant cell lines of human (K562R) and mouse
(DA1-3b/M2
BCR-ABL
) leukemia (IC
50
values 12.4 and 7.8 µM, respectively) [14]. In a panel
investigation of the cytotoxicity of 16 daucane esters isolated from two Ferula and one Ferulago
species on seven human tumor cell lines, 14 of them showed activity against at least one of the tumor
cell lines [7]. Pallinin, a 6α,10α diangeloyl daucane ester, was the most active compound with IC
50
values in the lower molecular range against six cell lines tested [7]. Structure-activity relationship
investigation showed that the β-orientation of ester group at position C(2) strongly enhances the
cytotoxic activity [7]. Furthermore, the presence of an α,β-unsaturated ketone (“enone”) groups in side
chains of sesquiterpenes and sesquiterpene lactones increases the cytotoxicity toward tumor cells [15].
Acetyldesoxodehydrolaserpitin is a tri-ester, with two angeloyloxi groups featuring an “enone” system
one of which has β-orientation at position C(2), thereby combining several structural factors
contributing to the observed potent cytotoxicity in human breast adenocarcinoma cell lines.
Laserin exerted a concentration dependent effect in both tests on both cell lines, with the most
potent activity in the MTT test for both cell lines (IC
50
values 4.57 and 2.46 µM on MCF 7/6 and MCF
7/AZ, respectively). This compound and specially its erythro isomer 2-epilaserin that was isolated
from carrot were characterized as cytotoxic phenylpropanoids in the human acute promyelocytic
leukemia cell line HL-60 [15].
Our results suggest that both the daucane esters and phenylpropanoid laserin may be, at least
in part, responsible for the demonstrated cytotoxic and antiproliferative activity of the investigated
L. latifolium extract. Among the isolated compounds, acetyldesoxodehydrolaserpitin, whose biological
Cytotoxic activity of Laserpitium latifolium
248
activity had not been tested before, was identified as the most potent cytotoxic and/or antiproliferative
agent, especially in the invasive MCF 7/6 cell line.
HO O
O
OH
O
O
O
H
HO
O
O
O
O
O
O
H
2
O
O
O
O
O
O
O
O
O
O
O
1 2 3 4
Figure 1. Structure of daucane esters laserpitin (1) acetyldesoxodehydrolaserpitin (2) and phenylpropanoids
laserin (3) and latifolon (4) isolated from the CHCl
3
extract of L. latifolium underground parts.
Figure 2. Percent of inhibition of the cell growth of the two human breast carcinoma cell lines MCF 7/6 and
MCF 7/AZ in MTT and SRB tests, in comparison to the negative controls after application of 5 µM of
acetyldesoxodehydrolaserpitin (ADODHL) and 10 nM of vinblastine sulphate as a reference compound. Results
are expressed as the mean values ± SD of four independent replicates. Statistical significance of inhibition was
determined in comparison to control groups at significance level * P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.
Table 1.
In vitro cytotoxic activity expressed as IC
50
values on MCF 7/6 and MCF 7/AZ cell lines for
L. latifolium CHCl
3
extract, compounds and reference compound (vinblastine sulphate) in MTT and SRB assays.
Compound MTT SRB
MCF 7/6 MCF 7/AZ MCF 7/6 MCF 7/AZ
1
a
31.80 87.13 >100 >100
2
a
0.60 2.29 0.51 31.87
3
a
4.57 2.46 62.04 7.36
Vinblastine
a
5.98
×
10
-
3
15.41
×
10
-
3
4.11
×
10
-
3
3.66
×
10
-
3
Extract
b
184.72 208.94 208.85 397.16
a
IC
50
values for laserpitin (1), acetyldesoxodehydrolaserpitin (2), laserin (3) and , vinblastine (reference
compound) are given in µM;
b
IC
50
values for L. latifolium CHCl
3
extract are given in µg/ml.
Acknowledgment
This research was supported by a Basileus grant (Application № 1011092), for a research stay
of PhD student Višnja Popović in Laboratory for Pharmacognosy and Phytochemistry, Faculty of
Pharmaceutical Sciences, Ghent University, as a part of the Erasmus Mundus Action 2 program of
European Comission, and Ministry of Education and Science of Republic of Serbia (Grant № 173021).
Statistical analyses were performed with the kind help of Prof. Dr. Dragić Banković, Faculty of
Mathematics, University of Kragujevac, Serbia.
Popović et al., Rec. Nat. Prod. (2013) 7:3 245-249
249
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