Pseudocnoside A, a new cytotoxic and antiproliferative triterpene glycoside from the sea cucumber Pseudocnus dubiosus leoninus

Article abstract of DOI:10.1080/14786419.2012.751596

A new triterpene glycoside, pseudocnoside A (1), was isolated from the sea cucumber Pseudocnus dubiosus leoninus. The structure of the new compound was established on the basis of extensive NMR spectroscopic analysis (¹H and ¹³C NMR, ¹H,¹H-COSY, HMBC, HSQC, TOCSY and NOESY), HR-ESI-MS data and chemical transformations. In addition, the cytotoxicity and antiproliferative activities of 1 and structurally related triterpene glycosides isolated from the sea cucumbers Psolus patagonicus and Hemioedema spectabilis were evaluated against cancer cell lines A-549 and HeLa.

Full text of DOI:10.1080/14786419.2012.751596

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Pseudocnoside A, a new cytotoxic and
antiproliferative triterpene glycoside
from the sea cucumber Pseudocnus
dubiosus leoninus
Valeria P. Careagaa, Carlos Buenob, Claudia Muniainc, Laura Alchéb
& Marta S. Maiera
a UMYMFOR (CONICET - UBA), Departamento de Química Orgánica,
Facultad de Ciencias Exactas y Naturales, Pabellón 2, Ciudad
Universitaria, 1428 Buenos Aires, Argentina
b Departamento de Química Biológica, Facultad de Ciencias
Exactas y Naturales, Pabellón 2, Ciudad Universitaria, 1428
Buenos Aires, Argentina
c Laboratorio de Ecología Química y Biodiversidad Acuática,
Instituto de Investigación e Ingeniería Ambiental, Universidad
Nacional de San Martín, Peatonal Belgrano 3563 1°, 1650 San
Martín, Buenos Aires, Argentina
Published online: 17 Dec 2012.
To cite this article: Valeria P. Careaga, Carlos Bueno, Claudia Muniain, Laura Alché & Marta S.
Maier (2014) Pseudocnoside A, a new cytotoxic and antiproliferative triterpene glycoside from the
sea cucumber Pseudocnus dubiosus leoninus, Natural Product Research: Formerly Natural Product
Letters, 28:4, 213-220, DOI: 10.1080/14786419.2012.751596
To link to this article: http://dx.doi.org/10.1080/14786419.2012.751596
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Natural Product Research, 2014
Vol. 28, No. 4, 213–220, http://dx.doi.org/10.1080/14786419.2012.751596
Pseudocnoside A, a new cytotoxic and antiproliferative triterpene glycoside
from the sea cucumber Pseudocnus dubiosus leoninus
a
Valeria P. Careaga , Carlos Bueno , Claudia Muniain , Laura Alche and Marta S. Maier *
b
c
b
a
´
a
´
UMYMFOR (CONICET - UBA), Departamento de Quımica Organica, Facultad de Ciencias Exactas y
Naturales, Pabellon 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina; Departamento de Quımica
´
b
´
´
´
Biologica, Facultad de Ciencias Exactas y Naturales, Pabellon 2, Ciudad Universitaria, 1428 Buenos
Aires, Argentina; Laboratorio de Ecologıa Quımica y Biodiversidad Acuatica, Instituto de Investigacion e
´
c
´
´
´
´
´
Ingenierıa Ambiental, Universidad Nacional de San Martın, Peatonal Belgrano 3563 18, 1650 San Martın,
Buenos Aires, Argentina
´
´
(Received 6 July 2012; final version received 5 November 2012)
A new triterpene glycoside, pseudocnoside A (1), was isolated from the sea cucumber
Pseudocnus dubiosus leoninus. The structure of the new compound was established on
1
the basis of extensive NMR spectroscopic analysis (1H and 13C NMR, H,1H-COSY,
HMBC, HSQC, TOCSY and NOESY), HR-ESI-MS data and chemical transform-
ations. In addition, the cytotoxicity and antiproliferative activities of 1 and structurally
related triterpene glycosides isolated from the sea cucumbers Psolus patagonicus and
Hemioedema spectabilis were evaluated against cancer cell lines A-549 and HeLa.
Keywords: Pseudocnus dubiosus leoninus; sulphated triterpene glycosides; cytotoxic;
antiproliferative
1. Introduction
Sea cucumbers (class Holothuroidea) have been shown to contain a variety of triterpene
glycosides based on a ‘holostanol’ skeleton ((3b,5a,20S)-3,20-dihydroxylanostano-18,20-
lactone) and a sugar chain of two to six monosaccharide units linked to the C-3 of the aglycon
(Maier, 2008). These substances are produced in the skin and in the Cuvier’s tubules of sea
cucumbers and are ejected when the animals are disturbed. This behaviour may be associated
with a defensive function due to the ability of holothurins to form complexes with cholesterol
and other D5-sterols from cell membranes. This membranolytic activity probably determines the
wide spectrum of their biological effects, including antifungal, cytotoxic, antiviral, haemolytic
and immunomodulatory properties (Kalinin, Aminin, Avilov, Silchenko, & Stonik, 2008).
Recently, we have reported on the antifungal activities of patagonicosides A–C, three sulphated
triterpene glycosides from the sea cucumber Psolus patagonicus (Careaga, Munian, & Maier,
2011).
As a continuation of our search for bioactive metabolites from cold-water echinoderms of the
South Atlantic (Chludil & Maier, 2005; Chludil, Seldes, & Maier, 2002; Maier et al., 2001;
Murray, Muniain, Seldes, & Maier, 2001), we have investigated the glycoside fraction of the sea
cucumber Pseudocnus dubiosus leoninus (Semper, 1867) (Dendrochirotida, Cucumariidae).
This small holothurian is frequently found from the patagonian intertidal rocks to 200 m in the
*Corresponding author. Email: maier@qo.fcen.uba.ar
q 2012 Taylor & Francis

214
V.P. Careaga et al.
South Atlantic. No chemical investigation of P. dubiosus leoninus has been reported as yet.
Here, we describe the isolation and structure elucidation of a new sulphated triterpene glycoside,
pseudocnoside A (1) (Figure 1), from the EtOH extract of P. dubiosus leoninus. Compound 1
and structurally related triterpene glycosides isolated from the sea cucumbers P. patagonicus
(Careaga et al., 2011) and Hemioedema spectabilis (Chludil et al., 2002) were evaluated for their
cytotoxicity and antiproliferative activities on two cancer cell lines (A-549 and HeLa) in order to
establish structure–activity correlations.
O
O
O
O
O
O
O
O
Sugars
Sugars
2 R1,2 = SO3Na R3 = H
3 R1,2,3 = SO3Na
1 R1,2 = SO3Na R3 = H
O
O
OH
OR1
CH3
OH
O
CH2OR
2 O
OH
O
CH2OR3
O
OCH3
O
OH
OH
OH
OH
O
HO O
OH
O
O
OH
OR1
CH3
O
4 R1 = SO3Na R2 = H R3 = H
5 R1,3 = SO3Na R2 = CH2OH
O
OH
R2
O
O
O
OH
CH2OR3
O
OCH3
OH
OH
OH
OH
Figure 1. Pseudocnoside A (1), hemoiedemosides A (2) and B (3), patagonicosides B (4) and C (5).

Natural Product Research
215
2. Results and discussion
Purification of the MeOH extract of P. dubiosus leoninus by vacuum dry column
chromatography on C18 reversed-phase (RP) silica gel and finally by RP-high performance
liquid chromatography (HPLC) on a Bondclone C18 yielded a new triterpene glycoside,
pseudocnoside A (1), as the major component.
1
An examination of the H and 13C NMR spectra of pseudocnoside A (1) indicated the
presence of a triterpenoid aglycon with one olefinic bond, a keto group and one lactone CvO
group bonded to an oligosaccharide chain composed of four sugar units. The structure of the
aglycon of 1 was determined on the basis of spectroscopic data and by its comparison with those
of triterpene glycosides isolated from the sea cucumber Cucumaria japonica (Chludil, Murray,
Seldes, & Maier, 2003). The aglycon of 1 belongs to the holostane type [based on the signals of
the 18(20)-lactone at dðCÞ 179.7 (C(18)) and 84.4 (C(20)) in the 13C NMR spectrum] and
contains a C(7)vC(8) bond (d(C) 122.7 (C(7)) and 145.1 (C(8)); d(H) 5.76 (H-C(7))) and a keto
group at C-16 (d(C) 213.6 (C(16)). The presence of a keto group at C(16) was evidenced by the
signals of C(15) and C(17) in the 13C NMR spectrum at d(C) 52.8 and 64.5, respectively,
together with signals at d(H) 2.57, 2.74 (H-C(15)) and 2.90 (H-C(17)) in the 1H NMR spectrum.
1
The H NMR spectrum also showed seven methyl groups, five characteristics of a holostane
skeleton at d 1.25 (s, H-32), 1.14 (s, H-30), 1.34 (s, H-31), 1.32 (s, H-19) and 1.50 (s, H-21), and
1
two of terminal methyl groups at d 0.90 (s, H-27) and 0.92 (s, H-26) in the side chain. H,1H-
COSY, HSQC and HMBC spectra allowed the assignment of all the proton and carbon
resonances of the aglycon. The relative stereochemistry of all chiral centres of the aglycon was
established with the aid of a NOESY experiment (Figure 2). The above data indicated that the
structure of the aglycon moiety was 16-keto-holost-7-en-3b-ol, the same as that of
cucumarioside A2-3, isolated from the sea cucumber C. japonica (Drozdova et al., 1993).
The carbohydrate chain of 1 consisted of four monosaccharide residues as deduced from the
13C NMR spectrum, which showed the signals of four anomeric C-atoms at 105.6–106.1 ppm,
correlated by the HSQC spectrum with the corresponding signals of anomeric H-atoms at 4.78
(d, J ¼ 7.2 Hz), 4.88 (d, J ¼ 8.1 Hz), 5.03 (d, J ¼ 7.8 Hz) and 5.41 (d, J ¼ 7.8 Hz). The coupling
constants of the anomeric H-atoms were indicative of b-configuration of the glycosidic bonds.
The presence of 3-O-methylglucose, glucose, quinovose and xylose in the ratio 1:1:1:1 was
established by acid hydrolysis with aqueous 2 N trifluoroacetic acid followed by GC analysis of
the corresponding peracetylated alditols (Maier et al., 2001). All H- and C-atom chemical shifts
of the oligosaccharide chain of 1 could be assigned by 1H,1H-COSY, HSQC, HMBC, TOCSY
and NOESY experiments. The four carbohydrate units belong to the D-series, as determined by
21CH3
19
CH3
O
O
H
30
CH3
H
17
H
9
H
H
1
12
5
3
H
O
O
15
1'
31
CH3
H
7
O
H
32CH3
H
H
H
H
Figure 2. NOESY correlations of the aglycon of pseudocnoside A (1).

216
V.P. Careaga et al.
the GC/MS analysis of the mixture of 1-{(acetyl)[(S)-2-hydroxypropyl]amino}-1-deoxyalditol
acetate derivatives following the procedure described in Maier et al. (2001).
The HR-ESI-MS (negative-ion mode) of pseudocnoside A (1) exhibited a pseudo-molecular
ion peak at m/z 1267.4288 ([M 2 Na]2; calcd 1267.4488). The MS/MS spectrum of this ion
indicated the peaks for fragment ions at m/z 1147.5565 ([M 2 NaSO4–H–Na]2) and 827.3620
([M 2 3-O-Me-Glc-O-Glc-OSO3Na]2), confirming the presence of two sulphated groups in the
carbohydrate chain and the presence of a terminal unit of a sulphated 3-O-methylglucose. This
and NMR spectroscopic data allowed the determination of the molecular formula as
C54H84O28S2Na2.
The interglycosidic linkages were deduced from NOESY (Figure 3) and HMBC correlations,
where cross-peaks between H-C(10) of the xylose residue and H-C(3) of the aglycon, H-C(100) of
the quinovose and H-C(20) of the xylose residue, H-C(1000) of the glucose and H-C(400) of the
quinovose unit, and H-C (10000) of 3-O-methylglucose and H-C(3000) of the glucose unit,
respectively, were observed. The carbohydrate chain of 1 is a linear tetrasaccharide consisting of
D-xylose (sulphated at C-4), D-quinovose, D-glucose (sulphated at C-6) and a terminal 3-O-
methyl-D-glucose. The oligosaccharide chain of 1 is identical to that of patagonicoside A
(Murray et al., 2001), hemoiedemoside A (Chludil et al., 2002) and cucumechinosides A and C
(Miyamoto, Togawa, Higuchi, Komori, & Sasaki, 1990).
On the basis of all the above data, the structure of pseudocnoside A (1) was established as
3-O-[3-O-methyl-b-D-glucopyranosyl-(1 ! 3)-6-O-sodiumsulphonato-b-D-glucopyranosyl-
(1 ! 4)-b-D-quinovopyranosyl-(1 ! 2)-4-O-sodiumsulphonato-b-D-xylopyranosyl]-holost-7-
en-16-one (Figure 1).
Studies on the structure–activity relationship for sea cucumber glycosides revealed that their
biological activities depend on both the aglycon and the carbohydrate chain structures (Kalinin
et al., 2008). Several triterpene glycosides containing a linear tetrasaccharide chain have shown
cytotoxic activity on tumour cell growth in vitro (Hang, Xu, Yi, Gong, & Jiao, 2010; Silchenko
et al., 2008; Sun et al., 2007). Usually, the literature deals mainly with sea cucumber triterpene
glycosides exhibiting cytotoxic activity in tumour cells rather than antiproliferative effects.
Recently, we have shown that patagonicoside A, a disulphated triterpene glycoside isolated from
the sea cucumber P. patagonicus and its desulphated analogue showed antiproliferative activity
in three tumour cell lines (Hep3B, MDA-MB231 and A549) and displayed lower levels of
´
cytotoxicity than most triterpene glycosides already reported (Careaga, Bueno, Muniain, Alche,
& Maier, 2009).
These results enabled us to test compound 1 for in vitro cytotoxicity and antiproliferative
activity on two human tumour cell lines (A549 and HeLa) following the procedure described in
Careaga et al. (2009) and compare these activities with those of four structurally related
triterpene glycosides, hemoiedemosides A (2) and B (3) and patagonicosides B (4) and C (5)
H
H
H
O
3
NaO3SO
HO
O
H
O
H
OSO3Na
H
H
H
OH
H
CH3
H
H
H
O
O
H
O
HO
O
O
HO
CH3O
HO
H
H
H
OH
H
OH
H
OH
H
H
H
H
Figure 3. NOESY correlations of the oligosaccharide chain of pseudocnoside A (1).

Natural Product Research
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Table 1. In vitro cytotoxic and antiproliferative activities of compounds 1–5 against human tumour cells
A549 and HeLa.
A549
HeLa
Cell line
CC50 (mM)
IC50 (mM)
CC50 (mM)
IC50 (mM)
1
2
3
4
5
39.17
29.71
5.96
8.87
4.04
14.53
7.43
3.16
9.73
5.56
74.41
21.96
2.80
8.58
3.39
57.36
9.95
2.15
7.94
3.57
(Figure 1) isolated from H. spectabilis (Chludil et al., 2002) and P. patagonicus (Careaga et al.,
2011), respectively. The IC50 and CC50 values were determined on the basis of the observed cell
viability after 24 h of drug exposure (Table 1).
The antiproliferative activity of 1 in A549 cells (IC50 ¼ 14.53 mM) was considerably higher
than in HeLa cells (IC50 ¼ 57.36 mM). Frequently, different tumour cell lines exhibit a
differential sensitivity to the cytotoxic effects of sea cucumber glycosides (Jin et al., 2009).
The differences in the antiproliferative activity of glycoside 1 may be ascribed to a higher
sensitivity of A549 tumour cells when treated with compound 1.
Hemoiedemosides A (2) and B (3) differ only in the degree of sulphation of the
oligosaccharide chain. Trisulphated glycoside 3 contains an additional sulphate group at C-6 of
the terminal 3-O-methylglucose unit. Glycosides 2 and 3 exhibited stronger antiproliferative
activity than compound 1 with similar IC50 values in A549 and HeLa cell lines (Table 1).
Besides, the higher cytotoxicity of glycoside 3 on both cell lines may be related to the presence
of a third sulphate group in the carbohydrate chain. Similar results were observed for
okhotosides B1, B2 and B3. Monosulphated okhotoside B1 showed lower cytotoxicity than their
disulphated analogues B2 and B3 in HeLa cell line (Silchenko et al., 2008).
Patagonicosides B (4) and C (5) exhibited similar antiproliferative and cytotoxic activities in
both cell lines with IC50 values comparable to those for hemoiedemoside B (3) (Table 1). It is
important to point out that compound 2 exhibited a low cytotoxicity not only for A549 and HeLa
cells but also for Vero cells, with a CC50 value of 30.02 mM. The fact that compound 2 displayed
a low level of cytotoxicity for tumoural and normal cells, together with its remarkable
antiproliferative effect, suggests that 2 is a more suitable molecule than other sea cucumber
triterpene glycosides to impede tumour cell growth in vitro.
3. Experimental
3.1. General
1-{(Acetyl)[(S)-2-hydroxypropyl]amino}-1-deoxyalditol acetate derivatives of monosacchar-
ides were analysed using a Shimadzu GCMS-QP5050 chromatograph equipped with a capillary
column (Ultra II Hewlett Packard, 50 m £ 0.20 mm). Preparative HPLC: SP liquid
chromatograph equipped with a Spectra Series P100 solvent delivery system, a Rheodyne
manual injector and a refractive index detector using a Bondclone 10m column (30 cm £ 7.8 mm
i.d.). The samples were eluted with CH3OH/H2O (55:45) with a flow of 2 ml/min. Thin layer
chromatography (TLC): pre-coated Si gel F254 (n-BuOH/HOAc/H2O 12:3:5) and C18 RP plates
(55:45% MeOH/H2O); detection by spraying with p-anisaldehyde (5% EtOH). Optical rotations:
Perkin-Elmer 343 polarimeter. 1H and 13C NMR spectra: in C5D5N/D2O (4:1) on a Bruker AM
500 spectrometer. HR-ESI-MS (negative-ion mode): Bruker Daltonics micrOTOF-QII mass
spectrometer (Bruker Daltonics, Billerica, MA, USA).

218
V.P. Careaga et al.
3.2. Animal material
Specimens of P. dubiosus leoninus were collected by dredge from beds of the scallop
Zygochlamys patagonica during May 2007 and October 2009 at a depth of 100–120 m in the
South Atlantic Ocean. Individuals of 20–40 mm were removed manually from each shell scallop
and immediately frozen. The organisms were identified by Dr C. Muniain. A voucher specimen
is deposited at the Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, Buenos
Aires, Argentina (MACN No. 39439).
3.3. Extraction and purification procedures
The sea cucumbers (39 g), frozen prior to storage, were homogenised in EtOH (1.5 l) and
centrifuged. The EtOH was evaporated and the residue was partitioned between MeOH/H2O 9:1
(300 ml) and cyclohexane (100 ml). The glassy material obtained after evaporation of the MeOH
extract was subjected to vacuum dry column chromatography on Davisil C18 RP (35–70 mm)
using H2O, H2O/MeOH mixtures with increasing amounts of MeOH and finally MeOH as
eluents. Triterpene glycosides were eluted with 60% and 70% MeOH. These fractions were
combined and submitted to RP-HPLC to give the pure glycoside 1 (3 mg; tR 47.5 min) as a white
20
amorphous powder. ½aꢀD 239.1 (c 0.18, pyridine).
1H NMR (500 MHz, C5D5/D2O (4:1)): d 1.50 (2H, m, H-1); 1.99, 2.19 (2H, m, H-2); 3.34
(1H, m, H-3); 1.11 (1H, m, H-5); 2.11 (2H, m, H-6); 5.76 (1H, m, H-7); 3.79 (1H, m, H-9); 1.71,
1.95 (2H, m, H-11); 2.14b, 2.34a (2H, m, H-12); 2.57 (1H, m, H-15); 2.74 (1H, d, J ¼ 15.8 Hz,
H-15); 2.90 (1H, s, H-17); 1.32 (3H, s, CH3-19); 1.50 (3H, s, CH3-21); 1.72, 1.87 (2H, m, H-22);
1.50 (2H, m, H-23); 1.22 (2H, m, H-24); 1.57 (1H, m, H-25); 0.92 (3H, s, CH3-26); 0.90 (3H, s,
CH3-27); 1.14, (3H, s, CH3-30); 1.34 (3H, s, CH3-31); 1.25 (3H, s, CH3-32); 4.78 (d, J ¼ 7.2 Hz,
C-10-H); 4.08 (m, C-20-H); 4.07 (t, J ¼ 8.9 Hz, C-30-H); 5.25 (m, C-40-H); 3.77 (m, 4.70 m, C-50-
H); 5.03 (d, J ¼ 7.8 Hz, C100-H); 3.97 (m, C200-H); 3.97 (m, C300-H); 3.52 (t, J ¼ 9.1 Hz, C400-H);
3.68 (m, C500-H); 1.62 (d, J ¼ 6.1 Hz, C100-3H); 4.88 (d, J ¼ 8.1 Hz, C1000-H); 3.81 (m, C2000-H);
4.36 (m, C3000-H); 3.88 (m, C4000-H); 4.30 (m, C5000-H); 4.87, 5.29 (m, C6000-2H); 5.41 (d,
J ¼ 7.8 Hz, C10000-H); 3.92 (m, C20000-H); 3.78 (m, C30000-H); 4.22 (m, C40000-H); 4.04 (m, C50000-H);
4.35 (m, C60000-H); 4.54 (brd, J ¼ 10.5 Hz, C60000-H); 3.93 (s OCH3).
13C NMR (500 MHz, C5D5/D2O (4:1)): d (C-1) 36.7; (C-2) 27.9; (C-3) 89.7; (C-4) 40.3;
(C-5) 49.2; (C-6) 24.1; (C-7) 122.7; (C-8) 145.1; (C-9) 47.7; (C-10) 36.4; (C-11) 23.2; (C-12)
30.4; (C-13) 57.7; (C-14) 46.5; (C-15) 52.8; (C-16) 213.6; (C-17) 64.5; (C-18) 179.7; (C-19)
23.5; (C-20) 84.4; (C-21) 26.9; (C-22) 39.9; (C-23) 23.7; (C-24) 40.1; (C-25) 28.4; (C-26) 23.6;
(C-27) 23.4; (C-30) 18.1; (C-31) 29.4; (C-32) 32.9; (C-10) 105.7; (C-20) 83.8; (C-30) 75.6; (C-40)
76.6; (C-50) 65.0; 64.55; (C-100) 105.6; (C-200) 76.7; (C-300) 74.4; (C-400) 89.1; (C-500) 72.3; (C-600)
18.8; (C-1000) 106.0; (C-2000) 72.3; (C-3000) 87.8; (C-4000) 71.2; (C-5000) 75.8; (C-6000) 68.6; (C-10000)
106.1; (C-20000) 74.9; (C-30000) 88.7; (C-40000) 71.1; (C-50000) 79.1; (C-60000) 62.6; OCH3 61.5.
(2) HR-ESI-MS, m/z 1267.4288 [M 2 Na]2, C54H84O28S2Na (1267.4488 calcd); (2) HR-
ESI-MS/MS of the ion [M 2 Na]2 at m/z 1147.5565 [M 2 NaSO4–Na–H] and 827.3620
[M 2 3-O-Me-Glc-O-Glc-OSO3Na]2.
3.4. Cell culture and reagents
Human lung carcinoma A549 cells were grown in Eagle’s minimal essential medium
supplemented with 10% inactivated fetal bovine serum (FBS) (MEM 10%). HeLa cells were
maintained in Dulbecco’s modified Eagle’s minimum essential medium and Ham’s F-12
medium containing 10% FBS (DMEM 10%). Vero cells, a cell line initiated from the kidney of a
normal adult African green monkey, were grown in Eagle’s minimal essential medium

Natural Product Research
219
supplemented with 5% inactivated FBS (MEM 5%). Cell cultures were maintained in a 4% CO2
humidified atmosphere at 378C.
3.5. Antiproliferative assay
We seeded 2.4 £ 106 cells in 96-well plates together with different concentrations of triterpene
glycosides 1–5 in duplicate, and incubated at 378C for 24 h in a 4% CO2 atmosphere. Then, cells
were fixed with 10% formaldehyde for 15 min at room temperature, washed once with distilled
water and stained with 0.05% crystal violet in 10% ethanol over 30 min. Afterwards, cells were
washed once and eluted with a solution of 50% ethanol and 0.1% acetic acid in water. The
absorbance of each well was measured on a Eurogenetics MPR-A 4i microplate reader using a
test wavelength of 590 nm.
3.6. Cytotoxicity assay
Cell viability was determined as previously reported (Careaga et al., 2009). Briefly, cell viability
in the presence of the compound was determined using the cleavage of the tetrazolium salt MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by the mitochondrial enzyme
succinate dehydrogenase to give a blue product (formazan). The absorbance of each well was
measured on a Eurogenetics MPR-A 4i microplate reader using a test wavelength of 570 nm and
a reference wavelength of 630 nm by duplicate.
Acknowledgements
This work was supported by the University of Buenos Aires, the National Research Council of Argentina
(CONICET) and the ANPCyT/FONCYT (PICT 34111). V.P.C. and C.B. thank the CONICET for a
doctoral fellowship. M.S.M., L.A. and C.M. are Research members of CONICET. We would like to thank
´
Dr S. Campodonico, Dr C. Bremec and Dr M. Lasta from INIDEP (Mar del Plata, Argentina) for collecting
the sea cucumber samples.
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