Experimental Parasitology 127 (2011) 222–227

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Plasmodium falciparum: Solanum nudum SN-1 steroid antiplasmodial activity

when combined with antimalarial drugs
Adriana Pabón a, Eric Deharo b,c, Silvia Blair a,⇑
a
Malaria Group, Universidad de Antioquia, Medellín, Colombia
b
Université de Toulouse, UPS, UMR 152 (laboratoire de pharmacochimie des substances naturelles et pharmacophores redox), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France
c
IRD, UMR-152, Mission IRD Casilla 18-1209, Lima, Peru

a r t i c l e i n f o a b s t r a c t

Article history: The effect of 16 alpha-acetoxy-26-hydroxycholest-4-ene-3,22-dione (SN-1) isolated from Solanum nudum
Received 4 December 2009 Dunal (a Solanaceae traditionally used for treating fever in Colombia) on Plasmodium falciparum erythro-
Received in revised form 11 July 2010 cyte stages and its in vitro antiplasmodial activity when combined with the following conventional drugs
Accepted 2 August 2010
was studied: chloroquine (CQ), amodiaquine (AQ), desethylamodiaquine (desethyl-AQ), quinine (QN),
Available online 22 August 2010
artemisinin (AR), atovaquone (ATV) and quinine (QN). It was found that SN-1 targeted trophozoites
and had a synergistic effect when combined with CQ and QN; however, it had an antagonist effect when
Keywords:
used with the other combinations.
Antimalarial
Solanum nudum
Ó 2010 Elsevier Inc. All rights reserved.
Drug interaction
Steroid

1. Introduction mutagenic and clastogenic activity assays revealed that none of

Malaria is the infectious parasitic disease causing the greatest
morbimortality worldwide; it leads to more than 300 million peo-
ple becoming ill and more than two million directly-related deaths
annually. It causes human and economic losses and places more
than 2400 million people at risk of contracting the disease (Fidock
et al., 2004; WHO, 2008; WHO/TDR, 2004). There were 110,389
cases of malaria in Colombia in 2007 according to the World Health
Organisation (WHO), 30,065 of them being caused by Plasmodium
falciparum (WHO, 2008). High percentages of resistance have been
reported in Colombia as well as therapeutic failure regarding the
most-used drugs such as 4-aminoquinolines (Arango et al., 2008;
Blair et al., 2006; Restrepo-Pineda et al., 2008). Therapeutic alter-
natives are thus urgently needed.
Ongoing work with a plant locally known as ‘‘zapata” (Solanum
nudum Dunal) has been carried out in the search for new treatment
strategies; folk-healers from the Colombian Pacific coast have re-
ported it as being one of the plants most widely used in the region
for curing malaria. Five steroid compounds have been isolated from
this plant as well as a steroidal sapogenin (diosgenone) presenting
the 4-en-3-one system as one of its characteristics (similar to that
of progesterone); they have presented in vitro antiplasmodial activ-
ity (Blair et al., 2001; Cardona, 1997; Saez et al., 1998). S. nudum
⇑ Corresponding author. Address: Grupo Malaria, Universidad de Antioquia, Calle
62 # 52-59, Sede de Investigación Universitaria (SIU), Torre 1, laboratorio 610,
Medellín, Colombia. Fax: +57 4 2106487.
E-mail address: silviablair@gmail.com (S. Blair).
the compounds being evaluated were mutagenic or clastogenic,
thereby representing a further advance in ongoing research (Alvarez
et al., 2004; Pabon et al., 2003). Whilst negative results have been re-
turned in cytotoxicity tests (Londono et al., 2006), other studies eval-
uating the inhibition of Plasmodium vivax sporozoites invading
HepG2 cells have revealed these steroids’ positive activity (Londono
et al., 2006). The SN-1 steroid has been shown to induce increased to-
tal glutathione and cysteine concentration and has inhibited more
than 80% of b-haematin formation at 5.0 mM (Pabon et al., 2009).
It has also been reported that two Solanum steroids have inhib-
ited isoosmotic sorbitol and alanine-induced lysis of parasitised
red blood cells (pRBC), suggesting that these metabolites act on
new pRBC permeability pathways (Lopez et al., 2009). A recent
report has stated that cell death in P. falciparum arising from
S. nudum compounds became evident by decreased mitochondrial
membrane potential, DNA fragmentation and cytoplasmic acidifi-
cation. Steroid treatment has been shown to induce some apopto-
tic-like and autophagic-like cell death characteristics in
P. falciparum asexual blood stages (López et al., 2010).
The above results regarding the S. nudum plant’s antiplasmodial
activity aroused interest in embarking on further studies aimed at
elucidating its mode of action. Research was thus begun on identi-
fying the P. falciparum intra-erythrocyte stage most sensitive to the
steroid offering the best in vitro antiplasmodial activity for com-
pounds isolated from S. nudum (SN-1). There was also great inter-
est in evaluating the antiplasmodial effect of combining steroids
with conventional drugs to support the development of combined
therapy or ethnopharmacological studies.

0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2010.08.009
 A. Pabón et al. / Experimental Parasitology 127 (2011) 222–227
2. Materials and methods
2.1. Plant material
S. nudum Dunal (commonly known as ‘‘zapata”) grows as a
weed or herbaceous plant on the banks of rivers and streams near
Tumaco and other towns on the Colombian Pacific coast; it has no
recognised commercial or ornamental value. A sample of the plant
is kept at the Herbarium of Universidad Antioquia (accession num-
ber 554, file 61181 in the Colombian flora catalogue); it was iden-
tified by the herbarium and confirmed by the Universidad Nacional
de Colombia, the Universidad de Nariño and professor Nee from
the New York Botanical Garden’s Herbarium (Blair et al., 2000;
Blair and Madrigal, 2005).
The Universidad de Antioquia’s Malaria Group has established a
verbal agreement with Afro-descendant communities from the
Colombian Pacific coast for gaining access to plants reported by lo-
cal communities as having antimalarial activity (Programa Inter-
nacional Tumaco, based in Cali, Colombia). This has led to
S. nudum being collected for several investigations. The most re-
cent samples of S. nudum stems and leaves were collected for this
work in June 2002 with the help of the native population and local
folk-healers along the banks of streams feeding the river Chagüí
near Tumaco (Nariño, Colombia) (78°300 log. W; 1°430 1500 lat. N.
15 m altitude).
SN-1 16a-acetoxy-26-hydroxycholest-4-ene-3,22-dione was
isolated from S. nudum and purified as previously described (Lond-
ono et al., 2006; Pabon et al., 2002; Saez et al., 1998); the steroid’s
structure was verified by proton nuclear magnetic resonance (1H
NMR).
2.2. Reagents
Chloroquine diphosphate (CQ), quinine sulphate (QN), amodia-
quine dihydrochloride dihydrate (AQ) and artemisinin (AR) were
obtained from Sigma. Monodesethylamodiaquine (DAQ) was do-
nated by Dr. Pascal Ringwald from the WHO’s Roll Back Malaria
department. Atovaquone (ATV) was a kind gift from Dr. Eric Le-
grand from the Pasteur Institute in French Guyana.
Sodium chloride, HEPES, hydroethidium (HE), thiazole orange
(TO), percoll, sorbitol, RPMI 1640 culture medium, hypoxanthine,
L-glutamine, sodium bicarbonate, D(+)-glucose and D-sorbitol re-
agents were obtained from Sigma whilst DMSO, petroleum ether,
ethyl ether, hexane and dichloromethane were obtained from
MERCK.
Ultra-pure MiliQ grade water or distilled water for injection (i.e.
parenteral use) was always used for suspending all reagents, pre-
paring buffer and culture media.
2.3. P. falciparum
The P. falciparum FCR-3 strain was used for the different exper-
iments; it is chloroquine-resistant and pyrimethamine-sensitive
(Jensen and Trager, 1978). It was kept in continuous culture in
the Universidad Peruana Cayetano Heredia’s Cell Parasitology
unit’s laboratory (Science and Philosophy Faculty) under the terms
of the cooperation agreement between Universidad Peruana Cayet-
ano Heredia (Lima, Perú) and the Institut de Recherche pour le
Développement (IRD, France). This strain was cultured and main-
tained according to Trager and Jensen’s 1976 method, using a sus-
pension of 5% human A+ erythrocytes in RPMI 1640 culture
medium (Sigma R4130) dissolved in sterile water with 25 mM
HEPES, 5% NaHCO3 and 10% fresh human A+ serum (inactivated
for 30 min at 56 °C). This was incubated in a 5% O2, 5% CO2 and
90% N2 atmosphere The medium was changed daily and fresh
223
red blood cells (RBC) were added twice a week (Ljungström
et al., 2004). A dilution of RBC infected with 1% parasitaemia, 2%
haematocrite, was prepared for carrying out the antiplasmodial
activity tests, combinations and determining the most sensitive
stage amongst the different strains assayed.
2.4. Synchronising P. falciparum strains
A P. falciparum continuous culture (non-synchronic), having
around 5% parasitaemia, was synchronised with consecutive 5%
D-sorbitol lysis to guarantee obtaining parasites having the same
parasite form; this destroyed the mature forms, leaving behind
an early ring stage population (<6 h after merozoite invasion)
(Lambros and Vanderberg, 1979; Rojas and Wasserman, 1987).
2.5. Determining which P. falciparum stage was most sensitive to the S.
nudum SN-1 steroid
Synchronised ring-stage parasites (1% parasitaemia, 2% haemat-
ocrite) were used for determining in vitro antiplasmodial activity
during several maturation periods (each treatment lasting for 4
or 8 h), in triplicate and at 7 dilutions (211.6–3.3 lM) (Garavito
et al., 2007). 96-well plates (FalconÒ) were filled with a 200 lL/
well suspension of synchronised ring-stage infected red blood cells
(RBC-R); 50 lL steroid dilution was added to a 96-well plate’s first
three columns (time 0). The plate was shaken gently and then incu-
bated at 37 °C in a 5% O2, CO2 and N2 balanced atmosphere. The
supernatant was removed from the first three columns once the
4-h or 8-h incubation period had elapsed and the RBC-R from these
columns was washed with RPMI 1640, the whole plate being cen-
trifuged (839g, 10 min). 200 lL RPMI-1640 medium labelled with
1.0 lCi/mL [3H] hypoxanthine (MP Biomedicals, USA) were then
placed in these wells. The steroid dilutions were placed in the next
three columns and the plate was incubated for a further 4 h. The
washing protocol was then repeated for the next three columns
(as described above). This procedure was repeated every 4 h (or
8-h) until the maturation period (48-h cycle) had been completed,
as described below. The plates were placed in a freezer at 20 °C to
provoke erythrocyte haemolysis; they were thawed out the next
day. The labelled nucleic acids were deposited in a fibreglass filter
with the help of a semi-automatic collector and read on a Beckman
LS 6000 scintillation counter. Readings were expressed in counts
per minute (cpm). Half maximal inhibitory concentration (IC50)
was determined by lineal interpolation (Huber and Koella, 1993).
2.6. Identifying P. falciparum stages by flow cytometry
Flow cytometry (as proposed by Makler et al. in 1987) was used
for double-labelling P. falciparum DNA with HE and TO for corrob-
orating P. falciparum intra-erythrocyte stage-sensitivity to the SN-1
steroid and standardising a sensitive, rapid and easily-executed
method. Briefly, P. falciparum FCR-strain continuous cultures (10%
parasitaemia) were synchronized by sorbitol treatment leading to
a ring-enriched culture, which was maintained for an additional
12 or 24 h to produce trophozoites or schizonts respectively
(Lambros and Vanderberg, 1979; Rowley et al., 1967). Also contin-
uous cultures were centrifuged at 839g for 5 min to obtain a pellet
which was taken to 25% haematocrite with RPMI 1640 and sepa-
rated on 80–60% Percoll-sorbitol gradients at 4 °C. This led to
RBC infected with mature forms becoming localised in the upper
layer and uninfected ones or those infected with young forms grav-
itating to the lower layers. The different layers were washed twice
with RPMI 1640 medium by spinning at 839g for 5 min; each of
them was smeared and stained with Giemsa to verify parasite
state. Twenty-five microliters were taken from each layer and
25 lL of a 0.4 mg/mL HE solution were added, the layer then being
224 A. Pabón et al. / Experimental Parasitology 127 (2011) 222–227
incubated in the dark for 20 min at 37 °C. They were then spun for
5 min at 839g, followed by 3 washes with PBS at 7.4 pH to elimi-
nate excess fluorochrome. The precipitate was then suspended in
1 mL TO at 0.05 lg/mL and incubated in the dark for 30 min at
room temperature; 50,000 cells were injected and analysed by flow
cytometry. Fluorescence-activated cell sorting (FACS), size detec-
tors, structure, FL1 (for detecting TO), FL2 (for detecting HE) were
used for data analysis and acquisition (Makler et al., 1987).
After identifying and localising P. falciparum parasite forms by
flow cytometry, the SN-1 compound’s antiplasmodial activity
(105.7 lM; 52.9 lM and 26.2 lM) and that of CQ (3.7 lM, 0.5 lM
and 0.2 lM) were then evaluated by treating parasites for 24 or
48 h, followed by HE and TO labelling.
2.7. Studying the SN-1 steroid’s in vitro interaction with conventional
drugs
The in vitro antiplasmodial activity of 200%, 100%, 75%, 50% and
25% combinations with SN-1 (for the IC50 previously calculated for
each of them) was evaluated for determining the type of SN-1 ste-
roid interaction with conventional drugs (i.e. CQ, AQ, DAQ, AR, ATV
and QN). The resulting IC50s were calculated for obtaining the other
fractional inhibitory concentrations (FICs) for each combination of
drugs and used for constructing the respective isobolograms (Fiot
et al., 2006; Garavito et al., 2007; Odds, 2003).
Fractional inhibitory concentrations (FICs) were obtained for
each drug combination as follows:
IC50 drug in mixture IC50 steroid in mixture
IC50 fractional ¼ þ
IC50 drug alone IC50 steroid alone
If the two molecules became mutually substituted and the
resulting curve in the isobologram was a diagonal, then this
showed additive activity. When both molecules mutually accentu-
ated their activity and the resulting curve approached the origin of
the axes and took on a concave aspect, then this showed synergis-
tic activity. If the curve was convex and accentuated above the
diagonal, this showed mutual inhibition of activities, indicating
an antagonic effect (Deharo et al., 2000; Garavito et al., 2007).
An additive interaction was also considered if the average FIC
concentration value = 1, synergistic < 1 and antagonist for > 1 aver-
age FIC value (Deharo et al., 2000; Fiot et al., 2006; Odds, 2003).
SPSS 11.5 was used for statistical analysis. The two-tailed t test
was used for testing the hypothesis that the average FIC value ob-
tained for each combination was different to theoretical value 1.0.
The t values were obtained according to degrees of freedom; the p
value corresponding to such t value was then calculated. The
hypothesis was rejected (FIC = 1) if the p value was statistically sig-
nificant at 95% confidence level (a 0.05 or p 6 0.05).
3. Results and discussion
3.1. P. falciparum sensitivity to the SN-1 steroid
Where P. falciparum FCR-3 strain treatment lasted 4 h (12 mat-
uration periods), around 137.7 ± 6.7 lM was found for the SN-1
IC50; greater sensitivity could not be shown, considering that the
historic IC50 for FCB-2-strain continuous 48-h culture treatment
is about 21 lM (Pabon et al., 2002). On the contrary, AQ control
treatment was 39.7 ± 4.2 nM IC50 for the early ring-form period
after 12 h invasion, this being very similar to that found after 42
h in continuous cultures (30.2 ± 8.2 nM). Bearing the above result
in mind, SN-1-treated synchronised ring-form cultures were re-
peated every 8 h, 5 periods being evaluated (0–8, 16, 24, 32 and
40 h maturation); the lowest IC50 was presented after 16 h, such
period corresponding to late ring-forms or young trophozoites
(Fig. 1). Even when the IC50 obtained was a bit far from the
30.4 ± 5.1 lM shown for continuous cultures treated for 42 h with
this strain or that reported for the FCB-2 strain, it may have been
that the nature of the SN-1 steroid influenced the time necessary
to produce an antiplasmodial effect in the synchronised cultures.
On the other hand, 24.3 nM and 23.4 nM IC50 were found when
administering AQ every 8 h after 8 and 16 h, respectively.
Treating P. falciparum cultures with CQ (another drug used as
control) also had effects on younger P. falciparum forms, effects
being presented which were very similar to those for AQ. Treating
cultures with CQ for 8 h and for 5 periods revealed 0.5 lM and
0.4 lM IC50 parasite sensitivity during the first hours of maturation
for 8 and 16 h, respectively.
3.2. Identifying P. falciparum stages using flow cytometry
P. falciparum DNA was double-labelled with HE and TO using
flow cytometry to validate whether the trophozoite stage was
the most sensitive to SN-1 treatment and to standardise a sensi-
tive, rapid and easily-executed method. The method was standard-
ised for characterising HE- and TO-labelled fluorochrome
P. falciparum culture signals. It was observed that samples (healthy
erythrocytes or synchronised ring-form, trophozoite or schizont
cultures) which had not been fluorochrome labelled did not pro-
duce any signal (i.e. all cells were located in the scatogram’s lower
left-hand quadrant, showing that none of the samples presented
autofluorescence which could have interfered with fluorochrome
labelling). HE-labelled Plasmodium cultures were displaced to
the right (corresponding to the amount of DNA) in samples con-
taining parasitised erythrocytes with ring-forms, mature trophozo-
ites and in non-synchronous cultures; however, such displacement
was greater in samples containing trophozoite-parasitised erythro-
cytes, correlating with the greater amount of DNA present during
this stage compared to ring-forms.
Displacement towards the upper right-hand quadrant was ob-
served (cell granularity) when samples were just labelled with
TO (a fluorochrome staining both DNA and RNA) without differen-
tiating a specific population or revealing changes regarding con-
trolling healthy (non-parasitised) erythrocytes. This result
indicated why this fluorochrome cannot be used alone since it does
not allow parasite stages or differences regarding control to be
identified.
Three populations in P. falciparum culture samples containing
ring-form or young trophozoite-parasitised erythrocytes (popula-
tions R1, R2 and R3) became displaced to the right when P. falcipa-
rum DNA was double-labelled with both fluorochromes (HE and
TO). R1 was located to the right of healthy RBCs (Fig. 2B and E).
The presence of a large healthy RBC population (R2) was observed
in the upper quadrant, as well as the R1 population. This popula-
tion predominated in cultures synchronised during trophozoite
and schizont stages (Fig. 2B–E). The R3 population (located in the
upper right-hand quadrant) was also revealed in synchronised ma-
ture trophozoite or schizont cultures (Fig. 2A–E).
Unsynchronised P. falciparum cultures (FCR-3 strain) were trea-
ted with three SN-1 steroid and CQ concentrations after it was
determined that ring-forms or young trophozoites corresponded
to population R1 and that parasitised erythrocytes having mature
forms (matures trophozoites or schizonts) were related to popula-
tions R2 and R3 respectively. The number of parasitised cells be-
came reduced (i.e. cells became displaced to the right of the
scatogram). It was observed that populations R1 and R2 became re-
duced by half when treating cultures with 52.9 lM SN-1 and pop-
ulation R3 by 8.1% after 24 h; a 94.6% and 87.2% reduction were
obtained for population R1 and R2 after 48 h and only 58.3% for
population R3. A 51.9% reduction for population R1 was observed
with 0.5 lM CQ; a 92.1% reduction was observed for population
 A. Pabón et al. / Experimental Parasitology 127 (2011) 222–227 225

ER LR ET LT ES LS- M

10000
8000 SN-1
6000 AQ
2000 CQ
1000
IC50

500 1,857.1
400
300
200 290.6
100 69.1 216.8
81.1 30.4

0- 8 8- 16 16- 24 24- 32 32- 40 48

Maturation periods (hours)
Fig. 1. P. falciparum stages’ sensitivity to SN-1 and controls AQ and CQ. Erythrocytes with ring P. falciparum stages were obtained as described in Section 2. Average inhibitory
concentration50 (IC50) obtained by treatment with SN-1, CQ or AQ concentrations, of SN-1, CQ or AQ, depending on P. falciparum erythrocyte cycle as described in Section 2.
IC50 for CQ and AQ concentration: nM and SN-1: lM. ER, early ring-forms; LR, late ring-forms; ET, early trophozoites; LT, late trophozoites; ES, early schizonts; LS, late
schizonts; M, merozoites. The red circle represents the IC50 for the P. falciparum FCR-3 strain culture treated with SN-1 for 48 h (complete cycle).

Fig. 2. Location of P. falciparum (FCR-strain) intra-erythrocyte stages labelled with fluorochromous hydroethidium (HE) and thiazole orange (TO) on the scatogram. Density
plot: R1: ring-forms or young trophozoites, R2: late trophozoites and R3: schizonts. Histogram: M1: healthy erythrocytes, M2: culture synchronised in labelled ring-forms;
M3: synchronised trophozoite and M4 unsynchronised culture.

R1 after 48 h treatment (Table 1). These data were corroborated
when SN-1 steroid affected the number of erythrocytes infected
in population R1 and R2 (trophozoites) and CQ affected preference
to R1 (young trophozoites), a total antiplasmodial effect being
achieved after 48 h.
3.3. Studying drug interaction with the SN-1 steroid
Experiments for ascertaining which type of interaction was pro-
duced when SN-1 was combined with conventional drugs revealed
that SN-1/CQ association showed a slight synergistic interaction
226 A. Pabón et al. / Experimental Parasitology 127 (2011) 222–227

Table 1
The effect of SN-1 and CQ treatment on P. falciparum (FCR-3 strain) continuous cultures on populations identified in the gscatogram.

Treatment Concentrationa % reduction in population

24 h 48 h
R1 R2 R3 R1 R2 R3
SN-1 105.7 NDb NDb NDb 99.3 93.5 70.8
52.8 49.4 44.5 8.1 94.6 87.2 58.3
26.4 35.8 41.6 20.3 77.9 69.8 59.7
CQ 3.7 ND ND ND 97.4 88.1 43.1
0.5 51.9 23.9 0.0 92.1 80.5 51.4
0.2 30.9 15.0 13.5 0.0 0.0 0.0

Unsynchronised P. falciparum cultures (FCR-3 strain) were treated with SN-1 steroid or CQ while controls were incubated with RPMI only as described in Section 2. Values
correspond to % reduction in population through comparisons with the control.
R1: population located in the lower right-hand quadrant of the scatogram (ring forms or young trophozoites).
R2: population located in the upper left-hand quadrant (late trophozoites).
R3: population located in the upper right-hand quadrant of the scatogram (schizonts).
a
Concentration: lM.
b
ND: not determined.

Table 2
Types of interaction resulting from combining SN-1 with conventional antimalarial drugs.

Combination FIC X ± SD SE t test p (t) 95% CI Interaction

LL UL
SN-1/CQ 0.903 ± 0.066 0.030 3.293 0.0301 0.8 0.9 Synergistic
SN-1/AQ 1.391 ± 0.182 0.091 4.303 0.0231 1.1 1.7 Antagonist
SN-1/DAQ 1.192 ± 0.072 0.036 5.335 0.0129 1.1 1.3 Antagonist
SN-1/QN 0.886 ± 0.054 0.024 4.758 0.0089 0.8 0.9 Synergistic
SN-1/ATV 1.344 ± 0.094 0.054 6.367 0.0238 1.1 1.6 Antagonist
SN-1/AR 1.179 ± 0.089 0.040 3.838 0.0312 1.1 1.3 Antagonist

CQ: chloroquine, AQ: amodiaquine, DAQ: desethyl-amodiaquine, AR: artemisinin, ATV: atovaquone, QN: quinine, FIC X ± SD: average and standard deviation for fractional
inhibitory concentrations, SE: standard error; p (t): p value for t test; 95% CI: 95% confidence interval; LL: lower limit; UL: upper limit.

Fig. 3. Isobolograms of the interaction of SN-1 isolated from the S. nudum plant with conventional antimalarial drugs. CQ: chloroquine, AQ: amodiaquine, DAQ:
desethylamodiaquine, AR: artemisinin, ATV: atovaquone, QN: quinine.

since all FICs were higher than 1.0, having a concave curve in isobo- curves, suggesting that carrying out in vivo assays combining this
lograms and significant p value (Table 2 and Fig. 3). On the contrary, steroid with these four antimalarial drugs would not be useful.
SN-1 combined with AQ, DAQ, ATV and AR showed an antagonist ef- Pabon et al. (2009) found that the SN-1 steroid inhibited more
fect, FICs being greater than 1.0 and having very pronounced convex than 80% of b-haematin formation, thereby agreeing with the slight
 A. Pabón et al. / Experimental Parasitology 127 (2011) 222–227
synergist effect shown by the SN-1/CQ combination. This sug-
gested that these two molecules may share a non-competitive role
regarding one of the proposed therapeutic targets for this drug. It
was also shown that this steroid increased both glutathione and
cysteine concentration, which is consistent with SN-1’s antagonist
effect when combined with antimalarial AQ, DAQ, ATV and AR as
oxygen-free radicals produced by these drugs would be neutralised
by SN-1-modulated endogenous oxidants in parasitised erythro-
cytes (Pabon et al., 2009).
These results show that SN-1 combined best with QN. This
could eventually lead to combination therapy being developed
with this drug and also with CQ, the latter being economically
and therapeutically more suitable than QN. A drug able to reverse
resistance to CQ would be of great interest in regions where this
drug’s effectiveness has been eroded by parasite resistance.
4. Conclusion
Whilst the in vitro antiplasmodial activity shown by steroids
isolated from the S. nudum plant was not very high, it should be
noted that these molecules have no mutagenic or clastogenic toxic
effects and may therefore be very useful as template molecules or
be seeded for synthesising new molecules as their chemical mod-
ification is straightforward, leading to them acquiring more anti-
genic power through their hemisynthesis. These results thus
shed light on continuing studies such as determining SN-1/CQ or
SN-1/CQ combinations’ in vivo response in mouse models orien-
tated towards searching for, developing and optimising new phar-
macologically-active molecules against malaria, as well as
providing indirect validation of S. nudum’s ethnomedical use.
Acknowledgments
This study had financial support from Colciencias (Grant 1115-
04-12944, RC-111-03), and the Universidad de Antioquia, Colom-
bia, CODI-UdeA. The authors would like to thank Ana Maria Mesa
for isolating compounds from S. nudum steroid, team members
from the Universidad Peruana Cayetano Heredia’s Cell Parasitology
unit’s laboratory for their technical assistance. We would also like
to thank Gonzalo Álvarez for help with statistical analysis and Ja-
son Garry for assistance in translation and editing.
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