Hindawi Publishing Corporation
Malaria Research and Treatment
Volume 2012, Article ID 381724, 5 pages
Efficacyof EosinBas a NewAntimalarialDrug ina MurineModel
FaridehVahabi,1AydaIravani,1and Mohammad Arjmand1
1Department of Biochemistry, Pasteur Institute of Iran, Tehran 1316943551, Iran
2Department of Biochemistry, Payame Noor University, Tahran 193195, Iran
3Department of Parasitology, Pasteur Institute of Iran, Tehran 1316943551, Iran
4Department of Clinical Research, Pasteur Institute of Iran, Tehran 1316943551, Iran
Correspondence should be addressed to Mohammad Arjmand, email@example.com
Received 21 May 2012; Revised 28 August 2012; Accepted 27 November 2012
Academic Editor: Donatella Taramelli
Copyright © 2012 Zahra Zamani et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The initial success of any adopted anti-infective strategy to malaria is followed by a descent due to the emergence of resistance to
it. The search for new drugs and drug targets is a consistent demand in this disease. Eosin B, a common laboratory dye, is reported
to have good antiparasitic properties in vitro. It was studied for its antiparasitic effect in vivo on chloroquine-sensitive Plasmodium
berghei murine malaria. Eosin B was administered in 2 different doses by either the oral or parenteral route, once or twice daily to
mice infected with Plasmodium berghei. Both the doses of eosin B 400mg/kg and 800mg/kg gave better results than the controls
whichwere40mg/kgchloroquineand100mg/kgofarteetherwithP < 0.005significance.PercentagesuppressiveactivitybyPeter’s
test of eosin B was better, though at a higher dose than both the controls. Survival rate of mice receiving the higher dose of eosin
B was longer than that of the controls. When administered twice daily, the mice were fully cured after 4 days. Eosin B seems to be
a promising drug exhibiting good antimalarial effects in the murine model of the disease.
Malaria is the major cause of morbidity and mortality in
tropical countries. In 2010, WHO reported 216 million cases
of malaria with an estimated 655,000 deaths, 86% of the
victims were children under 5 years of age, and 91% of the
deaths occurred in Africa. It should; however, be noted that
the number of disease cases has decreased for the first time
in years and malaria mortality rates have fallen by more
than 25% since 2000 . Different reasons can be evinced
for the reemergence of malaria, it could be due to spread of
between members of the limited number of drug families
available; in some areas, multidrug resistance. The search for
new drugs is imperative as discovery of new treatments and
appearance of resistance to it takes a certain period of time.
In the past decade, Chinese researchers have discovered
artemisinin (qinghaosu), the active component of Artemisia
annua, a herbal remedy used in Chinese folk medicine
for 2000 years. This molecule with its oil-soluble (e.g.,
artemether and arteether), water-soluble (e.g., artesunate),
and semisynthetic derivatives has shown excellent anti-
Plasmodium efficacy in vitro. They are being used in com-
bination with traditional antimalarials drugs such as meflo-
to recent reports of resistance to artemisinin in Plasmodium
falciparum on the Cambodian border .
Laboratory dyes have been used as antimalarial drugs
for at least a century. Guttmann and Ehrlich described the
clinical cure of two patients after oral administration of a
time, but, disappeared for nearly a century, before surfacing
in reports from 1995 onwards and interest has been revived
due to its low price. Several clinical trials are in progress,
trying to find a suitable drug combination. Methylene blue
has been tested both in vitro and in vivo for antimurine
malaria activity and on humans in Africa . It has recently
been shown that its combination with chloroquine (CQ) is
. However, oral MB given twice daily (4mg/kg/day)
2Malaria Research and Treatment
together with a standard dose of CQ over three days was
not effective in the treatment of uncomplicated malaria in
young children of Nouna town in Burkina Faso in 2003
. In another study, a combination of MB and amidoqine
(MB-AQ), MB and artesunate (MB-AS), and AS-AQ was
tested on patients in Burkina Faso. Parasite clearance time
differed significantly among the groups and was shortest
with MB-AS. By day 14, the rates of adequate clinical
and parasitological response after PCR-based correction for
recrudescence were 87% for MB-AS, 100% for MB-AQ (P =
0.004), and 100% for AS-AQ (P = 0.003). The respective
figures were the lowest for MB-AS (62%), intermediate for
standard treatment AS-AQ (82%, P = 0.015), and highest
for MB-AQ (95%, P < 0.001; P = 0.03) by day 28 .
In the same study, strong activity against gametocytes was
demonstrated by a combination of MB-AQ and MB-AS
together, which showed better activity than AS-AQ during
both the incubation period and active malaria .
Using molecular docking methods, which predicts the
preferred orientation of one molecule to another, eosin B has
been identified as a potential antiprotozoan drug . The
sensitivity of eosin B was tested in vitro on the apicomplexan
parasites, Toxoplasma gondii and a kinetoplastid Leishmania
replication inside host cells by 50%, as measured by [3H]
uracil incorporation, but proved to be a weak inhibitor in
Leishmania major. However, further in vitro studies have
shown eosin B to be a highly selective, potent inhibitor of
a variety of drug-resistant malarial strains, with an average
IC (50) of 124nM. Furthermore, there is no indication of
cross-resistance with other clinically utilized compounds,
suggesting that eosin B is acting via a novel mechanism
and it has been suggested that it could be an effective
lead compound for development of new, more effective
antimalarial drugs .
The relative safety of eosin B has been established
previously with Food and Drug Administration (FDA)
reports of it not being carcinogenic to mice after a dietary
exposure as high as 2.0% of their weight and approved for
use in drugs and cosmetics . We studied its effect on
Plasmodium berghei in mice. To our knowledge, this is the
first study of its kind wherein its potent inhibitor properties
on murine malaria are demonstrated.
Chemicals. Chloroquine (CQ) and arteether (Art) were
obtained from J.B. Chemicals and Pharmaceuticals Ltd.
(Vatva, Ahmedabad, India) and Themis Medicare Limited
(Gujarat, India), respectively. Eosin B, ethanol, and Tween
80 were purchased from Merck (Germany).
Malaria Parasite. P. berghei Haffkine strain was kindly
donated by Dr. Nateqpour, School of Public Health, Tehran
University, Tehran, Iran. As per the requirement for active
parasites, they were maintained by blood passage in NMRI
mice when active parasite; otherwise they were stored at
−70◦C in Alsever’s solution (2.33% glucose, 0.525% NaCl,
and 1% sodium citrate in deionised water) and glycerol (9:1
Animals. Animals used in this study were CBA/J (in-bred
strain) mice. Maintenance of animals and protocols used
were approved by the Ethics Committee of Pasteur institute
of Iran. Each group comprised of five mice which were
housed in plastic cages under standard laboratory conditions
of temperature (22 ± 1◦C), humidity (50–60%) and main-
tained on a commercially available pellet diet. The course
of parasitaemia was monitored by microscopic examination
of Giemsa-stained thin blood smears obtained from the tail
Preparation of Drugs. A solution of 70% Tween 80 and
30% ethanol was prepared, which was diluted tenfold
with sterile distilled water. This solution of 7% Tween
80 and 3% ethanol was used as drug vehicle (DV).
160mg of eosin B was dissolved in 1mL of DV to give
a stock solution. 100µL of the stock was used undi-
luted to give a dosage of 800mg/kg/100µL. It was then
to give dosages of 400mg/kg/100µL, 200mg/kg/100µL,
and 100mg/kg/100µL. The four doses were administered
intraperitoneally (ip) or given orally (op) to each group of
mice. 40mg of CQ was dissolved in 5mL of DV to give a
solution of injection containing 8mg/mL of CQ; 100µL was
injected ip or fed op at a dosage of 40mg/kg to another
group. In addition, 40mg of Art was dissolved in 2mL of DV
to give a 20mg/mL solution; 100µL was administered ip and
orally into each mouse to give a dosage of 100mg/kg in a
further group. DV was used as control both orally and ip in
the control group. Mice were divided into two groups A and
B. Group A was treated by ip and named as A(ip) and op as
A(op) once a day, whereas group B was treated twice a day by
the same routes B(ip) and B(op).
LD50 Test. LD50 test was carried out on CBA/J mice using
different dosages of eosin B: 100, 200, 500, 1000, 1400,
1500, 2000, 2500, and 3000mg/kg ip and the animals were
observed for 7 days .
Toxicity Assay. Two groups of mice were taken, one group
was injected with eosin B (800mg/kgip) and the other used
as controls. Close attention was paid to any external changes
and weight of internal organs. Microscopic preparations of
liver; heart; stomach; intestine; kidney; spleen were prepared
and examined histopathologically 5, 30, and 60 days after
eosin B administration .
Mean Survival Time. Mice in both the groups were treated
with four different doses of eosin B. Positive control mice
were treated with CQ and Art on day 0, 24h and 48h and
the negative control group of mice was tested with DV. These
tests were carried out by both the routes (ip) and (op) and
the mean survival of each group was noted.
Malaria Research and Treatment3
Table 1: Percent suppression activity in CBA/J mice with a single dose of drugs as per Peters four-day suppressive test in group A.
Mean survival time
in days in ip mice
Mean survival time
in days in op mice
Results were significant as analyzed by ANOVA P < 0.005.
Peters’ 4-Day Suppressive Test. Peters’ 4-day suppressive test
was carried out on the above-mentioned mice after deter-
of Giemsa-stained blood films taken on day 4 and twice
weekly till the 30th day or death of the animal . Dif-
ferences in parasitaemia percentage between treated groups
and untreated animals were analyzed by a one-way ANOVA
test using GraphPad Prism 4 and differences considered
significant if P < 0.05. Furthermore, the difference between
the mean value of the control group (taken as 100%) and
those of the experimental groups is expressed as percent
reduction activity using the following equation:
?mean parasitemia treated
Activity = 100 −
mean parasitemia control× 100
Results of Toxicity Assay. No external changes were observed
in the two groups of mice. The weight and microscopic
specimen of internal organs of infected animals and that of
toxicity of eosin B even after two months of administration.
LD50 Test. CBA/J mice died at 3000mg/kg eosin B and
could tolerate 1500mg/kg. However, at 2000mg/kg, half the
population of mice died. Different doses from 100mg/kg
were tested on CBA/J mice, but high suppression activity was
seen with 800mg/kg and 400mg/kg dosage of eosin B.
In Vivo Antimalarial Activity of Eosin B as Compared to CQ
and Art. Table 1 shows Peters’ 4-day suppressive test against
P. berghei. The suppression activity exhibited by eosin B was
better than CQ and Art, though it was a higher dose as
compared to the other two. After 96h by the ip route, eosin B
at 800mg/kg showed the highest antiparasitic activity (65%)
as compared to Art and CQ (60%) and (50%), respectively.
and CQ and Art (70%) and (74%).
Mean survival time (MST) of different drugs was
recorded by ip route in group A (Figure 1). Eosin B 800mg
showed the highest MST of 19 days, CQ and Art 16 days
whereas the controls lived only for 12 days. In group A, by
op route (Figure 2) 800mg eosin B had the longest MST
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Number of days
Eosin B 800 mg/kg ip
Eosin B 400 mg/kg ip
Art 100 mg/kg ip
CA 40 mg/kg ip
drugs once daily ip for four consecutive days (5 mice per group).
Results between test and control were significant by P < 0.005 as
analyzed by ANOVA.
of 21 days and Art, CQ, and eosin B 400mg 19 days and
control MST was for 12 days. All the results were significant
(P < 0.005) as analyzed by ANOVA (Figures 1 and 2).
However, in group B in which eosin B was administered
twice daily at 400 and 800mg/kg for four days in B(ip) and
B(op) groups and the mice were monitored from the fifth
day onwards for 30 days, all the mice were cured except for
controls which died on the 13th day. All the results were
significant(P < 0.005) asanalyzedbyANOVA(Figures3and
Eosin B is a dibromodinitrofluorescein used for cytoplasmic
staining (Figure 4). Molecular docking strategies have pre-
dicted it as a good antimalarial drug and in vitro studies have
supported the prediction. We carried out an in vivo study on
the effect of eosin B on P. berghei, a CQ-sensitive murine
malaria strain using it as a new drug. Even though lower
doses of chloroquine and artemisine gave protection, but
higher doses of the two were used so as to make our studies
comparable to the high dose of eosin B utilized. Eosin B
has an LD50 of 2000mg/kg much higher than the two drugs
4Malaria Research and Treatment
13579 11 13 15 1719 21
Eosin B 800 mg/mL op
Eosin B 400 mg/mL op
Art 100 mg/kg op
CQ 40 mg/kg op
Number of days
Figure 2: Mortality test on CBA/J mice in group A(op) adminis-
tered drugs once daily op for four consecutive days (5 mice per
group). Results between test and control were significant by P <
0.005 as analyzed by ANOVA.
13579 11 13 15 17 19 21 23 25 27 29
Number of days
Eosin B 800 mg/kg ip
Eosin B 400 mg/kg ip
Art 100 mg/kg ip
CQ 40 mg/kg ip
Figure 3: Percent parasitemia in CBA/J mice group B(ip) in ip
administered two daily doses of drugs for four consecutive days (5
mice per group). Results between test and control were significant
by P < 0.005 as analyzed by ANOVA.
tested. CQ has an LD50 330mg/kg for op and ip 190mg/kg.
LD50 of Art is reported from 1250mg/kg im in Swiss mice
as compared to LD50 of artimisinin and artesunate which
are 263mg/kg im and 475mg/kg im, respectively. As LD50
of eosin blue is the highest as compared to the other drugs,
hence, higher doses can be tolerated and used in case of
resistance development. Eosin B was shown to have a similar
if not better effect at the high doses given ip and op (Figures
2 and 3).
Methylene blue, another laboratory dye is a heterocyclic
aromatic chemical compound with many uses in various
fields, such as biology and chemistry (Figure 4). It has been
Figure 4: Chemical structure of eosin B and methylene blue.
used for malaria in human trials in Burkina Faso . Earlier
studies on P. yoelii nigeriensis, a murine malaria, showed
similar results when given with mefloquine . The mice
blue or 5mg/kg mefloquine by the 6th day, not letting the
parasitemia increase more than 6%. LD50 of methylene blue
is reported to be 1180mg/kg op and 180mg/kg ip, much
lower than the LD50 reported for eosin B. Side effects of
methylene blue have been widely reported including nausea,
chest pain, fever, and dizziness but so far for eosin B, besides
skin irritation no other side effect has been reported. Also, it
has been used in cosmetics with FDA approval, its toxicity
dose is 2% body weight, again much higher than all the
other drugs. Dosage trials of methylene blue showed that
two daily doses for three days cured the mice infected with
P. berghei, and a single daily dose had the same antiparasitic
effect as Art and CQ. Different dosages of eosin B, from
100mg/kg onwards, were studied for antimalarial activity
and the effective doses were 400mg/kg and 800mg/kg much
higher than that of methylene blue.
Bioinformatics using docking strategies have shown that
eosin B exerts selective antimalarial effects and there is no
indication of cross-resistance with other clinically utilized
compounds, suggesting that eosin B is acting via a novel
mechanism. The antimalarial mode of action appears to be
multifaceted and includes extensive damage to membranes,
the alteration of intracellular organelles, and enzymatic
inhibition not only of DHFR-TS but also of glutathione
reductase and thioredoxin reductase. In addition, prelimi-
nary studies suggest that eosin B is also acting as a redox
cycling compound [10, 16].
Eosin B is known as a glutathione-s-transferase inhi-
bitory  and preliminary work done byour laboratoryhas
Malaria Research and Treatment5 Download full-text
also shown its effect on Plasmodium falciparum glutathione-
s-transferaseenzyme (data not shown).However, more work
will have to be carried out before its mechanism of activity
can be determined. Methylene blue has been shown to affect
FDA reports have shown that eosin B is not carcinogenic
to mice after a dietary exposure as high as 2% of body
weight  and also reported LD50 of eosin B in rats
as 3000mg/mL, similar to our findings. This high LD50,
similarity of effective dose by both the routes and tolerance
of high doses can be looked upon as an advantage of eosin
B, as with emergence of resistance, higher doses can be
administered. Our studies, like those of FDA, have shown
lack of toxicity even in doses up to 800mg/kg. Eosin B seems
have shown its effect on Plasmodium falciparum though
more research will have to be carried out before this drug
can be used for human malaria.
This project has been funded by the Pasteur Institute of Iran
during the years 2008–2010. All the authors certify that they
or indirect financial relation with the commercial identity
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