Asian Pac J Trop Biomed 2014; 4(12): 947-951 947

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Asian Pacific Journal of Tropical Biomedicine

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

Document heading doi:10.12980/APJTB.4.2014APJTB-2014-0435 ©2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

In vitro and in vivo trypanocidal action of aescin and aescin liposomes against Trypanosoma evansi
in experimental mice

Matheus Dellaméa Baldissera1,2, Nathieli Bianchin Bottari2, Thirssa Helena Grando1, Roberto Christ Vianna Santos2,3, Ana Júlia Figueiró
Dalcin3, Patrícia Gomes3, Renata Platcheck Raffin3, Carine Eloise Prestes Zimmerman1, Janio Morais Santurio1, Silvia Gonzalez Monteiro1,
Aleksandro Schafer Da Silva4*
1
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil
2
Laboratory of Microbiology, Centro Universitário Franciscano, Santa Maria, RS, Brazil
3
Laboratory of Nanotechnology, Centro Universitário Franciscano, Santa Maria, RS, Brazil
4
Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil

PEER REVIEW A B S T R AC T

Peer reviewer Objective: To verify the trypanocidal effectiveness of aescin and aescin liposomes against
Dr. Humphrey Simukoko, School of Trypanosoma evansi in vitro and in vivo.
Veterinary Medicine, University of
Methods: Aescin and aescin liposomes were used in vitro on trypomastigotes at different
Zambia, P. O. Box 32379, Lusaka,
Zambia. concentrations (0.5%, 1.0% and 2.0%) and exposure times (0, 1, 3, 6 and 9 h). In vivo tests
Tel: +260 979 043 157 were performed using mice as the experimental model. Trypanosome evansi infected mice
E-mail: h.simukoko@unza.zm were treated with aescin and aescin liposomes with doses of 60 and 100 mg/kg during 4 d.
Results: The three concentrations tested in free form and nanoencapsulated showed
Comments
trypanocidal activity in vitro, completely eliminating the parasites in small concentration after
The present study has addressed an
important aspect of trypanosomosis 6 h of assay. Animals treated with aescin (100 mg/kg) and aescin liposomes (100 mg/kg)
research by evaluating the effectiveness showed increase in longevity, however without curative effect.
of a potential adjuvant to trypanosomosis Conclusions: Active compounds present in natural products, such as aescin, may potentiate
treatment in animals, aescin and aescin the treatment of trypanosomosis when used in association with other trypanocidal drugs.
liposomes. Undoubtedly, the rearch results
will add value to the current knowledge in
this field. KEYWORDS
Details on Page 950 Surra, Nanotechnology, Aesculus hippocastanum, Trypanosome

1. Introduction aceturate (DA), but this drug is ineffective for umpteen animals[7,8].
Most of the drugs used for the treatment of the disease do not
Trypanosoma evansi (T. evansi) is the etiologic agent of a provide total elimination of the infection and are associated with
disease known as Mal das cadeiras or surra in horses[1]. Surra is recurrence and mortality[8]. In many cases, DA treatment may
an important disease in a wide geographic region (Africa, Asia, not be effective, leading to recurrent parasitemia[8-10], as well as
and South and Central America), and infects mainly camels, hepatotoxicity, and nephrotoxicity[1,11]. As a result, researchers have
cattle, horses, buffalos and some wild animals[2-4]. The parasite tested natural products such as oils of copaiba, andiroba, aroeira, tea
is transmitted by infected blood through haematophagous insects tree[12-14], and propolis extract[15].
such as tabanid flies[5,6]. The disease is endemic in some regions Aescin is the predominant active constituent of Aesculus
(Mato Grosso, Pantanal in Brazil,) and in other regions it occurs in hippocastanum seed extract, which is a mixture of triterpene,
outbreaks, hindering the prophylaxis and control. saponins, consisting of A, B, C and D aescin[16]. Research has
The treatment of this disease in Brazil is based on diminazene been focused on plant secondary compounds, such as saponins,

*Corresponding author: Aleksandro S. Da Silva, Department of Animal Science, Article history:

Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil. Received 27 Aug 2014
E-mail: leksandro_ss@yahoo.com.br Received in revised form 15 Sep 2014
Foundation Project: Support by Fundacão de Amparo a Pesquisa do Rio Grande Accepted 28 Sep 2014
do Sul (FAPERGS), Grant No. 002071-2551/13-6. Available online 21 Oct 2014
948 Matheus Dellaméa Baldissera et al./Asian Pac J Trop Biomed 2014; 4(12): 947-951

for the control of parasite in sheep[17,18]. According to Carrasco (diluted in culture medium) at concentrations of 0.5%, 1.0% and
and Vidrio aescin has been shown to be effective in the treatment of 2.0%. The aescin liposomes also were used at concentrations of
inflammatory conditions[19]. Actually, some studies have indicated 0.5%, 1.0% and 2.0 %. A positive control (DA at a dilution of 0.5%)
that aescin is also a potential anticancer agent[20,21]. was also used, at the same volume (25 μL). The tests were performed
The field of nanoparticle synthesis, assembly, and application in duplicates and the parasites were counted at 1, 3, 6 and 9 h after
in biology is a fast growing area of nanotechnology and the onset of the experiment in Neubauer chambers. The microtiter
nanomedicine [22]. Through the development of materials that plates were placed in a 5% CO2 incubator at 37 °C according to
exhibit novel optical, chemical, and electrical properties at Baltz[26].
the nanometer-sized scale, it is hoped that it will be possible
improve therapies for disease [23] . In this context, it can be 2.4. In vivo test
highlighted the liposomes. Liposomes are spherical self-closed
structures, composed of curved lipid bilayers, that encapsulate 2.4.1. Animal model
both hydrophilic and lipophilic substances[24]. The simplicity of Forty-two, female, 60-day-old-mice weighing an average of
production, their biocompatibility, low toxicity, size and similar (23.0±0.7) g were used as the experimental model. They were kept in
composition to cells make them a revolutionary tool in biomedical cages with six females each, housed on a light/dark cycle of 12 h, in an
domain. Based on the needs for curative therapy of T. evansi and experimental room with controlled temperature (23±1) ºC and humidity
on the properties of aescin and the antiparasitic properties of 70%. They were fed with commercial feed, and water ad libitum. All
saponins mentioned above, the present study analyzed for the first animals were subjected to a period of 10 d for adaptation.
time the in vitro and in vivo activity of aescin and aescin liposomes
against T. evansi. 2.4.2. Experimental design and parasitemia estimation
The mice were divided into seven groups (A to G). Group A
2. Materials and methods consisted of uninfected mice and untreated (negative control); Group
B consisted of infected mice and untreated (positive control); Group
2.1. T. evansi isolate C was composed of animals infected and treated with aescin 60 mg/kg;
Group D was composed of animals infected and treated with aescin
This study was set up in two consecutive experiments (in vitro and 100 mg/kg; Group E was composed of animals infected and treated
in vivo). The same T. evansi isolate was used in both experiments[10]. with aescin liposomes 60 mg/kg; Group F was composed of animals
Two rats (R1 and R2) were infected intraperitoneally with infected and treated with aescin liposomes 100 mg/kg; Group G was
trypomastigotes contaminated blood kept cryopreserved in liquid composed of animals infected and treated with DA. Infected animals
nitrogen. This procedure was performed to obtain a large amount of were inoculated intraperitoneally with 0.05 mL of blood from one
viable parasites for in vitro tests (R1), and to infect the experimental rat containing 1.1伊106 trypanosomes.
groups (R2). The DA was administered in a single dose of 7.0 mg/kg,
intraperitoneally injection, and 1 h after infection of the animals.
2.2. Aescin and aescin liposomes Aescin and aescin liposomes were administered orally for 4 d,
starting the 1 h following infection.
Aescin was purchased from (Sigma-Aldrich®, St. Louis, USA). The evolution of parasitemia and the effect of the treatment were
Aescin liposomes were prepared with 0.5% aescin using a daily monitored through blood smear. Each slide was prepared with
proprietary method from Inventiva®. Particle size and polydispersion fresh blood collected from the tail vein, stained by the panoptic
index were evaluated by dynamic light scattering. Zeta potential was method, and visualized at a magnification of 1000伊 according to Da
evaluated using electrophoretic mobility technique. The samples Silva[27].
were diluted in Milli-Q water (500×) and the assays were performed
using Zeta Sizer Nanoseries, Malvern. The pH was assessed by 2.4.3. Treatment efficacy
direct use of Digimed potentiometer according to Da Silva[25]. Treatment efficacy was determined by the number of mice that
did not show clinical signs of T. evansi infection after treatment.
2.3. In vitro tests Prepatent period, longevity and animal mortality were also observed.

The culture medium for T. evansi was adapted from Baltz as

previously published by Baldissera [13,26]. The trypomastigotes 2.5. Statistical analysis
were acquired from the infection of one rat (R1) with a T. evansi
isolate. Five days post-infection rat showed high parasitemia (7.5伊 Data from in vitro were analyzed by analysis of variance for
106 trypanosomes/µL). The rat was was anesthetized with isoflurane repeated measures and comparison of concentrations tested for
for blood collection by cardiac puncture, and blood was stored aescin. Data of the prepatent period and longevity were submitted
in ethylene diamine tetraacetic acid tubes. For trypanosomes for analysis of variance according to Duncan test (P<0.05).
separation, each 200 µL blood was diluted in complete culture
medium (200 µL), stored in microcentrifuge tubes and centrifuged 3. Results
for 10 min at 1 550 r/min. The supernatant was removed and
resuspended in culture medium and the number of parasites was 3.1. Aescin liposomes
counted in a Neubauer chamber.
The culture medium with the parasites was distributed in microtiter The prepared suspension was evaluated regarding to their physical-
plates (270 μL/well), followed by the addition of 25 μL of aescin chemical properties. The particle size was (223±6) nm and the
 Matheus Dellaméa Baldissera et al./Asian Pac J Trop Biomed 2014; 4(12): 947-951
949
polydispersion index was (0.211±0.059) with a zeta potential of Table 1
(-18.1±1.7) mV. In vivo test-mean and standard deviation of the prepatent period, longevity
and mortality using treatment with aescin, aescin liposomes and DA in mice
3.2. In vitro test experimentally infected by T. evansi.
Groups Treatment# Prepatent Longevity (d) Mortality
The results showed a trypanocidal effect of aescin and aescin (n=6) period (d) (n)
liposomes on T. evansi directly proportional to the concentration A Negative control - 40.00±0.00a 0/6
used (Figure 1). After 1 h, there were no living trypomastigotes B Positive control 1.16±0.40a 4.00±0.63d 6/6
C Aescin (60 mg/kg) 1.83±0.75a 5.10±0.98cd 6/6
in 1.0% and 2.0% concentrations. A reduction of live
D Aescin (100 mg/kg) 2.00±0.89a 11.50±1.01b 6/6
trypomastigotes was observed at the concentration of 0.5% when E Aescin liposomes (60 mg/kg) 2.11±0.75a 6.00±1.22c 6/6
compared with the control group. After 3 h of the assay, there F Aescin liposomes (100 mg/kg) 1.66±1.03a 10.50±1.04b 6/6
were no living trypomastigotes in 0.5% concentration and DA G DA (7.0 mg/kg) 0.00±0.00a 40.00±0.00a 0/6
treatments (Figure 1A). Means followed by same letter in the same column do not differ significantly
4 000 A in the Duncan test; The experiment lasted 40 d post-infection; #: Survival
Number of trypanosomes/mL

animals in the end of experiment, showed negative PCR and blood smears.
3 000

2 000 4. Discussion
1 000
This study observed a trypanocidal action in vitro and in vivo of
0 aescin and aescin liposomes. The results showed a dose-dependent
0 1 3 6 9 trypanocidal effect of aescin and aescin liposomes against T. evansi
Hours post-treatment
trypomastigotes in vitro. The aescin had apparently a faster trypanocidal
4 000 B
Number of trypanosomes/mL

effect in vitro than the aescin liposome. This was expected, since
3 000
nanotechnology process usually provides a more slow and gradual
properties of release. Saponins have been cited by researchers because
2 000 of their active substances with various therapeutic properties and
have a range of biological and pharmacological properties such as
1 000
antimicrobial, anti–inflammatory and anti-cancer[28]. In vivo and in
0 vitro, studies have shown the effect of saponins against protozoa in the
0 1 3 6 9 rumen of lambs[29,30]. There are also as reports the saponins extracts
Hours post-treatment from Maesa balansae, Careya arborea and Astragalus oleifolius having
Control 1.0% Aceturate 0.5% 0.5% 2.0% antileishmaninal activity[31].
Figure 1. In vitro, trypanocidal activity in concentrations 0.5%, 1.0% Based on these previous promising in vitro results, we have
and 2.0% of aescin (A) and aescin liposomes (B) against T. evansi when designed an in vivo experiment using mice infected by T. evansi as
compared to the control (not-treated) and diminazene aceturate (anti- a model. However, the therapeutic protocol used with aescin had no
protozoa drug). curative effect for all the groups, but in a group’s treated with aescin
The results within a circle are not statistically different (P>0.05), at the and aescin liposomes (Groups D, E and F), an increase in longevity
same time (h). of animals was observed. According to Kedzierski[32], β-aescin,
the major compound found in Aesculus hippocastanum, showed
The aescin liposomes results are shown in Figure 1B. After 1 h, a moderate active on the intracellular amastigote stage of Leishmania
reduction of live trypomastigotes was observed at the concentrations infantum and highest active on the extracellular promastigote stage.
of 0.5%, 1.0% and 2.0%, respectively, when compared with The accurate mechanism the active of β-aescin is uncertain, but
the control group. After 3 h of the assay, there were no living saponins show anti-leishmaninal activity through the induction of
trypomastigotes in 2.0% and 1.0% concentration and DA, and a apoptosis or programmed cell death in the parasite[33,34].
reduction of live trypomastigotes at the concentration of 0.5%. After Biological compounds with trypanocidal activity incorporated
6 h of the assay, there were no living trypomastigotes forms in 0.5% into liposomes are described since 1987. Liposomes containing
concentration. To the contrary, in control tests (not using drugs), the stearylamine rapidly killed epimastigotes, amastigotes and
parasites were all alive (Figure 1), which validates our experiment. trypomastigotes forms of Trypanosome cruzi in vitro[35]. Van de
Ven also demonstrate in vitro that β-aescin nanoparticles showed
3.3. In vivo test leishmanial activity[36]. This study also looked for action against of
T. evansi, a protozoon of the same family of Trypanosome cruzi and
There were no differences between groups regarding the prepatent Leishmania sp. The best therapeutic response observed with use of
period (Table 1). Longevity of the group A was exactly represented liposomes is mainly the ability to modify the surface of target cells
by the days that the experiment lasted (10 d). Longevity in the and tissues, and the absorption peak or phagocytic mechanisms and
groups B, C, D, E, F and G were 4.0, 5.1, 11.5, 6.0, 10.5 and 40 the ability to overcome intracellular barriers and to massively deliver
days, respectively. The groups C, D, E and F had no curative efficacy, trypanocidal drugs into an extremely small volume[37].
but the groups D, E and F increased longevity compared to the group The utilization of liposomes is advantageous because these
B. The animals treated with DA showed negative blood smear during nanoparticles are very similar to the cell membranes interacting
the 40 days of the period of our study. more closely and with greater efficiency with cells and tissues[38].
950 Matheus Dellaméa Baldissera et al./Asian Pac J Trop Biomed 2014; 4(12): 947-951

In addition, liposomes are nontoxic, biodegradable and can be Innovations and breakthroughs
produced easily and quickly on a large scale. Finally, they can be In the present study, the authors have demonstrated the trypanocidal
administered by intravenous, ocular, pulmonary, or dermal route[24]. effects of aescin and aescin liposomes on T. evansi, a disease of
It is also important to emphasize the great advantage of overcoming economic importance.
physicochemical properties of encapsulated drugs (such as water
solubility or membrane), thus improving the pharmacodynamics Applications
(therapeutic effect potentiation), pharmacokinetics (absorption The saponins present in aescin can potentiate the treatment of
control and tissue distribution) and their toxicological effects trypanosomosis when used with other trypanocidl drugs. This may also
(reduction of local and systemic toxicity)[39]. Barratt also showed alleviate the potential for drug resistance.
that some liposomes may lead to dose reduction, decreasing the
frequency used without loss of effectiveness, reducing costs of Peer review
therapy and the risk of toxicity, thus demonstrating that the strategy The present study has addressed an important aspect of
to modify the biodistribution profile of the drug is more effective trypanosomosis research by evaluating the effectiveness of a potential
than the free drug use[40]. adjuvant to trypanosomosis treatment in animals, aescin and aescin
Based on the result it was concluded that pure aescin pure and liposomes. Undoubtedly, the rearch results will add value to the current
liposome aescin has trypanocidal action against T. evansi in culture knowledge in this field.
medium, to cause induction to apoptosis or programmed cell death
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