In vitro and in vivo antileishmania activity of sesquiterpene lactone-rich

dichloromethane fraction obtained from Tanacetum parthenium (L.)

Schultz-Bip

Mirela Fulgencio Rabito1; Elizandra Aparecida Britta1; Bruna Luiza Pelegrini1;

Débora Botura Scariot1; Mariana Bortholazzi Almeida2; Suzana Lucy Nixdorf2;

Celso Vataru Nakamura1; Izabel Cristina Piloto Ferreira1*

1 Programa de Pós-graduação em Ciências Farmacêuticas da Universidade

Estadual de Maringá, 87020-900 Maringá-PR

2 Programa de Pós-graduação em Química da Universidade Estadual de

Londrina, 86057-970 Londrina-PR

* Corresponding author: Izabel Cristina Piloto Ferreira. Phone number: +55 (44) 30114876. Fax number: +55 (44) 30115050. Complete postal address: Universidade Estadual de Maringá, Centro de Ciências da Saúde, Departamento de Farmácia, Avenida Colombo, 5790 block K80. Zona Sete, 87020-900- Maringá - PR, Brasil. e-mail address: icpferreira@uem.br

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Abstract

Find new treatments for neglected diseases, including leishmaniasis; consist in

a big challenger for scientific research. On the other hand, plant extracts has

shown potential selectively on treating tropical diseases. This study evaluated

in vitro and in vivo antileishmania activities of a dichloromethane fraction (DF), a

sesquiterpene lactone-rich extract, obtained from aerial parts of Tanacetum

parthenium (L.) Schultz-Bip. Applying DF at in vitro studies, indicates an IC50 of

2.40±0.76 µg mL-1 against promastigote form and of 1.76±0.25 µg mL-1 against

axenic amastigote form of Leishmania amazonensis. Besides, in vivo treatment

with intramuscularly DF, showed lowest mouse footpad lesions growth and size,

presenting also a considerable decrease on parasite population, compared to

animals undergoing treated by reference drug. Malondialdehyde levels

increases slightly using DF, attributed to its parthenolide-rich composition,

which causes cell apoptosis compared to control group, demonstrating the

efficacy of treatment, without toxicity and genotoxicity. Since isolate and purify

plant compounds are costly, takes time, generating low-yields, extract fractions

like DF, represent a promising alternative for leishmaniasis treatment.

Keywords: Leishmania amazonensis; phytochemical; Leishmaniasis.

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1. INTRODUCTION

Plant extracted compounds are novel agents with great potential to treat

important tropical diseases, such as amebiasis, leishmaniasis, Chagas disease,

and malaria. Alkaloids, terpenes, quinones, sesquiterpene lactones, and

flavonoids are compounds found in innumerous plants, which present selective

activity against Leishmania sp. major etiological agent that causes

leishmaniasis [1-3]. Researches of herbal with potential antileishmania activity

be justified due to results achieved using extracts, fractions, and isolated

compounds against Leishmania sp [2-4].

Several studies have demonstrated antileishmania activity of isolated

compounds extracted from plants, including Tanacetum parthenium, whose

basic chemical composition comprises essential oils and sesquiterpene

lactones [5]. These lactones consist of a large group of structurally diverse

molecules, including secondary metabolites of wide plants varieties presenting

anti-inflammatory, antitumor, and anticephalic activities [6, 7].

Tiuman et al. [8] found that parthenolide, the most abundant sesquiterpene

lactone found in T. parthenium, has significant activity against the promastigote

form of Leishmania amazonensis, with 50% inhibition (IC50) of cell growth at a

concentration of 0.37 µg mL-1, and an IC50 of 0.81 µg mL-1 against the

amastigote form. The purified compound showed no cytotoxic effects in J774G8

macrophages in cell culture and causes no lysis in sheep erythrocytes,

administered at high concentrations.

Silva et al. [9] had isolated another sesquiterpene lactone, 11,13-

dehydrocompressanolide a type of guaianolide from T. parthenium. The isolated

compound had an IC50 of 2.6 µg mL-1 against cell growth of promastigote form

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of Leishmania amazonensis. For intracellular amastigote form, this guaianolide

reduced the survival index of parasites by 10% in macrophages at a

concentration of 20.0 µg mL-1.

However, isolation and purification of plant compounds are costly, time

consuming, uses large volume of organic solvents, causing environmental

troubles concerning waste disposal. So, development to produce medicines

based on isolated compounds is a complex process. Thus, an alternative can

be to synthetize of such compounds. Another way consists in using

phytochemicals from fractions of plant crude extracts.

Given the promising activity exhibited by compounds isolated from T.

parthenium and difficulties in getting high-yields, the present study evaluated in

vitro and in vivo antileishmania activity of a sesquiterpene lactone-rich DF from

aerial parts of T. parthenium.

2. MATERIALS AND METHODS

2.1. Dichloromethane fraction of Tanacetum parthenium

Hydroalcoholic extract was prepared using 1000 g of powder of aerial

parts of T. parthenium (batch 396253; supplied by Herbarium), extracted by

dynamic stirring at room temperature using ethanol: distilled water (9:1, v/v).

After depletion, the hydroalcoholic extract was filtered and concentrated by

rotavap at 35-40ºC until complete elimination of the organic solvent, which was

frozen and lyophilized.

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To obtain the DF, hydroalcoholic extract was passed through a

chromatography column under reduced pressure. As adsorbent for stationary

phase was used silica gel 60 (70-230 mesh; Merck) with eluents of increasing

polarity: hexane, ethyl acetate, dichloromethane, and methanol. Each fraction

was collected and concentrated in a rotary evaporator.

2.2. Parasites

Promastigote forms of L. amazonensis (MHOM/BR/75/Josefa) were

isolated from a human case of diffuse cutaneous leishmaniasis. This strain was

maintained in a tissue flask by weekly transfers, in Warren’s medium

supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 25ºC.

Infective promastigotes were cultured in Warren’s medium, supplemented with

20% FBS, penicillin (100 U/mL), and streptomycin (0.1 mg mL) at 32ºC. Axenic

amastigotes cultures were obtained by in vitro transformations of infective

promastigotes and cultured in Schneider’s insect medium (Sigma, St. Louis,

MO, USA; pH 4.6) supplemented with 20% FBS at 32∘C.

2.3. Animals

Male BALB/c mice, between 4 and 6 weeks of age and weighing 20-25 g,

and male and female Mus musculus (Swiss) outbred mice, between 8 and 12

weeks of age and weighing 25-30 g, were obtained from Universidade Estadual

de Maringá (UEM, PR, Brazil). All animals were acclimatized to laboratory

conditions for 10 days before beginning the experiments. Animals were housed

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under standard laboratory conditions on a constant 12 h/12 h light/dark cycle

with controlled temperature (22 ± 2ºC). Food (Nuvilab Cr1) and water were

available ad libitum.

UEM Institutional Ethics Committee approved all procedures adopted in

this study (protocol no. 092/2011).

2.4. Antiproliferative activity of dichloromethane fraction of Tanacetum

parthenium (L.) Schultz-Bip

Promastigote forms of L. amazonensis from a 48-h-old culture, were

cultured in Warren's medium supplemented with 10% inactivated FBS at 25ºC

for 72 h in a 24-well plate in absence or presence of different concentrations of

DF (0.1, 1.0, 5.0, 10.0, 50.0, and 100.0 µg mL-1). In all tests, 0.5% dimethyl

sulfoxide (DMSO; Sigma, St. Louis, MO, USA) was used as a control -

concentration that was used to dissolve the highest dose of compound, but had

no effect on cell proliferation. IC50 (concentration that inhibits 50% of parasite

growth) was then evaluated by directly counting the number of cells in a

Neubauer chamber.

An initial inoculum of 1 106 cells/mL of amastigote forms of L.

amazonensis from a 72-h-old culture, were cultured in Schneider medium, was

added to a 12-well plate in absence or presence of different concentrations of

DF (0.1, 1.0, 5.0, 10.0, 50.0, and 100.0 µg mL-1) solubilized in 0.5% DMSO

(Sigma, St. Louis, MO, USA). Parasite growth was evaluated by directly

counting the number of cells in a Neubauer chamber after 72 h incubation at

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32ºC. Antileishmania activity is expressed as percentage of growth inhibition

compared with control.

2.5. Cytotoxicity assay

Adherent J774A1 macrophage cells in the logarithmic growth phase were

suspended to yield 5 105 cells/mL in RPMI 1640 medium supplemented with

10% FBS, and added to each well of a 96-well microwell plates. Plates were

incubated in a 5% CO2-air mixture at 37ºC to obtain confluent cell growth. After

24 h, medium was removed. Cells were treated with DF of T. parthenium.

Concentrations used were of 1, 10, 100, and 1000 µg mL-1. Wells without DF

were included as a control. Plates were incubated in a 5% CO2-air mixture at

37ºC for 48 h. Cultures were then fixed with 10% trichloroacetic acid for 1 h at

4ºC, stained for 30 min with 0.4% sulforhodamine B (SRB) in 1% acetic acid,

and subsequently washed five times with deionized water. Bound SRB was

solubilized with 200 µL of 10.0 mmol L-1 unbuffered Tris-base solution.

Absorbance was read in a 96-well plate reader (BIO-TEK Power Wave XS) at

530 nm. Dose-response curves were plotted, and values of which are

expressed as a percentage of control optical density and CC50 values (50%

cytotoxicity concentration) were estimated by regression analysis.

2.6. Micronucleus test

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Male and female Swiss mice were divided into three groups (n = 8; 4

animals/sex) and treated with following: negative control (saline solution),

positive control (40 mg kg-1 cyclophosphamide), and test (overdose of 2000 µg

kg-1 DF).

Animals were sacrificed 24 h after administration. Femurs were exposed

and sectioned, and bone marrow was gently flushed using fetal calf serum.

After centrifugation at 3000 g for 5 min, bone marrow cells were smeared on

glass slides, coded for blind analysis, and air-dried. Smears were stained with

May-Grunwald-Giemsa to detect micronucleated polychromatic erythrocytes

(MNPCE) [10]. For each animal, three slides were prepared, and 2,000

polychromatic erythrocytes (PCEs) were counted to determine frequency of

MNPCE.

2.7. In vivo evaluation

BALB/c mice were divided into four groups, with eight animals per group.

Each mouse was subcutaneously inoculated once with 1 107 metacyclic

promastigotes in 25 µL PBS into left rear footpad. Lesion development of this

single infection experiment was monitored weekly by measuring thickness of

infected footpad with a caliper (DIGIMESS 150 mm). Treatments began after

lesion development (40 days post-infection) and continued for 4 weeks. Lesion

size is expressed as the difference between thickness of parasite-inoculated

footpad on first day of treatment and thickness on day of measurement

(weekly).

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There were four groups formed: Group 1 (uninfected and untreated

control), Group 2 (20 µL DMSO administered intramuscularly; i.e., same volume

used to dilute test drug), Group 3 (100 mg/kg/day of reference drug

Glucantime administered intramuscularly), and Group 4 (30 mg/kg/3 days of

test drug T. parthenium DF dissolved in PBS and administered intramuscularly).

At end of 30-day treatment period, animals were anesthetized with an

intraperitoneal injection of 100 mg kg-1 sodium pentobarbital, and popliteal

lymph node, spleen, kidneys, and liver were collected.

2.8. Leishmania load in lymph nodes of experimentally infected mice

Parasite load in lymph nodes of mice experimentally infected was

calculated according to Honda et al. [11]. Popliteal lymph nodes that drained

from infected footpad were aseptically removed, weighed, macerated, and

ruptured by needle passages. Serial four-fold dilutions were prepared from

suspension and distributed in duplicate in 96-well microtiter plates. After 12

days of incubation at 26ºC, wells were examined under an inverted microscope

(Olympus CKX 41) for presence or absence of promastigotes. Plates were

maintained for another 7 days and examined once more. Final titer was last

dilution for which well contained at least one parasite. Parasite load (number of

parasites per gram of tissue) was calculated as geometric mean of reciprocal of

positive titers from each duplicate/weight of lymph node. Value obtained was

multiplied by reciprocal fraction of homogenized organ inoculated into first well.

2.9. Histopathology

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After 30 days, mice were sacrificed. Liver, spleen, and kidneys were

weighed and washed in PBS, fixed in Bouin’s solution, and processed for

histopathology by paraffin inclusion. Blocks were cut at 7 µm thickness with a

microtome (Leica Microsystems, Berlin, Germany), mounted on slides, and

stained with hematoxylin-eosin.

2.10. Toxicity evaluation

At end of treatment, blood samples were taken from control animals

(treated or uninfected). Plasma levels of alanine aminotransferase, aspartate

aminotransferase, creatinine, glucose, total protein, and urea, were evaluated.

2.11. Determination of malondialdehyde levels in plasma samples by

HPLC

At end of treatment, blood samples were taken from control animals

(treated or uninfected). Samples were centrifuged at 10,000 g for 5 min to

separate fibrin. An aliquot of plasma sample (200 µL) was introduced in an

Eppendorf microtube with addition of 18 µL of 0.2% butylated hydroxytoluene

(BHT) and 6.25 µL of 10.0 mol L-1 NaOH, homogenized by vortexing. The

alkaline hydrolysis of protein bound to malondialdehyde (MDA) was achieved by

incubating mixture for 30 min at 60ºC in a water bath. Sequential samples were

placed under ice cubes for 10 min. Samples protein was precipitated with 750

µL of a solution of 7.2% TCA (Sigma-Aldrich) + 1% KI and centrifuged at 3,000

rotations per min for 10 min. Supernatant (500 µL) was transferred to screw-

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capped cryotubes with 250 µL of 0.6% TBA (Merck, Darmstadt, Germany) and

incubated for 45 min at 90ºC in a water bath. An aliquot of 20 µL of this reaction

mixture was injected into a high-performance liquid chromatography (HPLC)

system (Alliance e2695, Waters, Milford, MA, USA).

For analysis, we used an isocratic mobile phase that consisted of 40%

CH3OH (J.T. Baker) and 60% potassium phosphate buffer (50.0 mmol L-1, pH

7.0) filtered through a 0.22 µm nylon membrane (Millipore, Merck KGaA,

Darmstadt, Germany) at a flow rate of 0.7 mL min-1. The PDA was set at 532

nm to detect adduct TBA–MDA obtained from reaction. Guard column (4.6

12.5 mm, 5 µm) and analytical column (4.6 250 mm i. d., 5 µm) consisted of a

Zorbax Eclipse XDB-C18 (Agilent technologies, Santa Clara, CA, USA). MDA

retention time under these chromatographic conditions was 7 min.

MDA stock solution (Acros Organics, Pittsburgh, PA, USA) to construct

calibration curve was prepared with 22 µL 1,1,3,3-tetramethoxypropane in 10.0

mL H2SO4 (1%). After 2 h protected from light, 5 µL of this MDA stock solution

was added to 1.5 mL H2SO4 (1%). Concentration of standard solution of MDA

was determined by reading absorbance at 245 nm in a spectrophotometer (ɛ245

= 13700 M-1 cm-1) [12].

Calibration curves for quantitation were generated by adding standard

solution to reach final concentrations of MDA (0.2, 0.5, 0.8, 1.6, 6.4, 12.8, and

20.3 µmol L-1) into a pool of plasma samples, following same procedure of

samples.

Chromatographic parameters for validating the method were calculated

based on two calibration curves. Method proved to be linear (R2 > 0.98) and

precise (RSD = 3.5% for an average of six consecutive injections) with recovery

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from 98.0 to 101.1%. Limit of quantitation was 0.46 µmol L-1 and limit of

detection was 0.14 µmol L-1.

2.12. Statistical analysis

Data are expressed as mean ± SD. Statistical significance of differences

in percentage, between treated and untreated groups, was evaluated using

Statistics 8.0 software.

Statistical analyses were performed using one-way analysis of variance

(ANOVA) followed by Tukey test. Differences were considered significant at p <

0.05.

For malondialdehyde levels of plasma determination, a multivariate

assay applying principal components analysis were also performed.

3. RESULTS AND DISCUSSION

In recent studies, our research group described antileishmania activity of

two sesquiterpene lactones found in the dried aerial parts of Tanacetum

parthenium: parthenolide [8] and guaianolide [9]. In the present study, we

investigated the antileishmania activity of T. parthenium DF, selected by the

highest amount of sesquiterpene lactones. DF was obtained using a modified

procedure from Tiuman et al. [8].

In vitro studies (performed in triplicate at different times) showed for DF

an IC50 of 2.40 ± 0.76 µg mL-1 for promastigotes and 1.76 ± 0.25 µg mL-1 for

amastigotes. In 2004, Tiuman et al. [8] reported an IC50 of 3.6 µg mL-1 using DF

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for promastigote forms. Thus, changes in present extraction process generated

a 50% increase in antiproliferative activity.

The cytotoxicity in J774A1 macrophages and activity against protozoan

were compared using selectivity index ratio (CC50 for J774A1 cells/IC50 for

protozoan). The CC50 of DF was 20.00 ± 3.16 µg mL-1, indicating that this

fraction was 8.00-times more selective for protozoa than for J774A1 cells.

Importantly, the isolated parthenolide exhibited a CC50 of 14 µg mL-1, which is

more cytotoxic than DF of T. parthenium [8].

These important in vitro results prompted us to further evaluate this

fraction of T. parthenium in animals by measuring mouse footpads weekly. By

comparing the increase in size of footpad lesions, Group 4 (i.e., animals treated

with DF intramuscularly) had the lowest lesion growth during treatment period

compared with other groups of infected animals, including Group 3 (i.e., animals

treated daily with reference drug Glucantime; Table 1). Extent of lesion in

fourth week showed that this group had a statistically similar increase in lesion

size (p = 0.1252) as uninfected group (Group 1; Table 1).

American tegumentary leishmaniasis is characterized by one or more

papules, nodules, or ulcers. Lesions are typically described as looking

somewhat like a volcano, with a raised edge and central crater [13]. Fig. 1A-I

show mouse footpads after 4 weeks of treatment for Group 2 (treated every 3

days with DMSO intramuscularly), Group 3 (treated daily with Glucantime

intramuscularly), and Group 4 (treated every 3 days with DF intramuscularly)

respectively. DF markedly reduced footpad swelling and injury in infected

animals. Reductions of lesions were observed on treated footpad.

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After sacrificing the animals, parasite load was investigated to determine

whether treatment is able to decrease population of parasitic lesions in mice.

Fig. 2 shows a considerable decrease in parasite population in popliteal region

of mice treated with intramuscular Glucantime and DF compared with control

group (intramuscular DMSO; p = 0.0029 and 0.0010, respectively).

DF caused a greater decrease in footpad size and healing papules

compared with Glucantime and a similar decrease in parasite load. Notably,

DF was administered every 3 days, whereas reference drug was administered

daily. This reduction of number of injections may generate a lower risk of

infection and greater adherence to clinical treatment. Decrease in footpad size

and healing papules is related to well-known anti-inflammatory activity of T.

parthenium and parthenolide [14-15].

An important criterion in the choice of active compounds for antiprotozoal

activity is toxicity. Thus, mice organs (i.e., kidneys, liver, and spleen) were

weighed (Table 2) and evaluated histopathologically. Weight of liver in Groups

2, 3 and 4 significantly increased compared with uninfected animals (Group 1),

indicating that the increase in blood flow generated by drug metabolism directly

interfered with weight of this organ and was associated with intramuscular drug

administration. Blood plasma was analyzed, and biochemical measurements

were performed to investigate whether treatment influences organ function and

whether the increase in kidney and liver weight is related to treatment

performance. No significant differences were found in biochemical plasma

analysis between infected and treated animals compared with uninfected

animals (Table 3).

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The histopathological evaluation did not reveal changes in kidneys, liver,

or spleen in treated animals compared with control animals. Figure 3 shows

comparison of histopathological study of Group 1 and 4. Parameters measured

in qualitative analysis of organs indicated that tissues presented normal

morphology. Genotoxicity was also evaluated, and results showed that DF

exerted no genotoxic effect at concentration tested, in which MNPCE frequency

was 19.2 ± 5.18 in 2,000 polychromatic erythrocytes. These levels were

significantly lower than in positive controls (28.6 ± 8.22, p = 0.0088) and did not

differ from negative control (15.2 ± 5.6, p = 0.3669).

Plasma MDA levels were evaluated in mice to detect possible changes in

oxidative stress (Table 4). Average MDA content in healthy animals was 1.08

mmol L-1. Infected groups that are treated by different kinds of drugs/herbal had

average levels of 1.39 - 2.31 mmol L-1. ANOVA statistical analysis showed no

significant difference in plasma MDA levels of Groups, considering 95% of

confidence. However, there was an increase in lipid oxidation level of treated

animals (G2-G4) compared to control group G1.

However, performing hierarchical cluster analysis and principal

component analysis (Fig. 4) distinction of four groups occurred in higher

Euclidean distance. Principal components analysis explained 100% of data

variability and principal component 1 provided a better distinction of clusters

(Fig. 4B).

Sen et al. [16] in studies with hamsters infected with leishmaniasis got no

distinction between control, infected and treated animals when using only the

MDA as an indicator of oxidative damage, although this marker is successfully

used in models of human leishmaniasis [17].

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On the other hand, studies by Wen et al. [7] indicated that parthenolide

present in DF administered to animals, not only provides good results in

treatment of inflammation, but also in treating tumor cells. Action of parthenolide

is by induction of apoptosis, and during this induction there is production of free

radicals and reactive species that cause oxidative stress, in this study, indicated

by MDA levels.

4. CONCLUSION

Present study found that sesquiterpene lactone-rich DF extracted from T.

parthenium showed in vitro and in vivo effectiveness and low toxicity, with

advantage of having a better yield in extraction process, with reduction of

solvent, lowering costs. Intramuscular route used in present study generated

impressive results, especially compared with reference drug Glucantime,

deserving future studies. Results reinforce proposition that plants can be

promising sources of potent antileishmania agents.

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Table 1. Footpad growth (%) of BALB/c infected mice once with Leishmania

amazonensis

Group 1: Control uninfected/untreated. Group 2: Control infected/treated with intramuscular DMSO. Group 3: Control infected/treated with reference drug Glucantime

®. Group 4: Test

Infected/treated with intramuscular lactone-sesquiterpenes-rich DF (n=8). * Shows statistically significant differences when compared to Group 1 (p> 0.05)

Week 1 Week 2 Week 3 Week 4

Group 1 0.32 ± 13.73 2.29 ± 9.02 -1.34 ± 13.48 10.60 ± 17.65 Group 2 11.25 ± 9.01 19.47 ±10.65 35.30 ± 11.47 45.72 ± 21.23 Group 3 14.03 ±1 2.14 18.22 ± 7.40 26.89 ± 20.88 44.25 ± 14.56 Group 4 - 2.36 ± 11.99 12.03 ± 8.48* 24.80 ± 9.52 33.10 ± 20.73*

20

Table 2. Weight (milligrams) of BALB/c organs in infected mice once with

Leishmania amazonensis Group 1: Control uninfected/untreated. Group 2: Control infected/treated with intramuscular DMSO. Group 3: Control infected/treated with reference drug Glucantime

®. Group 4: Test

Infected/treated with intramuscular lactone-sesquiterpenes-rich DF (n=8). * Shows statistically significant differences when compared to Group 1 (p> 0.05)

Kidney Liver Spleen

Group 1 0.20±0.02 1.10±0,07 0.11±0.007

Group 2 0.24±0.03 * 1.35±0,09 * 0.13±0.02

Group 3 0.23±0.01 1.28±0,06 * 0.13±0.02

Group 4 0.24±0.01* 1.27±0,08 * 0.12±0.02

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Table 3. Biochemical measurements of plasma collected from BALB/c mice

infected once with Leishmania amazonensis

Group 1

Group 2 Group 3 Group 4

AST 40.39 ± 19.16 26.72 ± 20.58 54.18 ± 40.29 29.06 ± 27.50

ALT 158.93 ±139.47 191.88 ± 83.39 519.00 ± 292.04 281.67 ±396.85

Creatinine 0.51 ± 0.43 0.61 ± 0.33 0.88 ± 0.51 0.50 ± 0.16

Total Protein 3.98 ± 0.48 4.38 ± 0.54 4.59 ± 0.41 4.80 ± 0.53

Urea 23.58 ± 4.57 26.66 ± 4.38 34.34 ± 2.19 24.74 ± 8.20

Glicose 92.49 ± 21.16 79.63 ± 21.67 87.20 ± 17.58 93.00 ± 13.29

Group 1: Control uninfected/untreated. Group 2: Control infected/treated with intramuscular DMSO. Group 3: Control infected/treated with reference drug Glucantime

®. Group 4: Test

Infected/treated with intramuscular lactone-sesquiterpenes-rich DF (n=8).

22

Table 4. Malondialdehyde assay (mmol L-1) in plasma of BALB/c infected mice

once with Leishmania amazonensis Group 1: Control uninfected/untreated. Group 2: Control infected/treated with intramuscular DMSO. Group 3: Control infected/treated with reference drug Glucantime

®. Group 4: Test

Infected/treated with intramuscular lactone-sesquiterpenes-rich DF (n=8). SD: Standard deviation

Animal Group 1

Group 2 Group 3 Group 4

1 1.19 ± 0.02 1.31 ± 0.01 1.64 ± 0.02 -

2 1.30 ± 0.01 1.11 ± 0.05 1.58 ± 0.03 -

3 1.24 ± 0.01 1.26 ± 0.02 1.65 ± 0.03 1.10 ± 0.03

4 0.57 ± 0.01 2.36 ± 0.02 1.36 ± 0.01 2.31 ± 0.04

5 0.84 ± 0.06 1.14 ± 0.04 1.07 ± 0.04 2.16 ± 0.02

6 1.10 ± 0.01 1.24 ± 0.02 1.31 ± 0.01 1.77 ± 0.03

7 1.10 ± 0.01 1.46 ± 0.02 1.59 ± 0.01 -

8 1.27 ± 0.06 1.24 ± 0.03 1.65 ± 0.02 1.70 ± 0.04

Mean ± SD

1.08 ± 0.25

1.39 ± 0.41

1.48 ± 0.21

1.52 ± 0.36