Experimental Parasitology 126 (2010) 191–197

Contents lists available at ScienceDirect

Experimental Parasitology

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

Toxocara canis: Potential activity of natural products against second-stage larvaein vitro and in vivo

Mariana Reis a,*, Alcione Trinca a, Maria José U. Ferreira b, Ana R. Monsalve-Puello c, Maria Amélia A. Grácio a

a Unidade de Helmintologia e Malacologia Médicas/UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugalb iMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugalc Nutrialimentos Jesana S.L., Barcelona, Spain

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

Article history:Received 14 January 2010Received in revised form 21 April 2010Accepted 26 April 2010Available online 4 May 2010

Keywords:Anthelmintic activityChenopodium ambrosioidesNematodePycnanthus angolensisToxocara canis

0014-4894/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.exppara.2010.04.023

* Corresponding author. Address: UHMM, InstituTropical, Rua da Junqueira 96, 1349-008 Lisboa, Portu

E-mail address: mariana.a.reis@gmail.com (M. Rei

The anthelmintic activity of extracts from Chenopodium ambrosioides, Pycnanthus angolensis and Nutrides-intox� was in vitro and in vivo investigated, against Toxocara canis larvae. The in vitro assays resultsshowed that the aqueous extract of Nutridesintox� was the most effective, followed by C. ambrosioidesextracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelminticactivity compared to the other natural products. For the in vivo assays, Nutridesintox�, the hexane extractand the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, dur-ing three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by count-ing T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histologicalanalysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influ-ence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observedin the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclu-sion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelminticactivity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvaein vivo.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Toxocara canis (Werner, 1782), the roundworm of dogs, is theetiologic agent of human toxocariasis. As the dog constitutes oneof our most common pets, this ensured the worldwide distributionof this zoonotic disease. Commonly the infection is acquired afteringestion of embryonated T. canis eggs that can be present in soilcontaminated with dog faeces. Children are the most susceptibleto infection because of their habits of pica (Despommier, 2003).As larvae hatch in the stomach they penetrate the mucosal epithe-lium and thereafter remain developmentally arrested in the tissuephase. Although they do not grow or differentiate in the paratenichost they maintain an active metabolism and migratory behaviour(Maizels et al., 2000). Their wandering around the body gives riseto two main syndromes; visceral larva migrans (VLM), in whichthe major organs are affected and ocular larva migrans (OLM),when it affects the eye, in some cases, it can lead to unilateralblindness (Magnaval et al., 2001).

The most common anthelmintic drugs used for the treatment ofhuman toxocariasis belong to the benzimidazole carbamates group

ll rights reserved.

to de Higiene e Medicinagal. Fax: +351 21 3632105.

s).

(Pawlowski, 2001). However, these drugs have a low bioavailabil-ity on the tissues, due to their extremely low solubility, and it re-sults in the administration of relatively high doses over longperiods of time (Hrckova and Velebny, 2001). In addition, the useof these drugs for treatment of other helminths may lead to drugresistance, as it is suspected for Necator americanus and Ancylos-toma duodenale (Geerts and Gryseels, 2000). Thus, new drugs fortreatment of helminthic diseases are urgently needed. The studyof plants used in traditional medicine as anthelmintics could givenew insights for active compounds. The co-evolution human-hel-minth has a long existence and the use of plants for treatment ofhelminth parasites is recorded since the prehistorical times. Sincethen, the popular knowledge of medicinal plants has been consol-idated throughout generations by trial and error experiences.

Chenopodium ambrosioides L. (Chenopodiaceae) is a species orig-inally from Central and South America, which grows wild in Portu-gal and in the Mediterranean region. In these regions, the infusion,decoction and juice prepared from the aerial part of the plant areused as vermifuge and for the treatment of asthmatic and nervousmanifestations (Tecedeiro, 1996; Lopez De Guimaraes et al., 2001;Gadano et al., 2006).

Pycnanthus angolensis (Welw.) Warb. (Myristicaceae), a tree thatgrows in West and Central Africa, has been used in traditionalmedicine of São Tomé and Príncipe islands for the treatment of ma-

192 M. Reis et al. / Experimental Parasitology 126 (2010) 191–197

laria and as an antipyretic (Mambu and Grellier, 2008). Prepara-tions of its stem bark are also used of as anthelmintic, analgesic,haemostatic and for the treatment of pneumonial infections (Diehlet al., 2004).

Nutridesintox� is a nutritional supplement composed of fruits,vegetables and seeds, designed to purify the organism from toxinsproduced by the metabolic activity of parasites (http://www.nutri-cioncuantica.com on the 21st April 2010).

The objective of the current study was to assay some plant ex-tracts from C. ambrosioides and P. angolensis and one nutritionalsupplement (Nutridesintox�) against T. canis larvae in order toevaluate their potential anthelmintic activity.

Table 1Criteria for evaluating the effect of drugs on Toxocara canis larvae.

State of larvae Score (n)

Fast movement using the whole body 5Intermediated movement using the whole bodya 4Slow movement using the whole bodya 3Moving with only a part of the body during the observation 2Immobile but not dead 1Deada 0

Mobility index (MI) =P

n Nn/P

Nn, where Nn: number of larvae with the score of n(1).Relative mobility (RM) = MIsample/MIcontrol � 100 (2).

a New scores added to the original method of Kiuchi et al. (1987).

2. Materials and methods

2.1. Parasite

Toxocara canis adult worms were collected from naturally in-fected puppies by using an anthelmintic (pyrantel pamoate, Pfizer).The eggs were removed from the worm uterus and were main-tained in 1% formalin at 27 �C until development to the infectivestage. Toxocara canis second-stage larvae were hatched accordingto De Savigny (1975). These larvae were maintained in HBSS med-ium at 37 �C with an atmosphere of 5% CO2, until in vitro nemato-cidal activity test.

2.2. Plant material and nutritional supplement preparations

C. ambrosioides was collected from Parque Botânico da Tapadada Ajuda in Lisbon, Portugal (August, 2005) and was identified bya botanist (voucher number 112/2008).

The plant extracts were obtained by sequentially extracting50 g of air-dried powdered plant material with 500 ml of hexane,dichloromethane, ethyl acetate and methanol for 48 h at roomtemperature. After filtration, the extracts were concentrated todryness, under reduced pressure at 40–45 �C, using a Büchi rota-tory evaporator, and then stored at 4 �C until used.

This species was also obtained in a traditional medicinal herbalshop and was authenticated based on literature data (Franco,1971). An infusion was prepared with this plant by adding100 ml of boiling water to 10 g of the dried aerial parts and leavingit standing for 10 min. This preparation produced a 100 mg of dryweight per ml of infusion. The infusion was sterilized through a0.2 lm filter (GyroDisc CA-PC. Orange Scientific) and stored at�20 �C.

The stem bark of P. angolensis was collected in São Tomé andPríncipe islands, identification and extraction procedures werepreviously described in Abrantes et al. (2008).

Nutridesintox� was obtained from Nutrialimentos Jesana S.L.,Barcelona (http://www.nutricioncuantica.com). This nutritionalsupplement is composed by Daucus carota L., Cucurbita pepo L., Al-lium sativum L. Sesamum indicum L. and Triticum aestivum L. It hasvitamin A (64 lg/100 g), D (19 lg/100 g), E (27 lg/100 g) and K(33 lg/100 g) and the minerals calcium (0.16%), iron (88 mg/kg),phosphorous (0.21%), magnesium (0.11%), sodium (102 mg/kg)and potassium (0.13%). One capsule (725 mg) was suspended in2.5 ml of distilled water for 1 h at 37 �C and then centrifuged at4000 rpm for 10 min. The supernatant was used directly in thein vitro experiments.

2.3. Nematocidal activity test on Toxocara canis larvae

Hexane and dichloromethane (DCM) extracts of C. ambrosioides;DCM, ethanolic (EtOH) and methanolic (MeOH) extracts of P. angol-ensis were diluted in dimethylsulphoxide (DMSO) to obtain the fol-

lowing concentrations 0.01; 0.05 and 0.1 mg/ml. Albendazole(Zentel, GlaxoSmithKline) was also diluted in DMSO to obtain solu-tions with the same concentrations. Infusion of C. ambrosioides andthe aqueous extract of Nutridesintox� were diluted in Hank’s Bal-anced Salts Solution (HBSS) (Sigma–Aldrich) to obtain the concen-trations of 2.5, 5 and 10 mg/ml.

The in vitro assay was performed in 24-well microplates (30 lar-vae/well) with the test substances, albendazole, a standard anthel-mintic, was used as positive control and HBSS medium and DMSOas solvent controls. All assays were performed in duplicate. Thenematocidal activity was evaluated in terms of relative mobility(RM). This method was originally developed by Kiuchi et al.(1987) however, due to the different types of movements observedduring the assays, we had to adapt this system, by adding morescores as it is shown in Table 1. After 48 h of exposure to the testsubstances, the larval mobility was examined under an invertedmicroscope. The score correspondent to a specific movement wasattributed to each larva. The mobility (MI) index was calculatedusing Eq. (1) and from these values, RM is calculated using Eq.(2) (Table 1). Larvae mortality was calculated as the number of lar-vae that received score 0 divided by the total number of larvae ineach well.

2.4. Animal treatment and infection

Male CD-1 mice with 8-week-old, weighing approximately 35 gwere obtained from the animal facilities of the animal house of thePortuguese Institute of Hygiene and Tropical Medicine, and main-tained under standard laboratory conditions, according to theEuropean Union requirements (86/609/CEE), recognised in Portu-guese law (DL 276/2001 and DL 314/2003). Mice were divided intoten experimental groups (Table 2), composed by seven mice each.According to the experimental groups (Table 2), mice were infectedwith 300 embryonated eggs by gavage. Negative control group ani-mals received only water.

The test substances were selected based on the in vitro assays.All the treatments were administered daily in a single dose fromday 10 post-infection (p.i.) to 12 p.i. The dosages used were chosenaccording to literature review (Table 2). The extracts of C. ambro-sioides and albendazole were administered orally, by gavage, usingthe doses presented in Table 2. Albendazole was diluted in 0.1 mlof water and the hexane extract of C. ambrosioides diluted in a mix-ture of PBS/DMSO (98:2, v/v). The administration of Nutridesintox�

by gavage was found to be difficult to perform, due to thickness ofthe product. For this reason, mice had to be separated into individ-ual cages and the content of two capsules of Nutridesintox� plus amixture of standard food was given to each mice, per day. On everynext day it was observed that all the mixture had been eaten. Forthe negative control animals that received treatment the same pro-tocol was used. All mice were euthanised on day 17th day p.i.

Table 2Experimental groups, daily doses and references of the doses.

Experimental groups Daily dose References

Negative control (no infection nor treatment) – –Positive control (infected but not treated)

Infection + Albendazole 800 mg/kg Delgado et al. (1989), Fok and Kassai (1998),Hrckova and Velebny (2001), Yarsan et al. (2002),Horiuchi et al. (2005)

Albendazole (no infection)

Infection + C. ambrosioides infusion 10% 1500 mg/kg Borba and Amorim (2004)C. ambrosioides infusion 10% (no infection)

Infection + C. ambrosioides hexane extract 30 mg/kg Monzote et al. (2007)C. ambrosioides hexane extract (no infection)

Infection + Nutridesintox� 2 capsules of Nutridesintox�/mice According to the manufacturer’s instructionsNutridesintox� (no infection)

Table 3In vitro anthelminthic activity against Toxocara canis second-stage larvae.

Test substance Relative mobility (%)Concentration (mg/ml)

0.01 0.05 0.1 2.5 5 10

Albendazole 91 77 77C. ambrosioides – hexane 33 21 20C. ambrosioides – DCM 72 30 22C. ambrosioides – infusion 52 38 24P. angolensis – DCM 71 98 85P. angolensis – EtOH 72 62 53P. angolensis – MeOH 95 67 42Nutridesintox� 76 44 15

DCM, dichloromethane; EtOH, ethanol; MeOH, methanol.

M. Reis et al. / Experimental Parasitology 126 (2010) 191–197 193

2.5. Larvae recovery from infected tissues

The brain, liver, lung and musculature were finely minced andincubated in a digestion fluid (pepsin 0.25 g, HCl 1 ml, H2O100 ml) during 24 h at 37 �C. The brain was incubated separatelyfrom the other organs. Larvae from the sediment were counted un-der a microscope.

2.6. ELISA test for determination of IgM and IgG antibodies in theserum

Blood was collected from mice by tail puncture at day 0 beforethe oral inoculation of parasites, day 10 p.i. (before the administra-tion of the treatment) and on the 17th day p.i.

Toxocara canis excretion–secretion (TES) antigen was preparedaccording to De Savigny (1975). The enzyme-linked immunosor-bent assay (ELISA) for detection of IgM and IgG was performed in96-well maxi-sorb plates (Nalgene Nunc). Plates were sensitisedwith 10 lg/ml of TES diluted in 0.1 M sodium carbonate buffer(pH 9.6; 100 ll/well) and incubated for 30 min at 37 �C, followedby overnight incubation at 4 �C. Plates were washed three timeswith phosphate-buffered saline (pH 7.2) containing 0.05% v/vTween-20 (PBS/Tween). Plates were blocked with a solution of1% bovine serum albumin during 1 h 30 min at room temperature;plates were then washed three times with PBS/Tween.

Serum samples were used at a dilution of 1:400 in PBS/Tween.After incubating 1 h at room temperature, plates were washed and100 ll of anti-mouse IgM or IgG conjugated to alkaline phospha-tase (Sigma–Aldrich, USA) at a dilution of 1: 10.000 in PBS/Tweenwere added for 1 h 30 min at room temperature. After the plateswere washed, substrate (p-nitrophenyl phosphate, Sigma–Aldrich)was added, according to the manufacturer’s instructions, to eachwell and the reaction was stopped by adding 3 N NaOH. The opticaldensity values were measured at 405 nm.

2.7. Histology

Livers and lungs of mice from the experimental groups were re-moved on 17th day p.i. Tissues were fixed in 10% formalin andembedded in paraffin blocks. Sections of 5 lm of each organ werestained with haematoxylin–eosin (HE). Analysis of the sectionswas performed in a blinded fashion, as the samples were numeri-cally coded and the pathologist who analysed the sections did notknow the provenience of each sample.

2.8. Statistical analysis

Statistical differences on larvae burden between the differentgroups were determined using Wilcoxon–Mann–Whitney test.The production of IgM and IgG antibodies between the groups

was statistically tested using Kruskal–Wallis test, a Spearman’scorrelation was also preformed. A probability value p < 0.05 wasconsidered statistically significant. Analysis was performed usingSPSS 16.0 statistical package.

3. Results

3.1. In vitro experiment

Albendazole, the plant extracts of C. ambrosioides and P. angol-ensis and an aqueous extract of the nutritional supplement Nut-ridesintox� were evaluated for their in vitro anthelmintic activityagainst T. canis second-stage larvae. The results of the nematocidalactivity are presented in Table 3. The value of the control (the med-ium) was set at 100 and it corresponds to score 5 of mobility. In theDMSO control assay a relative mobility of 100% was measured,which indicates that this substance did not have any anthelminticeffect. In the albendazole assays high values of RM were observedbecause more than 85% of the larvae exhibited mobility correspon-dent to score 4. The extracts of P. angolensis had the lowest RM val-ues compared to the other natural products, but except for theDCM extract of P. angolensis, EtOH and MeOH extracts had highernematocidal activity than albendazole (Table 3). At 0.1 lg/ml theEtOH extract had 6.2% of dead larvae (score 0) and 5.1% was mea-sured for the MeOH extract. All the extracts of C. ambrosioidesshowed similar relative mobility values at the highest concentra-tion tested. However, no mortality was observed and to the major-ity of the larvae was attributed score 1. Interestingly, the hexaneextract of C. ambrosioides was the one that exhibited nematocidaleffect even at the lowest concentration (Table 3). The aqueous ex-tract of Nutridesintox� was the natural product that exerted thehighest nematocidal activity. At 0.1 lg/ml a mortality of 65% wasmeasured.

Thus, Nutridesintox�, the hexane extract and the infusion of C.ambrosioides were chosen for the in vivo assay.

Table 4Recovery of Toxocara canis larvae from the brains and other organs (liver, lungs and muscles) of the different groups of mice, on the 17th day post-infection.

Groups of mice Brain Other organs

Median Mean (SD) pa Median Mean (SD) pa

Control 6.0 6.0 (2.2) – 4.0 4.0 (3.8) –Albendazole 25.0 28.7 (21.2) 0.002 9.5 12.2 (7.4) 0.026C. ambrosioides infusion 11.4 12.8 (6.4) 0.041 30.0 30.3 (11.1) 0.002C. ambrosioides hexane 4.5 4.7 (2.4) 0.240 3.0 3.8 (2.9) 0.937Nutridesintox� 35.5 30.7 (18.2) 0.009 21.0 31.8 (37.7) 0.009

n = 6 (for each group).a Significantly different number of larvae between control and treated group of mice at p level < 0.05 (Wilcoxon–Mann–Whitney test).

194 M. Reis et al. / Experimental Parasitology 126 (2010) 191–197

3.2. In vivo experiment

The effect of the treatments was evaluated on the day 5 post-therapy. At this day, except for the group treated with infusion ofC. ambrosioides, the accumulation of larvae in the brain was higherthan in any other organ studied (Table 4). The groups treated,respectively, with albendazole, infusion of C. ambrosioides and withNutridesintox� showed significantly higher number of larvae inthe studied organs in comparison to the positive control (Table 4).The Wilcoxon–Mann–Whitney statistics was also applied for thecomparison of larvae burden on the natural products groups vsabendazole group. Only larvae burden values of the C. ambrosioideshexane extract group were significantly different from albendazole(data not shown). No parasites were observed in the negative con-trol mice and in the uninfected controls that received treatment(data not shown).

The levels of IgM and IgG antibodies in the serum of mice frompositive and negative control and treated groups are shown inFig. 1. There was no production of these classes of antibodies inthe negative control mice (Fig 1). Using the non-parametric Krus-kal–Wallis test, there were no significant differences in the levelsof IgM (v2 = 4.048; df = 4; p = 0.399) between the different treat-ments and positive control and the same was verified for IgG levels(v2 = 9.202; df = 4; p = 0.056). In the groups of mice treated but notinfected, production of IgM and IgG was not observed (data notshown).

In addition, the analysis with the non-parametric Spearmancorrelation shows a non-linear association between the IgM levels

Fig. 1. Levels of IgM (A) and IgG (B) antibodies specific to T. canis TES in control mice andC. ambrosioides and Nutridesintox� (NTX). Values are median (n = 7 per group).

and the number of larvae (Rs = �0.200; p = 0.747) and the samewas verified for the IgG levels and the number of larvae(Rs = 0.100; p = 0.873), which may show a certain independenceof these two variables.

The livers and lungs from mice of all experimental groups wereanalysed by histology. The uninfected controls that received treat-ment showed a normal hepatic and pulmonary structure and didnot exhibit any observable signs of toxicity (data not shown). Theliver sections of T. canis-infected mice treated with the infusionof C. ambrosioides and Nutridesintox� showed the typical inflam-matory reaction produced by the migrating larvae, in comparisonto the control this reaction seems to be more moderate (Fig. 2B,D and F). The infected mice treated with albendazole and withthe hexane extract of C. ambrosioides did not show signs of inflam-matory infiltrates (Fig. 2C and E).

Regarding to the lung sections, the effects of the treatmentswere not so evident, as it was observed the presence of inflamma-tory infiltrates in all experimental groups (Fig. 3). However, aslightly inflammatory reaction was observed for the group treatedwith the hexane extract (Fig. 3E).

4. Discussion

Medicinal plants and other natural products have long beenused in traditional medicine. Together with other natural products,they have been playing a crucial role in drug discovery and devel-opment (Newman and Cragg, 2007).

in mice treated with albendazole (ABZ), infusion of C. ambrosioides, hexane extract of

Fig. 2. Photographs of liver sections taken from the different experimental groups, on the 17th day post-infection (HE). (A) Uninfected, normal liver (100�). (B) Control, livershowing inflammation of the portal space (w) (400�). (C) Albendazole (100�). (D) Infusion of C. ambrosioides, liver showing a periportal inflammation (w) (100�). (E) Hexaneextract of C. ambrosioides, liver with the absence of inflammatory infiltrates and showing hepatocyte regeneration (�) (400�). (F) Nutridesintox�, liver showing lobularinflammation (w) (400�).

M. Reis et al. / Experimental Parasitology 126 (2010) 191–197 195

In the present study we describe for the first time the anthel-mintic activity of some plant extracts and a nutritional supplementagainst T. canis larvae.

Although, there is no consensual drug for the treatment of vis-ceral larva migrans caused by T. canis, albendazol is the most com-monly used drug (Sturchler et al., 1989; Magnaval et al., 2001;Pawlowski, 2001). However, it seems to have a weak action againstthe T. canis larvae in vitro, as it was demonstrated on our in vitroassays as well as in other studies (Satou et al., 2005; Marquez-Nav-arro et al., 2009).

From all the natural products tested the aqueous extract of Nut-ridesintox� was the one that showed the highest anthelminticactivity in vitro. A synergistic effect between the components ofthis supplement might have been the responsible for the highanthelmintic activity observed. However, physiological and ultra-structural studies should be addressed in order to understandthese results in more detail.

It is long known that the anthelmintic properties of C. ambrosio-ides are due to the monoterpene ascaridole (Nelson, 1920). Thein vitro assay with this species extracts showed interesting results,as the hexane and DCM extracts, which are rich in this compound,showed an anthelmintic activity similar to the infusion of C. ambro-sioides. Some authors suggest that the anthelmintic activity of theaqueous infusions of this species is due to more hydrophilic com-pounds and not to ascaridole (MacDonald et al., 2004; Gadanoet al., 2006). MacDonald et al. (2004) verified that ascaridole-free

infusions of C. ambrosioides retained the anthelmintic propertiesagainst Caenorhabditis elegans.

Even though the extracts of P. angolensis showed a lower anthel-mintic activity compared to the other natural products tested, theycannot be considered to be completely inactive. These results couldhave been due to the concentrations tested, as the MeOH and EtOHextracts not only showed a higher activity compared to albenda-zole but also mortality values. For this species other authors ob-served that EtOH extracts had anthelmintic activity againstHaemonchus contortus (Diehl et al., 2004) and antimalarial activityagainst Plasmodium falciparum (do Céu de Madureira et al., 2002;Abrantes et al., 2008). Methanolic extracts were shown to haveanthelmintic activity against Eudrilus eugeniae (Gbolade and Adey-emi, 2008) and leishmanicidal activity against Leishmania major(Ajaiyeoba et al., 2008).

The experimental toxocarisis is a good model to study of host–parasite interactions and to test for prospective anthelmintics. Thecourse of infection and immunologic responses are similar to whathappens in humans infected with T. canis (Beaver, 1956). Up to thisdate, no studies that describe the in vivo activity of extracts of C.ambrosoiodes or Nutridesintox� against T. canis larvae were found.

Under our experimental conditions the tested substances werenot particularly effective in the reduction of larvae burden.

In comparison to the positive control none of the substancestested were able to reduce the larvae burden and none of the nat-ural products presented an advantage to albendazole. The signifi-

Fig. 3. Photographs of lung sections taken from the different experimental groups, on the 17th day post-infection. (A) Uninfected, normal lung (100�). (B) Control, showingperibronchial inflammatory infiltrates (w) (100�). (C) Albendazol, intense peribronchial (w) and parenchymatous ( ) inflammatory infiltrates (100�) (D) Infusion, lungshowing inflammatory infiltrates (w), alveolar desquamation and hemosiderophages (arrow) (400�). (E) Hexane, slight peribronchial inflammatory infiltrates (400�). (F)Nutridesintox�, lung showing peribronchial inflammatory infiltrates (w) (400�).

196 M. Reis et al. / Experimental Parasitology 126 (2010) 191–197

cant difference observed between the parasitic burden of C. ambro-sioides hexane extract group and albendazole cannot be validated,as there were no significant differences between this and the posi-tive control larvae burden.

The brain was the most parasitised organ on the 17th day p.i.,except for the group treated with infusion of C. ambrosioides, thisfact confirms that the substances were administered when larvaewere in myotropic–neurotropic phase of migration. Some authorssuggest that larvae in this migratory phase are less susceptible todrugs than those in hepato-pulmonary phase (Abo-Shehada andHerbert, 1984; Fok and Kassai, 1998). This could be one explana-tion for the lack of larvae reduction observed in the results. Horiu-chi et al. (2005) administrated albendazole (100 mg/kg) on themyotropic–neurotropic phase (13th–21st day p.i.) and did not findsignificant differences between the numbers of larvae recoveredfrom the brain in comparison to the control. Abo-Shehada and Her-bert (1984) verified that the dose of 100 mg/kg of albendazoleadministrated on the 10th–13th day p.i. did not have anthelminticefficacy.

The fact that the groups treated with albendazole, infusion of C.ambrosioides and Nutridesintox� showed significantly higher num-ber of larvae than the positive control and the hexane extract of C.ambrosioides, lead to the hypothesis that these substances exertedan immunosuppressant effect, that reduced the natural capacity ofmice to eliminate the parasites. Lescano et al. (2004) observed thatin BALB/c mice treated with cyclosporine A (immunosuppressant

drug) the burden of T. canis larvae was higher when compared tothe control. Although, in the present study there were no signifi-cant differences between the groups, in IgM and IgG production,up to the fifth day post-treatment. The results also show that theproduction of antibodies seems to be independent from the larvaeburden.

No conclusions concerning the anthelmintic effectiveness of thetested substances can be taken from the results of larvae counts orfrom the levels of immunoglobulins, however the histological anal-ysis showed interesting results. The doses used were not lethal andno signs of toxicity were observed in the control groups. In thegroup treated with albendazole, the typical inflammatory infil-trates caused by the migration of larvae were not observed in theliver sections. This could be related to the fact that albendazole ismetabolized in the liver to albendazole sulfoxide, which has ananthelmintic effect (Lacey, 1990). In the group treated with thehexane extract of C. ambrosioides, in the liver sections no inflam-matory infiltrates were observed and the regeneration of the hepa-tic tissue was recorded. A reduction of the inflammatory infiltratesin the lungs in comparison to the positive control and albendazolewas also observed. These observations suggest that the study of thecomponents of hexane extract of C. ambrosioides might have inter-est for future research on anthelmintic and/or anti-inflammatorycompounds.

In conclusion, the results presented here point out to the factthat further studies, on the hexane extract of C. ambrosioides are

M. Reis et al. / Experimental Parasitology 126 (2010) 191–197 197

of interest, as it showed not only an anthelmintic activity in vitrobut also a reduction on the inflammatory reaction produced bythe infection of T. canis larvae in vivo. These facts also correlatewith ethnobotanical data, in which the use of preparations C.ambrosioides is described for the treatment of nematode infections.This study also highlights for the necessity to test a wider range ofdoses, to compare the two phases of larval migration and to have amultifactorial analysis (larvae counts, immunology, histology),when planning any search for new drugs for use in the treatmentof human toxocariasis.

Acknowledgments

The authors are grateful to Prof. Dr. M. Correia and Dr. A. Belo(FCM, UNL) for the preparation of the histological sections andimages, Dr. T. Vasconcelos (ISA, UTL) for the identification of C.ambrosioides. The authors also thank Prof. M. Madureira for P.angolensis collection and to Dr. A. Afonso for the critical review ofthis paper.

References

Abo-Shehada, M.N., Herbert, I.V., 1984. Anthelmintic effect of levamisole,ivermectin, albendazole and fenbendazole on larval Toxocara canis infection inmice. Research in Veterinary Science 36, 87–91.

Abrantes, M., Mil-Homens, T., Duarte, N., Lopes, D., Cravo, P., Madureira, M., Ferreira,M.J., 2008. Antiplasmodial activity of lignans and extracts from Pycnanthusangolensis. Planta Medica 74, 1408–1412.

Ajaiyeoba, E.O., Ali, M.S., Onocha, P.A., 2008. In vitro antileishmaniasis, phyto andcytotoxicity of Pycnanthus angolensis methanolic extracts. Research Journal ofMedical Sciences 2, 178–181.

Beaver, P.C., 1956. Larva migrans: parasitological reviews. ExperimentalParasitology V, 587–621.

Borba, H.R., Amorim, A., 2004. Ação anti-helmíntica de plantas XIV. Avaliação daatividade de extratos aquosos de Chenopodium ambrosioides L. (erva-de-santa-maria) em camundongos naturalmente infectados com Syphacia obvelata eAspiculuris tetraptera. Revista Brasileira de Parasitologia Veterinária 13 (4), 133–136.

De Savigny, D.H., 1975. In vitro maintenance of Toxocara canis larvae and a simplemethod for the production of Toxocara ES antigen for use in serodiagnostic testsfor visceral larva migrans. The Journal of Parasitology 61, 781–782.

Delgado, O., Botto, C., Mattei, R., Escalante, A., 1989. Effect of albendazole inexperimental toxocariasis of mice. Annals of Tropical Medicine andParasitolology 83 (6), 621–624.

Despommier, D., 2003. Toxocariasis: clinical aspects, epidemiology, medicalecology, and molecular aspects. Clinical Microbiology Reviews 16, 265–272.

Diehl, M.S., Atindehou, K.K., Tere, H., Betschart, B., 2004. Prospect for anthelminthicplants in the Ivory Coast using ethnobotanical criteria. Journal ofEthnopharmacology 95, 277–284.

de Madureira, M., Paula Martins, A., Gomes, M., Paiva, J., Proenca da Cunha, A., doRosario, V., 2002. Antimalarial activity of medicinal plants used in traditionalmedicine in S Tome and Principe islands. Journal of Ethnopharmacology 81, 23–29.

Fok, E., Kassai, T., 1998. Toxocara canis infection in the paratenic host: a study on thechemosusceptibility of the somatic larvae in mice. Veterinary Parasitology 74(2–4), 243–259.

Franco, J.A., 1971. Nova flora de Portugal (Continente e Açores). I Sociedade Astória,Lisboa. pp. 648.

Gadano, A.B., Gurni, A.A., Carballo, M.A., 2006. Argentine folk medicine: genotoxiceffects of Chenopodiaceae family. Journal of Ethnopharmacology 103, 246–251.

Gbolade, A.A., Adeyemi, A.A., 2008. Investigation of in vitro anthelminticactivities of Pycnanthus angolensis and Sphenocentrum jollyanum. Fitoterapia79, 220–222.

Geerts, S., Gryseels, B., 2000. Drug resistance in human helminths: current situationand lessons from livestock. Clinical Microbiology Reviews 13, 207–222.

Horiuchi, A., Satou, T., Akao, N., Koike, K., Fujita, K., Nikaido, T., 2005. The effect offree and polyethylene glycol–liposome-entrapped albendazole on larvalmobility and number in Toxocara canis infected mice. Veterinary Parasitology129, 83–87.

Hrckova, G., Velebny, S., 2001. Treatment of Toxocara canis infections in mice withliposome-incorporated benzimidazole carbamates and immunomodulatorglucan. Journal of Helminthology 75, 141–146.

Kiuchi, F., Miyashita, N., Tsuda, Y., Kondo, K., Yoshimura, H., 1987. Studies on crudedrugs effective on visceral larva migrans I. Identification of larvicidal principlesin betel nuts.. Chemical & Pharmaceutical Bulletin 35, 2880–2886.

Lacey, E., 1990. Mode of action of benzimidazoles. Parasitology Today 6, 112–115.Lescano, S.A., Chieffi, P.P., Ikai, D.K., Ribeiro, M.C., 2004. Effects of cyclosporin A or

betamethasone on experimental murine toxocariasis. Revista da SociedadeBrasileira de Medicina Tropical 37, 22–24.

Lopez De Guimaraes, D., Neyra Llanos, R.S., Romero Acevedo, J.H., 2001. Ascariasis:comparison of the therapeutic efficacy between paico and albendazole inchildren from Huaraz. Revista Gastroenterologia Peru 21 (3), 212–219.

MacDonald, D., VanCrey, K., Harrison, P., Rangachari, P.K., Rosenfeld, J., Warren, C.,Sorger, G., 2004. Ascaridole-less infusions of Chenopodium ambrosioides containa nematocide(s) that is(are) not toxic to mammalian smooth muscle. Journal ofEthnopharmacology 92, 215–221.

Magnaval, J.F., Glickman, L.T., Dorchies, P., Morassin, B., 2001. Highlights of humantoxocariasis. The Korean Journal of Parasitology 39, 1–11.

Maizels, R.M., Tetteh, K.K., Loukas, A., 2000. Toxocara canis: genes expressed by thearrested infective larval stage of a parasitic nematode. International Journal forParasitology 30, 495–508.

Mambu, L., Grellier, P., 2008. Antimalarial compounds from traditionally-usedmedicinal plants. In: Colegate, S.M., Molyneux, R.J. (Eds.), Bioactive NaturalProducts: Detection, Isolation, and Structural Determination, second ed. CRCPress, Boca Raton, FL, pp. 491–530.

Marquez-Navarro, A., Nogueda-Torres, B., Hernandez-Campos, A., Soria-Arteche, O.,Castillo, R., Rodriguez-Morales, S., Yepez-Mulia, L., Hernandez-Luis, F., 2009.Anthelmintic activity of benzimidazole derivatives against Toxocara canissecond-stage larvae and Hymenolepis nana adults. Acta Tropica 109, 232–235.

Monzote, L., Montalvo, A.M., Scull, R., Miranda, M., Abreu, J., 2007. Activity, toxicityand analysis of resistance of essential oil from Chenopodium ambrosioides afterintraperitoneal, oral and intralesional administration in BALB/c mice infectedwith Leishmania amazonensis: a preliminary study. BiomedicalPharmacotherapy 61 (2–3), 148–153.

Nelson, E.K., 1920. The composition of oil of Chenopodium from various sources.Journal of the American Chemical Society 42, 1204–1208.

Newman, D.J., Cragg, G.M., 2007. Natural products as sources of new drugs over thelast 25 years. Journal of Natural Products 70, 461–477.

Pawlowski, Z., 2001. Toxocariasis in humans: clinical expression and treatmentdilemma. Journal of Helminthology 75, 299–305.

Satou, T., Horiuchi, A., Akao, N., Koike, K., Fujita, K., Nikaido, T., 2005. Toxocara canis:search for a potential drug amongst beta-carboline alkaloids – in vitro andmouse studies. Experimental Parasitology 110, 134–139.

Sturchler, D., Schubarth, P., Gualzata, M., Gottstein, B., Oettli, A., 1989.Thiabendazole vs Albendazole in treatment of toxocariasis: a clinical trial.Annals of Tropical Medicine and Parasitology 83, 473–478.

Tecedeiro, L.A.V., 1996. Plantas Medicinais do Ribatejo. Garrido artes gráficas,Alpiarça. pp. 317.

Yarsan, E., Celik S., Eraslan G., Aycicek, H., 2002. Effects of albendazole treatment onlipid peroxidation of healthy and Toxocara canis infected mice. Israel Journal ofVeterinary Medicine 57 (1). Available from: <http://www.isrvma.org/article/57_1_3.htm>.