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20 février 2011 7 20 /02 /février /2011 10:39

NUTRIDESINTOX (Chenopodium ambrosioides and Pycnanthus angolensis)

 

Title: In vitro and in vivo anthelmintic activity of plant extracts and a nutritional supplement against Toxocara canis

Authors:

Mariana Reisa, Maria J. Ferreirab, Ana R. Monsalve-Puelloc, Miguel Correiad, Alcione Trincaa, Maria A.A. Grácioa

 

Address:

a Unidade de Helmintologia e Malacologia Médicas/ UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal

b iMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal

c Nutrialimentos Jesana S.L., Barcelona, Espanha

d Departamento de Histologia, Embriologia e Biologia Celular, Faculdade de Ciências Médicas Universidade Nova de Lisboa, Lisboa, Portugal

 

Abstract:

The aim of this study was to search for new treatments for human toxocariasis using in vitro and in vivo experiments and ethnobotanical data. Thus, extracts from two plants (Chenopodium ambrosioides and Pycnanthus angolensis) used in folk medicine as anthelmintics, and a nutritional supplement (Nutridesintox®) have been evaluated against Toxocara canis larvae. Albendazole was used as a standard reference. In the in vitro experiments, the nutritional supplement Nutridesintox® was the most effective, followed by the hexane and the dichloromethane extracts and the infusion of C. ambrosioides.

The methanolic, ethanolic and the methylene chloride extracts of P. angolensis showed no anthelmintic efficacy. Due to their in vitro anthelmintic activity, the nutritional supplement, the hexane extract and the infusion of C. ambrosioides were selected for the in vivo experiments.

These products were administered orally to T. canis-infected CD1 mice in single doses during three consecutive days, commenced on day 10 post-infection. The efficacy of treatment was evaluated on day 5 after the last dose, by counting the numbers of T. canis larvae in the brain, liver, lungs and muscles, detection of IgM and IgG antibodies by ELISA and histological analysis of liver and lungs. The dynamics of the antibodies on the treated groups showed a similar profile as compared to the control group, statistically significant differences were not found. The different treatments did not show a clear evidence of reduction of larvae burden in the infected tissues. The level of infection in the brain was higher than in the other organs examined, except for the group treated with the infusion of C. ambrosioides.

Interestingly, a reduction of inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. The in vitro and in vivo results call for further studies on these natural products, in order to find alternative therapeutic regimens for human toxocariasis.

 

Keywords: Anthelmintic activity; Chenopodium ambrosioides; Pycnanthus angolensis Toxocara canis

 

1. Introduction

 Toxocara canis(Werner, 1782), the roundworm of dogs, is the etiologic agent of human toxocariasis. As the dog constitutes one of our most common pets, this ensured the worldwide distribution of this zoonotic disease. Commonly the infection is acquired after ingestion of embryonated T. canis eggs that can be present in soil contaminated with dog faeces. Children are usually the most susceptible to infect because of their habits of pica (Despommier, 2003). As larvae hatch in the stomach they penetrate the mucosal epithelium and thereafter remain developmentally arrested in the tissue phase. Although they do not grow or differentiate in the paratenic host they maintain an active metabolism and migratory behaviour (Maizels et al., 2000). Their wandering around the body gives rise to two main syndromes; visceral larva migrans (VLM), in which the major organs are affected and ocular larva migrans (OLM), when it affects the eye, in some cases, it can lead to unilateral blindness (Magnaval et al., 2001)

 The most common anthelmintic drugs used for the treatment of toxocariasis belong to the benzimidazole carbamates group (Pawlowski, 2001). However, these drugs have a low bioavailability for tissues, due to their extremely low solubility, resulting in relatively high doses over a long periods of time (Hrčkova and Velebný, 2001). In addition, the use of these drugs for treatment of other helminths leads to drug resistance, as it was already verified in Mali for Necator americanus (De Clercq et al., 1997). Thus, new drugs for treatment of helminthic diseases are urgently needed. The study of plants used in traditional medicine as anthelmintics could give new insights for active compounds. The co-evolution human-helminth has a long existence and the use of plants for treatment of helminth parasites is recorded since the prehistorical times (Reinhard et al., 1985). Since then, the popular knowledge of medicinal plants has been consolidated throughout generations by trial and error experiences.

Chenopodium ambrosioides L. (Chenopodiaceae) is a species originally from Central and South America, which grows wild in Portugal and in the Mediterranean region. In Portugal is popularly known as “formigueira” or “erva-de-Santa-Maria”, the infusions made of leaves and inflorescences are used as vermifuge and for the treatment of asthmatic and nervous manifestations (Tecedeiro, 1996).

Pycnanthus angolensis(Welw.) Warb. (Myristicaceae), is known as “pó-casson” in the islands of São Tomé e Principe. Preparations of its stem bark are used of as anthelmintic, analgesic, haemostatic and for the treatment of pneumonial infections (Betti, 2002; Diehl et al., 2004).

Nutridesintox® is a nutritional supplement composed of fruits, vegetables and seeds, designed to purify the organism from toxins produced by the metabolic activity of parasites.

The objective of the current study was to assay some plant extracts from Chenopodium ambrosioides and Pycnanthus angolensis and one nutritional supplement (Nutridesintox®) against T. canis larvae in order to evaluate the potential in vitro and in vivo anthelmintic activity.

 

2. Materials and Methods

2.2 Parasite

Toxocara canis adult worms were collected from naturally infected puppies by using an anthelmintic (pyrantel pamoate, Pfizer). The eggs were removed from the worm uterus and were maintained in 1% formalin at 27 ºC until the development of the infective stage.

 

2.1 Plant material and nutritional supplement preparations

Chenopodium ambrosioides was collected in Portugal (authenticated by voucher number). The plant extracts were obtained by sequentially extracting 50 g of air-dried powdered plant material with 500 ml of hexane, dichloromethane, ethyl acetate and methanol for 48 h at room temperature. After filtration, the extracts were concentrated to dryness, under reduced pressure at 40-45 ºC, using a Büchi rotatory evaporator, and then stored at 4ºC until used.

This species was also obtained in a traditional medicinal herb shop and was authenticated using Franco (1971). An infusion was prepared with this plant by adding 100 ml of boiling water to 10 g of the dried aerial parts and leaving it standing for 10 min. The infusion was sterilized through a 0.22 µm filter and stored at -20 ºC.

The stem bark of Pycnanthus angolensis was collected in São Tomé and Príncipe islands and extraction procedures were previously described in Abrantes et al (2008).

Nutridesintox® was obtained from Nutrialimentos Jesana S.L., Barcelona (http://www.nutricioncuantica.com). This nutritional supplement is composed by carrot, pumpkin, garlic, sesame and wheat. One capsule (725 mg) was dissolved in 2.5 ml of distilled water for 1 hour at 37 ºC and then centrifuged at 4000 rpm for 10 min. The supernatant was used directly in the in vitro experiments.

 

2.3 In vitro experiments

2.3.1. Sample preparation

Hexane and dicloromethane (DCM) extracts of C. ambrosioides; DCM, ethanolic (EtOH) and methanolic (MeOH) extracts of P. angolensis were diluted in dimethylsulphoxide (DMSO) to obtain the following concentrations 0.01; 0.05 and 0.1 mg/ml.

Albendazole (Zentel, GlaxoSmithKline) was also diluted in DMSO to obtain the same concentrations.

Infusion of C. ambrosioides and the aqueous extract of Nutridesintox® were diluted in Hank’s Balanced Salts Solution (HBSS) (Sigma-Aldrich) to obtain the concentrations of 25, 50 and 100 µl/ml.

2.3.1 Nematocidal activity test on T. canis larvae

Toxocara canissecond-stage larvae were hatched according to De Savigny (1975). These larvae were maintained in HBSS medium at 37 ºC with an atmosphere of 5 % CO2. The in vitro test was preformed in 24-well microplates (30 larvae/well) with the test substances, albendazole as standard anthelmintic and HBSS medium and DMSO as solvent controls. All assays were preformed in duplicate. After 48 h of exposure the mobility of the larvae were examined using an inverted microscope. Nematocidal 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 observed during the assays, we had to adapt this system, by adding more scores as it is shown in Table 1.

 

2.4. In vivo Experiments

2.4.1 Animal infection

Male CD-1 mice with 8-week-old, weighing 35 g were obtained from the animal facilities of the Biotério do Instituto de Higiene e Medicina Tropical, and maintained under standard laboratory conditions, according to the European Union requirements (86/609/CEE). Mice were infected with 300 embryonated eggs by gastric intubation via a metal cannula coated with silicon. Seven mice were used in each experimental group.

 

2.4.2. Animal treatment

The test substances were selected based on the in vitro test results. All the treatments were administered daily from day 10 post-infection (p.i) to 12 p.i.. The extracts of C. ambrosioides and albendazole were administered orally using the following doses: hexane extract of C. ambrosioides (30 mg/Kg/ 0.1 ml); infusion of C. ambrosioides (15 ml/Kg/0.5 ml) and albendazole (800 mg/Kg/0.1 ml). For the nutritional supplement administration, mice were separated into individual cages with a mixture of standard food and the content of two capsules of Nutridesintox® per day.

All mice were sacrificed at 17th day p.i..

 

2.4.3. Larvae recovery from infected tissues

The brain, liver, lung and musculature were finely minced and incubated in a digestion fluid (pepsin 0.25 g, HCl 1 ml, H2O 100 ml) during 24 h at 37 ºC. The brain was incubated separately from the other organs. Larvae from the sediment were counted under a microscope.

 

2.4.4. ELISA test for determination of IgM and IgG antibodies in the serum

Blood was collected from the tail of mice from the different groups before the infection, before the treatment and on the 17thday p.i..

Toxocara canis excretion-secretion (TES) antigen was prepared according to De Savigny (1975).

The enzyme-linked immunosorbent assay (ELISA) for detection of IgM and IgG was performed in 96-well maxi-sorb plates (Nalgene Nunc). Plates were sensitised with 10 μl/ml of TES diluted in 0,1 M sodium carbonate buffer ( pH 9.6; 100 µl/well) and incubated for 30 min at 37 ºC, followed by overnight incubation at 4 ºC. Plates were washed three times with phosphate-buffered saline (pH 7.2) containing 0.05% v/v Tween-20 (PBS/Tween). Plates were blocked with a solution of 1 % bovine serum albumin during 1h 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 and 100 µl of anti-mouse IgM or IgG conjugated to alkaline phosphatase (Sigma-Aldrich, USA) at a dilution of 1: 10.000 in PBS/Tween were added for 1h 30 min at room temperature. After the plates were washed, substrate (p-nitrophenyl phosphate, Sigma-Aldrich) was added to each well and the reaction was stopped by adding 3N NaOH. The optical density values were measured at 405 nm.

 

2.4.5. Histology

Livers and lungs of mice from the experimental groups were removed on 17thday p. i.. Tissues were fixed in 10 % formalin and embedded in paraffin blocks. Sections of 5 µm of each organ were stained with hematoxylin-eosin (HE). Analysis of the sections was performed in a blinded fashion.

 

2.4.6. Statistical analysis

Statistical differences on the larvae burden between the groups 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’s correlation was also preformed. A probability value p < 0.005 was considered statistically significant. Analysis was performed using SPSS 16.0 statistical package.

 

Results

3.1. In vitro experiment

Albendazole, extracts of C. ambrosioides, extracts of P. angolensis and an aqueous extract of Nutridesintox® were evaluated for their in vitro anthelmintic activity against T. canis second-stage larvae. The results of the nematocidal activity are presented in Table 2. In the negative control assays (HBSS medium and DMSO) a relative mobility of 100 % was measured, which indicates that these substances did not have any anthelmintic effect. At the highest concentration tested, except for the DCM extract of P. angolensis, all the other tested extracts had higher nematocidal activity than albendazole (Table 2). The aqueous extract of Nutridesintox® exerted the highest nematocidal activity, with 65 % of dead larvae (Fig. 1). All the extracts of C. ambrosioides showed similar relative mobility values at the highest concentration tested. Interestingly, the hexane extract of C. ambrosioides was the only that exhibited nematocidal effect even at the lowest concentration. The extracts of P. angolensis showed a low anthelmintic effect (Table 2).

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

 

3.2. In vivo experiment

The effect of the treatments was evaluated on the day 5 post-therapy. At this day, the accumulation of larvae in the brain was higher than in the other organs studied, except for the group treated with infusion of C. ambrosioides (Table 3). The groups treated respectively with albendazole, infusion of C. ambrosioides and with Nutridesintox® showed significant differences between larval numbers in the studied organs in comparison with the control (Table 3). However, these results did not show a reduction of larvae burden post therapy.

The levels of IgM and IgG antibodies in the serum of mice from control and treated groups are shown in Fig. 2. Using the non-parametric Kruskal-Wallis test, no significant differences in the levels of IgM (χ2= 4,048; df = 4; p = 0,399) and IgG (χ2= 9,202; df = 4; p = 0,056) between the different treatments were found. In addition, the analysis with the non-parametric Spearman correlation shows a non-linear association between the IgM levels and the number of larvae (Rs=-0,200; p=0,747) and the same was verified for the IgG levels and the number of larvae (Rs=0,100; p= 0,873), which may show a certain independence of these two variables.

The livers and lungs from mice of all experimental groups were analysed by histology. The uninfected controls that received treatment (albendazole, hexane extract and infusion of C. ambrosioides and Nutridesintox®) showed a normal hepatic and pulmonary structure and did not exhibit any observable signs of toxicity (data not shown). The liver sections of T.canis-infected mice treated with the infusion of C. ambrosioides and Nutridesintox® showed the typical inflammatory reaction produced by the migrating larvae, in comparison to the control this reaction seems to be more moderate (Fig. 3 B, D and F). The infected mice treated with albendazole and with the hexane extract of C. ambrosioides did not show signs of inflammatory infiltrates (Fig. 3 C and E).

Regarding to the lung sections, the effects of the treatments were not so evident, as it was observed the presence of inflammatory infiltrates in all experimental groups (Fig. 4). However, a slightly inflammatory reaction was observed for the group treated with the hexane extract (Fig. 4 E).

 

Discussion

Medicinal plants and other natural products have long been used in folk medicine. In the developed countries the depopulation of rural areas, led to the loss these ancient practises. Nowadays, there is an increasing interest in natural products so as to have alternatives to conventional medicine and for the development of new drugs. In the present study we have investigated the anthelmintic activity of some plant extracts and a nutritional supplement against T. canis larvae.

Although, there is no consensual drug for the treatment of visceral larva migrans caused by T. canis, albendazol is the most commonly used drug (Sturchler et al., 1989; Magnaval et al., 2001; Pawlowski, 2001). However, it seems to have a weak action against the T. canis larvae in vitro, as it was demonstrated on our in vitro assays as well as in other studies (Satou et al., 2005; Márquez-Navarro et al., 2008).

From all the natural products tested the nutritional supplement was the one that showed the highest anthelmintic activity in vitro. This supplement is composed of carrot, garlic, pumpkin, sesame and wheat bran that are used in folk medicine as anthelmintics (Elisha et al.,1987; Waller et al., 2001; Salgueiro, 2005 Cunha et al., 2006). A synergistic effect between the components of this supplement might have been the responsible for the high anthelmintic activity observed in vitro. However more studies should be addressed in order to understand this in more detail.

The in vitro assay of the C. ambrosioides extracts showed interesting results.It is long known that the anthelmintic properties of this species are due to the monoterpene  ascaridole (Nelson, 1920). The hexane and DCM extracts, which are rich in this compound, showed an anthelmintic activity similar to the infusion of C. ambrosioides. Some authors suggest that the anthelmintic activity of the aqueous infusions of this species is due to more hydrophilic compounds and not to ascaridole (MacDonald et al., 2004; Gadano et al., 2006). MacDonald et al. (2004) verified that ascaridole-free infusions of C. ambrosioides retained the anthelmintic properties against Caenorhabditis elegans.

Even though the extracts of P. angolensis did not show anthelmintic activity they cannot be considered to be completely inactive. These results could have been due to the concentrations tested as the methanolic and ethanolic extracts showed to have a higher activity compared to albendazole. For this species other authors observed that ethanolic extracts had anthelmintic activity against Haemonchus contortus (Diehl et al., 2004) and antimalarial activity against Plasmodium falciparum (do Céu de Madureira et al., 2002). Methanolic extracts were shown to have anthelmintic activity against Eudrilus eugeniae (Gbolade & Adeyemi, 2008) and leishmanicidal activity against Leishmania major (Onocha et al., 2008).

Regarding to the in vivo evaluation the tested substances were not particularly effective. The brain was the most parasitized organ on the 17thday p. i., which indicates that the migratory larvae were in myotropic-neurotropic phase. Horiuchi et al. (2005) administrated albendazole (100 mg/kg) between the 13thto the 21st day p.i. and did not find significant differences between the numbers of larvae recovered from the brain in comparison to the control. Abo-Shehada & Herbert (1984) verified that the dose of 100 mg/kg of albendazole administrated on the 10th-13thday p.i. did not have anthelmintic efficacy. These authors suggested that larvae in this migratory phase are less susceptible to drug actions than those in hepato-pulmonary phase.

In the groups treated respectively with albendazole, infusion of C. ambrosioides and Nutridesintox® the number of recovered larvae was significantly higher than the number of recovered larvae from the control. Lescano et al. (2004) observed that in BALB/c mice treated with cyclosporine A (immunosuppressant drug) the burden of T. canis larvae was higher when compared to the control and that there was a delay in IgG production. One explanation to our results could be that these substances had exerted an immunosuppressant effect, therefore reducing the natural capacity of elimination of parasites. However, up to the fifth day post-treatment there were no significant differences in IgM and IgG production between the groups.

Interestingly, despite the fact that in the group treated with the hexane extract of C. ambrosioides a reduction of the larvae burden has not been verified; it was observed an absence of inflammatory infiltrates in the liver sections and a reduction of the inflammatory infiltrates in the lungs. In the group treated with albendazole an absence of inflammatory infiltrates in the liver sections was also observed. This could be related to the fact that albendazole is metabolized in the liver to albendazole sulfoxide, which has an anthelmintic effect (Lacey, 1990).

The present results showed a correlation between the in vitro and in vivo experiments and ethnobotanical data, namely to the hexane extract of C. ambrosioides. This study also highlights for the necessity of comparison of the two phases of larval migration and to have a multifactorial analysis (larvae counts, immunology, histology) when planning any search for new drugs for use in the treatment of toxocariasis.

 

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

State of larva

Score (n)

Fast movement using the whole body

5

Intermediated movement using the whole body

4

Slow movement using the whole body

3

Moving with only a part of the body during the observation

2

Immobile but not dead

1

Dead

0

Motility index (MI) = ∑n Nn/ ∑Nn, where Nn : number of larvae with the score of n.

Relative mobility (RM) = MIsample/ MIcontrolx 100

 

 

Table 2. In vitro anthelminthic activity against Toxocara canis second-stage larvae.

Test substance

Relative Mobility (%)

 

 

Concentration (µg/ml)

Concentration (µl/ml)

 

0,01

0,05

0,1

25

50

100

 

Albendazole

91

77

77

 

 

 

 

C. ambrosioides - hexane

33

21

20

 

 

 

 

C. ambrosioides - DCM

72

30

22

 

 

 

 

C. ambrosioides – infusion

 

 

 

52

38

24

 

P. angolensis - DCM

71

98

85

 

 

 

 

P. angolensis - EtOH

72

62

53

 

 

 

 

P. angolensis - MeOH

95

67

42

 

 

 

 

Nutridesintox®

 

 

 

76

44

15

 

DCM: dicloromethane; EtOH: ethanol; MeOH: methanol

 

 

 

 

 

 

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