Inhibition of Trypanosoma cruzi by plant extracts used in Chinese medicine

used in Chinese medicine

D. Lirussia, J. Lib, J.M. Prietob, M. Gennaria, H. Buschiazzoa,

J.L. Rı´osb, A. Zaidenberga,c,*

aCa´tedra de Farmacologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de La Plata,

Calles 60 y 120, La Plata 1900, Argentina

bDepartamento de Farmacologia, Facultat de Farma` cia, Universitat de Vale`ncia, Vale`ncia, Spain

cComision de Investigaciones Cientı´ficas de la Provincia de Buenos Aires, La Plata, Argentina

Received 21 June 2004; accepted 3 September 2004

Available online 27 October 2004

Abstract

In this work, we assessed the effect of extracts obtained from 17 plants used in traditional Chinese

medicine. These extracts were tested in vitro with the epimastigote form of Trypanosoma cruzi, clone

Bra C15 C2, at 27 8C in F-29 medium at a concentration of 100 Ag/ml in axenic cultures. Allopurinol

was used as reference drug. Seven plant extracts showed inhibitory activities lower than 25%. Pueraria

lobata, Mahonia beaei, Dictamus dasycarpus, Kochia scoparia, Sophora flavescens and Ligustrum

lucidum showed effects with inhibition values between 25% and 60%, whereas Lithospermum

erythrorhizon, Saussurea lappa, Melia toosendan and Cinnamomum cassia showed the greatest

inhibitory activity of 100%. The IC50 of these extracts were: 0.4, 2.4, 1.8 and 3.9 Ag/ml, respectively.

The MTT assay was made and did not show cytotoxic activity. These results allowed us to

suggest that L. erythrorhizon, S. lappa, M. toosendan and C. cassia could be a source of new

compounds against T. cruzi.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Antiprotozoan drugs; Trypanosoma cruzi; Melia toosendan; Lithospermum erythrorhizon; Saussurea

lappa; Cinnamomum cassia

0367-326X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.fitote.2004.09.017

* Corresponding author. Ca´tedra de Farmacologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de

La Plata, Calles 60 y 120, La Plata 1900, Argentina. Tel.: +54 221 421 6932; fax: +54 221 423 6710.

E-mail address: azaidenberg@hotmail.com (A. Zaidenberg).

Fitoterapia 75 (2004) 718–723

1. Introduction

The etiologic agent of Chagas disease, Trypanosoma cruzi, affects 20 million people in Latin American countries ^[1] and is after malaria and schistosomiasis, the most prevalent vector-borne illness. All the drugs used in the treatments of Chagas disease have limitations concerning effectiveness and drug-related side effects. Some drugs already used for treating this parasitic infection may have a promising future ^[2,3] but the lack of selective cytotoxicity against T. cruzi makes the search for new products one of the priority in the medical field ^[4,5]. Novel possibilities for the search of new compounds against the parasite can be obtained from plant extracts ^[6]. In this work, we assessed the effect of extracts obtained from 17 plants used in traditional Chinese medicine.

2. Experimental

2.1. Plants

All the botanical species used are detailed in Table 1. They were collected in China,identified by one of the author (Dr. Li) and each specimen was deposited in the Herbarium of the Department of Pharmacology, Faculty of Pharmacy, University of Valencia, Spain.

Table 1

2.2. Extraction

Each plant (10 g) was stirred in MeOH (100 ml) at r.t. After filtration and concentration in vacuo, the extracts were dissolved in DMSO. The obtained yields are reported in Table 2. 2.3. Chemicals Methanol used for the extraction was PA Merck Lab. The culture medium base was 199, Gibco, Life Technologies, NY; Fetal bovine serum was Natacor, Argentina. All the chemicals for assays were analytical grade (Sigma, St. Louis). 2.4. Animals Male Wistar rats (250–300 g) were used. They were fed rodent diet with tap water adlibitum. They were kept in standard environmental conditions. 2.5. In vitro assays Epimastigotes of clone Bra C15 C2 of T. cruzi ^[7] were grown in F-29 medium modified by adding the supplement of 10% (v/v) FCS heat-inactivate. The incubation temperature was 27 8C. The inoculum was 5.6_105 cells/ml ^[8]. Plant extracts were added at a final concentration of 100 Ag/ml. All experiments were made by triplicate.

Table 2

Final DMSO concentration was less than 0.5%. The count of the parasites was madeafter 72 h by Neubauer chamber and stained with Wright Giemsa. The extracts activity was assessed by comparison with the MSO used as negative control. Allopurinol was used as reference drug. 2.6. Cell viability and cytotoxicity Elicited peritoneal leukocytes obtained from rats ^[9] were used to assess plant extracts cytotoxicity with viability greater than 95% observed by the trypan blue exclusion test.

The cell viability was assessed by the MTT test (Table 2). This assay measured the capacity of PMN mitochondrial dehydrogenase enzymes to convert the 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) into a dark blue formazan, which is assessed spectrophotometrically at 490 nm [^10]. 、hlorpromazine (200 AM) was used as a reference compound like a positive control. 2.7. Light microscopy A sample of each culture of 72 h of incubation was observed under light microscopy

with and without Wright Giemsa stain. 2.8. Statistical analysis Data were expressed as meansFS.D. Statistical analysis was performed by one way analysis of variance (ANOVA), followed by S.C.-P.T.S. Differences were considered significant at Pb0.05 ^[11,12]. The inhibitory concentration 50% (IC50) was alculated from the concentration/effect regression line. In each case, an appropriate range of four tofive concentrations was used. 3. Results and discussion In this work, we tested the effect of plant extracts obtained from China, used commonly in medicine, against T. cruzi, the etiologic agent of Chagas disease. The plant extracts were tested against the epimastigote stage in axenic cultures. This stage of T. cruzi has different sensitivity compared to other stages, and thus it is useful to identify active compounds against the parasite ^[4]. On the other hand, a considerable number of plant extracts with positive inhibitory results in vitro, for different reasons, did not become a new chemotherapeutic alternative. The results of our study are reported in Tables 2 and 3. Ilex pubescens, Paeonia suffruticosa, Tripterigium sp., Cyperus rotundus, Gardenia jasminoides and Erythrina variegata showed inhibition activities under 25%, while Pueraria lobata, Mahonia beaei, Dictamus dasycarpus, Kochia scoparia, Sophora flavescens, Tinospora capillipes and Ligustrum lucidum had inhibition activities between 25% and 60%. Finally, Lithospermum erythrorhizon, Saussurea lappa, Melia toosendan and Cinnamomum cassia showed the D. Lirussi et al. / Fitoterapia 75 (2004) 718–723 721

greatest inhibitory activity, 100%. The IC50 of these extracts (Fig. 1) were 0.4, 2.4, 1.8 and 3.9 Ag/ml, respectively; these activities are 10 and 100 times greater than Benznidazole (IC50i50 Ag/ml), the current drug used for the treatment of Chagas disease ^[4]. The MTT assay did not reveal any cytotoxic activities for all the extracts (Table 2).

These plant species, in general, are already known to have pharmacological properties: C. cassia, antimicrobial ^[13,14], anticarcinogenic ^[15], insecticide ^[16], antipyretic ^[17], and stimulant of human linfocitary activity in vitro ^[18]; L. erythrorhizon, anticarcinogenic ^19], antioxidant ^[20]; M. toosedan, insecticide ^[21,22]; S. lappa, antiviral ^[23], antiinflammatory ^[24], and gastroprotective ^[25]. To our knowledge, this is the first time that these 17 species are tested against T. cruzi in vitro, and the results obtained concerning a high inhibitory rate for L. erythrorhizon, S. lappa, M. toosedan and C. cassia, and the